11.Witzig07 by Howard Junca

Environmental Microbiology (2007) 9(5), 1202–1218

doi:10.1111/j.1462-2920.2007.01242.x

Molecular detection and diversity of novel diterpenoid dioxygenase DitA1 genes from proteobacterial strains and soil samples

Robert Witzig,1 Hamdy A. H. Aly,1 Carsten Strömpl,1 Victor Wray,2 Howard Junca1 and Dietmar H. Pieper1* 1 Department of Environmental Microbiology, HZI – Helmholtz Centre for Infection Research, Inhoffenstraße 7, D-38124 Braunschweig, Germany. 2 Department of Structural Biology, HZI – Helmholtz Centre for Infection Research, Inhoffenstraße 7, D-38124 Braunschweig, Germany. Summary Resin acids are tricyclic diterpenoids naturally synthesized by trees that are released from wood during pulping processes. Using a newly designed primer set, genes similar to that encoding the DitA1 catalytic a-subunit of the diterpenoid dioxygenase, a key enzyme in abietane resin acid degradation by Pseudomonas abietaniphila BKME-9, could be amplified from different Pseudomonas strains, whereas ditA1 gene sequence types representing distinct branches in the evolutionary tree were amplified from Burkholderia and Cupriavidus isolates. All isolates harbouring a ditA1-homologue were capable of growth on dehydroabietic acid as the sole source of carbon and energy and reverse transcription polymerase chain reaction analysis in three strains confirmed that ditA1 was expressed constitutively or in response to DhA, demonstrating its involvement in DhA-degradation. Evolutionary analyses indicate that gyrB (as a phylogenetic marker) and ditA1 genes have coevolved under purifying selection from their ancestral variants present in the most recent common ancestor of the genera Pseudomonas, Cupriavidus and Burkholderia. A polymerase chain reactionsingle-strand conformation poylmorphism fingerprinting method was established to monitor the diversity of ditA1 genes in environmental samples. The molecular fingerprints indicated the presence of

Received 25 July, 2006; accepted 31 October, 2006. *For correspondence. E-mail dpi@helmholtz-hzi.de; Tel. (+49) 531 6181 4200; Fax (+49) 531 6181 4499.

a broad, previously unrecognized diversity of diterpenoid dioxygenase genes in soils, and suggest that other bacterial phyla may also harbour the genetic potential for DhA-degradation.

Introduction Resin acids are tricyclic diterpenoids synthesized by many softwood tree species and, constituting up to a few per cent of the trees’ biomass, contribute to a significant proportion of the global organic carbon pool. These compounds are released from wood during pulping processes and are commonly found in pulp and paper mill effluents (PPME) (Liss et al., 1997; Quinn et al., 2003). Their release into aquatic systems has led to considerable environmental concern as they are significant contributors to the toxicity of PPME to aquatic organisms (Liss et al., 1997; Ellis et al., 2003). In contrast, diterpene resin acid derivatives were demonstrated to maintain pharmaceutically useful properties such as antimicrobial (Savluchinske Feio et al., 1999), antiviral (Ohtsu et al., 2001), or antitumoral (Kinouchi et al., 2000) activity. Thus, the identification and characterization of enzymes transforming this class of compounds is of great interest for both biotechnological and environmental applications. The biodegradation of resin acids has been investigated for almost four decades [reviewed by Liss and colleagues (1997) and Martin and colleagues (1999)] and consistent with the ubiquitous nature of resin acids, microorganisms with the ability to mineralize these compounds have been isolated from numerous sources [reviewed by Martin and colleagues (1999)]. The metabolic pathways of degradation of abietic (AbA) and dehydroabietic (DhA) acid, which are the most abundant types of resin acids among those commonly found in PPME (Liss et al., 1997; Ellis et al., 2003), have initially been proposed on the basis of intermediates isolated from culture media (Biellmann et al., 1973a,b) and were largely in agreement with recent molecular genetic studies on DhA-degradation by Pseudomonas abietaniphila BKME-9 (Martin and Mohn, 1999; 2000; Smith et al., 2004). Convergent pathways for the abietane diterpenoid [AbA, DhA and palustric acid (PaA)] metabolism have been suggested, which lead to

Molecular detection and diversity of DitA1 genes 1203 the key intermediate 7-oxo-dehydroabietic acid (7-oxoDhA) (Martin and Mohn, 1999; 2000; Smith et al., 2004). Smith and colleagues (Smith et al., 2004) proposed that a P450 monooxygenase catalyses the hydroxylation of DhA at C-7 to form 7-hydroxy-DhA, which is then further oxidized to 7-oxo-DhA, prior to dihydroxylation of the aromatic ring. The latter reaction is catalysed by a Rieske non-haem iron oxygenase (Martin and Mohn, 1999), resulting in the formation of a dihydrodiol, 7-oxo-11,12dihydroxy-8,13-abietadienic acid, which is dehydrogenated to form 7-oxo-11,12-diol and cleaved by an extradiol ring-cleavage dioxygenase (Martin and Mohn, 2000). Evidence that the gene ditA1 encoding the a-subunit of the terminal oxygenase component (ISPaDit) of the diterpenoid dioxygenase is essential for the degradation of PaA, AbA and DhA by this strain has been provided by inactivation experiments (Martin and Mohn, 2000). Bacteria harbouring a ditA1 gene have been previously detected in various PPME biotreatment systems (Yu et al., 1999). However, while it was reported that such strains are only minor members of the resin aciddegrading population (Yu et al., 1999), the primers employed by the authors failed to detect ditA1homologous genes in a number of characterized resin acid-degrading strains (Yu et al., 1999; 2000). This shortcoming prevents the identification of other key bacterial taxa that contribute to the degradation of resin acids in these systems. Thus, a more comprehensive analysis of the diversity and distribution of diterpenoid dioxygenase genes would be of crucial importance to gain insight into the ecology and diversity of microorganisms involved in resin acid degradation and optimization of the biological PPME treatment systems. Previous studies have demonstrated that the iron sulfur proteins of the oxygenase component of Rieske non-haem iron oxygenases (ISPa) provide excellent molecular targets for genetic analysis of aromatic hydrocarbon-degrading enzymes (Taylor et al., 2002; Kahl and Hofer, 2003), and polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis has been demonstrated to be a powerful tool for the molecular analysis of the structures of microbial communities and functional genes (Schwieger and Tebbe, 1998; Junca and Pieper, 2004; Witzig et al., 2006). In the present study, we report on the design of new PCR primers for the molecular analysis of genes encoding the a-subunits of diterpenoid dioxygenases and the application of a PCR-SSCP method for the screening of ISPaDit gene polymorphisms in reference strain cultures and environmental samples. Furthermore, cultivation studies in conjunction with reverse transcription (RT)-PCR analysis of a subset of the cultivated strains were performed to confirm that PCR-positive strains were capable of degrading DhA.

Results PCR amplification of ditA1-homologues from aromatic hydrocarbon-degrading reference strains and environmental isolates When the degenerate primer set bphAf371B/bphAr115-2, which had previously been used for amplification of ISPa genes of the toluene/biphenyl oxygenase subfamily (ISPaTol/Bph) (Witzig et al., 2006), was applied to a set of 21 characterized aromatic hydrocarbon-degrading bacteria (Table 1), a second product of 892 bp was observed with Burkholderia xenovorans LB400 and Cupriavidus sp. PS12 template DNA (Supplementary material Fig. S1). Similarly, PCRs with Burkholderia spp. WBF3 and WBF4 as template resulted in simultaneous amplification of approximately 820 bp and 890 bp products. Single product bands (890 bp) were observed with Cupriavidus sp. WBF7, Burkholderia sp. WBF1, Burkholderia sp. WBF2, Burkholderia sp. WBF5 and Burkholderia sp. WBF6. The DNA sequences of the approximately 890 bp PCR fragments had, among ISPa genes with validated activity of the gene product, highest nucleotide sequence similarity (71–77% DNA sequence identity) to the ISPaDit gene ditA1 of P. abietaniphila BKME-9 (Martin and Mohn, 1999). These results indicated that the DNA regions used as primer binding sites for amplification of the ISPaTol/Bph gene sequences were, at least to some extent, also conserved in ISPa subunits of the diterpenoid oxygenase branch of Rieske non-haem iron oxygenases. However, when the primer set bphAf371B/bphAr1153-2 was used for amplification of ISPa genes from eight references strains reported to be capable of growth on dehydroabietic and/or isopimaric acid (Wilson et al., 1996; Mohn et al., 1999; Yu et al., 2000), a 892 bp PCR amplicon was observed only with Cupriavidus sp. BKME-6 (Supplementary material Fig. S1, Table 1). Sequencing revealed that this fragment was highly homologous (> 96.8% DNA sequence identity) to the ISPaDit gene fragments of Cupriavidus spp. PS12 and WBF7. Design of a new primer set for PCR amplification of ditA1-homologues from reference strains and environmental isolates Because the primer set bphAf371B/bphAr1153-2 failed to amplify ISPaDit gene fragments from seven characterized resin acid degraders, a new degenerate primer set (ditAf543/ditA1186) was designed for amplification of gene sequences encoding ISPa peptides of the diterpenoid dioxygenase branch. Using this primer set, single products of 684–693 bp could be amplified from four reference strains previously reported to be capable of degrading dehydroabietic acid (DhA-51, DhA-54, DhA-91 and BKME-6) (Table 1), whereas no amplification was

1204 R. Witzig et al. Table 1. Bacterial strains used in this study and results of the two PCR assays targeting Rieske non-haem iron oxygenase ISPaTol/Bph and/or ISPaDit genes. PCR primer seta

Organism Resin acid degraders Burkholderia sp. DhA-54 Cupriavidus sp. BKME-6 Pseudomonas vancouverensis DhA-51 Pseudomonas multiresinivorans IpA-1 Pseudomonas sp. IpA-2 Pseudomonas sp. IpA-92 Pseudomonas sp. IpA-93 Pseudomonas sp. DhA-91 Aromatic hydrocarbon degraders Burkholderia xenovorans LB400 Cupriavidus sp. PS12 Cupriavidus necator H850 Pandoraea sp. JB1* Rhodococcus globerulus P6 Pseudomonas pseudoalcaligenes KF707 Pseudomonas sp. Cam-1 Pseudomonas sp. JR1 Pseudomonas sp. CF600 Pseudomonas sp. IC Pseudomonas fluorescens IP01 Pseudomonas aeruginosa JI104 Pseudomonas stutzeri AN10 Pseudomonas stutzeri OM1 Pseudomonas stutzeri OX1 Pseudomonas putida F1 Pseudomonas putida G7 Pseudomonas putida mt-2 Pseudomonas putida MT53 Pseudomonas putida HS1 Pseudomonas putida 3,5X Isolates from BTEX-contaminated soil Pseudomonas sp. IA1YICDA Pseudomonas sp. IA1YICDB Pseudomonas sp. 3YC2 (3)g Pseudomonas sp. 1YXyl1 (5)g Pseudomonas sp. 1XB2 (1)g Pseudomonas sp. 1XC1 (2)g Pseudomonas sp. 3YdBTEX2 Pseudomonas sp. 1YB2 (7)g Pseudomonas sp. 3YXyl1 (3)g Pseudomonas sp. 1XB1 Arthrobacter sp. 3YC3 Sphingomonas sp. 1XXyl1b Isolates from PCB-contaminated soil Burkholderia sp. WBF1 Burkholderia sp. WBF2 Burkholderia sp. WBF3 Burkholderia sp. WBF4 Bukrholderia sp. WBF5 Burkholderia sp. WBF6 Cupriavidus sp. WBF7

Reported ISPa gene(s)

ditA1

bphA1 tecA1 bphA1 bphA1 bphA1 bphA1 bphA1 ipbA1 bphX, bphA1 cumA1 bnzA nahAc carAa todC1, cmtAb nahAc xylX, benA xylX xylX

ipbA1 ipbA1 ipbA1 ipbA1 ipbA1 ipbA1 ipbA1 ipbA1 ipbA1 ipbA1

bphAf371B bphAr1153-2

ditAf543 ditAr1186

Growth on DhA and transformation of DhAc

Mohn et al. (1999) Bicho et al. (1995) Mohn et al. (1999); Yu et al. (1999) Wilson et al. (1996) Wilson et al. (1996) Yu et al. (2000) Yu et al. (2000) Yu et al. (2000)

– lb –

+ + +

+d +d +d

– – sb – –

– – – – +

(+)d (+)d –e –e +e

Bopp (1986) Beil et al. (1997) Bedard et al. (1987) Witzig et al. (2006) Asturias et al. (1995) Furukawa et al. (1987) Master and Mohn (1998) Pflugmacher et al. (1996) Shingler et al. (1989) Carrington et al. (1994); Witzig et al. (2006) Aoki et al. (1996) Kitayama et al. (1996) Bosch et al. (1999) Ouchiyama et al. (1998) Bertoni et al. (1998) Gibson et al. (1970) Simon et al. (1993) Burlage et al. (1989) Keil et al. (1985) Kunz and Chapman (1981) Ng et al. (1994)

s/lb s/lb s sb s s s s – sb

+ + – – – + + + + –

+f + – ND – + + + + ND

sb sb – – – s – – – – –

– – – – + – – – – – –

– ND – ND + – ND ND ND ND –

Junca Junca Junca Junca Junca Junca Junca Junca Junca Junca Junca Junca

– – s s s s s s s s s s

– + – – – – – + – + – –

ND + ND ND ND ND ND + ND + ND ND

lb lb s/lb s/lb lb lb lb

+ + + + + + +

+ ND + + + + +

Reference(s)

and and and and and and and and and and and and

Pieper Pieper Pieper Pieper Pieper Pieper Pieper Pieper Pieper Pieper Pieper Pieper

(2004) (2004) (2004) (2004) (2004) (2004) (2004) (2004) (2004) (2004) (2004) (2004)

a. s, short fragment (805–829 bp); l, long fragment (883–892 bp); s/l, two product bands detectable in agarose gel analysis; –, no PCR product. b. The identity of the PCR products was confirmed by cloning and DNA sequencing. c. Liquid cultures containing 0.24 mM of DhA were monitored for substrate depletion and intermediate accumulation of pathway intermediates, and growth was assessed by determining the increase in protein concentration. +, growth; (+), poor growth; –, no growth; ND, not determined. d. Data from Mohn and colleagues (Mohn et al., 1999). e. Data from Yu and colleagues (Yu et al., 2000). f. Reported by Smith and colleagues (Smith et al., 2004). g. Numbers in parentheses indicate the number of isolates that were previously shown to be indistinguishable from the given isolate based on the catechol 2,3-dioxygenase, ISPaTol/Bph and 16S rRNA ARDRA genotype (Witzig et al., 2006).

Molecular detection and diversity of DitA1 genes 1205 1

Table 2. H NMR data of dehydroabietic acid, 7-oxodehydroabietic acid and 5-hydroxy-7-oxodehydroabietic acid. H Ha 18

CH3

He He

20 CH 3

HOOC Ha

H 8

CH3

Ha CH3

CH3

He He

HOOC Ha

J1a,1e = 13.2 J1a,2a = 13.2 J1a,2e = 3.0 J1a,1e = 12.8

He 10

1 5

He Ha

2.44 1.67–1.89a 1.67–1.89a 1.67–1.89a 1.67–1.89a 2.62

bd m m m m dd

2.91

1.69–1.89a

2.37

H7a,e H11 H12

2.83–2.94b 7.36 7.16

m d dd

– 7.55 7.67

– d dd

H14 H15

7.08 2.83–2.94b

d m

7.87 3.02

d sept

H16 H17 H18 H20

1.22c 1.22c 1.21d 1.21d

d d bs bs

1.26c 1.26c 1.28d 1.29d

d d bs bs

ddd

H1e H2a H2e H3a H3e H5

2.36 1.69–1.89a 1.69–1.89a 1.69–1.89a 1.69–1.89a 2.02

H6a

1.37–1.45

H6e

J5a,6a = 12.5 J5a,6e = 2.0

J11,12 = 8.2 J11,12 = 8.2 J12,14 = 1.3 J12,14 = 1.3 J15,16 = 7.0 J15,17 = 7.0 J15,16 = 7.0 J15,17 = 7.0

H 8

CH3

J5a,6a = 14.2 J5a,6e = 3.6 J5a,6a = 14.2 J6a,6e = 18.5 J6a,6e = 18.5 J5a,6e = 3.6 J11,12 = 8.2 J11,12 = 8.2 J12,14 = 2.1 J12,14 = 2.1 J15,16 = 6.9 J15,17 = 6.9 J15,16 = 6.9 J15,17 = 6.9

He 10

HOOC

11 13

CH3

J1a,1e = 12.5 J1a,2a = 12.5 J1a,2e = 3.2 J1a,1e = 12.5

CH3

He He

20CH 3

ddd

1.40

16CH3

1.59

H1a

20 CH 3

CH3

1.53–2.17a

1.53–2.17a 1.53–2.17a 1.53–2.17a 1.53–2.17a 1.53–2.17a –

–

3.39

J6a,6e = 18.2

2.65

J6a,6e = 18.2

– 7.46 7.64

– d dd

7.86 3.00

d sept

1.27c 1.27c 1.43d 1.46d

d d bs bs

J11,12 = 8.1 J11,12 = 8.1 J12,14 = 2.0 J12,14 = 2.0 J15,16 = 6.9 J15,17 = 6.9 J15,16 = 7.0 J15,17 = 7.0

a. Overlap of various protons in the given region. b. Not resolved because of overlap of protons H7a,e with H15. c. Non-equivalent (Dd < 0.01). d. Interchangeable. d, doublet; m, multiplet; bs, broad singlet; bd, broad doublet; dd, double doublet; ddd, double double doublet; sept, septet.

observed with four reference strains originally isolated on isopimaric acid (IpA). In addition, amplicons of the expected size were detectable with various reference strains reported to be capable of degrading different aromatic hydrocarbons and isolates obtained from soil contaminated with benzene, toluene, ethylbenzene, and xylenes (BTEX) or polychlorinated biphenyl (PCB) (Table 1). Based on the peptide sequence similarities of the translation products (see below), all PCR products were identified as homologues of ditA1 of P. abietaniphila BKME-9, indicating that the primers and PCR conditions used are specific for amplification of Rieske non-haem iron oxygenase ISPaDit genes. Growth on DhA and transformation of DhA The presence of a Rieske non-haem iron oxygenase ISPaDit gene has been demonstrated to be crucial for DhA-degradation in P. abietaniphila BKME-9 (Martin and Mohn, 1999). To analyse if the presence of a ditA1homologous gene is correlated with DhA-degradation

activity, 16 strains from which a ISPaDit gene was amplified by PCR with primer set ditAf543/ditAr1186 and six strains that did not yield such an amplification product, were tested for their ability to grow on DhA as the sole source of carbon and energy. All strains for which a ditA1homologue was detected by PCR were able to grow on DhA as indicated by protein yields of 0.05–0.1 g of protein per gram of DhA (Table 1), whereas the other strains were not (growth yield < 0.01 g of protein per gram of DhA). During growth, the intermediate accumulation of two metabolites, exhibiting retention volumes of 40% (metabolite M1) and 20% (metabolite M2) that of DhA was observed and high-performance liquid chromatographymass spectrometry (HPLC-MS) analysis of the culture supernatants indicated that these metabolites had molecular ions of m/z 314 and 330, respectively, suggesting that they were oxo- and oxo-hydroxy-derivatives of DhA. The in situ 1H nuclear magnetic resonance (NMR) spectrum of DhA (Table 2) was very similar to that previously reported (Gigante et al., 1995; Martin and Mohn, 1999) and comparison of its spectrum with that of a

1206 R. Witzig et al. sample containing both DhA and metabolite M1 allowed the identification of signals originating from this metabolite (Table 2). The 1H NMR spectrum of metabolite M1 was very similar to that previously reported for 7-oxo-DhA (Martin and Mohn, 1999). The large low field chemical shift of H-14 and smaller shifts of H-11 and H-12 compared with DhA were indicative of the introduction of a carbonyl group at C-7. Similar effects were observed for the signals of the neighbouring aliphatic protons, H-6a, H-6e and H-5. The coupling constants observed (Table 2) were indicative of the axial (H-5 and H-6a) and equatorial (H-6e) dispositions of the protons within this system. The analysis of the 1H NMR spectrum of a sample containing M2 in addition to DhA and M1 allowed the identification of signals originating from M2. Compared with M1, M2 showed only small differences in chemical shifts of the aromatic protons, indicating that this compound contained a 7-oxo-substituent, similarly to M1. However, the signals of only two protons were observed to low field compared with three in M1. The magnitude of the coupling constant (18.2 Hz) indicated these must belong to an isolated methylene group at C-6. This, together with the increase in molecular weight of 16 mass units compared with M1 could only be rationalized by the introduction of an axial hydroxyl group at C-5 in M2 suggesting this compound is 5-hydroxy-7-oxo-DhA. Comparison of the integrals of the resonance lines of the 11-H protons of DhA, 7-oxo-DhA and 5-hydroxy-7-oxoDhA allowed their relative abundances to be determined and their use, together with a defined standard of DhA, as a quantitative standard in HPLC analysis, for assessing the transient accumulation of 7-oxo- and 5-hydroxy-7-oxoDhA during growth of isolates and reference strains on DhA. All strains transiently accumulated significant amounts of 7-oxo-DhA, accounting for up to 20% of the applied substrate when the strain grew on 0.24 mM of substrate. Intermediate excretion of 5-hydroxy-7-oxo-DhA was significant in Pseudomonas and Cupriavidus strains and accounted for up to 15% of applied substrate, whereas Burkholderia strains excreted only minor amounts of 5-hydroxy-7-oxo-DhA (< 2% of applied substrate). Expression of ditA1-like genes in Pseudomonas pseudoalcaligenes KF707, Pseudomonas sp. Cam-1 and Burkholderia sp. WBF4 To confirm that the identified ditA1-like genes are expressed in response to DhA, RT-PCR experiments were performed with total RNA extracted from cultures of P. pseudoalcaligenes KF707, Pseudomonas sp. Cam-1 and Burkholderia sp. WBF4. Amplification products of the expected size (approximately 690 bp) were observed in cultures grown on DhA, whereas no such products were detectable with RNA extracts of P. pseudoalcaligenes

KF707 and Pseudomonas sp. Cam-1 cells grown on fructose (Supplementary material Fig. S2). In addition, no amplification products were observed in controls devoid of either reverse transcriptase (Supplementary material Fig. S2, lane 15) or template cDNA (Supplementary material Fig. S2, lane 16). These results suggested that in KF707 and Cam-1 the respective ditA1 genes were specifically induced in the presence of DhA. In contrast, amplification of an RT-product in both fructose and DhA cultures of Burkholderia sp. WBF4 suggested that expression of ditA1-mRNA was constitutive in this strain. Sequencing of the 690 bp products confirmed that they were identical to the corresponding ditA1-like sequences amplified from genomic DNA of these strains. PCR-SSCP analysis of ditA1-homologues of characterized reference strains and bacterial isolates obtained from BTEX- and PCB-contaminated soils A SSCP fingerprinting method allowing for the sequencedependent differentiation of ISPaDit gene fragments amplified with primer set ditAf543/ditAr1186 was established to rapidly obtain an overview of the ISPaDit gene sequence diversity in pure culture strains and environmental samples. Using a MDETM gel concentration of 0.8¥ and a gel temperature of 40°C, the electrophoretic mobilities of ISPaDit single-strand products obtained from four characterized dehydroabietic acid degraders and seven characterized aromatic hydrocarbon degraders could be distinguished from each other (Fig. 1A), suggesting that these strains contained different ISPaDit gene variants. Sequencing of the re-amplified single-strand products confirmed that all gene segments encoded ISPa peptides homologous to DitA1 of P. abientaniphila BKME-9 (Martin and Mohn, 1999). Single-strand conformation polymorphism analysis of Pseudomonas sp. 1YB2 and seven additional Pseudomonas strains harbouring an identical 16S rRNA phylotype and ISPaTol/Bph/C23O genotype (Table 1) revealed that these strains obviously harboured two distinct ISPaDit gene sequence types (Fig. 1B). Cluster analysis indicated that the partial 212–215 aa peptide sequences deduced from the ISPaDit gene fragments (Fig. 1) fell into distinct lineages of the diterpenoid dioxygenase ISPa sequence cluster (Fig. 2). With the exception of the putative ISPaDit peptide of strain LB400 and one of the two ISPaDit peptides derived from the BTEX-degrading isolate Pseudomonas sp. 1YB2, all the remaining peptide sequences obtained from strains of the same genus grouped together and shared more than 90% amino acid sequence similarity. The ISPaDit peptides deduced from the SSCP single-strand products of Cupriavidus spp. and Burkholderia spp. grouped within distinct branches that shared less than 82% identical amino acid positions with previously reported ISPaDit peptide sequences.

Molecular detection and diversity of DitA1 genes 1207 Fig. 1. PCR-SSCP analysis of putative ditA1 gene segments generated from (A) characterized aromatic-hydrocarbon- and/or DhA-degrading bacteria (B) strains isolated from BTEX-contaminated soils, and (C) strains isolated from PCB-contaminated soil. The strain designations are given above the gel image. NTC1, NTC2, NTC3: PCR non-template controls. Marker, Molecular Weight Marker III (250 ng per lane, Roche) was used to normalize the band patterns. Single-strand products exhibiting low electrophoretic mobilities, as those generated from Cupriavidus sp. BKME-6 or Cupriavidus sp. PS12 resulted in the detection of two minor bands representing alternative single-strand conformers of the same sequence type.

Fig. 2. Cluster analysis of ISPaDit peptide sequences (comprising 212–215 amino acids, corresponding to positions 182–395 of DitA1 of P. abietaniphila BKME-9) deduced from putative ISPaDit segments isolated from SSCP profiles of reference strains and environmental isolates. GenBank accession numbers of known ISPaDit peptides are given in parentheses. The DNA sequence of the ISPaDit gene fragment obtained from P. vancouverensis DhA-51 differed in 12 positions from the previously published sequence (AF145210); however, the translation product of the sequence identified here was devoid of a reading frame shift observed in the previously published sequence. Homologous sequences identified in the unfinished genomes of P. aeruginosa 2192 (NZ AAKW01000022; locus tags Paer2_01002043 and Paer2_01002059) and Sphingomonas sp. SKA58 (NZ AAQG01000001) were retrieved from GenBank, and the sequence of strain SKA58 was used as an out-group. The bootstrap consensus tree was constructed by using the neighbour-joining method and bootstrap values above 75% (calculated from 500 re-samplings) are indicated at the nodes. The scale bar corresponds to an estimated evolutionary distance of 0.1 amino acid substitutions per site. Cluster designations are indicated at the right-hand side of the figure.

1208 R. Witzig et al.

Fig. 3. Evolutionary trees reconstructed from nucleotide sequences of (A) gyrB genes and (B) ditA1 genes. The trees were constructed by using the neighbour-joining method. Bootstrap values above 75% (calculated from 500 re-samplings) are indicated at the nodes. The scale bar corresponds to an estimated evolutionary distance of 0.05 nucleotide substitutions per site. Genus-specific clusters are indicated by brackets: Ps, Pseudomonas-cluster; Cu, Cupriavidus-cluster; Bu, Burkholderia-cluster.

Pseudomonas sp. 1YB2 contained two distinct ISPaDit gene copies; with one allele (Fig. 1A, lower SSCP band) affiliated to the Pseudomonas-cluster and the other (Fig. 1A, upper SSCP band) more closely related to the putative DitA1 peptide sequences of Zoogloea resiniphila DhA-35 and Mycobacterium sp. DhA-55 (Yu et al., 1999) (Fig. 2). Interestingly, two distinct putative ISPaDit sequence types could be also identified in the unfinished genome of Pseudomonas aeruginosa 2192, with one of them (type I) affiliated to the Pseudomonas-cluster and the other (type II) more closely related to the upper-SSCPband-product of Pseudomonas sp. 1YB2 (Fig. 2). Nucleotide sequence polymorphisms of the 16S rRNA, gyrB and ditA1 genes of strains harbouring ditA1-homologues Because the tree inferred from DitA1 peptide sequences (Fig. 2) suggested a close linkage between host phylogeny and ditA1 gene evolution, the phylogenetic relationships between ditA1-harbouring isolates were inferred on the basis of 16S rRNA gene sequence analysis (Supplementary material Fig. S3). The 16S rRNA genes of the ditA1-harbouring Pseudomonas, Cupriavidus and Burkholderia strains were generally closely affiliated to type species of the respective genera. However, while pulse field gel electrophoresis demonstrated slight genomic differences between the three Cupriavidus strains PS12, BKME-6 and WBF7 and the four Burkholderia strains WBF1, WBF2, WBF3 and WBF4, respectively (data not shown), no differences between the 16S rRNA sequences of strains belonging to either Burkholderia or Cupriavidus were observed.

Because 16S rRNA sequence analysis may not be sufficiently discriminatory to permit resolution of intrageneric relationships (Yamamoto and Harayama, 1995; Yamamoto et al., 2000), the DNA gyrase subunit B genes (gyrB) were also used for phylogenetic analysis (Yamamoto et al., 2000). In fact, the gyrB nucleotide sequence variability allowed the closely related Cupriavidus and Burkholderia strains to be distinguished (Fig. 3A). Moreover, there was a significant congruence with regard to a genus-specific sequence clustering of gyrB and ditA1 genes (Fig. 3 A and B). Major differences were observed with the putative ditA1 sequence of LB400, which did not group together with ditA1 gene sequences of other Burkholderia strains, and the outgrouping putative ditA1 alleles of Pseudomonas strains 1YB2 and 2192. It has previously been reported that the gyrB genes of several bacterial taxa have evolved mainly by synonymous substitutions (that is, under ongoing purifying selection against deleterious non-synonymous mutations) (Yamamoto and Harayama, 1998; Dauga, 2002; Cladera et al., 2004). To determine whether the ditA1 genes are under similar negative selection pressure, the mean values of synonymous (pS) and non-synonymous distances (pN) within the genera Pseudomonas, Cupriavidus and Burkholderia were compared (Table 3). Congruent with previous findings (Yamamoto and Harayama, 1998; Cladera et al., 2004), strong purifying selection was observed for the gyrB genes of each of the three genera, as indicated by pN/pS ratios well below 1. Similarly, strong purifying selection has been operating during evolution of ditA1 genes of members of each genus (Table 3).

Molecular detection and diversity of DitA1 genes 1209 PCR-SSCP analysis of ditA1-homologues in BTEX- and PCB-contaminated soils The presence of ditA1-homologous genes in various reference strains and isolates enriched for independent purposes suggested that such genes are widespread (at least in the Proteobacteria analysed in this study). To obtain more information on the natural diversity of genes homologous to ditA1, the developed PCR-SSCP method was used to screen for the presence of ISPaDit sequence types in bacterial communities of four soil samples, which had previously been used for isolation of strains from contaminated soils. ISPaDit-PCR-SSCP analyses (Fig. 4A and B) indicated a broad diversity of ditA1-like genes to be present in each of the soils. From the BTEX-contaminated soil fingerprints, a total of 30 different ISPaDit gene segment types were isolated and cluster analysis revealed that the ISPaDit peptides deduced from re-amplified single strands were distributed into six different major lineages (Fig. 4C). The majority of the peptide sequences grouped within either the Pseudomonas-cluster or the BTEX soil-cluster I, which was more closely related to ISPaDit sequences identified in Burkholderia and Cupriavidus strains. The ISPaDit peptide sequences inferred from three bands (6, 28 and 29) of the BTEX-contaminated soil 1Y fingerprint profile (Fig. 4A) were identical in mobility and sequence to the corresponding ditA1-homologues identified in the benzene-degrading Pseudomonas isolate 1YB2. From the PCB-contaminated soil fingerprint, a total of 12 ISPaDit gene segment types differing in at least one nucleotide position were recovered and cluster analysis revealed that the deduced ISPaDit peptide sequences were distributed into three distinct major lineages (Fig. 4C). Three bands (7, 8 and 9) and band 17 (Fig. 4B) contained nucleotide sequence types similar to those of Burkholderia spp. WBF5 and WBF6 respectively. The peptide sequence deduced from band 1, however,

showed no clear relationship to ISPaDit types derived from pure culture strains. Nine highly similar ISPa sequence types were obtained from bands migrating at different positions in the SSCP gel, the translation products of which converged to three different deduced amino acid sequences (Fig. 4C). Because the same segregating nucleotide positions were identified in at least three individual clones exhibiting identical or nearly identical sequences, sequencing and/or PCR errors appeared to be unlikely to account for the observed sequence variation. Discussion The results of this study demonstrate that the metabolic capability for degradation of the abietane diterpenoid DhA is widespread in a collection of reference strains and isolates, which have previously been enriched from diverse sources, based on their ability to degrade different aromatic hydrocarbons rather than resin acids. Studies of the biochemistry of the aerobic biodegradation of resin acids had suggested convergent pathways for abietane diterpenoid metabolism that channels the non-aromatic abietanes and dehydroabietic acid into the central metabolic intermediate 7-oxo-DhA (Martin and Mohn, 1999; 2000; Smith et al., 2004). This compound was also detected as an intermediate during growth of all strains analysed in this study. Moreover, all strains capable of degrading DhA were found to harbour a gene segment encoding a peptide homologous to DitA1 of P. abietaniphila BKME-9 and expression analyses of ditA1-mRNA in P. pseudoalcaligenes KF707 and Pseudomonas sp. Cam-1 are in accordance with a substrate-induced resin acid-degrading enzyme system in these strains, similar to the one previously reported for P. abietaniphila BKME-9 (Martin and Mohn, 2000). Even though diverse bacteria were found to be capable of degrading DhA and structurally related resin acids (Martin et al., 1999; Mohn et al., 1999), only a few

Table 3. Evolutionary distances of gyrB and ditA1 gene sequences between members of the genera Pseudomonas, Burkholderia and Cupriavidus.a

gyrB Pseudomonas Burkholderia Cupriavidus ditA1 Pseudomonas c Burkholderiad Cupriavidus a. b. c. d.

% average pairwise distanceb

No. of polymorphic sites

pN/pS ratio

0.150 0.043 0.011

304 75 14

0.044 (0.046) 0.007 (0.009) 0.003 (0.003)

0.419 (0.650) 0.151 (0.177) 0.037 (0.038)

0.105 (0.071) 0.046 (0.051) 0.081 (0.079)

0.145 0.034 0.021

205 53 19

0.028 (0.028) 0.003 (0.003) 0.000 (0.000)

0.460 (0.802) 0.129 (0.153) 0.085 (0.090)

0.061 (0.035) 0.023 (0.020) 0.000 (0.000)

Numbers in parentheses were calculated using the Jukes–Cantor model (Jukes and Cantor, 1969). Calculated using the Jukes–Cantor model (Jukes and Cantor, 1969). Excluding the upper-SSCP-band-product of Pseudomonas sp. 1YB2 and the type II sequence of P. aeruginosa 2192 (see Fig. 3B). Excluding the putative ISPaDit gene sequence of B. xenovorans LB400.

1210 R. Witzig et al.

Molecular detection and diversity of DitA1 genes 1211 Fig. 4. PCR-SSCP profiles of putative ISPaDit gene segments amplified from contaminated soils and ditA1-harbouring bacterial strains isolated from the corresponding sites (A and B) and distance analysis of deduced amino acid sequences (comprising 212–215 amino acids, corresponding to positions 182–395 of DitA1 of P. abietaniphila BKME-9), of ISPaDit gene segments derived from SSCP profiles of BTEX- and PCB-contaminated soil samples (C). The following DNA sources were used as a template in PCR: (A) BTEX soil 1Y (lane 1), BTEX soil 1X (lane 2), BTEX soil 3Y (lane 3), Pseudomonas sp. 1YB2 (lane 4), Pseudomonas sp. 1XB1 (lane 5), Pseudomonas sp. IA1YICDB (lane 6); (B) PCB-contaminated soil (lane 7), Burkholderia sp. WBF3 (lane 8), Burkholderia sp. WBF4 (lane 9), Burkholderia sp. WBF6 (lane 10), Burkholderia sp. WBF2 (lane 11), Burkholderia sp. WBF1 (lane 12), Burkholderia sp. WBF5 (lane 13), Cupriavidus sp. WBF7 (lane 14). M, Molecular Weight Marker III (250 ng per lane, Roche) was used to normalize the band patterns. Numbered lines indicate selected bands for cloning and sequencing. Single-strand products were identified by direct sequencing (white dots) or cloning and sequencing (black dots) of the PCR-reamplification product. Bands for which direct sequencing indicated the presence of underlying sequence types (marked by hatched dots) were also subjected to cloning. C. The putative ditA1-homologues were designated according to the sample origin (BTEX-1Y-Dit, BTEX-1X-Dit, BTEX-3Y-Dit, or PCB-Dit), the procedure of sequence recovery, i.e. direct sequencing of re-amplified SSCP products (-d) or sequencing of cloned re-amplification products (-c), followed by the band position number. In cases where more than one consensus sequence was retrieved by cloning, the different sequence types are indicated by lowercase letters (a–c) after the band position number. The bootstrap consensus tree was reconstructed from a JTT-model-based distance matrix (Jones et al., 1992) using the neighbour-joining method. Bootstrap values above 75% (calculated from 500 re-samplings) are indicated at the nodes. The homologous sequence identified in the unfinished genome of Mycobacterium sp. JLS (NZ AAQC00000000) was retrieved from GenBank and used as an out-group (not shown). The scale bar corresponds to an estimated evolutionary distance of 0.1 amino acid substitutions per site. The different subclusters of putative diterpenoid dioxygenase ISPaDit peptides are indicated at the right-hand side of the figure: Ps, Pseudomonas-cluster; Cu, Cupriavidus-cluster; Bu, Burkholderia-cluster; BTEX I, BTEX-soil-cluster I; BTEX II, BTEX-soil-cluster II; BTEX III, BTEX-soil-cluster III; PCB, PCB-soil-cluster.

attempts have been undertaken to investigate the abundance and diversity of the catabolic genes involved in resin acid degradation. In a survey using primers designed to specifically amplify the ditA1 gene of strain BKME-9, Yu and colleagues (Yu et al., 1999) could amplify products from various biotreatment systems for PPME and confirm by restriction digestion analysis that the amplified genes were similar to ditA1. However, because the numbers of strains estimated to harbour a ditA1-homologue could not account for the observed performance of the PPME treatment systems, the authors speculated that the detected ditA1 gene-containing microorganisms were quantitatively only minor members of the resin acid-degrading populations. In fact, the applied primer set failed to amplify ditA1-like gene sequences from resin acid-degrading bacteria except strain BKME-9 (Yu et al., 1999; 2000) and, because of the high number of mismatching positions within the forward primer ditA1719f (data not shown), it seems unlikely that any of the sequences identified here could have been amplified with the primer set ditA1-719f/ditA1-1212r used by Yu and colleagues (Yu et al., 1999). In a second PCR approach, targeting conserved DNA regions of ditA1 encoding the [2Fe-2S] Rieske cluster-coordinating site and a conserved Asp residue that has been shown to be involved in gating electron transport in Rieske non-haem oxygenase reactions, Yu and colleauges (Yu et al., 1999) were able to amplify putative ditA1-homologues from six other resin acid-degrading bacteria, but still some of the analysed DhA-degraders failed to result in PCR amplification. Using the new degenerate primer set ditAf543/ditAr1186 (which was designed to complement conserved regions of an updated alignment of the putative ISPaDit gene segments), it was possible to amplify and characterize putative ditA1-homologues from 18 bacterial species of three different genera, including three characterized DhA-

degraders (Pseudomonas sp. DhA-91, Cupriavidus sp. BKME-6 and Burkholderia sp. DhA-54) for which previous attempts to amplify ISPaDit gene segments by PCR were unsuccessful (Yu et al., 1999; 2000). Moreover, the PCR results were in perfect accordance with results from growth experiments (Table 1), suggesting that the detected gene segments are involved in DhA-degradation in the PCR-positive strains, as demonstrated for the strains KF707, Cam-1 and WBF4 (Supplementary material Fig. S2). However, the primer binding sites targeted here appear not to be conserved in the genomes of four resin acid degraders (Pseudomonas strains IpA-1, IpA-2, IpA-92 and IpA-93), which had originally been isolated on isopimaric acid (Wilson et al., 1996; Yu et al., 2000) (Table 1). Studies on the biodegradation of resin acids by Gramnegative bacteria have suggested distinct biochemical pathways for the degradation of abietane and pimarane diterpenes (Wilson et al., 1996; Martin et al., 1999; Yu et al., 2000). The failure of PCR primers ditAf543/ ditAr1186 to amplify a diterpenoid dioxygenase a-subunit gene in resin acid degraders exhibiting higher specificity towards IpA degradation indicates that these strains contain genes encoding an a-subunit differing in sequence from those previously reported for Gramnegative strains capable of degrading DhA, at least at the regions targeted here. Possibly, these a-subunits belong to Rieske non-haem iron oxygenases of another yet to be identified subfamily. Previous analyses (Martin and Mohn, 1999; Yu et al., 1999) have suggested that DitA1 of strain BKME-9 and the homologous sequences of six other DhA-degrading strains formed a distinct group within the Rieske nonhaem iron oxygenase ISPa family and three divergent lineages had been recognized. Cluster analysis of the partial putative ISPaDit protein sequences deduced in this

1212 R. Witzig et al. study indicates that the ISPaDit gene sequences obtained from Burkholderia and Cupriavidus strains and, even more noticeable, those recovered from soil samples represent new major branches within the ISPaDit sequence cluster (Fig. 4C). Interestingly, the results presented in Fig. 3 suggest the ditA1 gene sequence divergence to be linked to the host phylogeny. This finding contrasts with the related Rieske non-haem iron oxygenase ISPa genes of the naphthalene and biphenyl subfamilies, which have often been reported to be localized on mobile elements and dispersed via horizontal transfer (Nojiri et al., 2004; Pieper, 2005), thus disconnecting catabolic gene evolution from the host cells phylogeny. Moreover, the Pseudomonas strains IA1YICDB, JR1, Cam-1 and DhA-91 contained considerably different ditA1 sequence types (differing in up to 24 base positions), which however, encoded identical ISPaDit peptide sequences. Similarly, identical peptides were encoded by the putative ISPaDit genes (differing in up to 16 base positions) of the Cupriavidus strains BKME-6, PS12 and WBF7. The fact that mainly synonymous substitutions contributed to the sequence variation of ditA1 genes (Table 3) suggests that there are structural and/or functional constraints on amino acid replacements in the analysed region of DitA1 and purifying selection was acting to remove deleterious amino acid mutations from the populations while neutral or nearly neutral silent variants could persist (Kimura, 1983). Reconstruction of the evolutionary history of ditA1 and gyrB genes further suggests that they were both present in the most recent common ancestor of Pseudomonas, Cupriavidus and Burkholderia before speciation occurred, and that they coevolved during the course of evolution (Fig. 3). The isolated position of the LB400-ditA1-homologue, however, indicates a different evolutionary history compared with the ditA1 genes of other Burkholderia strains. The evolutionary analysis of ISPaDit homologues further raises the question of the function of the second ditA1homologue in Pseudomonas spp. 1YB2 and 2192. Both strains harbour a ditA1-homologue of the Pseudomonascluster (Fig. 2), which is presumed to be involved in DhAdegradation. Because the primers used by Yu and colleagues (Yu et al., 1999) could not amplify a broad range of ditA1 gene segments, the presence of a second ISPaDit gene with higher similarity to the Pseudomonas-, Cupriavidus-, or Burkholderia-clusters in strains harbouring the outlying sequence types, i.e. the non-classified b-Proteobacterium DhA-73, Schlegelella thermodepolymerans DhA-71; Mycobacterium sp. DhA-55 and Z. resiniphila DhA-35 (Fig. 2), analogous to the case of Pseudomonas sp. 1YB2 and P. aeruginosa 2192, cannot be excluded. PCR-SSCP fingerprinting of the soil samples demonstrated the presence of a broad, previously unrecognized

diversity of ditA1-homologues in these environments. Earlier characterizations of both the 16S rRNA (Hendrickx et al., 2005) and functional (C23O and ISPaTol/Bph) gene structures (Junca and Pieper, 2004; Witzig et al., 2006) of the BTEX-degrading bacterial communities, using culture-dependent and -independent methods, indicated that Pseudomonas spp. dominated the BTEXcontaminated soils. In particular, phylotypes and genotypes identical to those of Pseudomonas sp. 1YB2 were identified as being strongly enriched in BTEX soil 1Y using these methods. The previous findings are further supported by the ISPaDit-PCR-SSCP analysis presented in this study, where ditA1 gene sequence types identical to those identified in strain 1YB2 were abundant in the highly contaminated soil. In view of the apparent correlation between bacterial host phylogeny and ditA1 gene phylogeny, at least for ditA1 genes sensu stricto, the sequence types identified in the BTEX soil ISPaDitfingerprints further suggest that diverse Pseudomonas strains sharing the ability to degrade resin acids are present at this site (Fig. 4C). Moreover, the detection of ISPaDit sequence types more closely related to those of Burkholderia spp. and Cupriavidus spp. (BTEX-soilcluster I), for which a cultured representative has not yet been described, indicates that bacterial phyla other than Pseudomonas spp. may also be abundant in the BTEXcontaminated soils. None of the reference strains shown in this study to harbour a ditA1-homologue had previously been reported to be capable of growth on DhA. However, they have all been recovered and characterized from diverse environments as aromatic hydrocarbon-degrading bacteria, being capable of degrading chlorobenzene (strain PS12), (polychlorinated) biphenyl (strains Cam-1, KF707 and JR1), or phenol (strains OX1, CF600). Thus, the occurrence of ditA1-homologues seems to be widespread in organisms sharing the capability of aromatic hydrocarbon degradation and apparently such genes can be detected with a high probability in strains affiliated to the genera Pseudomonas, Cupriavidus and Burkholderia. Yu and colleagues (Yu et al., 2000) observed that hydrocarbon contamination in Arctic tundra soil (in which no resin acids were detectable) promoted the survival and/or selection of resin acid degraders and pointed out that this might have been the result of selection for hydrocarbon degraders that coincidentally use resin acids. Indeed, their hypothesis is corroborated by this study, demonstrating that numerous aromatic hydrocarbondegrading strains affiliated to the genera Burkholderia, Cupriavidus and Pseudomonas, members of which are among the most versatile aromatic hydrocarbon degraders and thus are often enriched at contaminated environments, and are capable of resin acid degradation. While the presence of both (aromatic hydrocarbon and abietane

ÂŠ 2007 The Authors Journal compilation ÂŠ 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 1202â&#x20AC;&#x201C;1218

Molecular detection and diversity of DitA1 genes 1213 resin acid degradative) genotypes appears to be purely coincidental, this feature could be an important factor relating to the microbial ecology of aromatic hydrocarbon degradation. When confronted with aromatic hydrocarbon pollutants the organisms’ ability to use the pollutant is likely the dominating factor in shaping the microbial community, while the presence of resin acids prior to pollution may have selected for resin acid-degrading populations and thus preconditioned the community structure.

Experimental procedures Bacterial strains and growth conditions The bacterial strains used in this study (Table 1) comprised Pseudomonas vancouverensis DhA-51, which has previously been described to possess a Rieske non-haem ISPa gene of the diterpenoid oxygenase subfamily (ISPaDit), seven further strains that have been reported to be capable of growing on resin acids, and 21 aromatic hydrocarbon-degrading reference strains. According to the 16S rRNA gene sequence data (see below), the previously characterized strains Ralstonia sp. PS12 (Beil et al., 1997) and Ralstonia sp. BKME-6 (Mohn et al., 1999) and Burkholderia sp. IpA-51 (Mohn et al., 1999) belong to the genera Cupriavidus (Vandamme and Coenye, 2004) and Pandoraea (Coenye et al., 2000) respectively. The strain collection further comprised seven Burkholderia and Cupriavidus strains, which had been randomly isolated on R2A medium (Difco) from PCB-contaminated soil (Nogales et al., 1999), and 36 bacterial isolates obtained from BTEXcontaminated soils (Junca and Pieper, 2004; Witzig et al., 2006).

Chemicals Dehydroabietic acid (99% purity) was purchased from Helix Biotech, New Westminster, BC, Canada.

Soil samples Three BTEX-contaminated soil samples were collected from the unsaturated (X) and capillary fringe (Y) horizons at two sampling sites (site 3 contained slightly BTEX-contaminated soil whereas site 1 was highly contaminated) of a BTEXcontaminated aquifer located in the Czech Republic. The chemical and microbiological characteristics of the soil samples have been described previously (Junca and Pieper, 2004; Hendrickx et al., 2005). The PCB-contaminated soil sample was taken from the upper few centimetres of the soil surface at a PCB-polluted site near Wittenberg (Germany) in 2000 and stored at 4°C until used for DNA extraction. Detailed chemical characteristics of the sampling site have been previously described (Nogales et al., 1999).

Extraction of DNA from pure cultures and soil Total genomic DNA was extracted and quantified from pure culture strains and BTEX-contaminated soil samples as pre-

viously described (Junca and Pieper, 2004; Witzig et al., 2006). Total DNA from the PCB-contaminated soil sample (10 g wet weight) was extracted in triplicate using a UltraCleanTM MegaPrep soil DNA isolation kit (MO BIO Laboratories, Carlsbad, CA) in combination with cell disruption by bead beating for 30 s using a MSK cell homogenizer (Braun, Melsungen, Germany). DNA was precipitated and purified using standard methods (Sambrook et al., 1989), followed by a further purification step with the Wizard DNA clean-up system (Promega, Madison, WI). DNA concentrations from soil extracts were quantified using a PicoGreen doublestranded DNA (dsDNA) quantitation kit (Molecular Probes, Leiden, the Netherlands). The DNA extracts from BTEXcontaminated soil samples containing approximately 2 ng ml-1 DNA were used either directly or 10-fold diluted in Tris-HCl buffer (10 mM, pH 8.0), while the DNA extracts from the PCB-contaminated soil containing approximately 380 ng ml-1 DNA were 50- or 100-fold diluted in Tris-HCl buffer (10 mM, pH 8.0) and used as template DNA in PCR.

PCR amplification of Rieske non-haem iron oxygenase ISPa gene segments Two primer sets were used for PCR amplification of Rieske non-haem iron oxygenase ISPaDit gene segments. ISPaTol/Bph and/or ISPaDit gene segments (of 805–829 bp and 883– 892 bp respectively) were amplified with the degenerate primer set bphAf371B/bphAr1153-2 using the previously reported protocol (Witzig et al., 2006) with an increased final concentration of MgCl2 (2 mM). Amplification products were separated by agarose gel electrophoresis (2.5% agarose, 1¥ TAE buffer) and visualized by ethidium bromide staining. PCR products of interest were purified from agarose gels using a QIAquick gel extraction kit (Qiagen, Hilden, Germany), cloned and sequenced (see below). Based on a DNA alignment using CLUSTALW (Chenna et al., 2003) of the 883–892 bp putative ISPaDit gene segments amplified from the Cupriavidus spp. BKME-6, PS12 and WBF7 and the Burkholderia spp. WBF1, WBF2, WBF3, WBF5 and WBF6 with the ISPaDit gene ditA1 of P. abietaniphila BKME-9 (GenBank Accession Number AF119621), the putative ditA1 sequence of B. xenovoransLB400 (CP000272) and six previously reported ditA1-homologous sequences of other resin acid-degrading bacteria (AF145209, AF145210, AF145211, AF145212, AF145213, AF145214), primers ditAf543 (5′GGC GAT GCS AAG TGG TAY TWC GAC-3′) and ditAr1186 (5′-CCA CGT GTC MG AGT CRT CCT GYTC-3′) were designed to target conserved alignment regions corresponding to nucleotide positions 520–543 and 1186–1209 in the ditA1 sequence of P. abietaniphila BKME-9. PCR for subsequent SSCP analysis was performed with a 5′ end phosphorylated reverse primer ditAr1186. PCR amplifications with primer set ditAf543/ditAr1186 were performed in a 50 ml final volume containing 0.5 mM of each primer (MWG-Biotech, Ebersberg, Germany), 200 mM of each deoxynucleotide (Bioline, Luckenwalde, Germany), 1¥ PCR buffer (Qiagen), supplemented with 1.5 mM MgCl2, 2.5 U of HotStarTaq polymerase (Qiagen), and 2 ml of template DNA. The following PCR conditions were used: an initial denaturation for 15 min at 95°C, followed by 32 cycles of 40 s at 94°C, 40 s at 58°C, 60 s at 72°C and a final elongation for 10 min at 72°C. PCR

1214 R. Witzig et al. amplicons (2 ml of the PCR) were verified by agarose gel electrophoresis (1.5% agarose, 1¥ TAE buffer) and ethidium bromide staining.

Growth on DhA Strains were maintained on R2A medium or M9 mineral salt medium (Sambrook et al., 1989) containing 0.4 mM DhA and 3% agar. Liquid cultures were grown in M9 mineral salt medium supplemented with DhA (0.24 mM) as the sole source of carbon and energy in fluted Erlenmeyer flasks at 30°C on a rotary shaker at 150 r.p.m. For monitoring growth on DhA, Erlenmeyer flasks containing 25 ml of medium were inoculated with 250 ml of cells pregrown in R2A or mineral medium supplemented with 0.4 mM DhA, and cell-free supernatants were regularly sampled for HPLC analysis (see below). Culture supernatants containing higher amounts of metabolites sufficient for in situ 1H NMR and HPLC-MS analyses were obtained from cell suspensions grown on 0.4 mM of DhA, to which additional 0.32–0.4 mM of substrate was added after complete DhA consumption. Growth on DhA was assessed by protein quantification after 48 h of incubation. For the protein assay, cells were harvested from 10 ml of culture, re-suspended in 900 ml of 0.4 M NaOH, heated for 10 min at 95°C and centrifuged at 16 000 g for 10 min. Supernatants were neutralized with 100 ml of 4 M HCl and protein was quantified using the Bio-Rad Protein Assay (BioRad, München, Germany) with bovine serum albumine as a standard.

Extraction of mRNA, cDNA synthesis and RT-PCR For gene expression studies, P. pseudoalcaligenes KF707, Pseudomonas sp. Cam-1 and Burkholderia sp. WBF4 grown on DhA (0.24 mM) were harvested and used to inoculate 25 ml of fresh M9 mineral medium containing DhA (0.24 mM). To assess constitutive expression, the strains were grown in parallel in M9 mineral medium supplemented with 5 mM fructose. After the cultures had degraded 67–84% of the substrate (DhA cultures) or reached the mid-log phase (fructose cultures), 25 ml of RNAprotect (Qiagen) was added to the cell suspensions. The samples were vortexed briefly and incubated on ice for 5 min, followed by centrifugation for 30 min at 7300 g. Total RNA was isolated by a modified version of the method of Siering and Ghiorse (Siering and Ghiorse, 1997). In brief, the cells were suspended in 700 ml of sterile nuclease-free water (Qiagen), transferred to lysing matrix tubes provided with the FastDNA spin kit for soil (Bio101 Systems, Q-BIOgene) and centrifuged for 10 min at 16 000 g. Supernatants were removed, and 750 ml of phosphate buffer (0.12 M, pH 7.0) and 500 ml of acidic phenol (pH 4.6) (Roth, Karlsruhe, Germany) added, then cells were disrupted by bead beating for 45 s in a FastPrep FP120A instrument (Bio101 Systems, Q-BIOgene). The nucleic acids were precipitated from the aqueous phase and purified using previously described methods (Siering and Ghiorse, 1997). Residual DNA was removed by incubating aliquots of the samples with RNase-free DNase I (Roche, Mannheim, Germany) at room temperature for 2 h, followed by RNA purification using a RNeasy kit (Qiagen) and quantification

using a RiboGreen RNA quantitation kit (Molecular Probes). Extracts of cells grown on DhA comprised RNA concentrations of 350 (WBF4), 680 (KF707) and 700 ng (Cam-1) while extracts of cells grown on fructose comprised 30 (WBF4), 50 (KF707) and 100 (Cam-1) ng ml-1 respectively. cDNA was synthesized from 1 to 3 ml of total RNA using a first-strand cDNA synthesis kit for RT-PCR (Roche, Mannheim, Germany). The reverse transcription reaction mixtures were serially diluted (3.2-fold) with nuclease-free water (Qiagen) and 1 ml of each dilution was subjected to amplification by PCR using the primer set ditAf543/ditAr1186 and the conditions described above. Amplification products were separated in 1.5% agarose gels and stained with ethidium bromide. Product bands were purified from agarose gels using a QIAquick PCR Purification Kit (Qiagen) and sequenced to verify their identity.

Analytical methods Transformation of DhA was monitored by HPLC analysis. Aliquots of 10 ml of cell-free supernatants were analysed with a Shimadzu HPLC system (LC-10AD liquid chromatograph, DGU-3 A degasser, SPD-M10A diode array detector and FCV-10AL solvent mixer) equipped with a Lichrospher RP8 column (125 mm by 4.6 mm, Bischoff, Leonberg, Germany) using an aqueous solvent system (flow rate, 1 ml min-1) containing 0.01% (v/v) H3PO4 (87%) and 30% (v/v) methanol. High-performance liquid chromatography-mass spectrometry was performed using an Agilent 1000 LC system (Agilent Technologies, Palo Alto, CA) equipped with a Nucleosil 1205-C18 column (125 mm by 2 mm), coupled to a Sciex API2000 mass spectrometer (Perkin-Elmer Sciex, Foster City, CA) equipped with a TurboIonSpray (ESI) source. Elution was performed at a flow rate of 0.3 ml min-1 using an aqueous solvent system with a linear gradient of 5 mM ammonium acetate (pH 5.5) in 5% acetonitrile to 5 mM ammonium acetate (pH 5.5) in 95% acetonitrile over 9 ml followed by isocratic elution with 5 mM ammoniumacetate (pH 5.5) in 95% acetonitrile over 3 ml. Mass spectrometry analysis was performed using the Sciex TurboIonSpray source at a temperature of 350°C in positive and negative ion mode. The one-dimensional 1H NMR spectra were recorded at 300 K on a Avance DMX 600 NMR spectrometer (Bruker, Rheinstetten, Germany) locked to the deuterium resonance of D2O in the solution. Spectra were recorded by using the standard Bruker 1D NOESY suppression sequence with 280 scans, each with a 1.8 s acquisition time and a 1.3 s relaxation delay.

PCR-SSCP analysis of ditA1-homologues PCR-SSCP with the single-strand removal approach (Schwieger and Tebbe, 1998) was used for detection and differentiation of ISPaDit gene segments and was carried out using a previously described protocol (Witzig et al., 2006), except for diluting the single strand with Tris-HCl buffer (10 mM, pH 8.0) to a final concentration of 0.9 ng ml-1 prior to SSCP. The influence of MDETM gel solution (Cambrex Bio Science, Rockland, ME) concentration and gel temperature on the resolution performance of the SSCP method was

Molecular detection and diversity of DitA1 genes 1215 tested with single-stranded ISPaDit gene segments generated from B. xenovorans LB400, Pseudomonas sp. Cam-1, P. pseudoalcaligenes KF707, Cupriavidus sp. PS12 and Cupriavidus sp. WBF7 in individual runs using the following conditions (gel temperature/MDETM gel solution concentration): (i) 30°C/0.7¥, (ii) 34°C/0.7¥, (iii) 36°C/0.7¥, (iv) 38°C/ 0.7¥ and (v) 40°C/0.8¥. Electrophoresis under optimized conditions was performed on 21 cm ¥ 21 cm 0.8¥ MDETM gels in 1¥ TBE as running buffer at 400 V and 10 mA for 16 h at 40°C in a Pharmacia Macrophor electrophoresis unit 2010001 connected to a circulating water bath (Lauda ecoline RE104). The gels were silver-stained according to Bassam and colleagues (Bassam et al., 1991) and dried at room temperature. Analysis of the silver-stained gels and isolation of selected single-strand products was performed as described previously (Witzig et al., 2006).

PCR amplification of 16S rRNA and gyrB genes Phylogenetic relationships of strains harbouring a ditA1homologue were derived using the nucleotide sequences of the genes coding for 16S rRNA and DNA gyrase subunit B (gyrB). Nearly complete sequences of the 16S rRNA genes (corresponding to positions 28–1481 in the Escherichia coli numbering system) were determined directly from PCR fragments by using primers and conditions described by Lane (Lane, 1991). PCR amplification of the gyrB gene was performed following the method of Yamamoto and colleagues (Yamamoto et al., 2000); however, gyrB primers (MWGBiotech) omitted the M13-forward and -reverse primer sequence moieties.

DNA sequencing and sequence analysis PCR products were purified with a QIAquick PCR Purification Kit and sequenced using an ABI PRISM BigDye Terminator v1.1 Ready Reaction Cycle Sequencing Kit (Applied Biosystems) and an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). Primers used for sequencing reactions were the same as those used in the original PCR. Re-amplified SSCP products that resulted in ambiguous sequences, and the ISPaTol/Bph and ISPaDit gene segments that were amplified with the primer set bphAf371B/bphAr1153-2, were analysed after cloning with the pGEM-T-vector system (Promega). Plasmid inserts were amplified and sequenced with vector-specific primers M13-forward and M13-reverse (Promega). To account for potential PCR and sequencing errors, at least eight clones from each ligation reaction were screened for similar sequence types and consensus sequences [deduced from at least three identical or nearly identical (ⱕ 1% difference) clones] were generated for further analysis. Raw sequence data from both strands were assembled with Sequencher software (4.0.5) (Gene Codes Corporation, Ann Arbor, MI). DNA and protein similarity searches were performed using BLASTN and BLASTP programs from the National Centre for Biotechnology Information website. Nucleotide sequences of ditA1 and gyrB genes were translated and aligned at the protein level using CLUSTALW implemented in MEGA software version 3.1 (Kumar et al., 2004), and backtranslated to obtain the corresponding DNA alignments. ISPa

sequences of the biphenyl and naphthalene subfamily and gyrB sequences of closely related strains were retrieved from GenBank and included in the alignments of ditA1 and gyrB genes respectively. Evolutionary trees were constructed with MEGA software, using the neighbour-joining (NJ) method (Saitou and Nei, 1987) with genetic distances computed using the Jukes–Cantor (Jukes and Cantor, 1969) and JTT (Jones et al., 1992) substitution model for DNA and protein data respectively. Primer sequences, ambiguous positions and regions of insertions and deletions of the alignments were excluded. Consensus trees were inferred from a total of 500 bootstrap trees generated for each data set. The numbers of synonymous substitutions per synonymous site (pS) and of non-synonymous substitutions per nonsynonymous site (pN) were estimated by the method of Nei and Gojobori (Nei and Gojobori, 1986) and evolutionary distances (dS and dN) were corrected for multiple substitutions by the Jukes–Cantor model (Jukes and Cantor, 1969) using MEGA software. Strong evidence for the occurrence of purifying selection is provided by the fact that the number of synonymous nucleotide substitutions per synonymous site exceeds the number of non-synonymous nucleotide substitutions per non-synonymous site (Li et al., 1985). Values for dS and dN were estimated for each pairwise comparison between homologous sequences and from these values, pS (the average value of all pairwise dS values) and pN (the average value of all pairwise dN values) were estimated for ditA1 and gyrB data sets of each genus. The 16S rRNA sequences obtained in this study were analysed using the sequence match tool of the Ribosomal Database Project II (RDP-II) (Cole et al., 2003) and aligned against the most similar 16S rRNA gene sequences of type strains obtained from the RDP-II using CLUSTALW. Evolutionary distances (Jukes and Cantor, 1969) of 16S rRNA genes were calculated using only unambiguously determined nucleotide positions and a phylogenetic tree was constructed using the neighbour-joining method (Saitou and Nei, 1987). A bootstrap consensus tree was inferred from a total of 1000 re-samplings.

Nucleotide sequence accession numbers The nucleotide sequences reported in this study were deposited in the DDBJ/EMBL/GenBank databases under the following accession numbers: DQ777727–DQ777739 (16S rRNA gene sequences), DQ844792–DQ844810 (gyrB gene sequences), DQ679936–DQ679946 (bphA1 and ditA1 gene sequences amplified with primer set bphAf371B/ bphAr1153-2) and DQ789330–DQ789350, DQ844811– DQ844910, DQ844726–DQ844791, DQ852290–DQ852307 (ditA1 gene sequences amplified with primer set ditAf543/ ditAr1186).

Acknowledgements The authors wish to thank Professor W.W. Mohn for kindly providing the resin acid-degrading reference strains used in this work. We also thank Julia Bötel, Silke Kahl, Christel Kakoschke and Beate Jaschok-Kentner for excellent technical assistance, and Heinrich Steinmetz for performing the

1216 R. Witzig et al. HPLC-MS analysis. We gratefully acknowledge Dr Melissa Wos and Dr Andrew Oxley for their thoughtful comments on the manuscript and Dr Hans-Jürgen Hecht for inspiring discussions. This research was supported by project BIOTOOL (GOCE-003998) from the European Commission.

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Supplementary material The following supplementary material is available for this article online: Fig. S1. PCR screening for the presence of genes encoding Rieske non-haem iron oxygenase ISP proteins in (A) previously characterized BTEX-degrading isolates, (B) aromatic hydrocarbon-degrading reference strains, (C) isolates from PCB-contaminated soil, and (D) resin acid-degrading reference strains (Table 1) using the primer set bphAf371B/ phAr1153-2. Only the PCR-positive strains are shown, and their strain designations are given above the gel image. Marker, 1 kb plus DNA ladder (Invitrogen). PCR products that were purified from the gel, cloned and sequenced are indicated in Table 1. The smaller fragments (805–829 bp) represent gene segments encoding ISP peptides of the toluene/ biphenyl subfamily ISPaTol/Bph, whereas the longer fragments (883–892 bp) represent gene segments encoding ISP peptides of the diterpenoid dioxygenase branch ISPaDit. Fig. S2. RT-PCR amplification of ditA1-mRNA extracted from strains KF707 (A, B), Cam-1 (C, D) and WBF4 (E, F), grown on either DhA (A, C, E) or fructose (B, D, F). M, HyperLadder I (Bioline, Luckenwalde, Germany). cDNA generated from template RNA was serially diluted (3.2-fold) with nuclease-free water and 1 ml of each dilution was subjected to amplification by PCR (lanes 1–14). Negative controls included RT and PCRs devoid of reverse transcriptase (lane 15) and template cDNA (lane 16) respectively. PCRs containing 1 ng of genomic DNA as a template were used as positive control (lane 17). Fig. S3. Phylogenetic tree based on 16S rRNA gene sequences of bacterial strains harbouring a ditA1-homologue (shown in bold) and related reference and/or type strains. The bootstrap consensus tree (1000 re-samplings) was reconstructed from a sequence alignment of 1350 unambiguously determined nucleotide positions by neighbour-joining analysis using MEGA software (Kumar et al., 2004). Bootstrap values > 75% are shown at the nodes. GenBank accession numbers of the 16S rRNA sequences are given in parentheses. The scale bar represents per cent estimated sequence divergence. This material is available as part of the online article from http://www.blackwell-synergy.com