International Microbiology

Volume 20 路 Number 3 路 September 2017 路 ISSN 1139-6709 路 e-ISSN 1618-1905

INTERNATIONAL MICROBIOLOGY www.im.microbios.org

20(3) 2017

Official journal of the Spanish Society for Microbiology

Publication Board

Editorial Board

Editor in Chief José Berenguer, Universidad Autónoma de Madrid

Ricardo Amils, Universidad Autónoma de Madrid, Madrid, Spain Albert Bordons, Rovira i Virgili University, Tarragona, Spain Albert Bosch, University of Barcelona, Barcelona, Spain Josep Casadesús, University of Seville, Sevilla, Spain Yehuda Cohen, The Hebrew University of Jerusalem, Jerusalem, Israel Rita R. Colwell, Universityof Maryland & Johns Hopkins University, MD, USA Katerina Demnerova, Inst. of Chem. Technology, Prague, Czech Republic Esteban Domingo, CBM, CSIC-UAM, Cantoblanco, Spain Mariano Esteban, Natl.Center for Biotechnology, CSIC, Cantoblanco, Spain Steven D. Goodwin, University of Massachussets- Amherst, MA, USA Juan C. Gutiérrez, Complutense University of Madrid, Madrid, Spain Juan Imperial, Technical University of Madrid, Madrid, Spain John L. Ingraham, University of California-Davis, CA, USA Juan Iriberri, University of the Basque Country, Bilbao, Spain Roberto Kolter, Harvard Medical School, Boston, MA, USA Michael T.Madigan, Southern Illinois University, Carbondale, IL, USA Beatriz S. Méndez, University of Buenos Aires, Argentina Diego A. Moreno, Universidad Politécnica de Madrid, Madrid, Spain Ignacio Moriyón, University of Navarra, Pamplona, Spain Juan A. Ordónez, Complutense University of Madrid, Madrid, Spain José M. Peinado, Complutense University of Madrid, Madrid, Spain Antonio G. Pisabarro, Public University of Navarra, Pamplona, Spain Carmina Rodríguez, Complutense University of Madrid, Madrid, Spain James A. Shapiro, University of Chicago, IL, USA John Stolz, Duquesne University, Pittsburgh, PA, USA James Strick, Franklin & Marshall College, Lancaster, PA, USA Gary A.Toranzos, University of Puerto Rico, San Juan, Puerto Rico Antonio Torres, University of Seville, Sevilla, Spain José A. Vásquez-Boland, University of Edinburgh, Edinburgh, UK Antonio Ventosa, University of Seville, Sevilla, Spain Tomás G. Villa, University of Santiago de Compostela, Santiago de Compostela, Spain Miquel Viñas, University of Barcelona, Barcelona, Spain Dolors Xairó, Biomat S.A., Grifols Group, Parets del Vallès, Spain, Fernando Rojo, National Centro of Biotechnology, Madrid, Spain Juncal Garmendia, Institute of Agrobiotechnology, Pamplona, Spain Olga Genoilloud, MEDINA fundation, Granada, Spain.

Mariano Gacto Fernández, University of Murcia, Spain Carmen Ruiz Roldán, University of Córdoba, Spain Gemma Reguera (Michigan State U.) Beatriz Diez. Catholic University of Chile, Santiago de Chile, Chile

Associate Editor Diego Moreno, Universidad Politécnica de Madrid Guest Editor Felipe Cava, University of Umea Managing Editors Ana M. García, Universidad Politécnica de Madrid Digital Media Coordinator Andrés Núñez, Universidad Politécnica de Madrid Specialized Editors Josefa Antón, University of Alicante Susana Campoy, Universidad Autónoma de Barcelona Josep Guarro, University Rovira Virgili Enrique Herrero, University of Lleida Emili Montesinos, University of Girona José R. Penadés, University of Glagow Jordi Vila, University of Barcelona

Addresses Editorial Office C/Rodríguez San Pedro, 2, 210 28015 Madrid, Spain E-mail: international.microbiology@semicrobiología.org Spanish Society for Microbiology C/Rodríguez San Pedro, 2, 210 28015 Madrid, Spain Tel. +34-915613381; Fax: +34-915613299 E-mail: secretaria.sem@microbiologia.org http://www.semicrobiologia.org/ © 2017 Spanish Society for Microbiology, Madrid, & Institute for Catalan Studies, Barcelona. Printed in Spain ISSN (printed): 1139-6709 e-ISSN (electronic): 1618-1095 D.L.: B.23341-2004

The Spanish Society for Microbiology (SEM) is a scientific society founded in 1946 at the Jaime Ferrán Institute of the Spanish National Research Council (CSIC) in Madrid. Its main objectives are to foster basic and applied microbiology, promote international relationships, bring together the many professionals working in this science in Spain, and contribute to the dissemination of science in general and of microbiology in particular among society. It is and interdisciplinary society, with around 1800 members working in different fields of microbiology.

CONTENTS

Volume 20, Number 3, September 2017 Special number on the Biology of Vibrio cholerae Guest Editor: Felipe Cava

EDITORIAL Cava F Biology of Vibrio cholerae: Overview

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RESEARCH REVIEWS Islam MT, Alam M, Boucher Y Emergence, ecology and dispersal of the pandemic generating Vibrio cholerae lineage

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Yen M, Camilli A Mechanisms of the evolutionary arms race between Vibrio cholerae and Vibriophage clinical isolates

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Espinosa E, Barre F-X, Galli E Coordination between replication, segregation and cell division in multi-chromosomal bacteria: lessons from Vibrio cholerae

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Kostiuk B, Unterweger D, Provenzano D, Pukatzki S T6SS intraspecific competition orchestrates Vibrio cholerae genotypic diversity

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Escudero JA, Mazel D Genomic Plasticity of Vibrio cholerae

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Cava F Divergent functional roles of D-amino acids secreted by Vibrio cholerae

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Journal Citations Reports 5-year Impact Factor of International Microbiology is 2,17.

The journal is covered in several leading abstracting and indexing databases, including the following ones: Agricultural & Environmental Biotechnology Abstracts; ASFA/Aquatic Sciences & Fisheries Abstracts; BIOSIS; CAB Abstracts; Chemical Abstracts; SCOPUS; Current Contents/Agriculture, Biology & Environmental Sciences; EBSCO; EMBASE/Elsevier Bibliographic Databases; Food Science & Technology Abstracts; ICYT/CINDOC; IBECS/BNCS; ISI Alerting Services; MEDLINE/ Invdex Medicus; Latindex; MedBio World; PubMed; SciELO-Spain; Science Citation Index Expanded; SciSearch.

Front cover legends

Center. Experimental avocado plot number 16, allocated at the Experimental Station “La Mayora”, The Institute for Mediterranean and Subtropical Horticulture “La Mayora” (IHSM-UMA-CSIC; Málaga, Spain). At the left, a 40 years-old healthy avocado tree. At the right, an avocado tree with typical symptoms of avocado white root rot caused

by Rosellinia necatrix. Photograph by Francisco M. Cazorla.

Upper left. Transmission electron micrograph of the marine phage H1 negatively stained. Phage was isolated from a strain of Pseudoalteromonas sp. from the Blanes Bay Microbial Observatory station (BBMO), a surface coastal site in the NW Mediterranean. Photo by Elena Lara, Marine Sciences Institute (ICM-CSIC) (Magnification, 330,000×) Upper right. Transmission electron micrograph of Sphingobacterium detergens during the process of cellular division. The bacterium was isolated from a soil sample from the Azorean Islands and was selected for its ability to reduce the surface tension of the culture medium. Photo by Ana M. Marqués and César Burgos-Díaz, Faculty of Pharmacy and Food Sciences, University of Barcelona. (Magnification, 10,000×) Lower left. Dark field micrograph of several individuals of the ciliate Vorticella sp. detached from its peduncles. Note the big and active macronucleus with the shape of a long and bluish band.

Photo by Rubén Duro, Center for Microbiological Research, Barcelona. (Magnification, 1000×)

Lower right. Transmission electron micrograph of the plasmolized yeasts (“lias”) of Saccharomyces cerevisiae from the elaboration of sparkling wines according to the “cava” method after second fermentation in closed bottles. Photo by Montserrat Riu and Rebeca Tudela, Faculty of Pharmacy and Food Sciences, University of Barcelona. (Magnification, 10,000×)

Back cover: Pioneers in Microbiology A light in the fight against infectious diseases: Ferran’s cholera vaccine Doctor Jaime Ferrán y Clua was probably one of the most brilliant, brave and intuitive minds that, despite a very conservative and distrustful atmosphere, made remarkable advances in the history of medical and microbiological science. Jaime Ferrán was born in Corbera de Ebro (Cataluña, Spain) in 1851 and following in his father’s footsteps, studied medicine and worked as a physician, eventually becoming head of the City Hospital of Tortosa. Open-minded and empowered by great curiosity (he was a photography enthusiast, seeing it as a powerful tool to explore the beauty of nature), he embraced the early works of Louis Pasteur and quickly was attracted to the newborn field of bacteriology research. Starting a laboratory with chemist Inocente Paulí, he initiated an unexpected journey into microbiological research, specifically immunization against infectious diseases. Promptly, his research yielded the first vaccines in Spain against diverse epizootic diseases such as the red disease of pigs or carbuncle, and his Memory on bacterial parasitism was honored by the Real Academy of Medicine of Madrid. It was the end of the 19th century, a turbulent period for public health, when continuous outbreaks of cholera ravaged populations all over the world. Koch’s discovery of cholera’s etiological agent, thennamed Bacillus virgulans, sparked a race to develop efficient sanitation methods aimed at its eradication, attracting the attention of physicians, governments, and researchers including Pasteur and later Jaime Ferrán. Urged by fresh outbreaks of Asian cholera in Egypt spreading to the French coast, Spanish officials dispatched a commission including Jaime Ferrán to learn the way French researchers dealt with this fatal disease. Having thus acquired practice in isolation and identifica-

tion of the bacterium from infected patients, Jaime Ferrán returned to Spain to start development of a vaccine against cholera, based on the concept of microbial attenuation developed by Pasteur and Jenner’s smallpox vaccine. He observed that subcutaneous injection of attenuated B. virgulans isolated from cholera patients or propagated in vitro did not kill guinea pigs; rather they became protected against topical exposure of a lethal dose of the related virulent strain. Excited by this finding, he repeated the same “immunization” protocol on himself and later on other volunteers, which survived a lethal dose of the “bacillus Koch”. The effectiveness of his invention was illustrated in Alcira (Valencia, Spain) in 1885, where 50,000 civilians were successfully immunized against cholera, a milestone in the field of public health and prevention of infectious diseases. It is worth noting that Ferrán’s anti-cholera vaccination protocol was successfully employed to suppress a cholera epidemic in the armies fighting during the First World War. This remarkable deed in the history of medical science was initially controversial, but finally found acclaim in 1902 with the Breant Prize awarded by the Academy of Science of Paris. Dr. Ferrán is recognized as the first to describe lethality of the cholera bacillus, and to develop an immunization protocol in humans. His dedication to the fight against infectious diseases extended to rabies, diphtheria, tetanus, bubonic plague, and tuberculosis. Dr. Jaime Ferrán, warmly remembered as “El Cartujo de la Sagrera” because of his dedication and enthusiasm in the study of bacteriology, was a brave and intuitive scientist, a bright light in humankind’s fight against infectious diseases, and a world-class pioneer for all future immunization therapies.

Diego Romero Universidad de Málaga

EDITORIAL International Microbiology 20(3):105 (2017) doi:10.2436/20.1501.01.290. ISSN (print): 1139-6709. e-ISSN: 1618-1095 www.im.microbios.org

Biology of Vibrio cholerae Editorial overview Felipe Cava The laboratory for Molecular Infection Medicine Sweden (MIMS), Department of Molecular Biology. Umeå University.

90187. Umeå. Sweden. Ph: +46(0) 90 785 6755.

In this monographic issue, we have the pleasure to present contributions from six of the leading laboratories at the forefront of Vibrio cholerae genetics, ecology and evolution, together with a brief tribute by Diego Romero to Doctor Jaime Ferrán y Clua, a pioneering Spanish bacteriologist who developed the first vaccine against this pathogen.

V. cholerae is a free-living aquatic bacterium that interacts with and infects a variety of organisms. In humans it causes cholera, the deadly diarrhoea that was responsible for millions of deaths during seven pandemics since 1817, and still thousands every year. The Boucher lab presents a study of the ecology, evolution and dispersal of pandemic V. cholerae biotypes in relation to environmental reservoirs. They show how both species-specific and lineage-specific genetic determinants play a role in the ability of V. cholerae strains to cause pandemics, having evolved gradually over centuries.

One of the key aspects that makes a particularly successful pathogen is its genomic plasticity. The V. cholerae genome contains a superintegron (SI) that is involved in development and dissemination of antibiotic resistance genes among diverse bacterial species, permitting population expansion in challenging conditions. Escudero and Mazel review the SI as a true hotspot of V. cholerae’s genomic diversity and low-cost memory of adaptive functions in its complex lifestyle and ecology. Another remarkable aspect of V. cholerae’s genetics is the presence of two chromosomes. Segregation and division in multi-chromosomal becteria is relatively complex, and V. cholerae remains the paradigm. Espinosa and colleagues review the cell cycle of V. cholerae, comparing and contrasting with that of E. coli.

In addition to genome plasticity, V. cholerae uses a variety of attack/defence strategies to compete and thrive in different niches, through interaction with bacteriophages, bacteria and eukaryotes.

The role of phages in the life cycle of V. cholerae has been increasingly recognized and investigated over the past decade. Andrew Camilli and colleagues take us through the exciting evolutionary arms race between V. cholerae and virulent bacteriophages, based both on mechanisms of phage resistance in the bacterium and a unique phage-encoded CRISPR-Cas system used to counteract this resistance. Finally, the authors discuss the impact of these predator-prey dynamics in the context of infection, and their use as a strategy to limit cholera transmission within a community.

In regards to its ability to coexist with other microbes, V. cholerae can produce effectors that are either released to the extracellular media, or delivered via intimate cell-to-cell contact such as those injected via the type VI secretion system (T6SS).

The Pukatzki lab reviews the versatility of the T6SS to produce different combinations of such effectors, which establishes the strains of V. cholerae that can co-exist in the environment. After killing a cell, its DNA is released and incorporated by natural competence into other living cells, thereby being a potential source of diversification for V. cholerae’s T6SS effectors.

Finally, I revisisit the discovery of non-canonical D-amino acids, recently identified effectors secreted by V. cholerae which have increasingly been shown to be important in enhancing the ability of the bacterium to colonize and persist in a particular niche. I focus on recent observations that suggest different D-amino acids influence distinct cellular processes in bacteria, and discuss their role in modulating environmental bacterial biodiversity.

Felipe Cava, Guest Editor

* For correspondence: Felipe Cava, E-mail: felipe.cava@umu.se ORCID: 0000-0001-5995-718X

RESEARCH REVIEW International Microbiology 20(3):106-115 (2017) doi:10.2436/20.1501.01.291. ISSN (print): 1139-6709. e-ISSN: 1618-1095 www.im.microbios.org

Emergence, ecology and dispersal of the pandemic generating Vibrio cholerae lineage Mohammad Tarequl Islam1, Munirul Alam2 and Yan Boucher1* Department of Biological Sciences, University of Alberta, Edmonton, Canada 2 International Centre for Diarrheal Disease Research (icddr,b), Bangladesh

1

Received 20 September 2017 · Accepted 30 September 2017 Summary. Although cholera is an ancient disease that first arose at least half a millennium ago, it remains a major health threat globally. Its pandemic form is caused by strains from a single lineage of the bacterium Vibrio cholerae. The ancestor of this lineage harbored several distinctive characteristics, the most notable being the O1 antigen polysaccharide. This lineage generated two biotypes, first Classical, responsible for six pandemics, and later El Tor, responsible for the seventh and ongoing pandemic.

Just as El Tor replaced Classical as the main cause of outbreaks in the last fifty years, several variants of El Tor have evolved and displaced their predecessors worldwide. Understanding the ecology, evolution and dispersal of pandemic V. cholerae is central to studying this complex disease with environmental reservoirs. Here, we present recent advancements of our knowledge on the emergence and spread of the pandemic generating lineage of V. cholerae in the light of established eco-evolutionary observations.

Specific ecological interactions shape seasonal cholera, playing a role in the abundance and distribution of its causative agent.

Both species-specific and lineage-specific genetic determinants play a role in the ability of V. cholerae strains to cause pandemics with seasonal outbreaks, having evolved gradually over centuries. On the basis of the current understanding, we outline future threats and changes in biogeographical and genomic-based investigation strategies to combat this global problem.

Keywords: Vibrio cholerae · cholera · pandemic · ecology · evolution

Introduction Microorganisms causing human disease often have a complex dynamics of transmission, persistence and dispersion within their natural reservoirs [39]. Vibrio cholerae, the causative agent of cholera, is a unique model system to study the effect of such environment-human interactions in shaping a deadly infectious disease from aquatic origin [23]. Cholera has been endemic for centuries in many countries in South Asia and Africa, where it occurs almost every year, infecting people encountering the pathogen through consumption of untreated water [42]. According to World Health Organization (WHO) reports, there are roughly 1.3 to 4.0 million cases, and 21 000 to 143 000 deaths worldwide due to cholera every year (WHO cholera fact sheet, 2017). Cholera can also become pandemic and so * Corresponding author: Yan Boucher Phone: 7802481180 Email: yboucher@ualberta.ca

far, seven recorded pandemics of this disease have shaken the world since 1817, killing millions of people worldwide [42, 32]. Unlike some other pandemic infectious diseases, cholera is unlikely to be eradicated. This is because its causative agent is an autochthonous member of marine and estuarine ecosystems around the world, and there is no clear transmission vector to serve as a means to control its human association cycle [23, 49].

Two main hypotheses have been proposed to explain the global spread of pandemic cholera. The first is that human travel has spread the bacteria from endemic countries to other parts of the world [61]. An alternative hypothesis is that ocean currents and maritime transfer of the pathogenic bacteria are responsible for the spread of cholera, with climatic events (i.e. El Niño, global warming) playing a major role in shaping pandemics [22, 35, 40]. Regardless of how V. cholerae spreads across the world, occurrence of the disease is significantly influenced by living standard of people in cholera prone regions as well as climatic conditions. For example, in countries with compromised water supply and sanitation infrastructure, excessive rainfall and

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flooding can lead to massive cholera epidemics if pathogenic variants of Vibrio cholerae are present in the environment [42, 22,40]. Environmental links are evident in the dynamics of the disease, as cholera incidence in endemic areas usually show seasonal patterns, i.e. number of cholera cases increases and reaches a peak in specific months every year [33, 2].

In the environment, V. cholerae exists as a diverse species.

However, a very small portion of the heterogeneous population of V. cholerae strains found in nature is capable of causing human disease [42]. The outer membrane lipopolysaccharides of V. cholerae have a region named O-antigen, the synthesis of which is encoded by a diverse combination of genes, giving rise to the remarkable diversity of more than 200 serogroups (starting from O1) of V. cholerae observed in nature [20]. Although isolated infections and outbreaks have been caused by various V. cholerae genotypes, pandemics are only caused by strains from a single lineage, most of which display the O1 antigen on their surface (Figure 1). Understanding cholera pandemics is therefore dependent on determining ecological characteristics of this specific lineage, not necessarily the entire V. cholerae species. This lineage has usually been referred to as V. cholerae O1/O139, which is misleading as it includes several strains displaying other serogroups (such as O37), and that many unrelated harmless strains display the O1 and O139 antigens [20]. Here we will refer to the monophyletic phylogenetic group containing all genotypes responsible for cholera pandemics as the Pandemic Generating (PG) lineage to avoid confusion. The factors affecting environmental persistence, survival during inter-epidemic periods, emergence and spread of pathogenic genotypes from this lineage are still poorly understood. This is because population-level analysis over a wide range of geographical locations and variety of potential niches requires large-scale sampling of a bacterium forming only a small proportion (usually <1%) of natural populations. Most of our current knowledge on V. cholerae ecology has been obtained from cultivation-based studies. Despite their limited sampling size, these studies have created a solid foundation to develop culture-independent population-level approaches that will enable investigations of pandemic V. cholerae local and global dynamics.

Seasonality of cholera is mediated by the ecological interactions of V. cholerae V. cholerae has been detected in aquatic habitats from the tropics to temperate waters world-wide [50], underscoring it’s highly adaptable and persistent nature over a broad range of environmental conditions [64]. However, cholera incidence patterns can vary greatly among geographic locations.

It is firmly endemic in some South Asian countries, where it appears in distinct seasonal patterns [30]. Other regions, such as parts of South America and Africa, have historically had only sporadic epidemics of cholera [30]. In Bangladesh, cholera maintains an annual cycle with two infection peaks;

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before monsoon and just after monsoon [1, 32, 2, 3]. This marked seasonality of cholera appears to be closely linked with the changes in flora and fauna of the coastal environment where pathogenic V. cholerae exists, mediated by micro- and macro-level environmental factors such as water temperature, salinity, organic matter concentrations, abundance of planktonic surface and water consumption [23, 30] and ultimately influenced by larger-scale climatic variables [49, 30]. In the aquatic environment, V. cholerae are known to be associated with phytoplankton, zooplankton, chitinous animals, aquatic plants, protozoa, bivalves, fish and water birds [50]. These associations could serve as environmental reservoirs where the pathogen can live over time, with the potential to be disseminated and cause cholera outbreaks in nearby human populations [9, 68]. Many of these studies looked at the entire V. cholerae species, but not specifically the PG lineage. Although the ecological parameters leading to high abundance of PG V. cholerae are not well understood, it is clear that during the annual cholera epidemic periods, the conditions in the coastal ecosystems are ideal for the multiplication and transmission of these bacteria. As a result, these water sources contain high enough concentrations of PG V. cholerae to cause human disease upon consumption of contaminated water or food that has come in contact with it [64, 38]. Those peak months display blooms of phytoplankton, which provide food for zooplanktons, both potential resources for growth of V. cholerae in water [27, 25, 66, 30]. Studies have found significant correlations with seasonal bloom in aquatic microorganisms and cholera incidence rates in nearby human populations [30, 37, 49]. During inter-epidemic periods, PG V. cholerae remains mostly undetectable by routine microbiological culture-dependent assays based on growth of the targeted bacteria on selective culture media [7]. However, using culture-independent techniques, including direct fluorescence antibody (DFA) assays and PCR, PG V. cholerae can readily be detected in the water year round, indicating a survival strategy making them either rare, unable to grow under laboratory conditions, or concentrated in specific reservoirs (host, sediments, particles, etc.) [7]. Indeed, V. cholerae possesses the ability to switch into a viable but non-culturable (VBNC) or dormant state in response to nutrient deprivation or other stresses [21, 24].

In its VBNC state, the V. cholerae cells become coccoid (as opposed to their normal curved rod shape) and do not respond readily to typical microbiological medium, hence cannot be detected by culture-based surveillance. These non-culturable cells have been found to retain pathogenic ability upon passage through animal intestine [21, 7]. Such a dormant state could serve as a survival strategy in inter-epidemic months, as resuscitation could occur once conditions are favorable again during epidemic months [22, 7].

In aquatic habitats, a possible reservoir of V. cholerae is chitin-containing organisms. Chitin can serve as a source of energy, carbon, and nitrogen and a substrate for biofilm formation for this microorganism [14, 70, 50]. Biofilms provide a

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Fig. 1. Emergence and evolution of pandemic V. cholerae in a phylogenetic context. The maximum likelihood phylogenomic tree was constructed from the alignment of locally collinear blocks (2,784,396 bp) using GTR gamma substitution model with 100 bootstrap replicates (all nodes had >98% bootstrap support).

Environmental non-toxigenic V. cholerae O1 strain 12129 was used as the outgroup to root the tree. The source and isolation year of the strains used are indicated next to the strain names. The timeline shows important events on the path of evolution toward pandemic V. cholerae from environmental origin. Important genetic elements including virulence factors are denoted in solid rectangles whereas putative genetic events are denoted in dotted rectangles. Stadium boxes denote for the emergence or appearance of lineages or pandemics.

microenvironment favoring survival and persistence, displaying increased resistance to various stresses, facilitating success in both of V. cholerae ecological niches, namely aquatic habitats and the human body [14, 70]. Association with plankton as biofilms could lead to the persistence of the pathogen during inter-epidemic periods [23], possibly an important part of the seasonal cycle of cholera in endemic areas. The infectious dose required to cause human disease is quite high (~ 104 to 1011) for cholera, the bacterium needing to pass the acidic stomach to reach small intestine where the cholera toxin is effective [56].

The association of the bacterium with biotic or abiotic surfaces could lower the infectious dose, as indicated by the observation that ingestion of V. cholerae along with food products decreases the number required to cause infection [42, 9]. Biofilm forma-

tion can also simply increase the likelihood of ingesting a larger dose of V. cholerae, thus increasing chance of successful human infection [9]. Moreover, biofilm-derived V. cholerae was found to show hyper-infectious phenotypes, leading to a reduction of the infectious dose by orders of magnitude in contrast to the ingestion of planktonic cells [65].

These characteristics have particular significance in maintaining seasonal cycle of the bacteria in the aquatic environment, especially in cholera endemic areas. Most of the evidence for V. cholerae association with hosts in the environment remains anecdotal and no systematic study has been done so far. Moreover, studies of its presence in aquatic niches are rarely specific to the PG lineage, but look at the species as a whole, which is likely to display significant ecological vari-

EMERGENCE, ECOLOGY AND DISPERSAL OF V. CHOLERAE

ations below the species level. A culture-based study of a V. cholerae population in two connected water bodies at a single coastal location in northeastern USA, encompassing extensive sampling and phylogenetic analysis, revealed subspecies-level divergence between competing genotypes [46]. Clonal complexes, which are groups of closely related V. cholerae strains as defined by multi-locus sequence typing, showed distinct spatial distributions across adjacent water bodies and water column size fractions (free-living, small and large particle associated), indicating likely subspecies-level ecological differentiations [46]. If this finding is confirmed in other ecosystems, it is possible that pathogenic V. cholerae such as members of the PG lineage, differ from non-pathogenic strains in their ecological preferences. One possibility is that PG lineage strains could show significant association with zooplankton, helping to explain the seasonal cholera epidemics correlating with planktonic blooms. Such blooms could increase the number of bacteria in the water and trigger the epidemic cycle. It would also help explain why filtration of water with Sari cloth, which can trap larger particles and their associated microbes, can reduce the incidence of cholera [37].

The role of human hosts in the V. cholerae life cycle In inter-epidemic periods, strains with pathogenic potential are rarely isolated in environmental surveillance [7, 2]. It seems that during the initiation of seasonal cholera epidemics, there is an enrichment period for pathogenic V. cholerae in the combined niche of aquatic habitats and human body [33, 9]. During inter-epidemic periods, most of the V. cholerae cells in nature have been found to be in the VBNC state [22, 7]. Passages in animal models showing resuscitation of V. cholerae from the VBNC state suggest a potential advantage for cells able to survive in a human/animal host [21, 9]. Furthermore, production of cholera toxin, which causes massive diarrhea, aids rapid spread/dissemination of the bacteria into the nearby water in very high concentrations [56]. This rapid increase is likely to be important to outnumber competing microbes, predators and lytic phages in natural reservoirs. Secreted bacteria from infected humans can also be hyper-infectious, requiring fewer bacteria to cause subsequent infection, fostering the epidemic cycle [53, 56]. Positive effect of human association was also evident in regulation of the type VI secretion system (T6SS), which has roles in competitive fitness of the bacteria in both the human host and the environment. Pandemic type V. cholerae strains were found to activate T6SS only inside the small intestine of animal host and not in vitro conditions [12]. Human association can also contribute to the survival and persistence of V. cholerae in the environment [48]. It was found that the transfer rate of CTXĎ&#x2020; genetic element in V. cholerae was higher within the mice gastrointestinal tract than under laboratory conditions [69]. Baharoglu et al. have shown that the SOS response in V.

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cholerae, triggered by pH changes, oxidative stress or exposure to DNA damaging antibiotics, can increase rate of gene cassette insertion in integrons, a gene capture element with the potential to acquire new advantageous genes [13]. The human intestine is an environment allowing for extensive interaction of diverse microorganisms at high densities, and could serve to induce acquisition of virulence and adaptive genes in V. cholerae. These observations make the human gastrointestinal tract a possible niche/hotspot for the exchange of crucial virulence or other advantageous gene clusters, which might have a significant role in the evolution of pathogenicity within V. cholerae populations.

Human hosts also serve as a vehicle to transfer the bacterium into new places through various forms of transportation.

Asymptomatic carriers are of special interest in this case [56], as they could play an important role in transporting pathogenic V. cholerae to a new habitat [45]. Asymptomatic carriage was proposed to have role in initiating the recent cholera epidemic in Haiti [58], where pandemic V. cholerae were introduced by UN troops originating from a cholera endemic area [43, 57].

Asymptomatic infections are mild enough to go undetected and estimates of the ratio of asymptomatic to symptomatic infections have ranged from 3:1 to 100:1 [45]. Asymptomatic carriers usually posses a certain level of immunity to the disease, can be physically healthy individuals and thus travel anywhere unnoticed and shed approximately 103Â bacteria per gram of stool [56]. Shed pathogenic V. cholerae can grow in numbers in the nearby environment if conditions are ideal; a scenario that would be consistent with the Haiti epidemic [40]. In areas where people do not have immunity to cholera at sufficient levels or at all (such as Haiti), disease can spread rapidly. Understanding the role of asymptomatic infections in the dynamics of endemic and epidemic cholera requires detailed investigation of a large number of people showing no symptoms, which as so far proved elusive.

Genetic factors influencing the dual stage life cycle of pandemic V. cholerae Pandemic V. cholerae have both environmental and human stages in their life cycle in cholera endemic areas [56]. To maintain a successful seasonal epidemic cycle, these V. cholerae strains need to adapt with two different competitive niches. The ability of V. cholerae to survive in two drastically different niches is largely due to their inherent and/or acquired resistance to environmental shifts [50]. As a species, V. cholerae has certain attributes which makes it predisposed to survive in the human body. This includes the ability to live in freshwater, grow well at human body temperature, utilize human intestinal biopolymers with aquatic analogs, form biofilms, resist acidic passage in the stomach and evade the host immune system [9, 16]. In addition to these inherent abilities of the species, pandemic Vibrio cholerae harbor several genetic elements directly contributing

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to virulence. These include the two major virulence factors cholera toxin (CT) and the toxin co-regulated pilus (TCP), as well as other genes thought to be associated with the infection process in humans, such as repeat in toxin (RTX), mannose sensitive hemagglutinin pillin (mshA), pillin gene (pilE), hemolysin gene (hlyA), and sialic acid degradation gene (nanH) [42, 32]. However, the exact role of all of these genes in the infection process is not clear. The gene set crucial for providing competitive advantage in environmental survival and human to environment transition to the pandemic V. cholerae are also not fully understood. However, regulation of virulence and fitness related genes are critical for the long-term viability of the bacterium in humans. A recent study using transposon mutagenesis combined with massively parallel sequencing (Tn-seq) revealed 133 genes including 76 genes previously unknown for having any role in human infection, as contributing to survival of pandemic V. cholerae O1 in the infant rabbit model [41]. When dissemination from host into the environment were studied, 165 genes were found to be important for survival in pond water, including genes having known or hypothetical roles in energy production and conservation, cell wall and outer membrane biogenesis, electron transport, flagellar biosynthesis, transcriptional regulation and transportation [41]. Fu et al. identified 400 genes potentially critical for the fitness of V. cholerae in the infant rabbit intestine [34]. Among these, genes for encoding outer membrane porin ompU were found important for the fitness of the bacterium inside the host and genes for glycogen utilization and storage were shown to be crucial for dissemination, survival and persistence of host released V. cholerae into the environment [41, 34, 18, 26]. Shapiro et al. also found evidence for allelic differentiations in ompU, linked to virulence and environmental survival; where there seems to be a trade-off between human adapted and environment specific roles of the gene among the V. cholerae populations [63]. One genetic system, which is believed to be significant in survival and fitness of pandemic V. cholerae in both human and environmental stages, is the type VI secretion system (T6SS) [59].

V. cholerae has been shown to contain three gene clusters, each harboring different combinations of effector–immunity proteins. In each effector-immunity module type, an effector gene encodes a protein that can kill other surrounding bacteria, and their corresponding immunity gene encodes a protein protecting the bacteria from their matching effector [67, 47].

Killing of non-compatible cells by T6SS might serve as source of readily available food and DNA from other bacteria with potentially beneficial or protective functions. In environments where V. cholerae deals with low nutrient conditions, type VI mediated killing could generate supplementary source of nutrients to maintain their physiological activities [59]. In the human intestine however, type VI mediated killing ability could give a selective advantage to a particular strain competing with the commensal host flora. It was found that mucin in human intestine can activate T6SS system in pandemic V. cholerae, whereas bile acids further modulate its activity [12]. After excretion with

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diarrheal stool, V. cholerae with activated T6SS are potentially better equipped to fight against bacterial and eukaryotic predators in the aquatic environment [54]. Thus, T6SS might give significant advantage to pandemic strains inside the human host as well in the environment by outcompeting others for the colonization of a desired niche and providing energetic benefits from lysed cells upon entry into and before exiting the human host [59].

All known PG lineage V. cholerae strains consistently possess at least three genomic islands which are not shared with all other V. cholerae: CTXφ, Vibrio Pathogenicity Island I (VPI1) and Vibrio Pathogenicity Island 2 (VPI2) [32, 20] (Figure. 1). Maintenance of these genetic elements and coexistence in the same genome can be crucial for the disease-causing ability. They are part of a virulence gene repertoire that has been acquired progressively over centuries by horizontal gene transfer (Figure. 1) [20]. Acquisition of these factors can give advantage to PG lineage V. cholerae over benign environmental strains in surviving and exploiting the human gut as an ecological niche (Fig. 1). The toxin co-regulated pilus (TCP), one of the two main virulence factors of pandemic V. cholerae, is encoded within the horizontally acquired VPI1 [42] and serves as the essential colonization factor and receptor for the CTXφ phage, which carries the second major virulence factor, cholera toxin (CT). Phylogenetic analysis suggests that VPI1 was acquired long before CTXφ by the ancestors of modern pandemic V. cholerae, making them capable of integrating CTXφ in their genome to become cholera causing agents (Figure 1).

Beside TCP, VPI1 also encodes metallo protease TagA, which can breakdown mucin glycoproteins, and cell-surface glycans, making them available as a source of nutrients for the bacterium [59]. Saccharides, mucins and the glycocalix on the surface of human gut epithelial cells provide energy sources necessary for the growth and multiplication of the bacterium during the early stages of infection [59]. VPI-2 encodes several genes for sialic acid transport and catabolism, some of which were found to provide V. cholerae with a competitive advantage against other bacteria in the mouse gut [9]. Two genomic islands present in 7th pandemic clade but mostly absent in other clades within the PG lineage, Vibrio Seventh Pandemic Island 1 and 2 (VSP1 and VSP2), encode yet to be fully described but potentially important functions for the pathogenesis and survival of the lineage. For example, VSP1 encodes a transcription factor required for efficient colonization of human epithelial cells [53, 9]. All these genetic elements likely provided advantages in aquatic populations before giving a fitness benefit inside the human host, which is a secondary niche for V. cholerae [16].

The toxin co-regulated pilus encoded in VPI-1 was shown to be crucial for bacterial interactions required for biofilm differentiation on chitinaceous surfaces and thus likely to have important role in ecological fitness [60]. As TCP also serves as a receptor for CTXφ, its expression during the formation of biofilms also fosters CTXφ transduction and thus represents an ecological setting outside the host in which selection for a

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host colonization factor may take place [60]. We have already mentioned that T6SS can serve as a weapon in defense against predation by eukaryotic grazers or other competing bacteria in the aquatic environment [59]. Several studies have also found factors involved in pathogenesis to be expressed or required in association of the bacterium with algae, i.e. an increase in toxin production was observed in V. cholerae when in association with the green alga Rhizoclonium fontanum [38]. These findings support the idea that these pathogenicity factors have environmental functions and are useful for bacterial survival and persistence outside of the human host [9, 60].

Transcriptional profiling of V. cholerae secreted in stool from cholera patients revealed that genes involved in nutrient acquisition and motility were highly expressed whereas genes for chemotaxis were expressed at lower levels [41]. It appears that V. cholerae differentially regulates gene expression inside the human body and during passage to the environment, i.e. turns off expression of particular virulence genes as part of a program for dissemination to the environment [41, 53]. These changes in gene expression are thought to be linked to efficient exit from the host, re-entry to the aquatic environment and maintenance of an hyper-infectious state which enhances subsequent water borne spread of the cholera by lowering the infectious dose significantly [53]. When V. cholerae is shed into water, it is likely to encounter a drastic change in physiological conditions, i.e. drop in osmolarity, temperature and nutrient availability. V. cholerae transitioned into pond water was found to repress genes for protein synthesis and energy metabolism and induction of phosphate and nitrogen scavenging genes indicating a adaptive program in response to the low nutrient condition [56, 41]. The glycogen utilization and storage program was found to have a central role in this adaption to transition between to vastly different niches [41].

Although it is assumed that PG Vibrio cholerae survive inter-epidemic periods in aquatic reservoirs, they could potentially also reside in the human gut during that time. After ingesting PG V. cholerae at low concentrations from the environment, human carriers may not show disease symptoms but could still be colonized as asymptomatic carriers. These carriers could shed pathogenic clones into nearby water bodies, eventually facilitating the initiation of a seasonal epidemic [33]. Fullblown cholera can be considered helpful for bacterial dispersion in the environment in large numbers, cholera stool containing between 1010 and 1012 bacteria per litre [56]. This enables a particular type of bacteria to outnumber other types in the environment and with a continuous annual cycle, they get a selective advantage over other non-pathogenic type to sustain in an environment-human-environment life cycle. This kind of competitive advantage is not uncommon, which can explain how over time pathogenic V. cholerae becomes endemic within a population associated with a natural reservoir. But the ability to become a successful pandemic agent capable of going through a human infection and environmental survival cycle requires a constellation of virulence, regulatory and survival genes to be acquired and maintained for a long time. Environmental V. chol-

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erae had to gradually change over a long period of time with continued selective pressure for such a genetic combination to evolve. The basic genetic backbone, which made the evolution of pandemic variants possible, apparently evolved a single time in the ancestor of the PG lineage (Figure 1). Actual pandemic variants evolved twice independently within this lineage, giving rise to two major pandemic biotypes [20, 16].

The rise and spread of a deadly pathogen Lethal variants capable of causing pandemic cholera emerged twice independently from two branches of the pandemic generating lineage (PG), the Classical biotype (possibly in Asia between 1500 and 1800) and the El Tor biotype (Indonesia, between 1930 and 1960) [20, 55, 36, 61]. How, where and when did this PG lineage with the capacity to generate extremely virulent variants emerge from heterogeneous environmental V. cholerae populations?

Despite being a widespread aquatic bacterium, V. cholerae as a species has several characteristics that make it predisposed for survival in the human gut [9, 16]. These traits seemingly provide a basic genetic background that fortuitously makes survival in a human host more likely, but are not sufficient for V. cholerae to become a human pathogen, which requires virulence factors and other fitness genes to be added to this background and enhance their potential to cause human disease on a stable basis [42, 32, 41]. However, no lineage-specific genomic region or genes exclusively present in PGs but absent in environmental groups (EGs) were found in genome wide analysis [63]. Thus no particular gene or gene families could be linked to the emergence and evolution of the PG group. Hence, virulence adaptive polymorphism were proposed to play a vital role in the process, which implied that the environmental ancestor of the PGs had a particular genomic back-bone containing alleles of core genes that served as ‘preadaptation’ and enhanced its potential to give rise to the pandemic clones [63]. A proposed conceptual model states that a variety of virulence related genes circulate in a diverse, recombining environmental gene pool, which is maintained in the population through various biotic and abiotic selective pressures. Upon encountering a new ecological opportunity, such as human consumption or transient colonization of other animal hosts, proliferation and gradual expansion of the clones encoding an advantageous combination of vital genes for virulence and pandemicity is selected.

These pre-adapted lineages can then serve as progenitors to acquire crucial virulence factors either in the environment or inside human body to mediate the emergence of pandemic V. cholerae [63].

Pandemic causing V. cholerae appears to have the optimized genetic systems to maintain a dual stage life cycle as opposed to most of their benign environmental counterparts. Epidemiological and genetic data suggests that only members of the PG lineage have been successful in evolving and maintaining these

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adaptive traits [20]. Sporadic cholera cases are caused by V. cholerae strains outside this lineage throughout the world [42, 32, 20], but none of them could be established as a long-term etiological agent to cause consistent seasonal cholera episodes.

This complex capability is unlikely to be created only in a single lineage without a consistent selection pressure over an extended period of time. This kind of evolutionary drive is most likely to have happened in Ganges delta, which has been endemic for cholera in at least the last three centuries and represents a unique ecosystem for V. cholerae [16]. It has been proposed that extensive contact between V. cholerae living in the coastal brackish waters and dense human population drinking from that water over centuries has created the circumstances for the emergence of pandemic lineage. Fecal-oral circulation of the bacterium in the local environmental reservoirs could have led to the selection and enrichment of variants capable of thriving both in the human gut and the environment [16]. This hypothesis implies that longterm association with human host is the driving factor for the emergence of V. cholerae with pandemic capabilities. Recent phylogenomic data suggests that the currently ongoing seventh pandemic of cholera might have originated from Bay of Bengal and from there spread to other parts of the world in several waves [55]. Hu hypothesized that the Middle East and Indonesia played essential roles in the evolution of seventh pandemic strains [36].

However, this hypothesis is based on the analysis of very few strains and remains highly speculative.

Even though this pandemic generating lineage, also termed phylocore genome (PG), is a distinct monophyletic group from an extremely diverse environmental pool, it can be divided into two main phylogenetic branches; PG1 and PG2 [20]. The PG1 branch contains strains of the El Tor biotype and the PG2 branch those of the Classical biotype. These biotypes differ from each other by certain phenotypic and molecular traits [62]. Strains of Classical biotype clade (PG2) are known to be responsible for the sixth and presumably the earlier pandemics, whereas strains from the El Tor clade (PG1) are the causative agent of the currently ongoing seventh pandemic of cholera starting in 1961 [62]. Classical biotype strains have not been isolated since the early nineties even from Southeast Asia, where they were last found and have thus been considered as completely outcompeted by the El Tor biotype both from clinical and environmental settings [62]. Expansion of the 7th pandemic has given rise to new variants of the prototype V. cholerae O1 El Tor regularly during the course of the pandemic. These variants include strains harboring Classical biotype features within an El Tor genetic backbone and/or other divergent genetic features including mutations in major virulence factors and hence are named atypical El Tor [62]. After the initial wave of the current pandemic spread prototype El Tor strains across the world, two additional waves spread the variants of El Tor strains, each wave mostly displacing bacteria from the preceding one and has been a feature of global cholera epidemiology [55].

The 7th pandemic of cholera struck South America in 1991 via the Peruvian coast and reached in Mexico the same year [5]. In

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2010, one of the most devastating cholera epidemics in history occurred in Haiti, killing thousands of people [35, 43]. Cholera has now set residency in the local environment of Mexico and Haiti, even though both the countries did not have any recorded cases in 100 years before the recent epidemics happened [6, 10, 8]. Cholera epidemics leading to endemicity of PG V. cholerae in the affected area have prompted extensive environmental sampling of natural waters in Mexico and Haiti. These have revealed remarkable diversity of pandemic-related V. cholerae for countries, which did not have any known history of cholera until recently [6, 11]. In Mexico, a recent series of retrospective studies have reported the discovery of Classical, prototype El Tor, atypical El Tor, and non-toxigenic O1 strains with some unusual genetic features in V. cholerae strains isolated from 1983 to 2008 [4, 5, 6]. Along with this surprising diversity, there was presence of strains grouping at the base of the PG lineage that led to Classical and El Tor biotype strains, hence candidates for being considered as previously undetected descendants of the ancestor of the two biotypes [19, 17] (Figure 1). In Haiti as well, where cholera cases could clearly be attributed to the atypical El Tor strains introduced from Nepal [43], presence of this divergent lineage (termed pandemic sister group) in the water was evident [10, 17]. Azarian et al. estimated the time for the divergence of this lineage from the common ancestor of pandemic V. cholerae around 1548 C.E. [10], long before the report of the first pandemic in 1817. These observations are consistent with the historic records suggesting that descendants of the V. cholerae PG lineage common ancestor have been globally distributed for centuries and that this dissemination happened long before the first recorded pandemic [15, 16]. Presence of V. cholerae strains belonging to the PG lineage but clearly distinct from the Classical and El Tor strains have been isolated sporadically from around the world over the last few decades, including some non-endemic regions i.e., US Gulf coast, Australia, Russia, Thailand and China [20, 36, 17] (Figure 1) and have been reported very recently from Haiti and Mexico [19, 17, 10, 44]. Presence of these non-pandemic members of the PG lineage in wide geographical locations underscores that genomic database of V. cholerae today is extremely biased by clinical isolates and large-scale environmental sampling over wide geographical areas is needed to get a better picture of the diversity and global distribution of the PG lineage. Even though these close relatives of pandemic causing strains in most case lack the main virulence factor CT, they harbour TCP, which can act as the receptor for CT. The rest of their genetic backbone is also very similar to pandemic strains [19, 10, 17, 44]. Therefore, the possibility for the emergence of novel V. cholerae with pandemic potential from this globally spread lineage cannot be discounted.

Combating cholera epidemics in the future Pathogenic bacteria with environmental reservoirs like PG V. cholerae, which has to survive in both host and environmental

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conditions, need to maintain a delicate balance between two very contrasting life styles. The drastic transition from environment to human and vice versa requires adaptations for both human body and the aquatic environment. The currently ongoing 7th pandemic is the longest in duration and largest in geographical span. During the course of this pandemic, cholera has struck countries in virtually every continent except Antarctica and has even become endemic in countries other than Asia and Africa, surviving in those geographic settings successfully and causing regular cholera outbreaks [6, 11, 8]. El Tor biotype strains are known to have better survival in the environment than Classical biotype strains [32], whereas the Classical type toxin is found to cause more severe cholera than the El Tor type [42]. Currently found variants of prototype El Tor strains possess Classical type toxin in the El Tor genetic backbone, which is likely to make them more potent pandemic causing agents. 7th pandemic isolates contain two genomic islands, VSP-1 and VSP-2, which were not found consistently in other lineages. Even though exact function of these elements is not well understood, VSP1 encodes a transcription factor, which was shown to be required for efficient intestinal colonization [53]. Their consistent and exclusive presence in current 7th pandemic isolates implies that they might well have significant roles in environmental fitness and pathogenic capabilities [9]. From 1992 and onwards, most El Tor strains have been found to harbor a integrative conjugative element called SXT, which is known to serve as hotspot for acquisition of genes including resistance to certain antimicrobials and environmental persistence [55]. Acquisition of antimicrobial resistance can be crucial for the success of the modern El Tor strains as a long lasting disease-causing agent. The high fitness of the currently circulating strains might have selected for traits constraining their evolution. Most of the 7th pandemic clinical V. cholerae strains isolated since 2000, including strains causing epidemic cholera in Haiti, were found to harbor an integrative conjugative element (ICE) containing a gene encoding an endonuclease which inhibits the uptake of foreign genetic elements [29]. Studies have also observed remarkably lower recombination rate in 7th pandemic strains in comparison to other lineages supporting that idea [36].

These observations suggest that the 7th pandemic of cholera is likely to continue in the near future. New atypical variants of the El Tor biotype are likely to emerge and could trigger new waves of the pandemic. In 1992, V. cholerae O139 emerged in the Ganges delta region and caused severe cholera outbreaks in various parts of Asia and was even suggested as a possible “Eight pandemic of cholera” by some investigators [42, 32, 62]. Even though serogroup O139 became rare since 2005, it is still being isolated sporadically from environmental and clinical samples [2, 62]. Of concern is also the possibility that a novel pandemic biotype, separate from El Tor or Classical but still belonging to the PG lineage, would emerge. As the PG lineage has already generated two pandemic biotypes independently, a third one is a real threat, especially given ongoing global warming and rapidly changing climatic conditions. In ideal transmission and dissemination settings, these novel biotypes or variants

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of current pandemic biotypes can adapt to the environment and spread to non-cholera endemic regions via human or environmental carriers to cause cholera outbreaks on a global scale.

Ecological niche modeling taking current and future climatic condition in consideration has predicted a latitudinal increase in potential areas of V. cholerae distribution in the future [31].

Effective methodologies to predict cholera outbreaks one to several months in advance would make controlling cholera outbreaks much easier. It is presumed that V. cholerae was originally a marine bacterium that could persist in estuarine, coastal waters over a broad range of environmental conditions [23, 49].

In cholera endemic areas, water current, flooding and human activity might carry the bacteria inland, where it can adapt and survive [40, 1] to infect human populations drinking contaminated water. Over the last decades, studies have identified potential environmental variables associated with V. cholerae occurrence.

Ocean chlorophyll has been found to have the most consistent association with number of cholera cases in nearby populations and thus is thought to be a potential indicator for cholera outbreak prediction [28, 30]. However, prediction models for a complex and dynamic environmental disease like cholera would require more in-depth understanding of the ecology and biogeography of this pathogen, especially of its pandemic-generating lineage.

Concluding remarks In 2010, cholera killed more than 8000 people in Haiti, a country that did not have any recent cholera history [57]. War torn Yemen is currently facing the devastation of cholera, one of the worst outbreaks on record, with nearly 2000 deaths and around 500,000 suspected cholera cases as of August 2017 (http://www. who.int/mediacentre/ news/releases/2017/cholera-yemen-mark/ en/). The Haiti and Yemen episodes underscore the massive threat cholera poses even in this modern time, showing the need for more effective approaches to prevention and control of this deadly disease. Thus, a global scale coordination of biogeographical and genome based studies is warranted to improve prevention and management of future cholera epidemics.

Competing interests. Authors declare that no competing interests exist.

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RESEARCH REVIEW International Microbiology 20(3):116-120 (2017) doi:10.2436/20.1501.01.292. ISSN (print): 1139-6709. e-ISSN: 1618-1095 www.im.microbios.org

Mechanisms of the evolutionary arms race between Vibrio cholerae and Vibriophage clinical isolates Minmin Yen and Andrew Camilli* Department of Molecular Biology & Microbiology and Howard Hughes Medical Institute, Tufts University School of Medicine, Boston, MA 02111, USA Received 5 September 2017 · Accepted 30 September 2017 Summary.This review highlights recent findings on the evolutionary arms race between the causative agent of cholera Vibrio cholerae and virulent bacteriophages (phages) ICP1, ICP2, and ICP3 isolated from cholera patient stool samples. We discuss mechanisms of phage resistance such as a unique phage-inhibitory chromosomal island and mutations that affect phage receptor expression. We also discuss the molecular characterization of ICP1 and its unique CRISPR-Cas system, which it uses to combat the phage-inhibitory chromosomal island. The role of phages in the life cycle of V. cholerae has been increasingly recognized and investigated in the past decade. This article will review hypotheses as to how the predator-prey relationship may have an impact on infections within individuals and on the self-limiting nature of cholera epidemics. In addition, we put forth a strategy of using phages as an intervention to reduce household transmission of cholera within a community.

Keywords: Vibrio cholerae · Vibriophage · evolution · cholera

Introduction Bacteriophages, or phages for short, are found throughout the biosphere. There are an estimated 1031 phages in the world [18].

As viruses of bacteria, phages play an important role in the regulation of bacterial populations. Indeed, when phages were first discovered by Felix d’Herelle in 1917, he proposed that the predator-prey relationship may contribute to controlling the natural population of pathogens such as Vibrio cholerae, the causative agent of cholera [6]. Cholera is an acute gastrointestinal disease that is characterized by the rapid onset of vomiting and profuse, secretory diarrhea. It is caused by ingestion of water or food that has been contaminated with V. cholerae, a Gram-negative bacterium that resides in brackish coastal waters and estuaries [14]. Cholera is a significant burden on global health, particularly in developing countries where water sanitation services are not readily accessible. The World

*To whom correspondence may be addressed: andrew.camilli@tufts.edu

Health Organization estimates that there are approximately 2.8 million cases of cholera each year in endemic countries, which are predominantly in Africa and Asia [1].

Cholera is an ancient disease; descriptions in Sanskrit of cholera-like symptoms have been found and dated back to the 5th century BC. Sometimes referred to as “Asiatic cholera”, it has been endemic in South Asia, especially the Ganges delta region, since recorded history [32]. Naturally, populations of phages capable of infecting V. cholerae also are present in cholera-endemic regions. Before phages were identified, historical reports note that there were certain elements in the Ganges and Yamuna Rivers in India that can protect against cholera. In 1896, Ernest Hankin passed the water through fine porcelain filters and suggested that there was an unidentified substance in the filtrate that is responsible for killing V. cholerae. He further hypothesized that it perhaps plays a role in limiting the spread of cholera epidemics [13,33].

D’Herelle also identified phages from cholera patient stool samples during his work in the 1920s and used them to launch a phage therapy trial in India known as “The Bacteriophage Inquiry” [34]. Initial reports showed consistent observations

ARMS RACE BETWEEN V. CHOLERAE AND ITS VIBRIOPHAGES

that oral phage therapy resulted in reduced severity and duration of cholera symptoms as well as a decrease in mortality rates [8].

When phages were added to water wells, there were no further cases in villages that previously had cholera [7]. The sum of these results suggested that the introduced phage populations were capable of controlling the V. cholerae population in both clinical and environmental contexts.

Since then, there have been a number of cholera phage collections maintained globally. Phage typing has been used to identify V. cholerae strains and has contributed greatly to understanding cholera epidemiology. In 1968, Basu and Mukerjee developed a typing scheme using five groups of phages, allowing them to successfully identify 3,464 strains from different epidemics between 1937 and 1966 [2]. Additional updated phage collection schemes [3,4]. There are also a large number of cholera phages stored at the Eliava Institute in Tbilisi, Georgia, the majority of which were isolated from aquatic environments. A recent publication mentions that there are 71 phages collected from 2006 to 2009 alone [9]. Similar collections are maintained at institutions in Russia and China as well.

In recent years, there has been a renewed interest in cholera phages and their study using modern molecular methods [10,23,28]. At the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), Faruque et al. have isolated at least 18 cholera phages, known as the JSF series [12]. Our lab has isolated and sequenced the genomes of three distinct phages, referred to as the ICP phages, from 31 Bangladeshi clinical stool samples that span a 10-year period from 2001 to 2010 [28]. This review will discuss the co-evolutionary dynamics between V. cholerae and the ICP phages as well as implications for the role of phage in cholera epidemics.

The discovery of the ICP phages Through a collaboration with the ICDDR,B and Massachusetts General Hospital (MGH), a postdoctoral scholar in our lab at the time, Dr. Kimberley Seed, tested clinical samples for phage presence by plaque assay. Three novel, virulent phages were identified and designated as ICP1, ICP2, and ICP3 (Table 1).

ICP1 is specific for O1 serogroup V. cholerae; however, the host ranges for ICP2 and ICP3 are broader and include some non-O1 serogroup V. cholerae strains. Using primers specific for the DNA

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polymerase gene of each phage, Dr. Seed screened total DNA from the cholera patient stool samples by PCR to determine prevalence. While the presence of ICP2 and ICP3 were more sporadic, ICP1 was present in all stool samples tested [28].

Co-evolutionary dynamics between the ICP phages and V. cholerae V. cholerae has evolved multiple strategies to evade phage infection. For instance, Zahid et al. have demonstrated how down-regulation of cyclic AMP and the cyclic AMP receptor protein through mutations in the cyaA or crp genes, respectively, can confer resistance to multiple environmental cholera phages from the JSF series [36]. The work in our lab and that of Dr. Seed’s in her lab at the University of California, Berkeley has focused on understanding the co-evolutionary arms race between the ICP phages and V. cholerae through the use of comparative genomics and molecular biology approaches. A major theme that has emerged from these studies, described below, is that the ICP phages have evolved to use cell surface receptors that are critical virulence factors of V. cholerae, thus limiting the ability of V. cholerae to escape phage predation during infection of humans.

ICP1 and the O1 antigen receptor. To identify a mechanism for ICP1 resistance, V. cholerae spontaneous mutants that formed small colonies within otherwise clear zones of plaques were genome sequenced and compared to the sequence of the parent strain [29]. Single nucleotide deletions were identified in the poly-A tracts of two lipopolysaccharide (LPS) O-antigen genes wbeL and manA, which are responsible for tetronate and perosamine biosynthesis, respectively. These mutations, which shift the reading frame of the genes they reside in and cause premature termination, were sufficient to confer ICP1 resistance. In addition, purified LPS preparations from O1 V. cholerae strains were able to reduce ICP1 plaque formation, while those from non-O1 strains were not [28]. Taken together, these lines of evidence suggest that the receptor for ICP1 infection is the O1 antigen of V. cholerae LPS. As in the example above, one mechanism to prevent phage infection is eliminating, altering, or reducing the amount of

Table 1. Genome characteristics of the sequenced ICP phages isolated from cholera patients at the ICDDR,B Phage

Taxonomic family

Genome size (bp)

G+C content (%)

No. of predicted CDSs

% CDSs similar to known proteins

ICP1

Myoviridae

125,956

37.1

230

12

ICP2

Podoviridae

49,675

42.7

73

19

ICP3

Podoviridae

39,162

42.9

54

48

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phage receptor on the bacterial surface. Bacteria can modify the availability of the receptor through phase variation, allowing for a heterogeneous population to ensure survival. The example above illustrates this phase variation mechanism, whereby V. cholerae undergoes slipped-strand mispairing in the wbeL and manA poly-A tracts to modify the availability of its O1 antigen.

The LPS O1 antigen is normally expressed constitutively and, for V. cholerae, is required for intestinal colonization and is therefore considered a major virulence factor [5]. Competition assays in the infant mouse model of V. cholerae colonization were performed to assess the ability of wbeL and manA mutants to colonize the small intestine. Both showed decreased fitness, with the wbeL mutant being the more severely compromised [29]. Therefore, under ICP1 predation, the V. cholerae population is forced to survive by shifting towards an attenuated virulence phenotype.

ICP1 and its CRISPR/Cas system.â&#x20AC;&#x201A; The consistent presence of ICP1 in the face of ongoing cholera epidemics in Bangladesh, however, implies that V. cholerae has evolved a mechanism to resist ICP1 infection while remaining virulent. A large fraction of V. cholerae clinical isolates from Bangladesh were found to encode an 18-kb phage-inhibitory chromosomal island, which is referred to as a phage-inducible chromosomal island-like element (PLE) [30]. The PLE is specifically activated by ICP1 infection and inhibits ICP1 replication. Activation includes excision from the chromosome and subsequent replication as an episome. PLEs can be horizontally transferred by natural transformation or by ICP1 transduction [24], the latter implying that PLE DNA is packaged into virions. This mechanism of phage resistance is akin to abortive infection, where the infected cell sacrifices itself to block phage reproduction, thus protecting the neighboring cells.

ICP1 has evolved a mechanism to overcome the PLE defense mechanism by encoding a CRISPR/Cas system. CRISPR/Cas systems are sequence-specific, adaptive immunity mechanisms typically used by bacteria and archaea to protect themselves from invading nucleic acids such as phage DNA. However, five ICP1 phages, which were isolated from cholera stool samples spanning multiple years at the ICDDR,B, encoded a CRISPR/ Cas system with spacers that were 100% identical to sequences found within the V. cholerae PLE. The ICP1 CRISPR/Cas is also capable of acquiring new spacers during the phage infection process, which confers specific targeting of new PLE sequences and the restoration of ICP1 replication. Therefore, ICP1 has successfully co-opted the use of a classically microbial adaptive immune system to allow for its own propagation within its host, a mechanism that has not previously been demonstrated in phages [30].

ICP2 and the OmpU receptor.â&#x20AC;&#x201A; We previously described ICP2 as a virulent phage found sporadically in cholera patient

MINMIN YEN AND ANDREW CAMILLI.

stool samples from Bangladesh. While testing for the presence of phages within Haitian cholera patient samples, our lab, in collaboration with others, identified one sample from 2013 with a high titer of phage [31]. Whole-genome sequencing revealed this phage to have 84% identity over 93% of its genome to an ICP2 isolate from Bangladesh in 2011. The Haitian ICP2 isolate is the first lytic phage reported to be associated with epidemic cholera in Haiti [31].

Most of the V. cholerae single colony isolates that were recovered from the same clinical sample as the Haitian ICP2 were resistant to its infection. By comparative genomics, we determined that the ICP2-resistant bacteria had mutations in the ompU gene, which encodes the major outer membrane porin OmpU. Using Western blotting, we determined that wild-type amounts of OmpU were present in the outer membranes of these mutants, but the mutations were sufficient to confer ICP2 resistance. The mutations were mapped onto a predicted membrane topology of OmpU and shown to lie within two outer loops, implying that they may disrupt the interaction between OmpU and ICP2 tail fibers [31]. OmpU expression is induced during infection where it plays a major role in resistance to organic acids [20], anionic detergents [27], bile [35], and antimicrobial peptides [19]. A number of assays were performed to determine whether the OmpU mutants were attenuated. No detectable reduction in fitness was observed in the presence of bile or when the mutants were competed with the wild type strain in pond water. There was a mild competitive defect for two of the mutants when passaged multiple times in LB medium, implying a mild defect in the context of rapid growth and replication [31]. This may explain why these ICP2-resistant OmpU variants have not become fixed in the V. cholerae population in Bangladesh or Haiti.

ICP2 and the ToxR major virulence gene regulator.â&#x20AC;&#x201A; Whole-genome sequencing also revealed several ICP2-resistant isolates from Bangladeshi cholera patient stool samples with null mutations in the toxR gene. ToxR is the direct transcriptional activator of a number of virulence factors, including OmpU. ICP2 sensitivity in the ToxR mutants was restored by expressing ompU in trans, indicating that ICP2 resistance is mediated through the reduced expression of OmpU. Competitions between each clinical ToxR mutant and its isogenic wildtype ToxR revertant strain were performed in the infant mouse model of cholera, and the null mutants were 100- to 1000-fold attenuated [31]. These results are consistent with the inability of these mutant ToxR proteins to activate downstream virulence genes reported by other labs [21,25], thereby the ICP2-resistant ToxR mutants would be attenuated for infection [15].

Role of virulent phages in cholera epidemics Seasonal variations of phage levels in the environment were discovered in Kolkata, India as early as 1930 [26]. In endem-

ARMS RACE BETWEEN V. CHOLERAE AND ITS VIBRIOPHAGES

ic settings, cholera epidemics are self-limiting in nature; it has been suggested that virulent phages play a role in modulating the course of epidemics [10,11,16,22,23]. Based on epidemiological data collected from Dhaka, Bangladesh, Faruque et al. developed a model suggesting that virulent phages can attenuate cholera epidemics [16]. This model, which is based on observations that the rise of V. cholerae in the environment at the peak of epidemics is usually followed by a rise of cholera phages, suggests that the bloom of cholera phages reduces the V. cholerae population to the point that the epidemic peters out. A critical assumption of this model is that the initial drop in cholera cases should coincide with high levels of ambient phage. This assumption has been challenged by King et al., as the data show that the number of cholera cases began to decline while the phage numbers were still low [17]. Doubtless, phage predation plays a critical role within patients during the course of cholera infection as well as in its transmission to others or to the environment. More detailed clinical data are needed, however, before a causal role for phages in limiting cholera epidemics can be drawn.

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Conclusions In this review, we have discussed recent literature regarding the arms race between V. cholerae and its phages in the context of infection. Adaptations to phage predation are shown to involve trade-offs in fitness, which can impact virulence, transmission, and seeding of environmental reservoirs. By further understanding and characterizing the molecular mechanisms of these predator-prey relationships, we envision using phages as a rapid-acting intervention for at-risk populations, such as household contacts of cholera patients, to immediately protect against contracting the disease themselves. In this manner, phage prophylaxis can represent a fast and specific tool to reduce the burden of bacterial infections on global health.

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Competing interests. Authors declare that no competing interests exist.

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Indian Medical Research Memoirs 14 Elbakidze T, Kokashvili T, Janelidze N, Porchkhidze K, Koberidze T, Tediashvili M (2015) Biological characterization of Vibrio cholerae-specific bacteriophages isolated from water sources in Georgia. Georgian Medical News 240: 65-72 Faruque SM, Naser IB, Islam MJ, Faruque AS, Ghosh AN, Nair GB, Sack DA, Mekalanos JJ (2005) Seasonal epidemics of cholera inversely correlate with the prevalence of environmental cholera phages. Proc Natl Acad Sci USA 102: 1702-1707 Faruque SM, Islam MJ, Ahmad QS, Faruque AS, Sack DA, Nair GB, Mekalanos JJ (2005) Self-limiting nature of seasonal cholera epidemics: Role of host-mediated amplification of phage. Proc Natl Acad Sci USA 102: 6119-6124 Faruque SM (2014) Role of phages in the epidemiology of cholera. In Cholera Outbreaks, Nair GB, Takeda Y (Ed). Springer.

Hankin EH (1986) L’action bactericide des eaux de la Jumna et du Gange sur le vibrion du cholera. Ann Inst Pasteur 10: 511-523 Harris JB, LaRocque RC, Qadri F, Ryan ET, Calderwood SB (2012) Cholera. The Lancet 379: 2466-2476 Herrington DA, Hall RH, Losonsky G, Mekalanos JJ, Taylor RK, Levine MM (1988) Toxin, toxin-coregulated pili, and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans. J Exp Med 168: 1487-1492 Jensen MA, Faruque SM, Mekalanos JJ, Levin BR (2006) Modeling the role of bacteriophage in the control of cholera outbreaks. Proc Natl Acad Sci USA 103: 4652-4657 King AA, Ionides EL, Pascual M, Bouma MJ (2008) Inapparent infections and cholera dynamics. Nature 454: 877-880 Kutter E, Sulakvelidze A (2005) Introduction. In Bacteriophages: Biology and Applications, Kutter A, Sulakvelidze A (Ed). CRC Press.

Mathur J, Waldor MK (2004) The Vibrio cholerae ToxR-regulated porin OmpU confers resistance to antimicrobial peptides. Infect Immun 72: 3577-3583 Merrell DS, Bailey C, Kaper JB, Camilli A (2001) The ToxR-mediated organic acid tolerance response of Vibrio cholerae requires OmpU. J Bacteriol 183: 2746-2754 Morgan SJ, Felek S, Gadwal S, Koropatkin NM, Perry JW, Bryson AB, Krukonis ES (2011) The two faces of ToxR: Activator of ompU, co–regulator of toxT in Vibrio cholerae. Mol Microbiol 81: 113-128 Nelson EJ, Harris JB, Morris JG Jr, Calderwood SB, Camilli A (2009) Cholera transmission: The host, pathogen and bacteriophage dynamic.

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RESEARCH REVIEW International Microbiology 20(3):121-129 (2017) doi:10.2436/20.1501.01.293. ISSN (print): 1139-6709. e-ISSN: 1618-1095 www.im.microbios.org

Coordination between replication, segregation and cell division in multi-chromosomal bacteria: lessons from Vibrio cholerae Elena Espinosa1, François-Xavier Barre1, * and Elisa Galli1, * 1

Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CNRS, Université Paris Sud, 1 avenue de la Terrasse, 91198 Gif sur Yvette, France Received 10 September 2017 · Accepted 30 September 2017

Summary. Bacteria display a highly flexible cell cycle in which cell division can be temporally disconnected from the replication/segregation cycle of their genome. The accuracy of genetic transmission is enforced by restricting the assembly of the cell division apparatus to the low DNA-density zones that develop between the regularly spaced nucleoids originating from the concurrent replication and segregation of genomic DNA. In most bacteria, the process is simplified because the genome is encoded on a single chromosome. This is notably the case in Escherichia coli, the most well studied bacterial model organism. However, ~10% of bacteria have domesticated horizontally acquired mega-plasmids into extra-numerous chromosomes. Most of our current knowledge on the cell cycle regulation of multi-chromosomal species derives from the study of replication, segregation and cell division in Vibrio cholerae, the agent of the deadly epidemic human diarrheal disease cholera. A nicety of this model is that it is closely related to E. coli in the phylogenetic tree of bacteria. Here, we review recent findings on the V. cholerae cell cycle in the context of what was previously known on the E. coli cell cycle.

Keywords: Vibrio cholerae · DNA replication · chromosome segregation · cell division

Introduction During vegetative proliferation, cell division must be coordinated with the duplication of genomic DNA and its equal repartition in opposite cell halves to avoid the formation of non-viable cells. In eukaryotes, it is achieved by coupling the formation of the division apparatus, the divisome, to the activity of the segregation machinery, the mitotic spindle, whose assembly is itself delayed to the end of replication by a checkpoint mechanism. In contrast, cell division can be disconnected from replication and segregation in bacteria, which can multiply faster than the time it takes to replicate their genome by running multiple replication cycles in parallel and can live as and/or transiently form polyploid filamentous cells as an adaptation to their environment. How such flexibility is achieved without putting in jeopardy the accuracy of genetic transmission is To whom correspondence should be sent: francois-xavier.barre@i2bc.paris-saclay.fr

*

linked to two features of the bacterial cell cycle. First, segregation of newly replicated DNA is progressive and concurrent with replication. Bacterial chromosomes carry a single origin of bidirectional replication, which defines two replication arms.

As replication progresses along the two arms, newly replicated loci rapidly segregate to opposite cell halves [6]. Second, the cellular arrangement of genomic DNA directly controls cell division. The genome of bacteria forms a nucleus-like region within cells, the nucleoid. A process termed nucleoid occlusion impedes divisome assembly over the bulk of the nucleoid, thus restricting cell division to the low DNA-density zone that develops between newly forming nucleoids during each replication/ segregation round [1].

As most bacteria harbour a single chromosome, nucleoids normally correspond to the territory occupied by individual chromosomes, as illustrated by studies in the 3 major bacterial models, Escherichia coli, Bacillus subtilis and Caulobacter crescentus. However, the genome of ~10% of sequenced bacteria is divided on multiple chromosomes, raising questions on

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the mechanism coordinating cell division to the replication/ segregation cycle of each of their chromosomes [18]. Several multi-chromosomal species are under scrutiny, including Burkholderia, Rhyzobium, Rhodobacter and Brucella. However, most of our current knowledge on the cell cycle regulation of multi-chromosomal species mainly derives from the study of replication, segregation and cell division in Vibrio cholerae, the agent of the deadly epidemic human diarrheal disease cholera.

some (Figure 1, [7,36]). A notable difference is the presence of a polar organizing factor, HubP, which directs the action of a partition machinery, ParAB1 [61](Figure 1). In addition, V. cholerae Chr1 carries a gene encoding for DciA, the primordial loader/activity regulator of the replication helicase, which was replaced by DnaC in E. coli [8] (Figure 1). In agreement with its plasmid origin, the 1.07 Mbp secondary chromosome of V. cholerae, Chr2, carries genes dedicated to its sole replication and segregation, rctB and parAB2, respectively (Figure 1).

The V. cholerae model Coordination of Chr1 and Chr2 replication

V. cholerae belongs to the Vibrionaceae family, a large family of fresh and salt water Îł-proteobacteria, which includes most of the bioluminescent bacteria, many sea animal symbionts, and many human and sea animal pathogens [10]. All of the species within the family carry two circular chromosomes of uneven size [30,47,63]. The largest of these is called primary chromosome because it carries almost all of the essential genes of the cell [26] and because its replication origin and partition machinery group with the replication origin and partition machinery of mono-chromosomal Îł-proteobacteria [64]. On the contrary, the smallest chromosome is called secondary chromosome because it only carries a few essential genes [26] and because its replication origin and partition machinery group with those of plasmids [64]. In other bacterial families, extra-numerous bacterial chromosomes also harbour plasmid features and it is now largely admitted that they derive from the domestication of horizontally acquired mega-plasmids. One such domestication event might have participated to the evolutionary separation of the Vibrionaceae from the Enterobacteriales and to their expansion in aquatic environments.

V. cholerae first attracted the attention of research scientists because of its worldwide importance as a human pathogen.

However, it soon became a reference model for basic research on multi-chromosomal management because its 2.96 Mbp primary chromosome, Chr1, carries homologues of most (if not all) of the genes implicated in replication, chromosome organization and cell division in E. coli (Figure 1). In particular, it encodes homologues of the E. coli DNA adenosine methylation (dam) restriction-modification system, SeqA and MatP proteins, which together contribute to the regulation of replication initiation and the organization and segregation of E. coli chromo-

Both Chr1 and Chr2 carry a single origin of replication, oriC1 and oriC2, respectively. oriC1 is very similar in sequence to the origin of replication of the E. coli chromosome, oriC [19].

It contains an AT rich region flanked by five putative high affinity binding sites for DnaA (Figure 2A). E. coli DnaA is a weak ATPase. DnaA-ATP promotes the unwinding of the oriC AT-rich region by binding to lower affinity sites within oriC [15,16]. Like oriC, oriC1 also harbours a putative binding site for IHF, which stimulates the action of DnaA-ATP [15,16]. In E. coli, several mechanisms prevent over-initiation by regulating the quantity and/or availability of DnaA-ATP (Figure 2A): (i) DnaA represses its expression; (ii) there are >10-fold more DnaA binding sites in the E. coli chromosome than in the origin region, which titrate DnaA away from it; (iii) a protein that specifically binds to hemi-methylated GATC sites, SeqA, sequesters the low affinity DnaA binding sites present on oriC and in the dnaA promoter; (iv) a cluster of DnaA binding sites, datA, catalyses the conversion of active DnaA-ATP to inactive DnaA-ADP; (v) the ATPase activity of DnaA is stimulated when it encounters the replisome, which prevents DnaA activity during the elongation phase of DNA replication (RIDA for regulatory inactivation of DnaA); (vi) two chromosomal loci termed DnaA reactivating sequences (DARS) help to recharge DnaA with ATP, which permits to trigger overlapping rounds of replication in rapidly growing cells [43]. oriC1 can functionally replace oriC, suggesting that similar regulatory circuits probably operate on Chr1 [14], as confirmed by studies on the role of dam and SeqA [14,19].

In contrast, oriC2 is similar in structure to the iteron-based replication origin of large low copy number plasmids such as

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Fig. 1. Cell cycle effectors. The main effectors of DNA replication, chromosome segregation and cell division in E. coli and V. cholerae.

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F and P1 (Figure 2B). It harbours a single DnaA binding site.

Unwinding of its AT-rich region results from the binding of its own replication initiator, RctB, to short 11-12mers motifs (Figure 2B). RctB presents structural similarities to plasmid initiators [40]. Like iteron-based origins, it is inactivated by dimerization, which is counteracted by the action of chaperones [28,29]. However, several features distinguish the regulation of Chr2 replication initiation from plasmids. First, oriC2 harbours structurally different RctB binding sites to activate or repress initiation, 11-12mers and 29-39mers, respectively (Figure 2B).

RctB dimers mask the 11-12mers by directly bridging them to three 29-39mers [52]. RctB represses its production by binding to a 29-39mer within its gene promoter [53]. The action of the 29-39mer on the other end of oriC2 is inhibited by transcription from the promoter of a small RNA, rctA, and by the binding of ParB2 to an adjacent parS2 site [51,54]. ParB2 can also inhibit

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the action of the central 29-39mers by directly binding to it [51]. Second, dam is essential in V. cholerae because of its role in Chr2 replication [14,50]. The 11-12mers of oriC2 contain a dam methylation site (Figure 2A) and need to be fully methylated for efficient RctB binding [14]. In contrast, RctB binds to the 29-39mers repressors independently of dam. Thus, dam methylation serves to prevent Chr2 over-initiation in a similar way to how dam/SeqA prevents Chr1 over-initiation. Third, Chr2 replication initiation is coupled to the cell cycle, unlike F and P1 replication. It occurs when ⅔ of Chr1 have been replicated [41]. As Chr2 is only ⅓ of Chr1 in length, it leads to synchronous termination of replication of the 2 chromosomes [41]. This is due to a short intergenic sequence located on one arm of Chr1, crtS, whose duplication acts as a timer for Chr2 replication initiation (Figure 2A). Thus, crtS couples Chr2 replication initiation to the progress of Chr1 replication in a similar

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Fig. 2. Control of Chr1 and Chr2 replication initiation. A. Chromosome-like regulatory mechanisms. The left panel depicts the origin of replication of Chr1, oriC1, and the demonstrated (dam/SeqA) or putative (RIDA, datA, ATP and phospholipid synthesis, DARS) mechanisms controlling its unwinding. The role of dam and SeqA in Chr1 replication initiation was analysed. The controls exerted by RIDA, datA, ATP and phospholipid synthesis and DARS in E. coli were added on the basis that they should operate in V. cholerae since oriC1 can functionally replace the origin of replication of the E. coli chromosome, oriC. The origin of replication of Chr2, oriC2, is depicted on the right panel. Two chromosome-like regulatory mechanisms control its unwinding, dam methylation, which directly affects RctB binding to its 11-12mers binding site and crtS, which places Chr2 replication initiation under the control of Chr1 replication elongation. B. Plasmid-like regulatory mechanisms of Chr2 replication (right panel). A scheme of the origin of replication of P1 and of the mechanisms regulating its unwinding is shown on the left panel for comparison. Black arrow-head and T-head lines: initiation activating mechanisms; red arrow-head and T-head lines: initiation inhibitory mechanisms; grey circles: DnaA; grey pentagons: DnaA boxes; black circles: methylated GATC sites; red rectangles with curved angles: SeqA; jelly-fish shapes: phospholipids; yellow circles: IHF; yellow squares: IHF binding site; pink circle: ParB2; pink square: parS2; orange circles: RctB (top and bottom right panels) or RepA (bottom left panel); small orange arrow box: rctA; large orange arrow box: rctB or repA, as indicated; cyan arrow boxes: 11-12mers; dark blue diamond boxes: 29-39mers.

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way to how RIDA prevents re-initiation of Chr1 replication during the elongation phase [2,49]. The molecular mechanism of how crtS replication triggers initiation of Chr2 replication is still unknown. However, RctB directly binds crtS and, by analogy to DARS, might help convert inhibitory RctB dimers to active RctB monomers [2].

DciA probably controls the loading and release of the replicative helicase, DnaB, on either side of the origins of Chr1 and Chr2, to start bidirectional replication [8]. As Chr1 and Chr2 are circular, replication terminates with the merging of the opposite replication forks. Marker frequency analysis and GC-skew studies suggest that termination generally occurs in a region opposite to their origins, ter1 and ter2, respectively.

Cellular arrangement and choreography of segregation of Chr1 and Chr2 The organization of bacterial chromosomes can be stereotypically divided into two categories: a transversal arrangement, as described in slow-growing E. coli cells [38,56,58] and a longitudinal arrangement, as described in C. crescentus and in B. subtilis during sporulation [58,59]. In the transversal arrangement, the origin of replication is located at mid-cell and is flanked by the left and right arms of the chromosome in newborn cells, which creates a left-oriC-right pattern. In the longi-

tudinal arrangement, the origin and terminus of replication are located at opposite poles and the two chromosome arms reside beside each other along the long axis of newborn cells, which creates an oriC-ter pattern. During a division event, each of the two daughter cells inherits one of the pre-existing poles of the mother cell, the “old pole” and one of the two poles originating from the constriction event at the division site, the “new pole”.

In the oriC-ter configuration, the origin of replication is specifically located at the old pole and the terminus of replication at the new pole in newborn cells [42,58].

Despite the close relationship of V. cholerae and E. coli, Chr1 and Chr2 are both longitudinally arranged [35]. Systematic cytological analysis of multiple chromosomal loci showed that Chr1 covers the entirety of the cell, with oriC1 at the old pole and ter1 at the new pole, whereas Chr2 only resides in the younger half of the cell, with oriC2 at mid-cell and ter2 towards the new pole [11] (Figure 3).

Specific factors and protein complexes contribute to determine and to maintain the chromosomal organization during the entire cell cycle, from the beginning of the replication cycle to the end of the division cycle. In particular, the localization of specific chromosomal regions such as the origin and terminus of replication often rely on dedicated systems that control their segregation timing and positioning. In bacteria displaying an oriC-ter arrangement, the origin regions are segregated to opposite cell halves and maintained in proximity of the old poles by

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Figure 3 Schematic representations of Chr1 and Chr2 segregation and arrangement in V. cholerae. The segregation cycle and chromosome arrangement are depicted for V. cholerae Chr1 and Chr2, respectively in the left and right panel. Left panel: in newborn cells the origin of replication of Chr1, oriC1, is anchored

by HubP and the ParAB1-parS1 system to the old pole and the terminus of replication ter1 is kept in proximity of the new pole by MatP. During the cell cycle it is HubP the first factor transitioning towards the opposite pole, soon followed by ParAB1 and one sister copy of the newly replicated oriC1. While the replicated oriC1 copies are segregated in opposite cell halves, the ter1 region bound by MatP relocates to mid-cell where the newly duplicated ter1 regions remain together until the end of the cell cycle. Right panel: in newborn cells Chr2 occupies the younger half of the cell, its origin of replication, oriC2, is maintained at mid-cell by the ParAB2-parS2 system and the ter2 region close to the new cell pole by MatP. After duplication, in older pre-divisional cells, the oriC2 sister copies are segregated at the quarter positions by the parAB2-parS2 system and the movements of segregated ter2 sister copies are restricted around the division site by MatP.

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an origin-specific partition system [42,58]. Partition systems consist of a Walker-type ATPase, ParA, and a protein, ParB, which binds to specific cis-acting centromere-like sites, parS, located in the oriC region of bacterial chromosomes [42,58].

ParA interacts with ParB-parS complexes and drives one of the newly duplicated oriC copies towards the opposite cell pole, initiating the chromosome segregation cycle [58]. The extent to which Par systems contribute to the organization and segregation of oriC sister copies differs widely between bacteria.

Inactivation of the Par system leads to small defects in origin segregation and positioning during the vegetative growth of B. subtilis [57] whereas it is critical for chromosome partitioning in C. crescentus [46]. In V. cholerae, two distinct ParAB-parS systems, ParAB1-parS1 and ParAB2-parS2, drive the localization and segregation patterns of oriC1 and oriC2, respectively [21,62] (Figure 3).

ParAB1-parS1 imposes an asymmetric segregation process similar to that described for the origin of replication of the C. crescentus chromosome [11,21,55]. A transmembrane protein, HubP, acts as a polar organization factor in V. cholerae, like TipN in C. crescentus and DiIVA in B. subtilis [61]. HubP interacts directly with ParA1, which in turn recruits the ParB1parS1 complexes [12] (Figure 3, left panel). Correspondingly, HubP, ParB1 and oriC1 co-localize at the old pole during the entire cell cycle [11,23,61]. As the cell cycle progresses, HubP proteins start accumulating at the new pole, shortly followed by ParB1 and in turn by one copy of the newly duplicated oriC1 [23]. Even though disruption of the HubP-ParAB1-parS1 partition system perturbs oriC1 localization, Chr1 segregation is not impaired [11,21,61]. In addition, Chr1 remains longitudinally arranged within the cell, with oriC1 near the old pole of newborn cells [11].

In contrast to oriC1, oriC2 follows a symmetric segregation process similar to that of P1 and F plasmids [25,39,62]. After duplication at the centre of the cell, the two oriC2 copies move to Ÿ and ž cell positions, i.e. to the future cell centres of the two daughter cells [62] (Figure 3, right panel). In addition, the ParAB2-parS2 partitioning system is essential for segregation of Chr2. In its absence, aberrant unviable Chr2-deficient cells are produced [62].

The organization, positioning and segregation dynamics of the E. coli chromosome terminus of replication, ter, depends on the MatP macrodomain protein. MatP binds to specific DNA motifs, matS, which are exclusively present and overrepresented in the chromosome terminus region [36]. MatP interacts directly with a specific component of the divisome machinery that co-localizes with the Z-ring [20]. As a result, MatP maintains newly replicated ter copies at mid-cell. Reciprocally, MatP plays a role in the selection of the division site and the licensing of divisome assembly [34]. V. cholerae codes for an ortholog of E. coli MatP. ter1 and ter2 both harbour E. coli matS motifs with a density similar to E. coli ter [16,36]. However, ter1 and ter2 behave differently. Sister ter1 copies remain together at mid-cell until a very late stage of the division cycle, when septa

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are clearly visible (Figure 3, left panel), whereas sister ter2 copies segregate in the two cell halves before initiation of septation [16] (Figure 3, right panel). Careful inspection of the segregation dynamics of ter1 and ter2 loci showed that even though sister copies of ter2 loci separate earlier than sister copies of ter1, they remain in the vicinity of the division site and keep colliding with each other during the septation process. When MatP is inactivated the position of ter2 sisters is no longer restricted, dramatically reducing collision events. In the absence of MatP, ter1 sister copies separate early in the cell cycle. However, they remain in the vicinity of the cell centre [16].

When a chromosome is circular, as it is the case for most bacterial chromosomes, homologous recombination events between sister chromatids can generate chromosome dimers, which threaten chromosome segregation [35]. In E. coli, chromosome dimers are resolved by the addition of a crossover at a specific site within the terminus region, dif, by two tyrosine recombinases, XerC and XerD [37]. A cell division protein, FtsK, plays two roles in the process. First, it uses the energy from binding and/or hydrolysis of ATP to pump DNA between daughter cell compartments after the assembly of the divisome but before final scission [15]. Polar DNA motifs, the KOPS, orient the loading of FtsK on DNA, which directs the direction of translocation [4,5]. KOPS are overrepresented in the E. coli genome and point from the origin of replication towards dif [5]. As a result, FtsK brings together the two dif sites of a chromosome dimer at mid-cell. Second, FtsK activates Xer recombination by a direct interaction with XerD [37]. The dimer resolution machinery described in E. coli is conserved in almost all bacteria. Chr1 and Chr2 harbour two specific and incompatible dif sites in their terminus region, dif1 and dif2, which are used for the resolution of chromosome dimers by a common Xer/FtsK machinery at the time of cell division [16,48]. In E. coli, FtsK contributes to the segregation of sister chromosomes independently of chromosome dimer formation under slow growth conditions [22,44]. However, the action of FtsK is mainly restricted to chromosome dimers in fast growth conditions in E. coli because sister ter separate before the onset of cell division [22]. In V. cholerae, FtsK also processes sister Chr1 copies independently of chromosome dimer formation in slow growing conditions. However, it remains implicated in the process also under fast growth because ter1 sister copies persist at mid-cell for a prolonged length of time independently of the growth rate [22]. Future work will be necessary to elucidate the behaviour of ter2 sister copies and how they are managed by FtsK at different growth rate.

Cell division cycle and division site placement The cell division process has been depicted in detail in E. coli, B. subtilis and C. crescentus. In these species, the divisome is a dynamic protein complex comprising at least a dozen highly conserved proteins, which are recruited to the division site in

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divisome complex at mid-cell at about 80% of the cell cycle.

The pre-divisional FtsZ structures concomitantly coalescence into a compact Z-ring. Cell wall constriction initiates at about 90% of the cell cycle, leaving a very short time to complete cell scission [23,24] (Figure 4A).

In E. coli, the combined action of two FtsZ-polymerization inhibitory systems, Min and nucleoid occlusion (NO), specifically licenses cell division at mid-cell at the end of each round of replication/segregation cycle. Min couples the longitudinal positioning of the Z-ring to the geometrical shape of the cell.

It prevents FtsZ polymerization at the cell poles, which directs it to mid-cell [33]. NO couples Z-ring formation to the replication/segregation cycle of the E. coli chromosome. It prevents Z-ring formation over the bulk of the nucleoid, which directs it to the low DNA-density zone that develops between newly forming nucleoids [3,60]. Min is the major regulator of division site placement. Min-deficient mutants form filamentous cells.

Z-rings can assemble at the poles of the filaments, which generates anucleated mini-cells [33,12,65]. In contrast, inactivation of NO does not generate noticeable phenotypes, unless it is

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an almost linear pathway [12,17]. Divisome assembly can be schematically divided into two distinct sequential steps [12,17].

First, a tubulin homologue, FtsZ, polymerizes into a ring-like structure, the Z-ring, at mid-cell at about 25-38% of the cell cycle. The Z-ring is stabilized and anchored to the membrane by a set of proteins that are recruited at the same time. Second, periplasmic and integral membrane proteins, which are involved in cell wall remodelling or in safe keeping sister chromosome replication termini (FtsK) are recruited at about 48-52% of the cell cycle. The septation process starts soon after the arrival of this second set of proteins and lasts throughout the remaining half of the cell cycle [12,17].

V. cholerae harbours homologues of most of the E. coli cell division proteins. However, their cell cycle choreography is considerably different. All divisome components are specifically located at the new pole at the beginning of the cell cycle. FtsZ molecules, soon followed by the other early cell division proteins, only relocate to mid-cell at about 50% of the cell cycle, where they form a loose pre-divisional Z-ring. The remaining cell division proteins leave the new pole and join the early

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Fig. 4 Divisome assembly and regulation of division site placement in V. cholerae. A. Schematic representation of the divisome assembly. All division proteins are located at the new pole in newborn cells. At about 50% of the cell cycle FtsZ and the early cell division proteins leave the cell pole and relocate to mid-cell where they form a loose pre-divisional structure. The Z-ring coalescences into a compact structure at about 80% of the cell cycle, concomitantly with the arrival of the late cell division proteins at mid-cell. Cell constriction characterized by visible cell wall indentations starts at about 90% of the cell cycle. B. Schematic representation of the Min system, spatial regulator of division site placement. Throughout the cell cycle MinCD oscillate between the cell poles creating a gradient of MinC, inhibitor of FtsZ polymerization, which is lowest at mid-cell and highest at the poles. Z-rings can only assemble at mid-cell, the geometrical centre of the cell, characterized by the lowest MinC concentration over time. C. Schematic representation of the NO system, spatiotemporal regulator of division site placement.

The effector of NO and inhibitor of Z-ring assembly SlmA binds to specific DNA sequences distributed all around Chr1 and Chr2 (SlmA-bound DNA) with the exception of ter1 and ter2 regions. During the cell cycle the spatial arrangement and segregation timing of Chr1 and Chr2 direct FtsZ molecules and assembly of divisional Z-rings to the SlmA-free zones. In newborn cells the SlmA-free ter1 and ter2 regions are both located at the new cell pole. Chr1 SlmA-free DNA is located at the centre of the cell starting from about 50% of the cell cycle, however Chr2 SlmA-bound DNA is still located at mid-cell at this stage, delaying the formation of compact Z-ring structures. It is only at about 80% of the cell cycle that both ter1 and ter2 regions co-localize at mid-cell, permitting assembly of divisional Z-rings at the future division site.

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combined with defects in initiation of replication, segregation, or the disruption of Min [3]. E. coli Min is composed of three proteins: MinC, MinD and MinE. MinC is the factor responsible for blocking Z-ring formation, MinD is the activator of MinC, and MinE is the topological regulator of MinCD [33].

Specific inhibition of FtsZ polymerization at the cell poles is achieved through the regulated oscillation of the Min proteins between the two cell poles. MinD is an ATPase. Its ATP-form binds to the membrane where it recruits MinC. MinE stimulates ATP hydrolysis, which releases MinD-ADP and MinC from the membrane. MinD and MinC then migrate towards the opposite pole where, after nucleotide exchange in the cytosol, MinDATP re-associates to the membrane [33]. Continuous shuttling of MinCD between the poles creates a concentration gradient of MinC with a minimum at mid-cell (Figure 4B). NO couples the timing and assembly of the Z-ring to the replication/segregation cycle. The nucleoid serves as a scaffold for the positioning of a DNA binding protein that inhibits FtsZ polymerization, SlmA. SlmA binding sites (SBS) are asymmetrically distributed on the E. coli chromosome and essentially absent from ter. As a result, cell division can only initiate at the very end of the chromosome duplication/segregation cycle when sister ter, devoid of SBS, are the only chromosomal regions left at mid-cell [9,12,45] (Figure 4C).

V. cholerae carries orthologs of both the Min and NO effectors, MinCDE and SlmA. V. cholerae MinD was shown to shuttle between poles as reported for E. coli [24]. However, Min-inactivation does not generate any apparent phenotype unless additional mutations perturbs the cellular arrangement of chromosomes, suggesting that NO is the major regulator of division site placement in V. cholerae [24]. Indeed, SBS sites were identified on both Chr1 and Chr2 and their distribution was shown to drive the choreography of the cell division proteins and the timing of assembly and maturation of the divisome [24] (Figure 4C). As the partition machinery of Chr1 is conserved in most bacteria with the notable exception of Enterobacteriales, it seems reasonable to argue that NO was probably the primary cell division regulation mechanism in the Enterobacteriales/Vibrionaceae ancestor and that Min superseded it in the Enterobacteriales because they lost their origin partition machinery in the course of evolution.

Concluding remarks and open questions A recurrent question about multi-chromosomal bacteria concerns the definition of a secondary domesticated chromosome and how they can be distinguished from plasmids. The presence of essential genes is not sufficient because large portions of the genome can be moved from one replicon to another. Likewise the size of the replicon is insufficient because of the existence of large mega-plasmids. Some criteria can be proposed based on the V. cholerae model. Preventing the over-initiation of Chr1 and Chr2 replication relies on dam while the final stages of

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Chr1 and Chr2 segregation depend on the same FtsK/XerCD machinery. Thus, a first criterion could be to exploit the replication/segregation regulatory systems of the primary chromosome. Duplication of a small sequence on Chr1, crtS, serves to license Chr2 replication. Thus, a second criterion could be to integrate replication cycle coordination. Third, Chr1 and Chr2 form a single nucleoid, with the territory occupied by Chr2 in the cell comprised within the territory occupied by Chr1, and Chr2 harbours SBS that are essential for the regulation of cell division by nucleoid occlusion. Thus, direct participation of secondary chromosomes to the regulation of the cell cycle could be added to the list of criteria. However, the validity of each of these criteria cannot be assessed without any insight in the domestication process. Chr2 harbours many features of Chr1: a dif site, KOPS directed towards dif on both replichores, matS sites in its terminus region, SBS sites outside of the terminus region and dam methylation sites to control RctB binding to its origin region. Were they all acquired during the domestication process or were some of them already present in the plasmid ancestor of Chr2 to permit its maintenance? It would be advantageous for large replicons to use FtsK oriented DNA translocation to align dimer resolution. It would mean acquiring properly oriented KOPS but probably also synchronizing replication termination with the formation of the divisome.

The addition of matS sites would help maintain sister ter in the vicinity of the divisome and harbouring SBS sites avoid septum closure before replication termination. In contrast, it is difficult to imagine how Chr1 crtS could have pre-existed. We can thus question if some of the “chromosome-like” features of Chr2 were acquired during domestication or pre-existed. RctB does not belong to the classical replication initiator family of plasmids and seems quite specific to Vibrionaceae. To answer this question, it would be interesting to find a plasmid relying on an RctB-like replication initiator and study the regulation of its replication and segregation.

Another recurrent question concerns the size of secondary replicons. As bacterial chromosomes harbour a single origin of replication, splitting the genome on several chromosomes reduces the length of time necessary for the duplication of genetic information. With a replication speed of 1000 bp/sec, replication of the 4.5 Mbp E. coli chromosome takes ~38’ while replication of Chr1 and Chr2 only takes ~25’ min and ~8’, respectively. V. cholerae can thus multiply as fast as E. coli in rich growth conditions while running less replication circles in parallel than E. coli [22]. From this point of view, it is surprising that no multi-chromosomal species was found that harboured chromosomes of similar size. It is now explained in the case of the Vibrionaceae: their secondary chromosome must be smaller than their primary chromosome for the crtS regulation mechanism to operate.

Finally, a complex unexpected mechanism has evolved to enforce synchronization of the replication termination of Chr1 and Chr2. As stated earlier, this is probably linked to the role FtsK plays in the management of ter1 and ter2. In this regard,

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it seems surprising that Vibrios lack a homologue of the E. coli replication fork trap machinery [27]. Did another system evolved in the Vibrios? Likewise, what differences between the E. coli and V. cholerae ter macrodomain organization system explain why MatP, which acts on both ter1 and ter2 and was shown to directly link sister copies of E. coli ter to the divisome, seems unable to maintain ter2 at mid-cell? In addition to further our understanding of the V. cholerae cell cycle, answering these questions could help unmask the primordial role of regulatory mechanisms common to E. coli and V. cholerae from any additional role they might have adopted during speciation, as illustrated by the cell division studies.

Acknowledgements. This work had financial support from the European Research Council under the European Community’s Seventh Framework Programme [FP7/2007-2013 Grant Agreement no. 281590] and the ANR [PhenX/16-CE12-0030-01].

Competing interests. No competing interests exist.

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RESEARCH REVIEW International Microbiology 20(3):130-137 (2017) doi:10.2436/20.1501.01.294. ISSN (print): 1139-6709. e-ISSN: 1618-1095 www.im.microbios.org

T6SS intraspecific competition orchestrates Vibrio cholerae genotypic diversity Benjamin Kostiuk1, Daniel Unterweger2, Daniele Provenzano3 and Stefan Pukatzki4* 1. Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB T6G 2S2, Canada 2. Department of Zoology, University of Oxford, Oxford OX1 3PS, UK 3. Department of Biology, University of Texas Rio Grande Valley, Brownsville, TX 78520, USA 4. Department of Immunology & Microbiology, University of Colorado School of Medicine, Aurora, CO 80045, USA Received 26 August 2017 · Accepted 30 September 2017 Summary. Vibrio cholerae is a diverse species that inhabits a wide range of environments from copepods in brackish water to the intestines of humans. In order to remain competitive, V. cholerae uses the versatile type-VI secretion system (T6SS) to secrete anti-prokaryotic and anti-eukaryotic effectors. In addition to competing with other bacterial species, V. cholerae strains also compete with one another. Some strains are able to coexist, and are referred to as belonging to the same compatibility group.

Challenged by diverse competitors in various environments, different V. choleare strains secrete different combination of effectors – presumably to best suit their niche. Interestingly, all pandemic V. cholerae strains encode the same three effectors. In addition to the diversity displayed in the encoded effectors, the regulation of V. cholerae also differs between strains. Two main layers of regulation appear to exist. One strategy connects T6SS activity with behavior that is suited to fighting eukaryotic cells, while the other is linked with natural competence – the ability of the bacterium to acquire and incorporate extracellular DNA. This relationship between bacterial killing and natural competence is potentially a source of diversification for V. cholerae as it has been shown to incorporate the DNA of cells recently killed through T6SS activity. It is through this process that we hypothesize the transfer of virulence factors, including T6SS effector modules, to happen. Switching of T6SS effectors has the potential to change the range of competitors V. cholerae can kill and to newly define which strains V. cholerae can co-exist with, two important parameters for survival in diverse environments.

Keywords: Vibrio cholerae · T6SS · competition · evolution

Introduction Over millions of years, bacteria have evolved mechanisms to compete against each other for limited resources, inhabiting nearly every environmental niche on the planet [19, 47]. Evolution of virulence strategies have permitted bacteria to infect higher vertebrates and expand their niche repertoire. Competitive tactics to fight for resources include secreted colicins, antibiotics, siderophores and contact-dependent secretion systems to engage in cell-cell mediated killing while avoiding detection of the immune system. These mechanisms have enabled bacteria to adapt to unique niches by acquiring genetic elements * Corresponding author stefan.pukatzki@ucdenver.edu

and developing strategies for protection from predation [19, 29, 35, 47, 50].

Mechanisms of predation protection have been studied extensively in Vibrio cholerae, the Gram-negative, marine bacterium that causes a dramatic form of diarrheal disease in humans known as cholera [41, 45, 46, 56]. Over 200 serogroups comprise the species V. cholerae, some of which are primary environmental and are present in the marine environment year round [58]. Other strains bloom during epidemics, and are optimized for causing disease in the human host [15, 16]. One consequence of this diversity is increasing the variety of organisms that V. cholerae must compete with.

In environmental reservoirs, V. cholerae’s ability to recycle N-acetylglucosamine, a carbon source sequestered in the chitin polymer, challenging these bacteria to inhabit multiple diverse

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microbial environments, including the surface of copepods [22, 37, 44]. V. cholerae contends for resources with competing strains of the same species, other Vibrios, and a range of additional bacteria and grazing eukaryotic amoeba. V. cholerae bacteria employ various techniques, including adherence molecules and biofilm production, iron scavenging molecules, such as vibriobactin, and toxins, including those produced by the type 6 secretion system (T6SS) to negotiate this social complexity [18, 24, 31, 34]. Numerous factors, including V. cholerae proliferation on copepods and changes in phage populations cause unpredictable V. cholerae blooms to reach titres sufficient to become infectious to humans [16, 22, 58]. Most notable are V. cholerae strains that belong to the O1/O139 serogroup, as these strains have been implicated in all V. cholerae pandemics [14]. Non-O1/O139 strains have been implicated in local outbreaks and also represent a significant health burden [43].

When susceptible human hosts consume contaminated water, V. cholerae confronts a considerably distinct host environment and activates acid-response pathways to survive passage through the stomach acid [38, 39]. In the small intestine, V. cholerae navigates a number of chemical and physical barriers such as mucin, bile detergents, and the host microbiome [5, 21, 45, 49]. Upon establishing intimate contact with the epithelial layer, V. cholerae cells co-agglutinate as a result of expression of a Type-IV pilus known as Toxin Co-regulated Pilus (TCP), and then secrete cholera toxin (CT) resulting in massive watery efflux characteristic of cholera diarrhea which functions to disperse V. cholerae back into the environment [20, 28]. How diverse Vibrio cholerae compete in such a wide array of environments with diverse competitors is currently unknown. We propose that among many factors, the T6SS secretes highly specific effectors contributes to the needs of individual strains.

Genes coding the T6SS have thus far been identified in all V. cholerae strains examined to date [26, 55]. Structurally, the T6SS is a molecular contractile toxin delivery device that bacteria engage to inject a protein spear decorated with effectors across the cell boundary of adjacent eukaryotic and prokaryotic cell, resulting in lysis of the targeted cell [3, 34, 46]. The T6SS of V. cholerae secretes effectors that degrade lipids, peptidoglycan, purportedly DNA, as well as form pores that act on the outer membrane of the prey bacteria [8, 32, 42]. The T6SS also secretes effectors that lead to pore formation and actin crosslinking in eukaryotic cells; however, how significant these observations are to V. cholerae pathogenesis remains to be clarified. Actin-crosslinking takes place in-vivo when V. cholerae bacteria traverse the small intestine [33]. Although the host cells subjected to actin crosslinking have not been identified, this mechanism likely serves to immobilize approaching immune cells, allowing V. cholerae to establish an infection [33].

Within bacterial communities, T6SS-mediated attacks can be protected. V. cholerae synthesizes immunity proteins that sequester cognate T6SS effectors expressed by sister, or kin cells [12, 55]. Immunity proteins that protect cells from effector alleles of kin cells are ineffective against effectors encoded by

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alleles of non-kin bacteria belonging to the same species. Taken together, these observations suggest that “compatibility rules” allow distinct strains of V. cholerae with identical effector modules to coexist, thereby giving rise to a unique self-recognition system [55]. Conversely, V. cholerae strains expressing dissimilar effector/immunity pairs are unable to share a niche as one of the two s trains will be excluded [51, 52, 55]. Each V. cholerae strain examined to date encodes three distinct effector/ immunity alleles within the three T6SS gene clusters. So far, we identified a total of nineteen effectors across the three clusters, but expect that number to increase as additional strain sequences become available [26]. We assigned each module a letter as an identifier to distinguish strains able to coexist from those that compete against each other. In addition to these three clusters, some strains, including pandemic O1 strains encode a fourth T6SS effector, tseH, in an additional cluster; strains that do not have this effector have no replacement [1]. All sequenced pathogenic V. cholerae strains harbored the same effector/ immunity module set, TseL/TsiV1, VasX/TsiV2 and VgrG3/ TsiV3, we called the AAA compatibility group regardless of serogroup; these included all pandemic O1/O139 strains [55].

Virtually all strains belonging to the AAA compatibility group we examined shared the presence of the horizontally-acquired genetic elements Virulence Pathogenicity Island I (VPI-1) and Cholera Toxin prophage (CTX-Φ) essential for pandemic spread [55]. In contrast, environmental strains available for examination displayed highly diverse effector/immunity allele pairs, belonged to a wide range of serogroups and did not harbor VPI-1 and CTX-Φ. Furthermore, laboratory experiments demonstrated the AAA effector/immunity allele pair to be by far the most effective at killing non-kin V. cholerae. Collectively, these results suggest that non-toxigenic strains are unable to coexist with each other or with toxigenic V. cholerae strains, but that toxigenic strains even belonging to different serogroups are compatible and can coexist.

The three T6SS effector/immunity allele pairs reside within three highly conserved gene clusters that have considerably lower GC contents compared to the surrounding sequences, supporting the hypothesis that they are horizontally mobile among V. cholerae strains [55]. Further evidence supporting this hypothesis is the observation that V. cholerae T6SS regulation and natural competence pathways are linked because V. cholerae growth on chitin activates both DNA acquisition and T6SS expression [7, 59].

Observations that T6SS compatibility groups define the competitive behaviour of V. cholerae strains and that effector/ immunity allele pairs may be freely exchanged in nature and in the host collectively suggest a critical role for the T6SS in evolution of not only the competitive behaviour of the pathogen, but also in the acquisition of virulence factors [51]. This review will outline the diversity of the T6SS in V. cholerae, both on a genetic and regulatory level and discuss the consequences of T6SS competition driven exchange of genetic information.

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The Conserved Structure of the T6SS In contrast to the remarkable diversity of T6SS effectors, the core structural components of the T6SS are highly conserved amongst distantly related V. cholerae strains and other Vibrio species [55]. T6SS genes are distributed over both V. cholerae chromosomes and consist of three clusters: a large cluster and two auxiliary clusters. The large cluster encodes the majority of the structural T6SS components, including the outer sheath proteins, VipA/B; key proteins for the tip of the T6SS, VgrG3 and a PAAR protein; and proteins that assemble at the inner and outer membranes [10]. Additionally, the large cluster encodes a gene necessary for disassembly of the T6SS, clpV, and an essential transcriptional regulator, vasH [6, 27]. The two auxiliary clusters also encode structural components; Hcp-1 and Hcp-2 as well as the tip proteins VgrG1 and VgrG3 respectively and the inner tube proteins. Also encoded in each cluster is an effector module, consisting of an adaptor, effector and immunity gene [53]. The fourth and last cluster appears in several V. cholerae strains, including pandemics, where it either encodes an amidase, or no effector at all. While structural T6SS components have >95% identity over 37 sequenced strains, effector module DNA sequences have <30% identity among the same strain set. Further genetic differences are highlighted by GC-content divergence between effector modules and core regions. Effector modules harbor a 6-13% lower GC-content than the core structural components indicating that these DNA sequences were acquired independently [55]. This initial observation provoked the hypothesis that effector modules mobilize and are freely exchanged among V. cholerae strains. Together, this describes a T6SS in V. cholerae that is highly conserved in regions coding for the core structural components and assembly yet highly diverse in effector module sequences.

Regulation of the T6SS The T6SS of V. cholerae is tightly regulated and subject to distinct layers of regulation in different strains. Briefly, among pandemic V. cholerae strains, T6SS regulation is controlled by three principal transcriptional regulators: VasH, TfoY and TfoX [27, 40]. The large cluster becomes transcriptionally activated first, by either TfoY or TfoX. While both of these activators act on the large T6SS gene cluster and thus effector modules, each also drives independent processes that depend on V. cholerae’s lifestyle. TfoX is activated in the presence of chitin and co-regulates chitin catabolism and DNA uptake, whereas TfoY’s response to decreased cyclic-di-GMP levels in the cell encourages anti-eukaryotic behavior such as upregulation of motility and hemolysin production, while inhibiting cell attachment [40]. These distinct pathways infer multiple roles for the T6SS based on the environment V. cholerae confronts; regardless, both pathways result in the transcription of the large T6SS gene cluster, including the regulator, vasH. VasH is a

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sigma-54 dependent transcription factor encoded in the large T6SS cluster that positively regulates the two auxiliary clusters essential for T6SS activity [40]. In addition to VasH, the quorum sensing-regulated transcription factor HapR - induced at high cell density - binds to hcp-1 and hcp-2 promoters and positively regulates the T6SS auxiliary clusters [48].

In V. cholerae, the T6SS is negatively regulated by sRNAs through two distinct mechanisms related to quorum sensing. In response to low cell densities, LuxO is phosphorylated thereby producing quorum regulatory sRNAs. These small RNAs bind to and negatively regulate the 5’ untranslated regions of the mRNA for hapR and the large T6SS cluster. This is a twopronged regulator silencing network that shuts down expression of genes residing in auxiliary clusters through the downregulation of hapR and also of large cluster genes directly. Interestingly, this layer of regulation also exists in non-pandemic strains suggesting a conserved relationship between quorum sensing and the T6SS in this species [48].

V. cholerae bacteria modulate T6SS activity in response to a wide variety of environmental cues; some of these function as “on/off” switches, while others modulate the intensity of the response. For example, mucin, chitin and high-osmolality have been shown to induce T6SS in a variety of toxigenic and non-toxigenic strains, while bile salts and thiourea influence the magnitude of an already active T6SS [2, 7, 23].

As a general rule, pandemic O1 strains appear to regulate T6SS expression differently than non-patient derived strains.

One comprehensive study showed that a constitutively active T6SS under laboratory conditions is rare amongst clinical El Tor O1 strains (<15%), but common among environmentally derived strains (<90%)[4]. This correlates with V. cholerae’s natural competence on chitin as more environmental than clinical strains incorporated exogenous DNA (33.3% vs 13.8%).

This profound regulatory difference might provide insights into how different sets of strains use the T6SS as it pertains to their individual lifestyles. Furthermore, the different regulatory cues that the strains respond to gives insight into where they use their T6SS. Effector Diversity and Compatibility groups The discovery that V. cholerae’s T6SS has antibacterial activity led to experiments showing that O1 V. choleare strains were immune to the fate of the bacterial killing by V52, a toxigenic non-O1 strain that expresses T6SS constitutively [34]. Curiously, V52 was able to kill environmental V. cholerae isolates endemic to the lower Rio Grande delta that also express T6SS constitutively, yet these same environmental strains were able to kill O1 [52]. We hypothesized that O1 strains express immunity proteins cognate to the effectors expressed by V52 (O37 serogroup), but not to the effector proteins of V. cholerae endemic to the Rio Grande delta. This was confirmed when two groups independently concurrently demonstrated that open reading

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frames downstream of T6SS effectors – VCA1419, VCA022 and VC0124 – encoded immunity proteins [12, 55]. Interestingly, O1 strains do not express T6SS constitutively under laboratory conditions, yet still retained immunity against V52 suggesting that immunity is regulated independently from the rest of the T6SS [42]. Later, a promoter region was identified in the 5’ region of the effector genes that constitutively provide T6SS immunity. Immunity genes appear to be expressed constitutively under laboratory conditions in O1 strains even in the absence of T6SS activation [42]. However, immunity gene regulation becomes less clear in the host. Using the infant rabbit model investigators found that both tsiV1 and tsiV3 are upregulated three-fold higher than their respective effector. Interestingly, tsiV2 and its cognate effector vasX were not significantly upregulated in the infant rabbit [17].

Next, we performed a comprehensive bioinformatics analysis to categorize V. cholerae strains based on immunity genes sequences to lay the roadmap of the competitive relationship between V. cholerae strains. Pairwise competition assays were performed to test the hypothesis that strains encoding the same immunity genes would be able to coexist while strains encoding a different complement of immunity genes will compete [55]. Strains that coexisted based on their T6SS immunity genes were said to belong to the same compatibility group. Sharing a compatibility group is hypothesized to allow strains to share DNA, a niche and interact with one another. Our study and others identified 19 distinct effectors across the three clusters, with 2 possible effectors in auxiliary cluster one, 5 possible effectors in auxiliary cluster two, and 12 possible effectors in the larger cluster – a total of 120 potential combinations [26].

Perhaps most important is the observation that all toxigenic V. cholerae strains that have caused epidemics, including pandemic strains, all encode the same effector/immunity pairs, given the designation AAA. Delivery of distinct effectors through a highly conserved structure requires adaptor proteins to mediate the biochemical/ physical interaction. Modular T6SS adaptor proteins (Tap or Tec) having a domain that bind effectors and another that interacts with VgrG trimers at the tip of the T6SS were reported by several investigators to be ubiquitous in all V. cholerae strains and other Gram-negative species [30, 54].

The notion of compatibility groups was recently expanded upon by an in-depth study examining over 400 V. cholerae strains isolated from five different locations within Oyster Pond, MA, USA. Kirchberger et al. found that V. cholerae isolated from the same collection site all shared the same compatibility group, yet compatibility groups were distinct across different sites [25]. The authors hypothesized that homogeneity of V. cholerae at any given site is driven by the T6SS, resulting in incompatible strains being excluded.

Another important aspect of compatibility are so-called orphan immunity genes consisting of open reading frames that bear considerable homology to immunity genes but are not positioned directly downstream of a cognate effector but still

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exist within a given T6SS gene cluster [26]. All AAA-module strains harbor a single orphan immunity gene downstream of the tsiV1 immunity gene outside of the T6SS auxiliary cluster 1, yet other V. cholerae strains have several orphan immunity genes in long arrays following all three T6SS gene clusters.

While it is not yet known if these purported genes are active and provide protection to other effector genes, RNAseq data from V. cholerae demonstrate that the orphan immunity gene downstream of tsiV1 is activated along with the rest of the cluster when T6SS is induced through tfoX overexpression [7].

Additional immunity genes could offer a resistance mechanism for V. cholerae to effectors other than the ones they encode, providing a mechanism by which incompatible strains could coexist in a heterogeneous environmental niche.

Membership to a compatibility group dictates the outcome of competition, occupation of a niche, ability to participate in co-infections and the ability to share DNA. Understanding how compatibility groups are acquired and maintained is critical to understanding V. cholerae biology. While the consequences of compatibility grouping are coming to light in environmental and laboratory conditions, the impact they have during colonization and pathogenesis remain unclear. However, the universality of the AAA compatibly group in pandemic strains suggests they are critical for in-vivo fitness.

Compatibility group switching Co-regulation of T6SS and natural competence invites an intriguing hypothesis whereby T6SS mediated killing causes release of extracellular prey DNA (eDNA), which could then be acquired by the predator strains [7]. This could result in the acquisition of potentially any gene sequence including new virulence factors and/or T6SS effector modules. This notion is supported by the observations that both T6SS and natural competence are activated when V. cholerae is grown on chitin under nutrient-limiting conditions. Natural competence following T6SS killing has been observed under laboratory conditions; effector modules marked with antibiotic resistance cassettes have been shown to be horizontally mobilized and integrated into the genome resulting in a change in competitive behavior [51].

When V. cholerae binds chitin, chitinases are upregulated and secreted. Oligomeric chitin is sensed by ChiS which activates TfoX – acting as a transcription factor for natural competence genes such as pilin, comEA, and dprA, as well as the T6SS [59]. The regulatory connection between the T6SS and natural competence is also functionally linked as chitin-mediated transformation following T6SS-mediated killing has been reported by multiple groups [7, 51, 59]. In addition to chitin, mucin appears to be sensed and responded to analogously in this manner [9]. We propose a model whereby V. cholerae in the environment competes and exchanges genetic information (including T6SS-effector modules) with members of the same

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species giving rise to a pool of diverse genotypes; akin to genetic card reshuffling. A surviving heterogeneous pool of V. cholerae ingested by the human host are then subjected to selective pressure that favors acquisition of virulence factors which provide an advantage in this host followed by amplification where those V. cholerae selected for multiply to high titers before being release back into the environment thereby giving rise to a clonal lineage sharing T6SS modules optimized for human infection. In addition to acquiring T6SS modules, we hypothesize that host-directed virulence factors, or any genetic elements increasing fitness in the host are also being exchanged and subsequently selected for (Figure 1). Evidence for the exchange of multiple genetic components in a single bacterium emerged from a technique called multiplex genome editing by natural transformation (MuGENT) which was developed to manipulate multiple loci on the same genome simultaneously [11]. V. cholerae bacteria are grown on chitin and antibiotic-marked and unmarked DNA is added to the culture. Over half of the bacterial population that integrates antibiotic-marked DNA also integrates unmarked DNA, providing evidence to support a model whereby V. cholerae recombines additional genetic elements into their genome while reconfiguring their T6SS effector clusters [11]. Experiments performed with V. cholerae grown on chitin suggest that non-toxigenic strains are more likely to become competent than toxigenic strains suggesting that the flow of genetic material may move preferentially from toxigenic genes to non-toxigenic strains [4].

Discussion Despite the diversity of T6SS effectors and regulation, many conserved elements of the T6SS both genetically and functionally conserved. All tested strains display antimicrobial activity against E. coli [4, 34, 52]. This ubiquitous feature implies a universal function for the T6SS to be used in competition with other Gram-negative species. Within the species, the T6SS is used for either self-recognition, phase separation or exclusion.

Self-recognition, through shared immunity genes, allows the strains to coexist and share a niche [51, 55]. Phase separation occurs when two bacteria compete with one another but no strain is able to overtake the other, instead they form their own clusters [34, 36]. Finally, if the T6SS of one strain overpowers the other, one V. cholerae strain can kill the other strain â&#x20AC;&#x201C; completely excluding them from the niche [52].

Beyond conserved functional roles, on the genetic level, the T6SS is highly conserved throughout all structural and assembly/disassembly genes. Additionally, this conservation could be exploited through the development of therapeutic drugs targeted towards the T6SS. In stark contrast, effector module genes display variability both on a genetic and functional level.

The interaction between highly conserved and variable protein domains requires the presence of adaptor proteins to link these two components. Indeed, a bipartite adaptor protein links

KOSTIUK ET AL.

Fig. 1. Model for the diversification of V. cholerae in the environment and selection in the host. 1. V. cholerae strains of different compatibility groups (indicated by differently coloured bacteria) encounter each other on chitin surfaces in environmental reservoirs. Because they have incompatible effector modules, they kill each other in a T6SS-dependent manner and incorporate new DNA when co-existing on chitin (black arrows). The net flow of genetic information is towards bacteria with a dominant T6SS effector set (AAA). 2.

Uptake of genetic traits results in heterologous bacteria from various compatibly groups with different combinations of genetic traits (coloured circles within bacteria). Compatible bacteria (despite differences in genomic content) can coexist in environmental reservoirs. 3. Infection of the human host by a mixed inoculum of strains. 4. Human host selects for toxigenic bacteria of one compatibility group. Previous acquisition of genetic traits beneficial for persistence in the host (blue circle within red bacterium) allows V. cholera to exit the host in increased numbers.

the conserved core T6SS structural tip with the diverse effector proteins [54]. This variability in T6SS effectors amongst V. cholerae species leads to competition between the strains.

Interestingly, despite this considerable divergence, all O1 pandemic strains encode the same effector set and can therefore coexist [55].

Regulation of the T6SS represents another source of diversity. Patient-derived strains tend to encode a tightly regulated T6SS that is activated by mucin and non-functional under laboratory conditions whereas environmental strains engage in T6SS-mediated killing under the same conditions; i.e. express the system constitutively [2, 4]. Additionally, environmental strains appear to activate tfoX expression when grown on chitin, which further induces the T6SS [7]. This observation is consistent with the life-style differences between pathogenic and environmental V. cholerae as the T6SS is likely utilized differently by the two groups of strains. Although the lines are blurred, TfoY-based regulation may benefit epidemic strains and TfoX-dependent regulation may favor environmental strains

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enhancing genetic exchange [40]. This would also explain the increased T6SS effector module diversity among non-pandemic strains compared to pandemic strains as their T6SS appears to be more likely regulated with natural competence [4, 25].

Horizontal gene transfer following T6SS-mediated attack may lead to the acquisition of eDNA that can recombine anywhere in the genome [7, 11]. This DNA may mediate a fitness advantage as it is acquired from living cells that were actively killed by the T6SS and not from dead cells that potentially died as a result of their low fitness [57]. This process might contribute to the diversity of the species in two ways: by mediating the uptake of new (T6SS-independent) traits and acquisition of novel T6SS effector modules.

The second scenario presents a conceptual paradox: a predator strain acquiring an effector/immunity allele pair from a defeated, lysed prey makes it vulnerable to its own kin and would be selected against. This problem is seemingly solved by the acquisition of orphan immunity genes – an array of genes that are found immediately downstream of T6SS effector modules [26]. These open reading frames are co-regulated with the T6SS and share high sequence identity to T6SS immunity genes found in other strains [7]. Such mechanisms would allow strains to develop immunity to several effectors, expanding their niche and microbial community by now coexisting with other compatibility groups. Nevertheless, through sequential rounds of exchange, cassettes of orphan immunity genes could be acquired downstream of the effector module, suggesting that successive rounds of competition and competence diversify V. cholerae’s T6SS effector/immunity modules [26].

The diversity of T6SS effectors could help different V. cholerae acquire competitive mechanisms beneficial in different environments. For example, for a strain that encounters eukaryotic phagocytes, the acquisition of the anti-eukaryotic effectors VasX and the actin-crosslinking domain of VgrG1 would provide a competitive advantage [41, 46]. VasX presents an interesting example of an effector that displays cross-kingdom toxicity targeting both eukaryotic and prokaryotic cells. Such an effector would presumably help V. cholerae in a wide range of environments. This presents an interesting example whereby a set of T6SS might expand the niche of V. cholerae.

Bioinformatics analysis of the distribution of T6SS effectors reveals a clear difference between clinical and environmental strains [26, 55]. This is an indication that the T6SS effectors, at minimum, correlate with the lifestyle of a V. cholerae strain.

The increased diversity amongst the T6SS repertoire of environmental strains could reflect the diversity of environmental reservoirs V. cholerae inhabits. V. cholerae could potentially constantly modify their compatibility group to best fit their environment as a result of the highly modular nature of the effector/immunity alleles. On the other end of the spectrum, the observation that all toxigenic strains encode the same T6SS effectors raises many interesting questions. For example, the AAA combination may provide the best competitive advantage amongst other V. cholerae isolates within a single host. In a

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host, competition between a heterogeneous inoculum of V. cholerae would lead to selection of a clonal lineage and subsequent expansion of toxigenic AAA strains, which may explain why cholera diarrhea has been described as a virtually pure culture of clonal bacteria [13]. Alternatively, this combination could be best suited for outcompeting commensals and phagocytic immune cells in the gut. Indeed, several groups have shown that the T6SS of pandemic strains is activated in-vivo supporting a potential role in pathogenesis.

Intraspecies competition has long been studied as a contributor to diversity [50]. That a diverse bacterial species like V. cholerae employs numerous mechanisms of intraspecific competition should therefore not be surprising. How the T6SS contributes to such diversity has yet to be clarified; however, if acquisition of a new effector set facilitates V. cholerae niche expansion, a given strain will likely accumulate secondary mutations that differ from those found in strains inhabiting other niches. In support of this theory, evolutionary trees built from bioinformatics analysis of conserved genes have shown separating branches of the tree correlates with changes in T6SS compatibility groups [26, 55].

The ability to exclude other strains from a bacterial niche is one important role of T6SS activity; however, the link with natural competence is a separate facet. This dual role places the T6SS at a key position in the natural history of a diverse species. This molecular mechanism facilitating diversity could continue to allow V. cholerae to adapt to the human host, selecting for novel strains that could pose new challenges to human health.

Competing interests. Authors declare that no competing interests exist.

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RESEARCH REVIEW International Microbiology 20(3):138-148 (2017) doi:10.2436/20.1501.01.295. ISSN (print): 1139-6709. e-ISSN: 1618-1095 www.im.microbios.org

Genomic Plasticity of Vibrio cholerae Jose Antonio Escudero1,2,3,4*, Didier Mazel1,2* 1. Institut Pasteur, Unité de Plasticité du Génome Bactérien, Département Génomes et Génétique, Paris, France 2. CNRS, UMR3525, Paris, France 3. Molecular Basis of Adaptation, Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain 4. VISAVET Health Surveillance Centre.

Universidad Complutense Madrid. Avenida Puerta de Hierro, s/n. 28040 Madrid. Spain Received 22 September 2017 · Accepted 30 September 2017 Summary. Vibrio cholerae is one of the deadliest pathogens in the history of humankind. It is the causative agent of cholera, a disease characterized by a profuse and watery diarrhoea that still today causes 95.000 deaths worldwide every year. V. cholerae is a free living marine organism that interacts with and infects a variety of organisms, from amoeba to humans, including insects and crustaceans. The complexity of the lifestyle and ecology of V. cholerae suggests a high genetic and phenotypic plasticity. In this review, we will focus on two peculiar genomic features that enhance genetic plasticity in this bacterium: the division of its genome in two different chromosomes and the presence of the superintegron, a gene capture device that acts as a large, low-cost memory of adaptive functions, allowing V. cholerae to adapt rapidly.

Keywords: Vibrio cholerae · genome plasticity · superintegron

Introduction Vibrio cholerae is a halophilic Gram-negative organism endemic to certain regions of Asia, such as the Ganges Delta. It is the causative agent of cholera, an infamous disease that produces a profuse watery diarrhoea with high mortality rates if untreated [25].

It is commonly found in saline coastal waters and estuaries, either as a free-living organism or forming biofilms on the chitinous surface of crustaceans [59,104]. This association to zooplankton seems central to its lifestyle, since chitin is the major carbon and nitrogen source for V. cholerae, as well as the signal that triggers a state of natural competence enabling extensive horizontal gene transfer (HGT) events [80]. Apart from the human intestine, V.

* Corresponding authors: José Antonio Escudero, E-mail: jaescudero@ucm.es. Phone: +34-91-39437-20.

Didier Mazel, E-mail: mazel@pasteur.fr. Phone: +33-1-40-61-32-84.

ORCID: José Antonio Escudero: 0000-0001-8552-2956 Didier Mazel: 0000-0001-6482-6002

cholerae can infect or colonise a variety of distant organisms, including yeast [9,10], amoeba [1,86], flies [15], and mice [47].

Such distant habitats highlight the adaptability of V. cholerae and are suggestive of a remarkable underlying genetic plasticity.

Indeed, the arsenal of genetic weapons that V. cholerae uses to thrive during infection varies depending on the host: for instance, the main virulence factors in humans and some animal models are the cholera toxin [81], encoded in the CTX phage [112], and the toxin co-regulated pilus (TCP) [57], while during intracellular infection of eukaryotic cells, V. cholerae translocates effectors into the cell to subvert cellular metabolism [9,86].

A key point in the evolvability of V. cholerae -as it is the case for many bacteria species-, is horizontal gene transfer (HGT) [14].

The best example of this is likely the fact that the cholera toxin is encoded in a phage [112], but HGT in this bacterium goes well beyond this passive form. V. cholerae is naturally competent and can internalize DNA from the environment in an active process that is triggered by the presence of chitin [80]. In marine environments, the chitinous exoskeleton of many animals represent an abundant surface where bacterial communities form. Given the common association of vibrios to zooplankton, natural competence is probably expressed frequently in these organisms, playing a central role

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in their lifestyle. The link to this signal makes ecological sense for a free-living marine bacterium: it is only worth paying the cost of expressing the natural competence machinery when living in community, where potentially interesting DNA is close enough to be captured. Strong support to this idea comes from the recent discovery that V. cholerae uses a type 6 secretion system as a weapon to kill non-kin surrounding bacteria and steal their DNA [16]. It is clear, hence, that V. cholerae has developed sophisticated genetic machineries to exploit HGT extensively as a powerful source of innovative functions.

The variety of ecological niches in which V. cholerae thrives, the different genetic armamentarium used for each occasion, and the sophisticated means used to acquire new DNA, are proof of a high degree of genetic plasticity. In an effort to underline the remarkable genetic peculiarities of V. cholerae, this review will focus on two genomic features of this bacterium that are especially uncommon in other species: the bipartite architecture of its genome, and the presence of an extremely large genetic memory of adaptive functions: the superintegron (Fig. 1).

The genome of V. cholerae is encoded in two chromosomes The origin of chromosome 2.â&#x20AC;&#x201A; Bacteria were initially thought to encode their genome in a single molecule of circular DNA. The discovery in 1989 that Rhodobacter sphaeroides has its genome split in two unequally sized chromosomes [103] fos-

Gene dosage effect ori1

ter1

ori2 crtS

ter2 superintegron

Triggers firing of ori2

Chr1 3 Mb

Chr2 1 Mb

Fig. 1. Representation of the genomic architecture of Vibrio cholerae. Chromosome 1 is larger than chromosome 2. Replication starts at the origin of replication (ori) and ends synchronously at the terminus (ter). ori2 is triggered by the replication of crtS, a small DNA segment encoded in Chr1. During fast growth, the multiple firing of ori1 leads to an increase in the dosage of genes encoded close to the replication origin. Chromosome 2 bears the 120Kb-long superintegron.

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tered investigations revealing that multipartite genomes are not uncommon among bacteria. Indeed, approximately 5% of bacterial species harbour more than one chromosome [105,110].

For instance, the Vibrionaceae family comprises 9 genera that share a bipartite genomic architecture [35]. The best characterised organism within this family is V. cholerae, that has become a model bacterium in the field of study of bacteria with multiple chromosomes. As in the case of R. sphaeroides, the two chromosomes of V. cholerae are of unequal size, with a 3Mb-long chromosome 1 (Chr1) and a 1Mb-long chromosome 2 (Chr2) [56] (Fig. 1). The origin of this singular genomic architecture seems to be the acquisition and domestication of a large plasmid in the ancestor of all the members of the radiation [56]. Two observations support this theory: First, the origin of replication of Chr2 (ori2) is similar to those of iteron-like plasmids and not of bacterial chromosomes. In these plasmids, a replication initiator protein binds to short direct repeats (iterons) in the origin of replication. Monomeric and dimeric conformations of the initiator protein allow it to act as a promoter or a repressor of replication [62]. Accordingly, Chr2 replication is governed by the initiator protein RctB [36], that is conserved within all Vibrionaceae, but shows no homology with other replication initiators [38]. RctB has monomeric and dimeric conformations and controls replication in a concentration dependent manner through its binding to six iterons in ori2 [62,111]. Control mechanisms for ori2 are similar to those of iteron plasmids, namely initiator autoregulation, initiator titration and origin handcuffing [33,67,110,111]. This replication system is different to the one of Chr1 (ori1), that resembles canonical bacterial chromosome replication origins [36]. ori1 contains DnaA boxes for the binding of DnaA, the main initiator of replication of bacterial chromosomes, as well as GATC sites for Dam-mediated regulation of replication. ori1 is similar to E. coliâ&#x20AC;&#x2122;s oriC to the point of being exchangeable [65,110]. Accordingly, the Chr1 partitioning protein ParA1, is phylogenetically related to other chromosomal ParAs, while the one encoded in Chr2 -ParA2- branches with plasmid, phage and megaplasmid homologs [56].

Second, there is an asymmetric distribution of the functions encoded in both chromosomes [77]. Chromosome 1 harbours most of the genes thought to be essential for growth and viability as well as genes encoding DNA replication and repair, transcription, translation, cell-wall biosynthesis and a variety of metabolic pathways; while in chromosome 2 there is a higher proportion of uncharacterized genes [56,77]. This observation could be potentially biased by the marked accumulation of genes of unknown function in the superintegron that we will discuss below. Yet, altogether, Chr2 shows a higher plasticity and seems to evolve faster than the more evolutionary stable Chr1, a feature that is also conserved in the case of Burkholderia spp. [30,56] and that suggests that secondary chromosomes, like multicopy plasmids, serve as evolutionary test beds for innovation [96].

The conserved genomic architecture among all genera of the Vibrionaceae family suggests that the domestication of the meg-

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aplasmid that has now become Chr2 likely occurred millions of years ago, before the radiation of the family. The ancient evolutionary link between both chromosomes is also supported by the fact that they both show very similar GC content (46.9% and 47% for Chr1 and 2 respectively) and codon usage bias, contrarily to what is observed when the genome is compared to plasmids eventually found in Vibrios [54]. Such a long common evolutionary history sets a scenario in which stochastic crossovers and genetic rearrangements between chromosome can occur. These events blur some of the rules presented above. Indeed, V. cholerae Chr2 does harbour some essential genes, such as the genes encoding ribosomal proteins L35 and L20, and the initiation factor IF3, as well as those encoding the D-serine dehydratase and the threonyl-tRNA synthetase [56]. The stochasticity of such gene movements is supported by the fact that there are 105 potential gene duplications in the V. cholerae genome in which copies are located in different chromosomes [56]. Also, the essential genes located in Chr2 in V. cholerae are still encoded in Chr1 in other species of the Vibrio genus, suggesting that Chr2 was already stable before these genetic rearrangements. Interestingly, there are also examples of regulatory pathways whose genes are split between both chromosomes like the luxOPQSU genes involved in the synthesis of the quorum sensing autoinducer molecule AI-2, with luxOSU located in Chr1 and luxPQ in Chr2 [56].

Altogether, it would seem that the ancestor of the Vibrionaceae acquired at some point a megaplasmid that was stable enough to become evolutionary linked to its host. This association was probably strengthened further by the transfer of essential genes from the main chromosome to the plasmid. Yet, since these rearrangements are not conserved among Vibrio species, it is unlikely that they were critical for the initial stabilisation of the plasmid, but rather the result of random events occurring between the main chromosome and the already stable megaplasmid.

Management of 2 chromosomes. In order to efficiently divide, bacterial cells have to replicate their chromosome, physically locate the two copies of each chromosome in precise regions of the cell and trigger the formation of a septum that will isolate the nucleoids and produce the division of two cells with equivalent genetic content. Distributing the genome among two replicons is therefore a complex evolutionary phenomenon, in which replication and segregation of both molecules have to become coordinated and synchronized processes. Indeed, both chromosomes have to be replicated once and only once per cell cycle, and segregated so that the offspring contains one copy of both chromosomes. This choreography occurs in a cell that is extremely smaller than the chromosomes it contains. The orchestration of this phenomenon in V. cholerae is complex, yet it is likely the simplest possible model to study the maintenance of multipartite chromosomes. Therefore, any advances in the understanding of V. cholerae’s management of its genomic architecture will likely have a profound impact in fields beyond Microbiology, where multipartite genomes are the common rule.

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Coordination of replication. Bacterial chromosomes replicate at a very precise moment within the cell cycle, while plasmids seem to replicate independently of it. Having a plasmid origin, Chr2 could potentially replicate independently of Chr1, but it has been shown that this is not the case [37]: co-evolution of the two replicons within the cell has led to a coordination in the replication timing of both molecules. This has provided the first example of communication between chromosomes for replication [87]. Interestingly, rather than starting simultaneously, replication is synchronized to terminate at the same time in both chromosomes [89]. Coherent with the difference in size between both molecules, ori2 is therefore fired when Chr1 has already replicated 2/3 of its total length. The specific cue triggering Chr2 replication has been cryptic for a long time, and only now we are starting to unveil the molecular basis of this synchronicity. The signal sparking the firing of ori2 is the replication of crtS, a 150bp-long sequence located at 2/3 of the right replichore of Chr1 [108] (Fig. 1). crtS is a binding site for RctB, the ori2 initiator, and it stablishes physical contacts with ori2 during all the cell cycle [4,108]. The exact mechanism by which crtS induces Chr2 replication is not yet understood, but it could involve a structural interplay. Hence, current data suggests that the mechanism is completely novel and its understanding will open new fields in biology.

Segregation choreography. The position and movement of replicating chromosomes along the cell is a well-orchestrated phenomenon governed by a dedicated machinery that assures its correct segregation into daughter cells. These partitioning systems are based on the interaction between ParAB proteins and specific binding sequences, the parS loci, encoded in the origin of replication of the chromosome. ParAB are mainly known for their role in replicon segregation, yet they can act as transcriptional repressors to control their own expression levels and that of adjacent genes [90]. Additionally they have an unrelated pleiotropic effect on the transcriptional levels of a variety chromosomal genes [5].

V. cholerae contains two sets of ParAB proteins -ParAB1 and ParAB2-, each recognising distinct parS sites and segregating its cognate chromosome [115,116]. Chromosomes are longitudinally arranged in the cell, but while Chr1 occupies its hole length, Chr2 seems to be restricted to the youngest half of it (the new pole) [32] (Fig. 2). The ori1 of Chr1 is anchored to the old pole through the interaction of ParA1 to HubP, a pole anchor protein [114]. After replication, ori1 starts migrating from the old pole to the new pole. ori2 follows the same path, but its replication is delayed until 2/3 of Chr1 replication, and its starting point is in the midcell region [32]. While the replication and segregation of both ori are separated in space and time, the replication and segregation of the terminus region (Ter) of both chromosomes are synchronized and locate together at midcell [32,89] (Fig. 2). In bacteria with a single chromosome, the Ter region is also located at midcell, and its segregation is synchronized with cell division. Before segregating, spontaneous cointegrates that might have formed through homologous recombination between sister chromatids, have to be resolved by the XerCD proteins. These recombinases release

GENOMIC PLASTICITY OF VIBRIO CHOLERAE

ori1

ter1

Chr1

ori2

ter2

Chr2

Replication

Fig. 2. Spatial distribution of both chromosomes in the V. cholerae cell and choreography of ori1, ori2, ter1 and ter2 during chromosome replication and cell division. Modified from [107].

monomeric sister chromatids through a site-specific recombination reaction at the dif site located within Ter. V. cholerae possesses a single set of XerCD proteins, that act upon both dif1 and dif2. These recombinases are activated by FtsK, a DNA propeller protein that is anchored to the cell membrane where the septum will be formed, and that is capable of searching and locating the dif sites by reading specific sequences on DNA [12,13]. This quick translocation of DNA away from where the septum is forming helps pull sister chromatids into their future cellular compartments. Once FtsK arrives at the dif site, it triggers XerCD dimer resolution [106], releasing monomeric chromosomes. Cells can then safely divide through the formation of the Z ring promoted by the FtsZ tubulin-like protein.

Advantages of a bipartite genomic architecture. Given the complexity of the maintenance of two chromosomes, the evolutionary advantage provided by a multipartite genome must be significant in order to drive such a major change. A key element here is that in fast growing organisms, the time needed to replicate the chromosome (taking into account the processivity of DNA polymerases and the size of each replichore of the chromosome) is larger than the doubling

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time of the bacterial cell. Bacteria with single chromosomes solve this paradox by firing the origin of replication several times per cell cycle (Fig. 1), so that when a given cell divides, each daughter cell receives a chromosome that has already been partially replicated. The presence of such a mechanism is proof of the adaptive value of fast growth. Hence, it was hypothesized that one of the main advantages of dividing the genome in two replicons could be a shorter replication time and a shorter doubling time. Coherent with this, some Vibrios replicate extremely quickly: V. cholerae shows doubling times of 17 minutes [97] -much shorter than the approximately 22 minutes it takes E. coli to divide- and Vibrio natriegens is the fastest growing organism described to date, with doubling times below 10 minutes [113].

Other traits of the organization of V. cholerae genome support the idea that this bacterium is streamlined for fast growth.

Bioinformatic studies suggest that in fast growing bacteria, highly used genes tend to locate close to the origin of replication where they can have an increased gene dosage effect during rapid growth [31] (Fig. 1). Indeed, as we saw before, bacteria can fire multiple times the oriC during fast growth, in order for chromosomal replication to keep up with cell division. This entails a transient increase in copy number of those genes that are located close to the oriC, providing a higher transcriptional activity. This observation was experimentally confirmed in V. cholerae [97,98]. The relocation of the S10 locus, that encodes half of the ribosomal proteins, to a variety of distances from the ori1 produced a distance-dependent increase in doubling time and an attenuation of virulence in an animal model [97]. Hence, gene order in V. choleraeâ&#x20AC;&#x2122;s chromosome is optimised for fast growth and this is key for colonisation. This supports the high adaptive value of such trait and the potential benefit of dividing the genome in two separate replicons.

Overcoming major technical difficulties, the hypothesis that a segmented genome allows V. cholerae for a faster growth was actually tested experimentally and ruled out. Val and collaborators produced a V. cholerae strain in which both chromosomes were fused together respecting the axial symmetry, gene synteny, strand bias and the polarities of the original replichores [109]. The effect on generation time of such rearrangement was found to be minimal, suggesting that a faster growth is not the force driving the evolution of multipartite genomes and leaving the subject still open for discussion. Coherently with the plasmid acquisition model it is possible that the question about the origin of a multipartite genome should be formulated the other way around: why have the two chromosomes not fused?

Indeed, chromosomes can spontaneously form cointegrates through homologous recombination between identical insertion sequences in both replicons. This has been observed in Dam methylase mutants, in which Chr2 replication is compromised [107]. Lethality can therefore be avoided through chromosomal cointegrates that allow Chr2 to be replicated from ori1. The viability of chromosome fusions when ori2 is inactive puts forward that bipartite genomes are probably stable because single chromosomes with more than one active ori are unstable.

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The superintegron What is an integron? Chromosome 2 of V. cholerae contains a large set of intergenic repeated sequences named Vibrio cholerae repeats (VCR), that flank sets of genes that were extremely variable. This structure turned out to be one of the largest integrons known to date, the superintegron [79] and the VCRs were in fact the recombination sites in cassettes.

Integrons are memories of adaptive functions that allow bacteria to adapt rapidly to changing environments [20,39,78].

These elements act as genetic platforms for the recruitment and stockpiling of new genes embedded in a small type of mobile genetic element called integron cassettes [50,51]. Integrons are composed of a stable platform and a variable region (Fig. 3).

The latter is formed by the array of cassettes that encode a variety of different functions, while the stable platform contains the intI gene that encodes the integrase, the insertion site (attI) and a dedicated promoter for the expression of cassettes, the Pc [28] (Fig. 3). Indeed, cassettes are normally composed of a promoterless gene and a recombination site (attC) so that genes are rendered functional upon integration in the attI site, where the Pc promoter ensures their transcription. Recombination in integrons is unique in many ways [19,74], and is the result of an evolutionary innovation process [40]. Cassette recruitment can occur many times, leading to the stockpiling of adaptive functions [26]. As a consequence, a gradient of expression from the Pc is established along the array [28], making the older functions (those acquired first chronologically) to be pushed apart from the Pc by new insertion events, and ultimately become silent [92]. These silent cassettes are therefore carried at the

PLATFORM

ARRAY

Pc

intI IntI

E X P R E S SION

Silent Cassettes

Pint

Insertion

Excision Reshuffling

Fig. 3. Diagram of the integron. Genes are represented by arrows and recombination sites as triangles. The intI gene encodes the integrase that governs cassette insertion and excision. Coupling of both reactions reshuffles cassette order. Cassettes are expressed from the Pc promoter in the integron platform.

Multiple insertion events lead to the stockpiling of cassettes, constituting a memory of adaptive functions. Expression of cassettes is weaker when located far from the PC. Integrases are expressed from the Pint when the SOS response is triggered and can reshuffle cassettes, changing their expression levels.

lowest possible cost â&#x20AC;&#x201C;the cost of replication- but remain available in case they are necessary. Indeed, under stressful conditions, integrases can randomly excise cassettes from the array and re-insert them in first position, next to the Pc, where their expression is maximal [27]. This reshuffling can help recover a function that was adaptive once, but has been silent for some time [6]. Integrase expression is under the control of the SOS response, a regulatory network that is triggered under stressful conditions and during HGT [6-8], linking the functionality of integrons to the hostâ&#x20AC;&#x2122;s needs [48]. The tight control of the integrase seems necessary to keep the fitness cost of integrons low [70]. Indeed, when integrons are found in bacterial species that lack the SOS response -like Acinetobacter spp.- integrase genes tend to accumulate disruptive mutations [53,100]. The same happens in the rare cases where the integrase activity is not under the control of the SOS response. This is the case of the class 2 integron integrase (see below), that has been known for many years to contain a premature stop (ochre) codon [52], and that has very recently been shown to escape SOS regulation [63]. The success of integrons is probably the consequence of the adaptive value they provide, their low cost, and the tight intertwining of its activity with bacterial physiology through the SOS response and other cellular mechanisms [39,73,76].

Integrons and resistance. Integrons were first discovered for their role in the rise of multidrug resistance in Shigella flexneri isolates during the 1950â&#x20AC;&#x2122;s in Japan [82,101]. These strains bore the NR1 plasmid, carrying, among other things, a Tn21 transposon that contained what was later classified as a class 1 integron, conferring resistance to aminoglycosides, sulphonamide and biocides [71]. Four more integrons were discovered later and classified as classes 2 to 5, attending to the sequences of the integrase-coding genes [2,29,52,58,99]. They were all related to antimicrobial resistance and were found associated to mobile genetic elements such as transposons and conjugative plasmids, earning them the name of mobile integrons (MI). The class 1 MI is the most clinically relevant integron, and has been studied in depth, quickly becoming the experimental model in the field. Further studies revealed the presence of integrons in the chromosomes of many bacterial species, leading to the current understanding that these sedentary chromosomal integrons (SCI) are actually the natural form of integrons and the ancestors of MIs [79,91,93]. Indeed, the stochastic mobilization onto plasmids of SCIs allowed them to reach the human environment through food [43,44], where those containing antibiotic resistance cassettes were selected through the high antibiotic pressure exerted by humans in the last decades. The dissemination of integrons is today a major cause of multidrug resistance in clinically relevant strains [69], and currently one of the major threats for modern medicine. The history of the mobilisation of integrons, from the environment through food to the hospital, exemplifies the need for a global view on the ecology of antimicrobial resistance determinants [46] in order to design efficient strategies to fight anti-

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microbial resistance. Such a framework is provided by the â&#x20AC;&#x153;One Healthâ&#x20AC;? concept, that postulates the interdependence of human health with the health of animals, food and the environment [21].

Chromosomal integrons: the superintegron.

The first sedentary chromosomal integron described was the superintegron (SI), an extremely long structure found in the secondary chromosome of V. cholerae (Fig. 4). It contains 179 cassettes and spans 126 kilobases, comprising 3% of the total DNA of the cell [79]. It is the most variable region of the genome to the point of being useful in the genetic characterization of isolates [24,68]. The superintegron is the best studied chromosomal integron and the paradigm in the field of SCIs. Arguably, this field has not been explored in depth, and clearly lacks experimental evidence for many of the observations performed. This is somewhat surprising if we take into account that integrons have been found in the chromosomes of a variety of major human pathogens, such as V. cholerae, Vibrio vulnificus [22] or Pseudomonas aeruginosa [18]; that they are also closely related to pathogenicity in bacteria affecting crops such as Xanthomonas, where cassette content seems to determine the pathovars of the strain [45]; and that they have driven bacterial evolution for eons, and might reveal a myriad of interesting aspects of their biology [78]. Still, most of the studies of SCIs, including the superintegron, are descriptive. Yet they reveal intrinsic and important differences with mobile integrons such as their difference in size, their streamlining to capture cassettes [75], the smaller size of the genes encoded in the SI cassettes, or the difference in the functions encoded. All these observations suggest that many aspects of the biology of these platforms will not be understood if we limit our studies to the class 1 integron.

Cassette functions. Cassette arrays of mobile integrons are almost exclusively devoted to genes conferring antimicrobial resistance. On the other hand, chromosomal integrons contain

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cassettes of broadly unknown function. 66% of cassettes in SCIs from Vibrio species encoded proteins with no homologs in the databases and 12% had homologs of unknown function [18,88].

The paucity of pseudogenes in cassettes, together with structural studies on proteins of unknown function, are proof that these cassettes are functional and subjected to purifying selection. The structure of some proteins encode in cassettes has been solved and show a variety of novel folds [102], suggesting that a myriad new protein families are awaiting to be discovered and characterized. The remaining 22% of cassettes encode genes related with a broad variety of functions such as virulence, DNA modification, toxin-antitoxin systems, phage-related functions, and acetyltransferases [18,88]. With superintegrons containing large arrays of extremely different cassette content from one strain to another, it is clear that the environmental pool of cassettes is a prodigious reservoir of novel protein families and functions of great biotechnological interest [18,95]. Despite our ignorance on the possible functions encoded in cassettes, a highly adaptive ecological value can be assumed. Indeed, while superintegrons from geographically distant isolates of the same species are distinct, highly similar superintegrons can be found among different Vibrio species that share the same ecological niche [17]. This suggests a strong selective advantage for cassette bearers and is also proof of an intense local circulation of cassettes. Given the high adaptive value they provide, cassette functions can reveal novel aspects of bacterial physiology, ecology and evolution.

The function of only a handful of the 179 cassettes encoded in the SI has been experimentally elucidated and serve as an example of the broad array of functions that can be encoded in cassettes. The function of several other cassettes found in a variety of other Vibrio species has also been elucidated (for a complete list see [88]). Here are the examples of functionally characterized cassettes in V. cholerae: 1.â&#x20AC;&#x201A; Sulphate binding protein (SBP). An SI cassette showing high homology with a SBP from E. coli was identified

Chr2 309.700

intIA

315.000

320.000

catB9

325.000

parD-1 parE-1

433.000

435.000

relD-4 relE-4

Fig. 4. Scheme of the superintegron. Genes are shown as arrows and VCRs (attC sites) as green triangles. Blue: genes of unknown function. Red: chloramphenicol resistance gene. Pink and green: toxin-antitoxins. Yellow: transposase. Grey: genes outside the SI.

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in V. cholerae [93]. Sulphate uptake is necessary for cysteine biosynthesis and in E. coli, it is granted by a sulphate (SBP) and a thyosulphate binding protein (TSBP), encoded respectively in the sbp and cysP genes. The sbp-/ cysP- double mutant is auxotrophic for sulphur. The SBP encoded in V. cholerae SI restored prototrophy in this mutant when growing on minimal media with sulphate as sole sulphur source, confirming the predicted role of the cassette gene [93].

2. Transcriptional regulator: an approach aiming to solve the crystal structure of cassette-encoded genes identified Cass2, a transcriptional regulator with a fold related to that of AraC/XylS transcriptional activators [34]. The closest homologs of Cass2 are drug-binding regulators.

Accordingly, Cass2 is capable of binding cationic drug compounds at nanomolar concentrations. Intriguingly, we ignore the gene or set of genes controlled by this regulator, but the fact that it is encoded in a mobile and accessory region of the genome makes it especially interesting. The structures of two other proteins encoded in V. cholerae cassettes have been solved, but their function remains unclear [102]. Instead, this study highlights the prodigious variety of new folds and functions held in integron cassettes.

3. A heat stable enterotoxin similar to that of E. coli was identified in non-O1 V. cholerae strains. This toxin produces the accumulation of fluids in the intestine of the suckling mouse [3] and is encoded in an integron cassette, as proved by the surrounding VCRs [84].

4. Mannose-fucose resistant hemagglutinin. Hemagglutinins are adhesins that help bacteria to adhere to cells and colonize their hosts. Their activity can be inhibited by certain saccharides, a feature that was used to characterize these proteins. A hemagglutinin that was insensitive to mannose and fucose was first identified in the genome of V. cholerae [42] and later linked to VCRs [11]. The gene encoding this protein was later identified as VCA0447 in the chromosome of strain N16961, and the cassette encoding it is located in position 163 within the superintegron. Compared to the WT strain, a mutant of this adhesin showed a drop of 3 orders of magnitude in total cell counts in the intestine of infant mice 20 hours post inoculation [42] proving its major role in the colonization process.

5. c atB9: The first available genome sequence of a V. cholerae isolate, that of O1 El Tor N16961 strain, revealed the presence of three cassettes potentially involved in drug resistance [56]. They encoded proteins showing high levels of homology with chloramphenicol acetyltransferases, fosfomycin resistance proteins and gluthatione transferases. Other acetyltransferases potentially related to antibiotic resistance were also identified [92]. Of all these, only the chloramphenicol acetyltransferase encoded in the catB9 gene was actually capable of conferring resistance [92].

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Interestingly, V. cholerae N16961 is susceptible to chloramphenicol because this cassette is located in ninth position of the array, and therefore too far away from the Pc promoter to be transcribed at the levels necessary to provide resistance.

Nevertheless, any of the stress signals that trigger integrase activity can produce the rearrangement of cassettes in the SI and lead the appearance of chloramphenicol resistant clones through the relocation of catB9 closer to the Pc [6].

This cassette can also be captured by mobile integrons borne by conjugative plasmids [92]. This finding provided empirical evidence for the origin of resistance cassettes in mobile integrons. We now understand that mobile integrons circulate among a variety of bacterial species recruiting integron cassettes from their SCIs and bringing them back to the clinical setting, where resistance cassettes provide a selective advantage and are selected for. This is further supported by the presence of attC sites in MI cassettes that are virtually identical to those in SCIs [94], as well as by strong differences in GC content and codon usage of genes encoded in MI cassettes, suggestive of cassettes originating in a diversity of genetic backgrounds.

6. Toxin antitoxins: a distinct class of cassettes in V. cholerae SI are toxin-antitoxin (TA) systems [49,60]. These modules are known to stabilize plasmids through a post segregational killing mechanism [61]: the antitoxin can counteract the activity of the toxin only as far as it is constantly produced. Indeed, the difference in half lives of the toxin and the antitoxin means that the loss of the TA genes produces a shortage in the supply of the labile antitoxin, that degrades rapidly and allows the more stable toxin to kill the cell. Therefore, any cell that loses a TA system will ultimately die, whether it is encoded in a plasmid or in a superintegron cassette. A variety of 17 TA systems are found distributed along the SI, suggesting that the structure needs strong stabilization systems to be streamlined for genetic capacitance [75] and become such a vast memory of adaptive functions (Fig. 4). Interestingly, since antitoxins need to be expressed constantly, TA systems encode their own promoters. This means that they can modify the transcription activity of genes located downstream, proving that the simplistic model of the integron in which the only promoter is the Pc is likely over-simplistic. Still, many TA modules are actually encoded in opposite orientation compared to the rest of cassettes, and therefore do not promote the expression of any other gene (Fig. 4). This exceptional inverted organization of genes within a cassette, as well as their autonomous transcription, are proof of the peculiar nature of TA systems. This has a profound impact in some fundamental questions of integrons, such as the origin of cassettes. Indeed the fact that TA modules encode their on promoter is one of the main arguments against the RNA-based model of cassette creation, the only hypothesis on the origin of cassettes (for a review see [39]). Also, given the streamlined con-

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trol of orientation in the integron [83], it remains cryptic how can a very specific subset of genes show an inverted orientation. TA systems are also relevant because they are known to have functions beyond DNA stabilization. Toxins can be activated through a variety of signals interfering with central metabolic activities in non-lethal ways [23].

TAs are involved in several important processes of bacterial physiology and are also involved in infection and antibiotic resistance. Some examples are the induction of persistence [41,85] (a dormancy state in which the low metabolism of bacteria entails an enhanced resistance to a broad variety of insults), biofilm formation [64], quorum sensing [66], phage resistance [55] and adaptation to physical conditions (temperature, pH...) [72].

Altogether, these six examples are proof of the broad adaptive functions that cassettes can encode. Since the vast majority of cassette functions remain cryptic, research aiming to unveil other functions will certainly produce major advances in our understanding of bacterial evolvability. With almost 200 extremely assorted cassettes per genome, Vibrios possess a virtually infinite reservoir of exchangeable adaptive functions.

The superintegron, with its continuous circulation of cassettes among bacteria, and the domestication of a large plasmid to become an intrinsic component of Vibrio cholerae’s genome are micro and macro-evolutionary proofs of the exceptionally high degree of genetic plasticity of this bacterium.

Acknowledgements. We would like to acknowledge the support received from the Atracción de Talento Program of the Comunidad de Madrid (2016-T1/ BIO-1105), the Institut Pasteur, the Centre National de la Recherche Scientifique (CNRS-UMR3525), the European Union Seventh Framework Program (FP7HEALTH- 2011-single-stage), the “Evolution and Transfer of Antibiotic Resistance” (EvoTAR) project, the FP7-FET Proactive “Plaswires” project; the French Government’s Investissement d’Avenir program Laboratoire d’Excellence “Integrative Biology of Emerging Infectious Diseases” (ANR-10-LABX-62-IBEID) and the French National Research Agency (ANR-12-BLAN-DynamINT).

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RESEARCH REVIEW International Microbiology 20(3):149-150 (2017) doi:10.2436/20.1501.01.296. ISSN (print): 1139-6709. e-ISSN: 1618-1095 www.im.microbios.org

Divergent functional roles of D-amino acids secreted by Vibrio cholerae Felipe Cava The laboratory for Molecular Infection Medicine Sweden (MIMS), Department of Molecular Biology. Umeå University.

90187. Umeå. Sweden. Ph: +46(0) 90 785 6755.

Received 29 September 2017 · Accepted 30 September 2017 Summary. The L-forms of amino acids are used in all kingdoms of life to synthesize proteins. However, the bacterium Vibrio cholerae, the causative agent of cholera, produces D-amino acids which are released to the environment at millimolar concentrations. We baptized these D-amino acids as non-canonical D-amino acids (NCDAAs) since they are different from those (i.e.

D-alanine and D-glutamate) normally present in the bacterial cell wall. In V. cholerae, production of NCDAAs relies on the BsrV enzyme, a periplasmic broad spectrum racemase. BsrV multispecific activity, produces of a wide range of distinct D-amino acids.

Using a combination of genetics and molecular physiology approaches we have demonstrated that NCDAAs target different cellular processes which may function as part of a cooperative strategy in vibrio communities to protect non-producing members from competing bacteria. Because NCDAA production is widespread in bacteria, we anticipate that NCDAAs are relevant modulators of microbial subpopulations in diverse ecosystems.

Keywords: Vibrio cholerae · D-amino acids · cell wall · D-methionine · D-arginine

Bacteria are terrific colonizers that can survive and progress in a wide range of environments due to their highly creative metabolisms and their rapid capacity to expand their populations. However, living in most niches requires their ability to socialize in complex communities both with friendly and not so friendly neighbours that range from animals, plants, fungi, protists, viruses to archaeas. In these environments, production of extracellular effectors has been largely appreciated as one of the classic strategies devised by bacteria to modulate the surrounding biodiversity by interfering with the growth and/or viability of nearby organisms [7]. One of such bacterial effectors only recognized in the last decade are D-amino acids, the enantiomeric forms of the (L)-amino acids that are the basic constituents of proteins in all kingdoms of life. Since the discovery that many phylogenetically unrelated bacteria secrete high levels of D-amino acids to the environment [6], * For correspondence: Felipe Cava, E-mail: felipe.cava@umu.se ORCID: 0000-0001-5995-718X

many laboratories around the world have been intrigued by the biological and ecological impact of these effectors. Today we are just beginning to uncover a few of the seemingly many regulatory roles of D-amino acids in distinct aspects of bacterial physiology such as cell wall biogenesis, biofilm integrity, and spore germination [3].

Although scientists have appreciated D-amino acids for a long time, their major importance in biology has come fundamentally associated to their role as main constituents of the peptidoglycan cell wall, a bacteria specific structure that envelopes the cell and provides the morphology and resistance to the internal turgor pressure [8]. With very few exceptions, cell wall D-amino acids are normally D-alanine and D-glutamate, both produced by cytoplasmic highly specific racemases [5].

However, in 2009, we discovered that Vibrio cholerae, the etiological agent of the diarrheal disease cholera, produced millimolar concentrations of mainly D-methionine and D-leucine to the extracellular media [6]. Given that these D-amino acids were different from D-Ala and D-Glu, we called them non canonical D-amino acids (NCDAAs). Interestingly, both D-Met and D-Leu are produced by a single enzyme, a broad spectrum racemase (Bsr), which in V. cholerae is expressed

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under the control of the stress sigma factor RpoS [2, 6].

According to this, NCDAAs production occurs in stationary phase and is followed by their efficient incorporation into the peptidoglycan structure replacing the terminal D-Ala of the peptide moieties. Such NCDAAs editing slows down peptidoglycan biosynthesis thereby permitting coordination between cell wall synthesis and V. cholerae population expansion when resources scarce [2, 6].

A key moment in our research was when we characterized the biochemical and structural properties of BsrV [4]. We found that BsrV can racemize a great variety (>10) of amino acids, which was not consistent with the limited types of D-amino acids we had detected in the V. cholerae extracellular media (i.e. mainly D-Met and D-Leu). As often happens in science, the discovery of the release of D-Met and D-Leu was somehow fortuitous. D-Met and D-Leu were identified in stationary-phase supernatant active fractions that induced a rod-to-sphere morphological transition of a cell wall-sensitive mutant (i.e., mrcA) [6]. As expected from BsrV in vitro multispecificity, a non-biased chemical analysis revealed the accumulation of other D-amino acids (such as e.g. D-Arg) at high concentrations in the extracellular medium of V. cholerae [1]. Given that D-Arg was not detected in the fractions that induced mrcA to turn from rod-to-spherical shape, it raised a fundamental question: Do all D-amino acids have the same biological role?. In principle, this made very little sense in our opinion: why V. cholerae should be making different D-amino acids if all share a unique function?

To address these questions, we performed genetic screenings to uncover V. cholerae (naturally resistant to D-amino acids) mutants sensitive to D-Met or D-Arg and found that several mutants on cell wall associated genes were synthetically lethal in the presence of D-Met while, no conditional lethality was observed in the presence of D-Arg [1]. Despite D-Arg is totally innocuous to V. cholerae, this D-amino acid was a potent bactericide for many bacterial species. We used this as an advantage to obtain deeper insights on the potentially different mechanism of action of D-Arg. Suppressor mutations against D-Arg lethality in two independent bacterial species (Caulobacter crescentus and Agrobacterium tumefaciens) targeted the phosphate uptake system, buttressing the idea that D-amino acids must be considered as functionally non redundant environmental effectors [1].

D-Arg bactericidal activity on a great diversity of species makes this D-amino acid a powerful chemical weapon for V. cholerae against microbes inhabiting the same niche. However, production of NCDAAs is not widely conserved in the genus.

The fact that virtually all vibrios are resistant to D-amino acids suggests that D-Arg could be used as an inter-species altruistic cooperation strategy to promote the expansion of vibrios population within challenging polymicrobial environments [1]. Indeed,

vibrio species co-inhabit diverse marine and fresh water niches and thus can certainly benefit from the production of D-Arg. The ability of some bacteria to efficiently generate suppressor mutations to overcome the deleterious effects of D-Arg might, at least to some extent, explain the production of distinct sets of NCDAAs to target different cellular processes and minimize the emergence of competing microbes. Whether the bactericidal activities of certain NCDAAs can be applied in combinatory antimicrobial therapies remains to be determined.

As there are diverse Bsr-producing bacterial species other than vibrios, we anticipate that D-Arg could govern additional microbial social interactions in other environments. The relative abundance of certain L-amino acids in a particular niche would also affect the final composition and amount of the secreted D-amino acids. Further research into the role of NCDAAs in other processes, such as signaling, development and metabolic interference would provide valuable mechanistic insights on the evolution of microbial ecosystems.

Acknowledgements. Thanks to the Spanish Society of Microbiology (SEM) for the concession of the Jaime Ferrรกn Award 2017. Research in the Cava lab is funded by The Knut and Alice Wallenberg Foundation (KAW), The Laboratory of Molecular Infection Medicine Sweden (MIMS), the Swedish Research Council and the Kempe Foundation.

Notes. The author declares no conflict of interests.

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Alvarez, L, Aliashkevich, A, de Pedro, M A, and Cava, F (2018). Bacterial secretion of D-arginine controls environmental microbial biodiversity.

ISME J 12:438-450 Cava, F, de Pedro, M A, Lam, H, Davis, B M, and Waldor, M K (2011a).

Distinct pathways for modification of the bacterial cell wall by non-canonical D-amino acids. EMBO J 30: 3442-3453 Cava, F, Lam, H, de Pedro, M A, and Waldor, M K (2011b). Emerging knowledge of regulatory roles of D-amino acids in bacteria. CMLS 68: 817-831 Espaillat, A, Carrasco-Lopez, C, Bernardo-Garcia, N, Pietrosemoli, N, Otero, L H, Alvarez, L, de Pedro, M A, Pazos, F, Davis, B M, Waldor, M K, et al (2014). Structural basis for the broad specificity of a new family of amino-acid racemases. Acta Crystallogr D Biol Crystallogr 70: 79-90.

Hernandez, S B, and Cava, F (2016). Environmental roles of microbial amino acid racemases. Environ Microbiol 18: 1673-1685 Lam, H, Oh, D C , Cava, F, Takacs, C N, Clardy, J, de Pedro, M A, and Waldor, M K (2009). D-amino acids govern stationary phase cell wall remodeling in bacteria. Science 325:1552-1555 Riley, M A, and Wertz, J E (2002). Bacteriocins: evolution, ecology, and application. An Rev Microbiol 56: 117-137 Vollmer, W, Blanot, D, and de Pedro, M A (2008). Peptidoglycan structure and architecture. FEMS Microbiol Rev 32: 149-167

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INTERNATIONAL MICROBIOLOGY Official jOurnal Of the SpaniSh SOciety fOr MicrObiOlOgy

Volume 20 · Number 3 · September 2017 · pp. 105-150

EDITORIAL Cava F Biology of Vibrio cholerae: Overview

105

RESEARCH REVIEWS Islam MT, Alam M, Boucher Y Emergence, ecology and dispersal of the pandemic generating Vibrio cholerae lineage

106

Yen M, Camilli A 116 Mechanisms of the evolutionary arms race between Vibrio cholerae and Vibriophage clinical isolates Espinosa E, Barre F-X, Galli E Coordination between replication, segregation and cell division in multi-chromosomal bacteria: lessons from Vibrio cholerae

Kostiuk B, Unterweger D, Provenzano D, Pukatzki S T6SS intraspecific competition orchestrates Vibrio cholerae genotypic diversity

130

Escudero JA, Mazel D Genomic Plasticity of Vibrio cholerae

138

Cava F Divergent functional roles of D-amino acids secreted by Vibrio cholerae

149

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