Virtual Special Issue:
Bacterial Cell Surfaces
June 2012
Overview Molecular Microbiology continues to publish papers at the cutting edge of bacterial cell biology. Our contributions are at the forefront of research in the fields of bacterial surfaces, protein secretion, cell division and morphogenesis. This Virtual Special Issue on Bacterial Cell Surfaces brings together a selection of articles from Molecular Microbiology to highlight the research and stimulate discussion of Bacterial Cell Surfaces.
Contents Targeting of Neisserial PorB to the mitochondrial outer membrane: an insight on the evolution of β-barrel protein assembly machines Jhih-Hang Jiang, John K. Davies, Trevor Lithgow, Richard A. Strugnell and Kipros Gabriel Super-resolution microscopy reveals cell wall dynamics and peptidoglycan architecture in ovococcal bacteria Richard Wheeler, StÊphane Mesnage, Ivo G. Boneca, Jamie K. Hobbs and Simon J. Foster The Vibrio cholerae VctPDGC system transports catechol siderophores and a siderophore-free iron ligand Elizabeth E. Wyckoff and Shelley M. Payne
Cover Image by Tracy Palmer
Contents Heterologous protein transfer within structured myxobacteria biofilms Xueming Wei, Darshankumar T. Pathak and Daniel Wall Mechanisms for maintaining cell shape in rod-shaped Gram-negative bacteria Leon Furchtgott, Ned S. Wingreen and Kerwyn Casey Huang An accessory protein required for anchoring and assembly of amyloid fibres in B. subtilis biofilms Diego Romero, Hera Vlamakis, Richard Losick and Roberto Kolter
High-throughput, subpixel precision analysis of bacterial morphogenesis and intracellular spatio-temporal dynamics Oleksii Sliusarenko, Jennifer Heinritz, Thierry Emonet and Christine Jacobs-Wagner YopK regulates the Yersinia pestis type III secretion system from within host cells Rebecca Dewoody, Peter M. Merritt, Andrew S. Houppert and Melanie M. Marketon The morphogene AmiC2 is pivotal for multicellular development in the cyanobacterium Nostoc punctiforme Josef Lehner, Yao Zhang, Susanne Berendt, Tobias M. Rasse, Karl Forchhammer and Iris Maldener
Escherichia coli low-molecular-weight penicillin-binding proteins help orient septal FtsZ, and their absence leads to asymmetric cell division and branching Lakshmi-Prasad Potluri, Miguel A. de Pedro and Kevin D. Young Isolation and identification of new inner membrane-associated proteins that localize to cell poles in Escherichia coli Gang Li and Kevin D. Young Disruption of the ESX-5 system of Mycobacterium tuberculosis causes loss of PPE protein secretion, reduction of cell wall integrity and strong attenuation Daria Bottai, Mariagrazia Di Luca, Laleh Majlessi, Wafa Frigui, Roxane Simeone, Fadel Sayes, Wilbert Bitter, Michael J. Brennan, Claude Leclerc, Giovanna Batoni, Mario Campa, Roland Brosch and Semih Esin
Contents The rod to L-form transition of Bacillus subtilis is limited by a requirement for the protoplast to escape from the cell wall sacculus Patricia Domínguez-Cuevas, Romain Mercier, Mark Leaver, Yoshikazu Kawai and Jeff Errington Surface contact stimulates the just-in-time deployment of bacterial adhesins Guanglai Li, Pamela J. B. Brown, Jay X. Tang, Jing Xu, Ellen M. Quardokus, Clay Fuqua and Yves V. Brun UV-inducible DNA exchange in hyperthermophilic archaea mediated by type IV pili Małgorzata Ajon, Sabrina Fröls, Marleen van Wolferen, Kilian Stoecker, Daniela Teichmann, Arnold J. M. Driessen, Dennis W. Grogan, Sonja-Verena Albers and Christa Schleper A structural motif is the recognition site for a new family of bacterial protein O-glycosyltransferases Marie-Ève Charbonneau, Jean-Philippe Côté, M. Florencia Haurat, Bela Reiz, Sébastien Crépin, Frédéric Berthiaume, Charles M. Dozois, Mario F. Feldman and Michael Mourez A σW-dependent stress response in Bacillus subtilis that reduces membrane fluidity Anthony W. Kingston, Chitra Subramanian, Charles O. Rock and John D. Helmann Function of the usher N-terminus in catalysing pilus assembly Nadine S. Henderson, Tony W. Ng, Iehab Talukder and David G. Thanassi Sorting of an integral outer membrane protein via the lipoprotein-specific Lol pathway and a dedicated lipoprotein pilotin Séverine Collin, Ingrid Guilvout, Nicholas N. Nickerson and Anthony P. Pugsley Specificity of motor components in the dual flagellar system of Shewanella putrefaciens CN-32 Sebastian Bubendorfer, Susanne Held, Natalie Windel, Anja Paulick, Andreas Klingl and Kai M. Thormann
Targeting of Neisserial PorB to the mitochondrial outer membrane: an insight on the evolution of β-barrel protein assembly machines Jhih-Hang Jiang, John K. Davies, Trevor Lithgow, Richard A. Strugnell and Kipros Gabriel Volume 82, Issue 4, November 2011, Pages 976–987
Summary Mitochondria originated from Gram-negative bacteria through endosymbiosis. In modern day mitochondria, the Sorting and Assembly Machinery (SAM) is responsible for eukaryotic βbarrel protein assembly in the mitochondrial outer membrane. The SAM is the functional equivalent of the β-barrel assembly machinery found in the outer membrane of Gram-negative bacteria. In this study we examined the import pathway of a pathogenic bacterial protein, PorB, which is targeted from pathogenic Neisseria to the host mitochondria. We have developed a new method for measurement of PorB assembly into mitochondria that relies on the mobility shift exhibited by bacterial β-barrel proteins once folded and separated under semi-native electrophoretic conditions.
We show that PorB is targeted to the outer mitochondrial membrane with a dependence on the intermembrane space shuttling chaperones and the core component of the SAM, Sam50, which is a functional homologue of BamA that is required for PorB assembly in bacteria. The peripheral subunits of the SAM, Sam35 and Sam37, which are essential for eukaryotic β-barrel protein assembly but do not have distinguishable functional homologues in bacteria, are not required for PorB assembly in eukaryotes. This shows that PorB uses an evolutionary conserved ‘bacterial like’ mechanism to infiltrate the host mitochondrial outer membrane.
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Super-resolution microscopy reveals cell wall dynamics and peptidoglycan architecture in ovococcal bacteria Richard Wheeler, StÊphane Mesnage, Ivo G. Boneca, Jamie K. Hobbs and Simon J. Foster Volume 82, Issue 5, December 2011, Pages 1096–1109
Summary Cell morphology and viability in Eubacteria is dictated by the architecture of peptidoglycan, the major and essential structural component of the cell wall. Although the biochemical composition of peptidoglycan is well understood, how the peptidoglycan architecture can accommodate the dynamics of growth and division while maintaining cell shape remains largely unknown. Here, we elucidate the peptidoglycan architecture and dynamics of bacteria with ovoid cell shape (ovococci), which includes a number of important pathogens, by combining biochemical analyses with atomic force and super-resolution microscopies. Atomic force microscopy analysis showed preferential orientation of the peptidoglycan network parallel to the short axis of the cell, with distinct architectural features associated with septal and peripheral wall synthesis. Super-resolution three-dimensional structured illumination fluorescence microscopy was applied for the first time in bacteria to unravel the dynamics of peptidoglycan assembly in ovococci. The ovococci have a unique peptidoglycan architecture and growth mode not observed in other model organisms.
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The Vibrio cholerae VctPDGC system transports catechol siderophores and a siderophore-free iron ligand Elizabeth E. Wyckoff and Shelley M. Payne Volume 81, Issue 6, September 2011, Pages 1446–1458
Summary Vibrio cholerae, the causative agent of cholera, has an absolute requirement for iron. It transports the catechol siderophores vibriobactin, which it synthesizes and secretes, and enterobactin. These siderophores are transported across the inner membrane by one of two periplasmic binding protein-dependent ABC transporters, VctPDGC or ViuPDGC. We show here that one of these inner membrane transport systems, VctPDGC, also promotes iron acquisition in the absence of siderophores. Plasmids carrying the vctPDGC genes stimulated growth in both rich and minimal media of a Shigella flexneri mutant that produces no siderophores. vctPDGC also stimulated the growth of an Escherichia coli enterobactin biosynthetic mutant in low iron medium, and this effect did not require feoB, tonB or aroB.
A tyrosine to phenylalanine substitution in the periplasmic binding protein VctP did not alter enterobactin transport, but eliminated growth stimulation in the absence of a siderophore. These data suggest that the VctPDGC system has the capacity to transport both catechol siderophores and a siderophore-free iron ligand. We also show that VctPDGC is the previously unidentified siderophore-independent iron transporter in V. cholerae, and this appears to complete the list of iron transport systems in V. cholerae.
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Heterologous protein transfer within structured myxobacteria biofilms Xueming Wei, Darshankumar T. Pathak and Daniel Wall Volume 81, Issue 2, July 2011, Pages 315–326
Summary Microbial biofilms represent heterogeneous populations of cells that form intimate contacts. Within these populations cells communicate, cooperate and compete. Myxobacteria are noted for their complex social interactions, including gliding motility and lipoprotein exchange. Here, we investigated cis protein sequence and cellular behaviour requirements for lipoprotein transfer between Myxococcus xanthuscells. Specifically, an outer membrane (OM) type II signal sequence (SS) fused to the heterologous mCherry fluorescent reporter resulted in OM localization. When donor cells harbouring SSOM–mCherry were mixed with GFP-labelled recipient cells they developed red fluorescence. Our results surprisingly showed that a type II SS for OM localization, but not inner membrane localization, was necessary and sufficient for rapid and efficient heterologous protein transfer. Importantly, transfer did not occur in liquid or on surfaces where cells were poorly aligned. We conclude that cell–cell contact and alignment is a critical step for lipoprotein exchange. We hypothesize that protein transfer facilitates cooperative myxobacteria behaviours.
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Mechanisms for maintaining cell shape in rod-shaped Gram-negative bacteria Leon Furchtgott, Ned S. Wingreen and Kerwyn Casey Huang Volume 81, Issue 2, July 2011, Pages 340–353
Summary For the rod-shaped Gram-negative bacterium Escherichia coli, changes in cell shape have critical consequences for motility, immune system evasion, proliferation and adhesion. For most bacteria, the peptidoglycan cell wall is both necessary and sufficient to determine cell shape. However, how the synthesis machinery assembles a peptidoglycan network with a robustly maintained micron-scale shape has remained elusive. To explore shape maintenance, we have quantified the robustness of cell shape in three Gram-negative bacteria in different genetic backgrounds and in the presence of an antibiotic that inhibits division. Building on previous modelling suggesting a prominent role for mechanical forces in shape regulation, we introduce a biophysical model for the growth dynamics of rod-shaped cells to investigate the roles of spatial regulation of peptidoglycan synthesis, glycan-strand biochemistry and mechanical stretching during insertion. Our studies reveal that rod-shape maintenance requires insertion to be insensitive to fluctuations in cellwall density and stress, and even a simple helical pattern of insertion is sufficient for over sixfold elongation without significant loss in shape. In addition, we demonstrate that both the length and pre-stretching of newly inserted strands regulate cell width. In sum, we show that simple physical rules can allow bacteria to achieve robust, shapepreserving cell-wall growth.
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An accessory protein required for anchoring and assembly of amyloid fibres in B. subtilis biofilms Diego Romero, Hera Vlamakis, Richard Losick and Roberto Kolter Volume 80, Issue 5, June 2011, Pages 1155–1168
Summary Cells within Bacillus subtilis biofilms are held in place by an extracellular matrix that contains cellanchored amyloid fibres, composed of the amyloidogenic protein TasA. As biofilms age they disassemble because the cells release the amyloid fibres. This release appears to be the consequence of incorporation of D-tyrosine, D-leucine, D-tryptophan and D-methionine into the cell wall. Here, we characterize the in vivo roles of an accessory protein TapA (TasA anchoring/assembly protein; previously YqxM) that serves both to anchor the fibres to the cell wall and to assemble TasA into fibres. TapA is found in discrete foci in the cell envelope and these foci disappear when cells are treated with a mixture of D-amino acids. Purified cell wall sacculi retain a functional form of this anchoring protein such that purified fibres can be anchored to the sacculi in vitro. In addition, we show that TapA is essential for the proper assembly of the fibres. Its absence results in a dramatic reduction in TasA levels and what little TasA is left produces only thin fibres that are not anchored to the cell.
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High-throughput, subpixel precision analysis of bacterial morphogenesis and intracellular spatio-temporal dynamics Oleksii Sliusarenko, Jennifer Heinritz, Thierry Emonet and Christine Jacobs-Wagner Volume 80, Issue 3, May 2011, Pages 612–627
Summary Bacteria display various shapes and rely on complex spatial organization of their intracellular components for many cellular processes. This organization changes in response to internal and external cues.
Quantitative, unbiased study of these spatio-temporal dynamics requires automated image analysis of large microscopy datasets. We have therefore developed MicrobeTracker, a versatile and high-throughput image analysis program that outlines and segments cells with subpixel precision, even in crowded images and mini-colonies, enabling cell lineage tracking. MicrobeTracker comes with an integrated accessory tool, SpotFinder, which precisely tracks foci of fluorescently labelled molecules inside cells. Using MicrobeTracker, we discover that the dynamics of the extensively studied Escherichia coli Min oscillator depends on Min protein concentration, unveiling critical limitations in robustness within the oscillator. We also find that the fraction of MinD proteins oscillating increases with cell length, indicating that the oscillator has evolved to be most effective when cells attain an appropriate length. MicrobeTracker was also used to uncover novel aspects of morphogenesis and cell cycle regulation inCaulobacter crescentus. By tracking filamentous cells, we show that the chromosomal origin at the old-pole is responsible for most replication/separation events while the others remain largely silent despite contiguous cytoplasm. This surprising position-dependent silencing is regulated by division.
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YopK regulates the Yersinia pestis type III secretion system from within host cells Rebecca Dewoody, Peter M. Merritt, Andrew S. Houppert and Melanie M. Marketon Volume 79, Issue 6, March 2011, Pages 1445–146
Summary The pathogenic Yersinia species share a conserved type III secretion system, which delivers cytotoxic effectors known as Yops into target mammalian cells. In all three species, YopK (also called YopQ) plays an important role in regulating this process. In cell culture infections,yopK mutants inject higher levels of Yops, leading to increase cytotoxicity; however, in vivo the same mutants are highly attenuated. In this work, we investigate the mechanism behind this paradox. Using a β-lactamase reporter assay to directly measure the effect of YopK on translocation, we demonstrated that YopK controls the rate of Yop injection. Furthermore, we find that YopK cannot regulate effector Yop translocation from within the bacterial cytosol. YopE is also injected into host cells and was previously shown to contribute to regulation of the injectisome. In this work we show that YopK and YopE work at different steps to regulate Yop injection, with YopK functioning independently of YopE. Finally, by expressing YopK within tissue culture cells, we confirm that YopK regulates translocation from inside the host cell, and we show that cells pre-loaded with YopK are resistant to Yop injection. These results suggest a novel role for YopK in controlling the Yersinia type III secretion system.
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The morphogene AmiC2 is pivotal for multicellular development in the cyanobacterium Nostoc punctiforme Josef Lehner, Yao Zhang, Susanne Berendt, Tobias M. Rasse, Karl Forchhammer and Iris Maldener Volume 79, Issue 6, March 2011, Pages 1655–1669
Summary Filamentous cyanobacteria of the order Nostocales are primordial multicellular organisms, a property widely considered unique to eukaryotes. Their filaments are composed of hundreds of mutually dependent vegetative cells and regularly spaced N2-fixing heterocysts, exchanging metabolites and signalling molecules. Furthermore, they may differentiate specialized spore-like cells and motile filaments. However, the structural basis for cellular communication within the filament remained elusive. Here we present that mutation of a single gene, encoding cell wall amidase AmiC2, completely changes the morphology and abrogates cell differentiation and intercellular communication. Ultrastructural analysis revealed for the first time a contiguous peptidoglycan sacculus with individual cells connected by a single-layered septal cross-wall. The mutant forms irregular clusters of twisted cells connected by aberrant septa. Rapid intercellular molecule exchange takes place in wild-type filaments, but is completely abolished in the mutant, and this blockage obstructs any cell differentiation, indicating a fundamental importance of intercellular communication for cell differentiation in Nostoc. AmiC2–GFP localizes in the cell wall with a focus in the cross walls of dividing cells, implying that AmiC2 processes the newly synthesized septum into a functional cell–cell communication structure during cell division. AmiC2 thus can be considered as a novel morphogene required for cell–cell communication, cellular development and multicellularity.
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Escherichia coli low-molecular-weight penicillin-binding proteins help orient septal FtsZ, and their absence leads to asymmetric cell division and branching Lakshmi-Prasad Potluri, Miguel A. de Pedro and Kevin D. Young Volume 84, Issue 2, April 2012, Pages 203–224
Summary Escherichia coli cells lacking low-molecular-weight penicillin-binding proteins (LMW PBPs) exhibit morphological alterations that also appear when the septal protein FtsZ is mislocalized, suggesting that peptidoglycan modification and division may work together to produce cell shape.
We found that in strains lacking PBP5 and other LMW PBPs, higher FtsZ concentrations increased the frequency of branched cells and incorrectly oriented Z rings by 10- to 15-fold. Invagination of these rings produced improperly oriented septa, which in turn gave rise to asymmetric cell poles that eventually elongated into branches. Branches always originated from the remnants of abnormal septation events, cementing the relationship between aberrant cell division and branch formation. In the absence of PBP5, PBP6 and DacD localized to nascent septa, suggesting that these PBPs can partially substitute for the loss of PBP5. We propose that branching begins when mislocalized FtsZ triggers the insertion of inert peptidoglycan at unusual positions during cell division. Only later, after normal cell wall elongation separates the patches, do branches become visible. Thus, a relationship between the LMW PBPs and cytoplasmic FtsZ ultimately affects cell division and overall shape.
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Isolation and identification of new inner membrane-associated proteins that localize to cell poles in Escherichia coli Gang Li and Kevin D. Young Volume 84, Issue 2, April 2012, Pages 276–295
Summary Several bacterial structures, processes and proteins are localized primarily to the poles of rod-shaped cells. To better understand this cellular organization, we devised a new method for identifying proteins that localize to the poles of Escherichia coli. Pole-derived membrane fragments were isolated by affinity capture of vesicles containing the chemotaxis protein, Tar; and for comparison, vesicles representing all parts of the cytoplasmic membrane were captured by expressing a Tar variant that was no longer pole-specific. A combination of one-dimensional SDS-PAGE and semi-quantitative mass spectrometry identified 31 proteins that were highly enriched in polar vesicles. Five were chemotaxis proteins known to be pole-specific and another, Aer, was an aerotaxis protein that had not yet been localized to the pole.The behaviour of these internal controls validated the overall approach. GFP-fused derivatives of four candidates (Aer, YqjD, TnaA and GroES) formed polar foci that were distinct from inclusion bodies. TnaA–GFP and GroES–GFP were functional, formed a single focus per cell, and competed for polar localization with the wild-type versions of these proteins. Polar localization of TnaA, GroES and YqjD was disrupted in cells lacking the MinCDE proteins, suggesting that this system may help localize proteins not involved in cell division.
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Disruption of the ESX-5 system of Mycobacterium tuberculosis causes loss of PPE protein secretion, reduction of cell wall integrity and strong attenuation
Daria Bottai, Mariagrazia Di Luca, Laleh Majlessi, Wafa Frigui, Roxane Simeone, Fadel Sayes, Wilbert Bitter, Michael J. Brennan, Claude Leclerc, Giovanna Batoni, Mario Campa, Roland Brosch and Semih Esin Volume 83, Issue 6, March 2012, Pages 1195–1209 Summary The chromosome of Mycobacterium tuberculosis encodes five type VII secretion systems (ESX-1–ESX-5). While the role of the ESX-1 and ESX-3 systems in M. tuberculosis has been elucidated, predictions for the function of the ESX-5 system came from data obtained inMycobacterium marinum, where it transports PPE and PE_PGRS proteins and modulates innate immune responses. To define the role of the ESX-5 system in M. tuberculosis, in this study, we have constructed five M. tuberculosis H37Rv ESX-5 knockout/deletion mutants, inactivating eccA5, eccD5, rv1794 and esxM genes or the ppe25-pe19 region. Whereas the Mtbrv1794ko displayed no obvious phenotype, the other four mutants showed defects in secretion of the ESX-5-encoded EsxN and PPE41, a representative member of the large PPE protein family. Strikingly, the MtbeccD5ko mutant also showed enhanced sensitivity to detergents and hydrophilic antibiotics. When the virulence of the five mutants was evaluated, the MtbeccD5ko and MtbΔppe25-pe19 mutants were found attenuated both in macrophages and in the severe combined immune-deficient mouse infection model. Altogether these findings indicate an essential role of ESX-5 for transport of PPE proteins, cell wall integrity and full virulence of M. tuberculosis, thereby opening interesting new perspectives for the study of this human pathogen.
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The rod to L-form transition of Bacillus subtilis is limited by a requirement for the protoplast to escape from the cell wall sacculus Patricia Domínguez-Cuevas, Romain Mercier, Mark Leaver, Yoshikazu Kawai and Jeff Errington Volume 83, Issue 1, January 2012, Pages 52–66
Summary L-forms are variants of common bacteria that can grow and proliferate without a cell wall. Little is known about their molecular cell biology but they undergo a remarkable mode of proliferation that is independent of the normally essential FtsZ-dependent division machinery. We have isolated a strain of Bacillus subtilis that can quickly and quantitatively convert from the walled to the L-form state. Analysis of the transition process identified an unexpected ‘escape’ step needed for L-form emergence from the rod. Mutations in two different genes,walR and sepF, contribute to the high frequency of escape: walR, a transcriptional regulator involved in cell wall homeostasis; and sepF, required for accurate and efficient cell division.
Time-lapse imaging shows that the mutations act by facilitating the release of the L-form from its walled parent cell but that they act in different ways. The walR mutation renders the activity of the protein partially constitutive, inappropriately upregulating the activity of autolytic enzymes that weaken the cell wall. The sepF mutation probably works by perturbing the formation of a properly constructed division septum, generating a mechanical breach in the wall. The new strain provides a powerful experimental system for studying the genetics and cell biology of Lforms.
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Surface contact stimulates the just-in-time deployment of bacterial adhesins Guanglai Li, Pamela J. B. Brown, Jay X. Tang, Jing Xu, Ellen M. Quardokus, Clay Fuqua and Yves V. Brun Volume 83, Issue 1, January 2012, Pages 41–51
Summary The attachment of bacteria to surfaces provides advantages such as increasing nutrient access and resistance to environmental stress. Attachment begins with a reversible phase, often mediated by surface structures such as flagella and pili, followed by a transition to irreversible attachment, typically mediated by polysaccharides. Here we show that the interplay between pili and flagellum rotation stimulates the rapid transition between reversible and polysaccharide-mediated irreversible attachment. We found that reversible attachment of Caulobacter crescentus cells is mediated by motile cells bearing pili and that their contact with a surface results in the rapid pili-dependent arrest of flagellum rotation and concurrent stimulation of polar holdfast adhesive polysaccharide. Similar stimulation of polar adhesin production by surface contact occurs in Asticcacaulis biprosthecum and Agrobacterium tumefaciens. Therefore, single bacterial cells respond to their initial contact with surfaces by triggering just-in-time adhesin production. This mechanism restricts stable attachment to intimate surface interactions, thereby maximizing surface attachment, discouraging non-productive self-adherence, and preventing curing of the adhesive.
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UV-inducible DNA exchange in hyperthermophilic archaea mediated by type IV pili Małgorzata Ajon, Sabrina Fröls, Marleen van Wolferen, Kilian Stoecker, Daniela Teichmann, Arnold J. M. Driessen, Dennis W. Grogan, Sonja-Verena Albers and Christa Schleper Volume 82, Issue 4, November 2011, Pages 807–817
Summary Archaea, like bacteria and eukaryotes, contain proteins involved in various mechanisms of DNA repair, highlighting the importance of these processes for all forms of life. Species of the order Sulfolobales of hyperthermophilic crenarchaeota are equipped with a strongly UVinducible type IV pilus system that promotes cellular aggregation. Here we demonstrate by fluorescence in situ hybridization that cellular aggregates are formed based on a species-specific recognition process and that UV-induced cellular aggregation mediates chromosomal marker exchange with high frequency. Recombination rates exceeded those of uninduced cultures by up to three orders of magnitude. Knockout strains of Sulfolobus acidocaldarius incapable of pilus production could not self-aggregate, but were partners in mating experiments with wild-type strains indicating that one cellular partner can mediate the DNA transfer. Since pilus knockout strains showed decreased survival upon UV treatment, we conclude that the UVinducible DNA transfer process and subsequent homologous recombination represents an important mechanism to maintain chromosome integrity in Sulfolobus. It might also contribute substantially to the frequent chromosomal DNA exchange and horizontal gene transfer in these archaea in their natural habitat.
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A structural motif is the recognition site for a new family of bacterial protein Oglycosyltransferases Marie-Ève Charbonneau, Jean-Philippe Côté, M. Florencia Haurat, Bela Reiz, Sébastien Crépin, Frédéric Berthiaume, Charles M. Dozois, Mario F. Feldman and Michael Mourez Volume 83, Issue 5, March 2012, Pages 894–907
Summary The Escherichia coli Adhesin Involved in Diffuse Adherence (AIDA-I) is a multifunctional protein that belongs to the family of monomeric autotransporters. This adhesin can be glycosylated by the AIDA-associated heptosyltransferase (Aah). Glycosylation appears to be restricted to the extracellular domain of AIDA-I, which comprises imperfect repeats of a 19-aminoacid consensus sequence and is predicted to form a β-helix. Here, we show that Aah homologues can be found in many Gram-negative bacteria, including Citrobacter rodentium. We demonstrated that an AIDA-like protein is glycosylated in this species by the Aah homologue. We then investigated the substrate recognition mechanism of the E. coli Aah heptosyltransferase. We found that a peptide corresponding to one repeat of the 19-amino-acid consensus is sufficient for recognition and glycosylation by Aah. Mutagenesis studies suggested that, unexpectedly, Aah recognizes a structural motif typical of β-helices, but not a specific sequence. In agreement with this finding, we observed that the extracellular domain of the Bordetella pertussis pertactin, a β-helical polypeptide lacking the 19-amino-acid consensus sequence, could be glycosylated by Aah. Overall, our findings suggest that Aah represents the prototype of a new large family of bacterial protein O-glycosyltransferases that modify various substrates recognized through a structural motif.
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A σW-dependent stress response in Bacillus subtilis that reduces membrane fluidity Anthony W. Kingston, Chitra Subramanian, Charles O. Rock and John D. Helmann Volume 81, Issue 1, July 2011, Pages 69–79
Summary Bacteria respond to physical and chemical stresses that affect the integrity of the cell wall and membrane by activating an intricate cell envelope stress response. The ability of cells to regulate the biophysical properties of the membrane by adjusting fatty acid composition is known as homeoviscous adaptation. Here, we identify a homeoviscous adaptation mechanism in Bacillus subtilis regulated by the extracytoplasmic function σ factor σW. Cell envelope active compounds, including detergents, activate a sense-oriented, σW-dependent promoter within the first gene of the fabHa fabF operon. Activation leads to a decrease in the amount of FabHa coupled with an increase in FabF, the initiation and elongation condensing enzymes of fatty acid biosynthesis respectively. Downregulation of FabHa results in an increased reliance on the FabHb paralogue leading to a greater proportion of straight chain fatty acids in the membrane, and the upregulation of FabF increases the average fatty acid chain length. The net effect is to reduce membrane fluidity. The inactivation of the σW-dependent promoter within fabHa increased sensitivity to detergents and to antimicrobial compounds produced by other Bacillus spp. Thus, the σW stress response provides a mechanism to conditionally decrease membrane fluidity through the opposed regulation of FabHa and FabF.
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Function of the usher N-terminus in catalysing pilus assembly Nadine S. Henderson, Tony W. Ng, Iehab Talukder and David G. Thanassi Volume 79, Issue 4, February 2011, Pages 954–967
Summary The chaperone/usher (CU) pathway is a conserved bacterial secretion system that assembles adhesive fibres termed pili or fimbriae. Pilus biogenesis by the CU pathway requires a periplasmic chaperone and an outer membrane (OM) assembly platform termed the usher. The usher catalyses formation of subunit–subunit interactions to promote polymerization of the pilus fibre and provides the channel for fibre secretion. The mechanism by which the usher catalyses pilus assembly is not known. Using the P and type 1 pilus systems of uropathogenic Escherichia coli, we show that a conserved N-terminal disulphide region of the PapC and FimD ushers, as well as residue F4 of FimD, are required for the catalytic activity of the ushers. PapC disulphide loop mutants were able to bind PapDG chaperone–subunit complexes, but did not assemble PapG into pilus fibres. FimD disulphide loop and F4 mutants were able to bind chaperone–subunit complexes and initiate assembly of pilus fibres, but were defective for extending the pilus fibres, as measured using in vivo copurification and in vitro pilus polymerization assays. These results suggest that the catalytic activity of PapC is required to initiate pilus biogenesis, whereas the catalytic activity of FimD is required for extension of the pilus fibre.
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Sorting of an integral outer membrane protein via the lipoprotein-specific Lol pathway and a dedicated lipoprotein pilotin Séverine Collin, Ingrid Guilvout, Nicholas N. Nickerson and Anthony P. Pugsley Volume 80, Issue 3, May 2011, Pages 655–665
Summary The lipoprotein PulS is a dedicated chaperone that is required to target the secretin PulD to the outer membrane in Klebsiella orEscherichia coli, and to protect it from proteolysis. Here, we present indirect evidence that PulD protomers do not assemble into the secretin dodecamer before they reach the outer membrane, and that PulS reaches the outer membrane in a soluble heterodimer with the general lipoprotein chaperone LolA. However, we could not find any direct evidence for PulD protomer association with the PulS–LolA heterodimer. Instead, in cells producing PulD and a permanently locked PulS–LolA dimer (in which LolA carries an R43L substitution that prevents lipoprotein transfer to LolB in the outer membrane), LolAR43L was found in the inner membrane, probably still associated with PulS bound to PulD that had been incorrectly targeted because of the LolAR43L substitution. It is speculated that PulD protomers normally cross the periplasm together with PulS bound to LolA but when the latter cannot be separated (due to the mutation in lolA), the PulD protomers form dodecamers that insert into the inner membrane.
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Specificity of motor components in the dual flagellar system of Shewanella putrefaciens CN-32 Sebastian Bubendorfer, Susanne Held, Natalie Windel, Anja Paulick, Andreas Klingl and Kai M. Thormann Volume 83, Issue 2, January 2012, Pages 335–350
Summary Bacterial flagellar motors are intricate nanomachines in which the stator units and rotor component FliM may be dynamically exchanged during function. Similar to other bacterial species, the gammaproteobacterium Shewanella putrefaciens CN-32 possesses a complete secondary flagellar system along with a corresponding stator unit. Expression of the secondary system occurs during planktonic growth in complex media and leads to the formation of a subpopulation with one or more additional flagella at random positions in addition to the primary polar system. We used physiological and phenotypic characterizations of defined mutants in concert with fluorescent microscopy on labelled components of the two different systems, the stator proteins PomB and MotB, the rotor components FliM1 and FliM2, and the auxiliary motor components MotX and MotY, to determine localization, function and dynamics of the proteins in the flagellar motors. The results demonstrate that the polar flagellum is driven by a Na+-dependent FliM1/PomAB/MotX/MotY flagellar motor while the secondary system is rotated by a H+-dependent FliM2/MotAB motor. The components were highly specific for their corresponding motor and are unlikely to be extensively swapped or shared between the two flagellar systems under planktonic conditions. The results have implications for both specificity and dynamics of flagellar motor components.
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