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  1. 1. 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 a broad, previously unrecognized diversity of diter- Victor Wray,2 Howard Junca1 and penoid dioxygenase genes in soils, and suggest that Dietmar H. Pieper1* other bacterial phyla may also harbour the genetic 1 Department of Environmental Microbiology, HZI – potential for DhA-degradation. Helmholtz Centre for Infection Research, Inhoffenstraße 7, D-38124 Braunschweig, Germany. 2 Introduction Department of Structural Biology, HZI – Helmholtz Centre for Infection Research, Inhoffenstraße 7, Resin acids are tricyclic diterpenoids synthesized by D-38124 Braunschweig, Germany. 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 com- Summary pounds are released from wood during pulping processes Resin acids are tricyclic diterpenoids naturally syn- and are commonly found in pulp and paper mill effluents thesized by trees that are released from wood during (PPME) (Liss et al., 1997; Quinn et al., 2003). Their pulping processes. Using a newly designed primer release into aquatic systems has led to considerable envi- set, genes similar to that encoding the DitA1 catalytic ronmental concern as they are significant contributors to a-subunit of the diterpenoid dioxygenase, a key the toxicity of PPME to aquatic organisms (Liss et al., enzyme in abietane resin acid degradation by 1997; Ellis et al., 2003). In contrast, diterpene resin acid Pseudomonas abietaniphila BKME-9, could be ampli- derivatives were demonstrated to maintain pharmaceuti- fied from different Pseudomonas strains, whereas cally useful properties such as antimicrobial (Savluchin- ditA1 gene sequence types representing distinct ske Feio et al., 1999), antiviral (Ohtsu et al., 2001), or branches in the evolutionary tree were amplified from antitumoral (Kinouchi et al., 2000) activity. Thus, the iden- Burkholderia and Cupriavidus isolates. All isolates tification and characterization of enzymes transforming harbouring a ditA1-homologue were capable of this class of compounds is of great interest for both bio- growth on dehydroabietic acid as the sole source of technological and environmental applications. carbon and energy and reverse transcription poly- The biodegradation of resin acids has been investi- merase chain reaction analysis in three strains con- gated for almost four decades [reviewed by Liss and firmed that ditA1 was expressed constitutively or in colleagues (1997) and Martin and colleagues (1999)] and response to DhA, demonstrating its involvement in consistent with the ubiquitous nature of resin acids, micro- DhA-degradation. Evolutionary analyses indicate that organisms with the ability to mineralize these compounds gyrB (as a phylogenetic marker) and ditA1 genes have have been isolated from numerous sources [reviewed by coevolved under purifying selection from their ances- Martin and colleagues (1999)]. The metabolic pathways of tral variants present in the most recent common degradation of abietic (AbA) and dehydroabietic (DhA) ancestor of the genera Pseudomonas, Cupriavidus acid, which are the most abundant types of resin acids and Burkholderia. A polymerase chain reaction- among those commonly found in PPME (Liss et al., 1997; single-strand conformation poylmorphism finger- Ellis et al., 2003), have initially been proposed on the printing method was established to monitor the basis of intermediates isolated from culture media (Biell- diversity of ditA1 genes in environmental samples. mann et al., 1973a,b) and were largely in agreement with The molecular fingerprints indicated the presence of recent molecular genetic studies on DhA-degradation by Pseudomonas abietaniphila BKME-9 (Martin and Mohn, 1999; 2000; Smith et al., 2004). Convergent pathways for Received 25 July, 2006; accepted 31 October, 2006. *For correspondence. E-mail dpi@helmholtz-hzi.de; Tel. the abietane diterpenoid [AbA, DhA and palustric acid (+49) 531 6181 4200; Fax (+49) 531 6181 4499. (PaA)] metabolism have been suggested, which lead to © 2007 The Authors Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd
  2. 2. Molecular detection and diversity of DitA1 genes 1203 the key intermediate 7-oxo-dehydroabietic acid (7-oxo- Results DhA) (Martin and Mohn, 1999; 2000; Smith et al., 2004). PCR amplification of ditA1-homologues from aromatic Smith and colleagues (Smith et al., 2004) proposed that a hydrocarbon-degrading reference strains and P450 monooxygenase catalyses the hydroxylation of DhA environmental isolates at C-7 to form 7-hydroxy-DhA, which is then further oxi- dized to 7-oxo-DhA, prior to dihydroxylation of the aro- When the degenerate primer set bphAf371B/bphAr115-2, matic ring. The latter reaction is catalysed by a Rieske which had previously been used for amplification of ISPa non-haem iron oxygenase (Martin and Mohn, 1999), genes of the toluene/biphenyl oxygenase subfamily resulting in the formation of a dihydrodiol, 7-oxo-11,12- (ISPaTol/Bph) (Witzig et al., 2006), was applied to a set of 21 dihydroxy-8,13-abietadienic acid, which is dehydroge- characterized aromatic hydrocarbon-degrading bacteria nated to form 7-oxo-11,12-diol and cleaved by an extradiol (Table 1), a second product of 892 bp was observed with ring-cleavage dioxygenase (Martin and Mohn, 2000). Evi- Burkholderia xenovorans LB400 and Cupriavidus sp. dence that the gene ditA1 encoding the a-subunit of the PS12 template DNA (Supplementary material Fig. S1). terminal oxygenase component (ISPaDit) of the diterpe- Similarly, PCRs with Burkholderia spp. WBF3 and WBF4 noid dioxygenase is essential for the degradation of PaA, as template resulted in simultaneous amplification of AbA and DhA by this strain has been provided by inacti- approximately 820 bp and 890 bp products. Single vation experiments (Martin and Mohn, 2000). product bands (890 bp) were observed with Cupriavidus Bacteria harbouring a ditA1 gene have been previ- sp. WBF7, Burkholderia sp. WBF1, Burkholderia sp. ously detected in various PPME biotreatment systems WBF2, Burkholderia sp. WBF5 and Burkholderia sp. (Yu et al., 1999). However, while it was reported that WBF6. The DNA sequences of the approximately 890 bp such strains are only minor members of the resin acid- PCR fragments had, among ISPa genes with validated degrading population (Yu et al., 1999), the primers activity of the gene product, highest nucleotide sequence employed by the authors failed to detect ditA1- similarity (71–77% DNA sequence identity) to the ISPaDit homologous genes in a number of characterized resin gene ditA1 of P. abietaniphila BKME-9 (Martin and Mohn, acid-degrading strains (Yu et al., 1999; 2000). This short- 1999). These results indicated that the DNA regions used coming prevents the identification of other key bacterial as primer binding sites for amplification of the ISPaTol/Bph taxa that contribute to the degradation of resin acids in gene sequences were, at least to some extent, also con- these systems. Thus, a more comprehensive analysis of served in ISPa subunits of the diterpenoid oxygenase the diversity and distribution of diterpenoid dioxygenase branch of Rieske non-haem iron oxygenases. However, genes would be of crucial importance to gain insight into when the primer set bphAf371B/bphAr1153-2 was used the ecology and diversity of microorganisms involved in for amplification of ISPa genes from eight references resin acid degradation and optimization of the biological strains reported to be capable of growth on dehydroabi- PPME treatment systems. Previous studies have dem- etic and/or isopimaric acid (Wilson et al., 1996; Mohn onstrated that the iron sulfur proteins of the oxygenase et al., 1999; Yu et al., 2000), a 892 bp PCR amplicon was component of Rieske non-haem iron oxygenases (ISPa) observed only with Cupriavidus sp. BKME-6 (Supplemen- provide excellent molecular targets for genetic analysis tary material Fig. S1, Table 1). Sequencing revealed that of aromatic hydrocarbon-degrading enzymes (Taylor this fragment was highly homologous (> 96.8% DNA et al., 2002; Kahl and Hofer, 2003), and polymerase sequence identity) to the ISPaDit gene fragments of chain reaction-single-strand conformation polymorphism Cupriavidus spp. PS12 and WBF7. (PCR-SSCP) analysis has been demonstrated to be a powerful tool for the molecular analysis of the structures Design of a new primer set for PCR amplification of of microbial communities and functional genes ditA1-homologues from reference strains and (Schwieger and Tebbe, 1998; Junca and Pieper, 2004; environmental isolates Witzig et al., 2006). In the present study, we report on the design of new Because the primer set bphAf371B/bphAr1153-2 failed to PCR primers for the molecular analysis of genes encod- amplify ISPaDit gene fragments from seven characterized ing the a-subunits of diterpenoid dioxygenases and the resin acid degraders, a new degenerate primer set application of a PCR-SSCP method for the screening of (ditAf543/ditA1186) was designed for amplification of ISPaDit gene polymorphisms in reference strain cultures gene sequences encoding ISPa peptides of the diterpe- and environmental samples. Furthermore, cultivation noid dioxygenase branch. Using this primer set, single studies in conjunction with reverse transcription (RT)-PCR products of 684–693 bp could be amplified from four ref- analysis of a subset of the cultivated strains were per- erence strains previously reported to be capable of formed to confirm that PCR-positive strains were capable degrading dehydroabietic acid (DhA-51, DhA-54, DhA-91 of degrading DhA. and BKME-6) (Table 1), whereas no amplification was © 2007 The Authors Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 1202–1218
  3. 3. 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 Growth on Reported DhA and ISPa bphAf371B ditAf543 transformation Organism gene(s) Reference(s) bphAr1153-2 ditAr1186 of DhAc Resin acid degraders Burkholderia sp. DhA-54 Mohn et al. (1999) – + +d Cupriavidus sp. BKME-6 Bicho et al. (1995) lb + +d Pseudomonas vancouverensis DhA-51 ditA1 Mohn et al. (1999); – + +d Yu et al. (1999) Pseudomonas multiresinivorans IpA-1 Wilson et al. (1996) – – (+)d Pseudomonas sp. IpA-2 Wilson et al. (1996) – – (+)d Pseudomonas sp. IpA-92 Yu et al. (2000) sb – –e Pseudomonas sp. IpA-93 Yu et al. (2000) – – –e Pseudomonas sp. DhA-91 Yu et al. (2000) – + +e Aromatic hydrocarbon degraders Burkholderia xenovorans LB400 bphA1 Bopp (1986) s/lb + +f Cupriavidus sp. PS12 tecA1 Beil et al. (1997) s/lb + + Cupriavidus necator H850 bphA1 Bedard et al. (1987) s – – Pandoraea sp. JB1* bphA1 Witzig et al. (2006) sb – ND Rhodococcus globerulus P6 bphA1 Asturias et al. (1995) s – – Pseudomonas pseudoalcaligenes KF707 bphA1 Furukawa et al. (1987) s + + Pseudomonas sp. Cam-1 bphA1 Master and Mohn (1998) s + + Pseudomonas sp. JR1 ipbA1 Pflugmacher et al. (1996) s + + Pseudomonas sp. CF600 Shingler et al. (1989) – + + Pseudomonas sp. IC bphX, bphA1 Carrington et al. (1994); sb – ND Witzig et al. (2006) Pseudomonas fluorescens IP01 cumA1 Aoki et al. (1996) sb – – Pseudomonas aeruginosa JI104 bnzA Kitayama et al. (1996) sb – ND Pseudomonas stutzeri AN10 nahAc Bosch et al. (1999) – – – Pseudomonas stutzeri OM1 carAa Ouchiyama et al. (1998) – – ND Pseudomonas stutzeri OX1 Bertoni et al. (1998) – + + Pseudomonas putida F1 todC1, cmtAb Gibson et al. (1970) s – – Pseudomonas putida G7 nahAc Simon et al. (1993) – – ND Pseudomonas putida mt-2 xylX, benA Burlage et al. (1989) – – ND Pseudomonas putida MT53 xylX Keil et al. (1985) – – ND Pseudomonas putida HS1 xylX Kunz and Chapman (1981) – – ND Pseudomonas putida 3,5X Ng et al. (1994) – – – Isolates from BTEX-contaminated soil Pseudomonas sp. IA1YICDA Junca and Pieper (2004) – – ND Pseudomonas sp. IA1YICDB Junca and Pieper (2004) – + + Pseudomonas sp. 3YC2 (3)g ipbA1 Junca and Pieper (2004) s – ND Pseudomonas sp. 1YXyl1 (5)g ipbA1 Junca and Pieper (2004) s – ND Pseudomonas sp. 1XB2 (1)g ipbA1 Junca and Pieper (2004) s – ND Pseudomonas sp. 1XC1 (2)g ipbA1 Junca and Pieper (2004) s – ND Pseudomonas sp. 3YdBTEX2 ipbA1 Junca and Pieper (2004) s – ND Pseudomonas sp. 1YB2 (7)g ipbA1 Junca and Pieper (2004) s + + Pseudomonas sp. 3YXyl1 (3)g ipbA1 Junca and Pieper (2004) s – ND Pseudomonas sp. 1XB1 ipbA1 Junca and Pieper (2004) s + + Arthrobacter sp. 3YC3 ipbA1 Junca and Pieper (2004) s – ND Sphingomonas sp. 1XXyl1b ipbA1 Junca and Pieper (2004) s – ND Isolates from PCB-contaminated soil Burkholderia sp. WBF1 lb + + Burkholderia sp. WBF2 lb + ND Burkholderia sp. WBF3 s/lb + + Burkholderia sp. WBF4 s/lb + + Bukrholderia sp. WBF5 lb + + Burkholderia sp. WBF6 lb + + Cupriavidus sp. WBF7 lb + + 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). © 2007 The Authors Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 1202–1218
  4. 4. 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 CH3 16 H H Ha H 12 16CH3 CH3 20 CH H H 16 3 15 Ha 20 CH 12 Ha 20CH 12 11 3 3 18 11 15 11 15 2 CH3 13 CH3 18 18 He 10 9 17 2 CH3 13 CH3 2 CH3 13 CH3 He 1 H He 10 9 17 He 10 9 17 Ha He 1 He 1 4 H H He 3 14 Ha Ha He 4 He 4 5 8 H 3 14 3 14 HOOC Ha 5 8 H 5 8 6 7 7 7 H 19 He H HOOC Ha 6 HOOC Ha 6 Ha 19 He 19 He Ha Ha Ha Ha O HO O H H1a 1.40 ddd J1a,1e = 13.2 1.59 ddd J1a,1e = 12.5 1.53–2.17a J1a,2a = 13.2 J1a,2a = 12.5 J1a,2e = 3.0 J1a,2e = 3.2 H1e 2.36 bd J1a,1e = 12.8 2.44 bd J1a,1e = 12.5 1.53–2.17a H2a 1.69–1.89a 1.67–1.89a m 1.53–2.17a H2e 1.69–1.89a 1.67–1.89a m 1.53–2.17a H3a 1.69–1.89a 1.67–1.89a m 1.53–2.17a H3e 1.69–1.89a 1.67–1.89a m 1.53–2.17a H5 2.02 dd J5a,6a = 12.5 2.62 dd J5a,6a = 14.2 – – – J5a,6e = 2.0 J5a,6e = 3.6 H6a 1.37–1.45 m 2.91 dd J5a,6a = 14.2 3.39 d J6a,6e = 18.2 J6a,6e = 18.5 H6e 1.69–1.89a m 2.37 dd J6a,6e = 18.5 2.65 d J6a,6e = 18.2 J5a,6e = 3.6 H7a,e 2.83–2.94b m – – – – H11 7.36 d J11,12 = 8.2 7.55 d J11,12 = 8.2 7.46 d J11,12 = 8.1 H12 7.16 dd J11,12 = 8.2 7.67 dd J11,12 = 8.2 7.64 dd J11,12 = 8.1 J12,14 = 1.3 J12,14 = 2.1 J12,14 = 2.0 H14 7.08 d J12,14 = 1.3 7.87 d J12,14 = 2.1 7.86 d J12,14 = 2.0 H15 2.83–2.94b m J15,16 = 7.0 3.02 sept J15,16 = 6.9 3.00 sept J15,16 = 6.9 J15,17 = 7.0 J15,17 = 6.9 J15,17 = 6.9 H16 1.22c d J15,16 = 7.0 1.26c d J15,16 = 6.9 1.27c d J15,16 = 7.0 H17 1.22c d J15,17 = 7.0 1.26c d J15,17 = 6.9 1.27c d J15,17 = 7.0 H18 1.21d bs 1.28d bs 1.43d bs H20 1.21d bs 1.29d bs 1.46d bs 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 activity, 16 strains from which a ISPaDit gene was ampli- isopimaric acid (IpA). In addition, amplicons of the fied by PCR with primer set ditAf543/ditAr1186 and six expected size were detectable with various reference strains that did not yield such an amplification product, strains reported to be capable of degrading different were tested for their ability to grow on DhA as the sole aromatic hydrocarbons and isolates obtained from soil source of carbon and energy. All strains for which a ditA1- contaminated with benzene, toluene, ethylbenzene, and homologue was detected by PCR were able to grow on xylenes (BTEX) or polychlorinated biphenyl (PCB) DhA as indicated by protein yields of 0.05–0.1 g of protein (Table 1). Based on the peptide sequence similarities of per gram of DhA (Table 1), whereas the other strains were the translation products (see below), all PCR products not (growth yield < 0.01 g of protein per gram of DhA). were identified as homologues of ditA1 of P. abietaniphila During growth, the intermediate accumulation of two BKME-9, indicating that the primers and PCR conditions metabolites, exhibiting retention volumes of 40% used are specific for amplification of Rieske non-haem (metabolite M1) and 20% (metabolite M2) that of DhA was iron oxygenase ISPaDit genes. observed and high-performance liquid chromatography- mass spectrometry (HPLC-MS) analysis of the culture supernatants indicated that these metabolites had Growth on DhA and transformation of DhA molecular ions of m/z 314 and 330, respectively, suggest- The presence of a Rieske non-haem iron oxygenase ing that they were oxo- and oxo-hydroxy-derivatives of ISPaDit gene has been demonstrated to be crucial for DhA. The in situ 1H nuclear magnetic resonance (NMR) DhA-degradation in P. abietaniphila BKME-9 (Martin and spectrum of DhA (Table 2) was very similar to that previ- Mohn, 1999). To analyse if the presence of a ditA1- ously reported (Gigante et al., 1995; Martin and Mohn, homologous gene is correlated with DhA-degradation 1999) and comparison of its spectrum with that of a © 2007 The Authors Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 1202–1218
  5. 5. 1206 R. Witzig et al. sample containing both DhA and metabolite M1 allowed KF707 and Pseudomonas sp. Cam-1 cells grown on fruc- the identification of signals originating from this metabolite tose (Supplementary material Fig. S2). In addition, no (Table 2). The 1H NMR spectrum of metabolite M1 was amplification products were observed in controls devoid of very similar to that previously reported for 7-oxo-DhA either reverse transcriptase (Supplementary material Fig. (Martin and Mohn, 1999). The large low field chemical S2, lane 15) or template cDNA (Supplementary material shift of H-14 and smaller shifts of H-11 and H-12 com- Fig. S2, lane 16). These results suggested that in KF707 pared with DhA were indicative of the introduction of a and Cam-1 the respective ditA1 genes were specifically carbonyl group at C-7. Similar effects were observed for induced in the presence of DhA. In contrast, amplification the signals of the neighbouring aliphatic protons, H-6a, of an RT-product in both fructose and DhA cultures of H-6e and H-5. The coupling constants observed (Table 2) Burkholderia sp. WBF4 suggested that expression of were indicative of the axial (H-5 and H-6a) and equatorial ditA1-mRNA was constitutive in this strain. Sequencing of (H-6e) dispositions of the protons within this system. The the 690 bp products confirmed that they were identical to analysis of the 1H NMR spectrum of a sample containing the corresponding ditA1-like sequences amplified from M2 in addition to DhA and M1 allowed the identification of genomic DNA of these strains. signals originating from M2. Compared with M1, M2 showed only small differences in chemical shifts of the PCR-SSCP analysis of ditA1-homologues of aromatic protons, indicating that this compound contained characterized reference strains and bacterial isolates a 7-oxo-substituent, similarly to M1. However, the signals obtained from BTEX- and PCB-contaminated soils of only two protons were observed to low field compared with three in M1. The magnitude of the coupling constant A SSCP fingerprinting method allowing for the sequence- (18.2 Hz) indicated these must belong to an isolated dependent differentiation of ISPaDit gene fragments ampli- methylene group at C-6. This, together with the increase fied with primer set ditAf543/ditAr1186 was established to in molecular weight of 16 mass units compared with M1 rapidly obtain an overview of the ISPaDit gene sequence could only be rationalized by the introduction of an axial diversity in pure culture strains and environmental hydroxyl group at C-5 in M2 suggesting this compound is samples. Using a MDETM gel concentration of 0.8¥ and a 5-hydroxy-7-oxo-DhA. gel temperature of 40°C, the electrophoretic mobilities of Comparison of the integrals of the resonance lines of the ISPaDit single-strand products obtained from four charac- 11-H protons of DhA, 7-oxo-DhA and 5-hydroxy-7-oxo- terized dehydroabietic acid degraders and seven charac- DhA allowed their relative abundances to be determined terized aromatic hydrocarbon degraders could be and their use, together with a defined standard of DhA, as distinguished from each other (Fig. 1A), suggesting that a quantitative standard in HPLC analysis, for assessing the these strains contained different ISPaDit gene variants. transient accumulation of 7-oxo- and 5-hydroxy-7-oxo- Sequencing of the re-amplified single-strand products DhA during growth of isolates and reference strains on confirmed that all gene segments encoded ISPa peptides DhA. All strains transiently accumulated significant homologous to DitA1 of P. abientaniphila BKME-9 (Martin amounts of 7-oxo-DhA, accounting for up to 20% of the and Mohn, 1999). Single-strand conformation polymor- applied substrate when the strain grew on 0.24 mM of phism analysis of Pseudomonas sp. 1YB2 and seven substrate. Intermediate excretion of 5-hydroxy-7-oxo-DhA additional Pseudomonas strains harbouring an identical was significant in Pseudomonas and Cupriavidus strains 16S rRNA phylotype and ISPaTol/Bph/C23O genotype and accounted for up to 15% of applied substrate, whereas (Table 1) revealed that these strains obviously harboured Burkholderia strains excreted only minor amounts of two distinct ISPaDit gene sequence types (Fig. 1B). Cluster 5-hydroxy-7-oxo-DhA (< 2% of applied substrate). 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 dioxy- Expression of ditA1-like genes in Pseudomonas genase ISPa sequence cluster (Fig. 2). With the exception pseudoalcaligenes KF707, Pseudomonas sp. Cam-1 of the putative ISPaDit peptide of strain LB400 and one of and Burkholderia sp. WBF4 the two ISPaDit peptides derived from the BTEX-degrading To confirm that the identified ditA1-like genes are isolate Pseudomonas sp. 1YB2, all the remaining peptide expressed in response to DhA, RT-PCR experiments sequences obtained from strains of the same genus were performed with total RNA extracted from cultures of grouped together and shared more than 90% amino acid P. pseudoalcaligenes KF707, Pseudomonas sp. Cam-1 sequence similarity. The ISPaDit peptides deduced from and Burkholderia sp. WBF4. Amplification products of the the SSCP single-strand products of Cupriavidus spp. and expected size (approximately 690 bp) were observed in Burkholderia spp. grouped within distinct branches that cultures grown on DhA, whereas no such products were shared less than 82% identical amino acid positions detectable with RNA extracts of P. pseudoalcaligenes with previously reported ISPaDit peptide sequences. © 2007 The Authors Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 1202–1218
  6. 6. 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. © 2007 The Authors Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 1202–1218
  7. 7. 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 Because 16S rRNA sequence analysis may not be gene copies; with one allele (Fig. 1A, lower SSCP band) sufficiently discriminatory to permit resolution of intrage- affiliated to the Pseudomonas-cluster and the other neric relationships (Yamamoto and Harayama, 1995; (Fig. 1A, upper SSCP band) more closely related to the Yamamoto et al., 2000), the DNA gyrase subunit B putative DitA1 peptide sequences of Zoogloea resiniphila genes (gyrB) were also used for phylogenetic analysis DhA-35 and Mycobacterium sp. DhA-55 (Yu et al., 1999) (Yamamoto et al., 2000). In fact, the gyrB nucleotide (Fig. 2). Interestingly, two distinct putative ISPaDit sequence variability allowed the closely related Cupria- sequence types could be also identified in the unfinished vidus and Burkholderia strains to be distinguished genome of Pseudomonas aeruginosa 2192, with one of (Fig. 3A). Moreover, there was a significant congruence them (type I) affiliated to the Pseudomonas-cluster and the with regard to a genus-specific sequence clustering of other (type II) more closely related to the upper-SSCP- gyrB and ditA1 genes (Fig. 3 A and B). Major differences band-product of Pseudomonas sp. 1YB2 (Fig. 2). were observed with the putative ditA1 sequence of LB400, which did not group together with ditA1 gene sequences of other Burkholderia strains, and the out- Nucleotide sequence polymorphisms of the 16S rRNA, grouping putative ditA1 alleles of Pseudomonas strains gyrB and ditA1 genes of strains harbouring 1YB2 and 2192. ditA1-homologues It has previously been reported that the gyrB genes of Because the tree inferred from DitA1 peptide sequences several bacterial taxa have evolved mainly by synony- (Fig. 2) suggested a close linkage between host phylog- mous substitutions (that is, under ongoing purifying eny and ditA1 gene evolution, the phylogenetic relation- selection against deleterious non-synonymous muta- ships between ditA1-harbouring isolates were inferred tions) (Yamamoto and Harayama, 1998; Dauga, 2002; on the basis of 16S rRNA gene sequence analysis Cladera et al., 2004). To determine whether the ditA1 (Supplementary material Fig. S3). The 16S rRNA genes genes are under similar negative selection pressure, the of the ditA1-harbouring Pseudomonas, Cupriavidus and mean values of synonymous (pS) and non-synonymous Burkholderia strains were generally closely affiliated to distances (pN) within the genera Pseudomonas, Cupria- type species of the respective genera. However, while vidus and Burkholderia were compared (Table 3). Con- pulse field gel electrophoresis demonstrated slight gruent with previous findings (Yamamoto and Harayama, genomic differences between the three Cupriavidus 1998; Cladera et al., 2004), strong purifying selection strains PS12, BKME-6 and WBF7 and the four was observed for the gyrB genes of each of the three Burkholderia strains WBF1, WBF2, WBF3 and WBF4, genera, as indicated by pN/pS ratios well below 1. Simi- respectively (data not shown), no differences between larly, strong purifying selection has been operating during the 16S rRNA sequences of strains belonging to evolution of ditA1 genes of members of each genus either Burkholderia or Cupriavidus were observed. (Table 3). © 2007 The Authors Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 1202–1218
  8. 8. Molecular detection and diversity of DitA1 genes 1209 PCR-SSCP analysis of ditA1-homologues in BTEX- and showed no clear relationship to ISPaDit types derived from PCB-contaminated soils pure culture strains. Nine highly similar ISPa sequence types were obtained from bands migrating at different The presence of ditA1-homologous genes in various ref- positions in the SSCP gel, the translation products of erence strains and isolates enriched for independent pur- which converged to three different deduced amino acid poses suggested that such genes are widespread (at sequences (Fig. 4C). Because the same segregating least in the Proteobacteria analysed in this study). To nucleotide positions were identified in at least three indi- obtain more information on the natural diversity of genes vidual clones exhibiting identical or nearly identical homologous to ditA1, the developed PCR-SSCP method sequences, sequencing and/or PCR errors appeared to was used to screen for the presence of ISPaDit sequence be unlikely to account for the observed sequence types in bacterial communities of four soil samples, which variation. had previously been used for isolation of strains from contaminated soils. ISPaDit-PCR-SSCP analyses (Fig. 4A Discussion and B) indicated a broad diversity of ditA1-like genes to be present in each of the soils. The results of this study demonstrate that the metabolic From the BTEX-contaminated soil fingerprints, a total capability for degradation of the abietane diterpenoid DhA of 30 different ISPaDit gene segment types were isolated is widespread in a collection of reference strains and and cluster analysis revealed that the ISPaDit peptides isolates, which have previously been enriched from deduced from re-amplified single strands were distrib- diverse sources, based on their ability to degrade different uted into six different major lineages (Fig. 4C). aromatic hydrocarbons rather than resin acids. Studies of The majority of the peptide sequences grouped within the biochemistry of the aerobic biodegradation of resin either the Pseudomonas-cluster or the BTEX soil-cluster acids had suggested convergent pathways for abietane I, which was more closely related to ISPaDit sequences diterpenoid metabolism that channels the non-aromatic identified in Burkholderia and Cupriavidus strains. abietanes and dehydroabietic acid into the central meta- The ISPaDit peptide sequences inferred from three bolic intermediate 7-oxo-DhA (Martin and Mohn, 1999; bands (6, 28 and 29) of the BTEX-contaminated soil 2000; Smith et al., 2004). This compound was also 1Y fingerprint profile (Fig. 4A) were identical in mobility detected as an intermediate during growth of all strains and sequence to the corresponding ditA1-homologues analysed in this study. Moreover, all strains capable identified in the benzene-degrading Pseudomonas of degrading DhA were found to harbour a gene isolate 1YB2. segment encoding a peptide homologous to DitA1 of From the PCB-contaminated soil fingerprint, a total of P. abietaniphila BKME-9 and expression analyses of 12 ISPaDit gene segment types differing in at least one ditA1-mRNA in P. pseudoalcaligenes KF707 and nucleotide position were recovered and cluster analysis Pseudomonas sp. Cam-1 are in accordance with a revealed that the deduced ISPaDit peptide sequences substrate-induced resin acid-degrading enzyme system in were distributed into three distinct major lineages these strains, similar to the one previously reported for (Fig. 4C). Three bands (7, 8 and 9) and band 17 (Fig. 4B) P. abietaniphila BKME-9 (Martin and Mohn, 2000). contained nucleotide sequence types similar to those of Even though diverse bacteria were found to be capable Burkholderia spp. WBF5 and WBF6 respectively. The of degrading DhA and structurally related resin acids peptide sequence deduced from band 1, however, (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 % average pairwise distanceb No. of polymorphic sites pN pS pN/pS ratio gyrB Pseudomonas 0.150 304 0.044 (0.046) 0.419 (0.650) 0.105 (0.071) Burkholderia 0.043 75 0.007 (0.009) 0.151 (0.177) 0.046 (0.051) Cupriavidus 0.011 14 0.003 (0.003) 0.037 (0.038) 0.081 (0.079) ditA1 Pseudomonas c 0.145 205 0.028 (0.028) 0.460 (0.802) 0.061 (0.035) Burkholderiad 0.034 53 0.003 (0.003) 0.129 (0.153) 0.023 (0.020) Cupriavidus 0.021 19 0.000 (0.000) 0.085 (0.090) 0.000 (0.000) a. Numbers in parentheses were calculated using the Jukes–Cantor model (Jukes and Cantor, 1969). b. Calculated using the Jukes–Cantor model (Jukes and Cantor, 1969). c. Excluding the upper-SSCP-band-product of Pseudomonas sp. 1YB2 and the type II sequence of P. aeruginosa 2192 (see Fig. 3B). d. Excluding the putative ISPaDit gene sequence of B. xenovorans LB400. © 2007 The Authors Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 1202–1218
  9. 9. 1210 R. Witzig et al. © 2007 The Authors Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 1202–1218
  10. 10. 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 abun- degraders (Pseudomonas sp. DhA-91, Cupriavidus sp. dance and diversity of the catabolic genes involved in BKME-6 and Burkholderia sp. DhA-54) for which previous resin acid degradation. In a survey using primers attempts to amplify ISPaDit gene segments by PCR were designed to specifically amplify the ditA1 gene of strain unsuccessful (Yu et al., 1999; 2000). Moreover, the PCR BKME-9, Yu and colleagues (Yu et al., 1999) could results were in perfect accordance with results from amplify products from various biotreatment systems for growth experiments (Table 1), suggesting that the PPME and confirm by restriction digestion analysis that detected gene segments are involved in DhA-degradation the amplified genes were similar to ditA1. However, in the PCR-positive strains, as demonstrated for the because the numbers of strains estimated to harbour a strains KF707, Cam-1 and WBF4 (Supplementary mate- ditA1-homologue could not account for the observed per- rial Fig. S2). formance of the PPME treatment systems, the authors However, the primer binding sites targeted here appear speculated that the detected ditA1 gene-containing micro- not to be conserved in the genomes of four resin acid organisms were quantitatively only minor members of the degraders (Pseudomonas strains IpA-1, IpA-2, IpA-92 resin acid-degrading populations. In fact, the applied and IpA-93), which had originally been isolated on isopi- primer set failed to amplify ditA1-like gene sequences maric acid (Wilson et al., 1996; Yu et al., 2000) (Table 1). from resin acid-degrading bacteria except strain BKME-9 Studies on the biodegradation of resin acids by Gram- (Yu et al., 1999; 2000) and, because of the high number of negative bacteria have suggested distinct biochemical mismatching positions within the forward primer ditA1- pathways for the degradation of abietane and pimarane 719f (data not shown), it seems unlikely that any of the diterpenes (Wilson et al., 1996; Martin et al., 1999; Yu sequences identified here could have been amplified with et al., 2000). The failure of PCR primers ditAf543/ the primer set ditA1-719f/ditA1-1212r used by Yu and ditAr1186 to amplify a diterpenoid dioxygenase a-subunit colleagues (Yu et al., 1999). In a second PCR approach, gene in resin acid degraders exhibiting higher specificity targeting conserved DNA regions of ditA1 encoding the towards IpA degradation indicates that these strains [2Fe-2S] Rieske cluster-coordinating site and a con- contain genes encoding an a-subunit differing in served Asp residue that has been shown to be involved in sequence from those previously reported for Gram- gating electron transport in Rieske non-haem oxygenase negative strains capable of degrading DhA, at least at the reactions, Yu and colleauges (Yu et al., 1999) were able to regions targeted here. Possibly, these a-subunits belong amplify putative ditA1-homologues from six other resin to Rieske non-haem iron oxygenases of another yet to be acid-degrading bacteria, but still some of the analysed identified subfamily. DhA-degraders failed to result in PCR amplification. Using Previous analyses (Martin and Mohn, 1999; Yu et al., the new degenerate primer set ditAf543/ditAr1186 (which 1999) have suggested that DitA1 of strain BKME-9 and was designed to complement conserved regions of an the homologous sequences of six other DhA-degrading updated alignment of the putative ISPaDit gene seg- strains formed a distinct group within the Rieske non- ments), it was possible to amplify and characterize puta- haem iron oxygenase ISPa family and three divergent tive ditA1-homologues from 18 bacterial species of three lineages had been recognized. Cluster analysis of the different genera, including three characterized DhA- partial putative ISPaDit protein sequences deduced in this © 2007 The Authors Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 1202–1218
  11. 11. 1212 R. Witzig et al. study indicates that the ISPaDit gene sequences obtained diversity of ditA1-homologues in these environments. from Burkholderia and Cupriavidus strains and, even Earlier characterizations of both the 16S rRNA (Hen- more noticeable, those recovered from soil samples rep- drickx et al., 2005) and functional (C23O and ISPaTol/Bph) resent new major branches within the ISPaDit sequence gene structures (Junca and Pieper, 2004; Witzig et al., cluster (Fig. 4C). Interestingly, the results presented in 2006) of the BTEX-degrading bacterial communities, Fig. 3 suggest the ditA1 gene sequence divergence to be using culture-dependent and -independent methods, linked to the host phylogeny. This finding contrasts with indicated that Pseudomonas spp. dominated the BTEX- the related Rieske non-haem iron oxygenase ISPa genes contaminated soils. In particular, phylotypes and geno- of the naphthalene and biphenyl subfamilies, which have types identical to those of Pseudomonas sp. 1YB2 were often been reported to be localized on mobile elements identified as being strongly enriched in BTEX soil 1Y and dispersed via horizontal transfer (Nojiri et al., 2004; using these methods. The previous findings are further Pieper, 2005), thus disconnecting catabolic gene evolu- supported by the ISPaDit-PCR-SSCP analysis presented tion from the host cells phylogeny. Moreover, the in this study, where ditA1 gene sequence types identical Pseudomonas strains IA1YICDB, JR1, Cam-1 and to those identified in strain 1YB2 were abundant in the DhA-91 contained considerably different ditA1 sequence highly contaminated soil. In view of the apparent corre- types (differing in up to 24 base positions), which lation between bacterial host phylogeny and ditA1 gene however, encoded identical ISPaDit peptide sequences. phylogeny, at least for ditA1 genes sensu stricto, the Similarly, identical peptides were encoded by the putative sequence types identified in the BTEX soil ISPaDit- ISPaDit genes (differing in up to 16 base positions) of the fingerprints further suggest that diverse Pseudomonas Cupriavidus strains BKME-6, PS12 and WBF7. The fact strains sharing the ability to degrade resin acids are that mainly synonymous substitutions contributed to the present at this site (Fig. 4C). Moreover, the detection of sequence variation of ditA1 genes (Table 3) suggests that ISPaDit sequence types more closely related to those of there are structural and/or functional constraints on amino Burkholderia spp. and Cupriavidus spp. (BTEX-soil- acid replacements in the analysed region of DitA1 and cluster I), for which a cultured representative has not yet purifying selection was acting to remove deleterious been described, indicates that bacterial phyla other than amino acid mutations from the populations while neutral Pseudomonas spp. may also be abundant in the BTEX- or nearly neutral silent variants could persist (Kimura, contaminated soils. 1983). Reconstruction of the evolutionary history of ditA1 None of the reference strains shown in this study to and gyrB genes further suggests that they were both harbour a ditA1-homologue had previously been reported present in the most recent common ancestor of to be capable of growth on DhA. However, they have all Pseudomonas, Cupriavidus and Burkholderia before spe- been recovered and characterized from diverse environ- ciation occurred, and that they coevolved during the ments as aromatic hydrocarbon-degrading bacteria, course of evolution (Fig. 3). The isolated position of the being capable of degrading chlorobenzene (strain PS12), LB400-ditA1-homologue, however, indicates a different (polychlorinated) biphenyl (strains Cam-1, KF707 and evolutionary history compared with the ditA1 genes of JR1), or phenol (strains OX1, CF600). Thus, the occur- other Burkholderia strains. rence of ditA1-homologues seems to be widespread in The evolutionary analysis of ISPaDit homologues further organisms sharing the capability of aromatic hydrocarbon raises the question of the function of the second ditA1- degradation and apparently such genes can be detected homologue in Pseudomonas spp. 1YB2 and 2192. Both with a high probability in strains affiliated to the genera strains harbour a ditA1-homologue of the Pseudomonas- Pseudomonas, Cupriavidus and Burkholderia. cluster (Fig. 2), which is presumed to be involved in DhA- Yu and colleagues (Yu et al., 2000) observed that degradation. Because the primers used by Yu and hydrocarbon contamination in Arctic tundra soil (in which colleagues (Yu et al., 1999) could not amplify a broad no resin acids were detectable) promoted the survival range of ditA1 gene segments, the presence of a second and/or selection of resin acid degraders and pointed out ISPaDit gene with higher similarity to the Pseudomonas-, that this might have been the result of selection for hydro- Cupriavidus-, or Burkholderia-clusters in strains harbour- carbon degraders that coincidentally use resin acids. ing the outlying sequence types, i.e. the non-classified Indeed, their hypothesis is corroborated by this study, b-Proteobacterium DhA-73, Schlegelella thermodepoly- demonstrating that numerous aromatic hydrocarbon- merans DhA-71; Mycobacterium sp. DhA-55 and degrading strains affiliated to the genera Burkholderia, Z. resiniphila DhA-35 (Fig. 2), analogous to the case of Cupriavidus and Pseudomonas, members of which are Pseudomonas sp. 1YB2 and P. aeruginosa 2192, cannot among the most versatile aromatic hydrocarbon degrad- be excluded. ers and thus are often enriched at contaminated environ- PCR-SSCP fingerprinting of the soil samples demon- ments, and are capable of resin acid degradation. While strated the presence of a broad, previously unrecognized the presence of both (aromatic hydrocarbon and abietane © 2007 The Authors Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 1202–1218
  12. 12. Molecular detection and diversity of DitA1 genes 1213 resin acid degradative) genotypes appears to be purely viously described (Junca and Pieper, 2004; Witzig et al., coincidental, this feature could be an important factor 2006). Total DNA from the PCB-contaminated soil sample relating to the microbial ecology of aromatic hydrocarbon (10 g wet weight) was extracted in triplicate using a Ultra- CleanTM MegaPrep soil DNA isolation kit (MO BIO Laborato- degradation. When confronted with aromatic hydrocarbon ries, Carlsbad, CA) in combination with cell disruption by pollutants the organisms’ ability to use the pollutant is bead beating for 30 s using a MSK cell homogenizer (Braun, likely the dominating factor in shaping the microbial com- Melsungen, Germany). DNA was precipitated and purified munity, while the presence of resin acids prior to pollution using standard methods (Sambrook et al., 1989), followed by may have selected for resin acid-degrading populations a further purification step with the Wizard DNA clean-up and thus preconditioned the community structure. system (Promega, Madison, WI). DNA concentrations from soil extracts were quantified using a PicoGreen double- stranded DNA (dsDNA) quantitation kit (Molecular Probes, Experimental procedures Leiden, the Netherlands). The DNA extracts from BTEX- contaminated soil samples containing approximately 2 ng ml-1 Bacterial strains and growth conditions DNA were used either directly or 10-fold diluted in Tris-HCl buffer (10 mM, pH 8.0), while the DNA extracts from the The bacterial strains used in this study (Table 1) comprised PCB-contaminated soil containing approximately 380 ng ml-1 Pseudomonas vancouverensis DhA-51, which has previously DNA were 50- or 100-fold diluted in Tris-HCl buffer (10 mM, been described to possess a Rieske non-haem ISPa gene of pH 8.0) and used as template DNA in PCR. 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 refer- PCR amplification of Rieske non-haem iron oxygenase ence strains. According to the 16S rRNA gene sequence data ISPa gene segments (see below), the previously characterized strains Ralstonia sp. PS12 (Beil et al., 1997) and Ralstonia sp. BKME-6 (Mohn Two primer sets were used for PCR amplification of Rieske et al., 1999) and Burkholderia sp. IpA-51 (Mohn et al., 1999) non-haem iron oxygenase ISPaDit gene segments. ISPaTol/Bph belong to the genera Cupriavidus (Vandamme and Coenye, and/or ISPaDit gene segments (of 805–829 bp and 883– 2004) and Pandoraea (Coenye et al., 2000) respectively. The 892 bp respectively) were amplified with the degenerate strain collection further comprised seven Burkholderia and primer set bphAf371B/bphAr1153-2 using the previously Cupriavidus strains, which had been randomly isolated on reported protocol (Witzig et al., 2006) with an increased final R2A medium (Difco) from PCB-contaminated soil (Nogales concentration of MgCl2 (2 mM). Amplification products were et al., 1999), and 36 bacterial isolates obtained from BTEX- separated by agarose gel electrophoresis (2.5% agarose, contaminated soils (Junca and Pieper, 2004; Witzig et al., 1¥ TAE buffer) and visualized by ethidium bromide 2006). 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 Chemicals DNA alignment using CLUSTALW (Chenna et al., 2003) of the 883–892 bp putative ISPaDit gene segments amplified from Dehydroabietic acid (99% purity) was purchased from Helix the Cupriavidus spp. BKME-6, PS12 and WBF7 and the Biotech, New Westminster, BC, Canada. 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 Soil samples sequence of B. xenovoransLB400 (CP000272) and six pre- Three BTEX-contaminated soil samples were collected from viously reported ditA1-homologous sequences of other resin the unsaturated (X) and capillary fringe (Y) horizons at two acid-degrading bacteria (AF145209, AF145210, AF145211, sampling sites (site 3 contained slightly BTEX-contaminated AF145212, AF145213, AF145214), primers ditAf543 (5′- soil whereas site 1 was highly contaminated) of a BTEX- GGC GAT GCS AAG TGG TAY TWC GAC-3′) and ditAr1186 contaminated aquifer located in the Czech Republic. The (5′-CCA CGT GTC MG AGT CRT CCT GYTC-3′) were chemical and microbiological characteristics of the soil designed to target conserved alignment regions correspond- samples have been described previously (Junca and Pieper, ing to nucleotide positions 520–543 and 1186–1209 in the 2004; Hendrickx et al., 2005). The PCB-contaminated soil ditA1 sequence of P. abietaniphila BKME-9. PCR for subse- sample was taken from the upper few centimetres of the soil quent SSCP analysis was performed with a 5′ end phospho- surface at a PCB-polluted site near Wittenberg (Germany) in rylated reverse primer ditAr1186. PCR amplifications with 2000 and stored at 4°C until used for DNA extraction. primer set ditAf543/ditAr1186 were performed in a 50 ml final Detailed chemical characteristics of the sampling site have volume containing 0.5 mM of each primer (MWG-Biotech, been previously described (Nogales et al., 1999). 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 poly- Extraction of DNA from pure cultures and soil merase (Qiagen), and 2 ml of template DNA. The following PCR conditions were used: an initial denaturation for 15 min Total genomic DNA was extracted and quantified from pure at 95°C, followed by 32 cycles of 40 s at 94°C, 40 s at 58°C, culture strains and BTEX-contaminated soil samples as pre- 60 s at 72°C and a final elongation for 10 min at 72°C. PCR © 2007 The Authors Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 1202–1218
  13. 13. 1214 R. Witzig et al. amplicons (2 ml of the PCR) were verified by agarose gel using a RiboGreen RNA quantitation kit (Molecular Probes). electrophoresis (1.5% agarose, 1¥ TAE buffer) and ethidium Extracts of cells grown on DhA comprised RNA concentra- bromide staining. tions 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 Growth on DhA synthesized from 1 to 3 ml of total RNA using a first-strand cDNA synthesis kit for RT-PCR (Roche, Mannheim, Strains were maintained on R2A medium or M9 mineral salt Germany). The reverse transcription reaction mixtures were medium (Sambrook et al., 1989) containing 0.4 mM DhA and serially diluted (3.2-fold) with nuclease-free water (Qiagen) 3% agar. Liquid cultures were grown in M9 mineral salt and 1 ml of each dilution was subjected to amplification by medium supplemented with DhA (0.24 mM) as the sole PCR using the primer set ditAf543/ditAr1186 and the condi- source of carbon and energy in fluted Erlenmeyer flasks at tions described above. Amplification products were sepa- 30°C on a rotary shaker at 150 r.p.m. For monitoring growth rated in 1.5% agarose gels and stained with ethidium on DhA, Erlenmeyer flasks containing 25 ml of medium were bromide. Product bands were purified from agarose gels inoculated with 250 ml of cells pregrown in R2A or mineral using a QIAquick PCR Purification Kit (Qiagen) and medium supplemented with 0.4 mM DhA, and cell-free super- sequenced to verify their identity. natants were regularly sampled for HPLC analysis (see below). Culture supernatants containing higher amounts of metabolites sufficient for in situ 1H NMR and HPLC-MS analy- Analytical methods ses were obtained from cell suspensions grown on 0.4 mM of DhA, to which additional 0.32–0.4 mM of substrate was Transformation of DhA was monitored by HPLC analysis. added after complete DhA consumption. Growth on DhA was Aliquots of 10 ml of cell-free supernatants were analysed with assessed by protein quantification after 48 h of incubation. a Shimadzu HPLC system (LC-10AD liquid chromatograph, For the protein assay, cells were harvested from 10 ml of DGU-3 A degasser, SPD-M10A diode array detector and culture, re-suspended in 900 ml of 0.4 M NaOH, heated for FCV-10AL solvent mixer) equipped with a Lichrospher RP8 10 min at 95°C and centrifuged at 16 000 g for 10 min. column (125 mm by 4.6 mm, Bischoff, Leonberg, Germany) Supernatants were neutralized with 100 ml of 4 M HCl and using an aqueous solvent system (flow rate, 1 ml min-1) con- protein was quantified using the Bio-Rad Protein Assay (Bio- taining 0.01% (v/v) H3PO4 (87%) and 30% (v/v) methanol. Rad, München, Germany) with bovine serum albumine as a High-performance liquid chromatography-mass spectrometry standard. was performed using an Agilent 1000 LC system (Agilent Technologies, Palo Alto, CA) equipped with a Nucleosil 120- 5-C18 column (125 mm by 2 mm), coupled to a Sciex Extraction of mRNA, cDNA synthesis and RT-PCR API2000 mass spectrometer (Perkin-Elmer Sciex, Foster City, CA) equipped with a TurboIonSpray (ESI) source. For gene expression studies, P. pseudoalcaligenes KF707, Elution was performed at a flow rate of 0.3 ml min-1 using an Pseudomonas sp. Cam-1 and Burkholderia sp. WBF4 grown aqueous solvent system with a linear gradient of 5 mM on DhA (0.24 mM) were harvested and used to inoculate ammonium acetate (pH 5.5) in 5% acetonitrile to 5 mM 25 ml of fresh M9 mineral medium containing DhA (0.24 mM). ammonium acetate (pH 5.5) in 95% acetonitrile over 9 ml To assess constitutive expression, the strains were grown in followed by isocratic elution with 5 mM ammoniumacetate parallel in M9 mineral medium supplemented with 5 mM (pH 5.5) in 95% acetonitrile over 3 ml. Mass spectrometry fructose. After the cultures had degraded 67–84% of the analysis was performed using the Sciex TurboIonSpray substrate (DhA cultures) or reached the mid-log phase (fruc- source at a temperature of 350°C in positive and negative ion tose cultures), 25 ml of RNAprotect (Qiagen) was added to mode. The one-dimensional 1H NMR spectra were recorded the cell suspensions. The samples were vortexed briefly and at 300 K on a Avance DMX 600 NMR spectrometer (Bruker, incubated on ice for 5 min, followed by centrifugation for Rheinstetten, Germany) locked to the deuterium resonance 30 min at 7300 g. Total RNA was isolated by a modified of D2O in the solution. Spectra were recorded by using the version of the method of Siering and Ghiorse (Siering and standard Bruker 1D NOESY suppression sequence with 280 Ghiorse, 1997). In brief, the cells were suspended in 700 ml of scans, each with a 1.8 s acquisition time and a 1.3 s relax- sterile nuclease-free water (Qiagen), transferred to lysing ation delay. 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 phos- PCR-SSCP analysis of ditA1-homologues phate buffer (0.12 M, pH 7.0) and 500 ml of acidic phenol (pH 4.6) (Roth, Karlsruhe, Germany) added, then cells were PCR-SSCP with the single-strand removal approach disrupted by bead beating for 45 s in a FastPrep FP120A (Schwieger and Tebbe, 1998) was used for detection and instrument (Bio101 Systems, Q-BIOgene). The nucleic acids differentiation of ISPaDit gene segments and was carried out were precipitated from the aqueous phase and purified using using a previously described protocol (Witzig et al., 2006), previously described methods (Siering and Ghiorse, 1997). except for diluting the single strand with Tris-HCl buffer Residual DNA was removed by incubating aliquots of the (10 mM, pH 8.0) to a final concentration of 0.9 ng ml-1 prior to samples with RNase-free DNase I (Roche, Mannheim, SSCP. The influence of MDETM gel solution (Cambrex Bio Germany) at room temperature for 2 h, followed by RNA Science, Rockland, ME) concentration and gel temperature purification using a RNeasy kit (Qiagen) and quantification on the resolution performance of the SSCP method was © 2007 The Authors Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 1202–1218
  14. 14. Molecular detection and diversity of DitA1 genes 1215 tested with single-stranded ISPaDit gene segments generated sequences of the biphenyl and naphthalene subfamily and from B. xenovorans LB400, Pseudomonas sp. Cam-1, gyrB sequences of closely related strains were retrieved from P. pseudoalcaligenes KF707, Cupriavidus sp. PS12 and GenBank and included in the alignments of ditA1 and gyrB Cupriavidus sp. WBF7 in individual runs using the following genes respectively. Evolutionary trees were constructed with conditions (gel temperature/MDETM gel solution concentra- MEGA software, using the neighbour-joining (NJ) method tion): (i) 30°C/0.7¥, (ii) 34°C/0.7¥, (iii) 36°C/0.7¥, (iv) 38°C/ (Saitou and Nei, 1987) with genetic distances computed 0.7¥ and (v) 40°C/0.8¥. Electrophoresis under optimized using the Jukes–Cantor (Jukes and Cantor, 1969) and JTT conditions was performed on 21 cm ¥ 21 cm 0.8¥ MDETM (Jones et al., 1992) substitution model for DNA and protein gels in 1¥ TBE as running buffer at 400 V and 10 mA for 16 h data respectively. Primer sequences, ambiguous positions at 40°C in a Pharmacia Macrophor electrophoresis unit 2010- and regions of insertions and deletions of the alignments 001 connected to a circulating water bath (Lauda ecoline were excluded. Consensus trees were inferred from a total RE104). The gels were silver-stained according to Bassam of 500 bootstrap trees generated for each data set. and colleagues (Bassam et al., 1991) and dried at room The numbers of synonymous substitutions per synonymous temperature. Analysis of the silver-stained gels and isolation site (pS) and of non-synonymous substitutions per non- of selected single-strand products was performed as synonymous site (pN) were estimated by the method of Nei described previously (Witzig et al., 2006). and Gojobori (Nei and Gojobori, 1986) and evolutionary dis- tances (dS and dN) were corrected for multiple substitutions by the Jukes–Cantor model (Jukes and Cantor, 1969) using PCR amplification of 16S rRNA and gyrB genes MEGA software. Strong evidence for the occurrence of puri- fying selection is provided by the fact that the number of Phylogenetic relationships of strains harbouring a ditA1- synonymous nucleotide substitutions per synonymous site homologue were derived using the nucleotide sequences of exceeds the number of non-synonymous nucleotide substi- the genes coding for 16S rRNA and DNA gyrase subunit B tutions per non-synonymous site (Li et al., 1985). Values for (gyrB). Nearly complete sequences of the 16S rRNA genes dS and dN were estimated for each pairwise comparison (corresponding to positions 28–1481 in the Escherichia coli between homologous sequences and from these values, pS numbering system) were determined directly from PCR frag- (the average value of all pairwise dS values) and pN (the ments by using primers and conditions described by Lane average value of all pairwise dN values) were estimated for (Lane, 1991). PCR amplification of the gyrB gene was per- ditA1 and gyrB data sets of each genus. The 16S rRNA formed following the method of Yamamoto and colleagues sequences obtained in this study were analysed using the (Yamamoto et al., 2000); however, gyrB primers (MWG- sequence match tool of the Ribosomal Database Project II Biotech) omitted the M13-forward and -reverse primer (RDP-II) (Cole et al., 2003) and aligned against the most sequence moieties. 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 DNA sequencing and sequence analysis using only unambiguously determined nucleotide positions and a phylogenetic tree was constructed using the PCR products were purified with a QIAquick PCR Purification neighbour-joining method (Saitou and Nei, 1987). A bootstrap Kit and sequenced using an ABI PRISM BigDye Terminator consensus tree was inferred from a total of 1000 v1.1 Ready Reaction Cycle Sequencing Kit (Applied Biosys- re-samplings. tems) 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 Nucleotide sequence accession numbers products that resulted in ambiguous sequences, and the ISPaTol/Bph and ISPaDit gene segments that were amplified with The nucleotide sequences reported in this study were depos- the primer set bphAf371B/bphAr1153-2, were analysed after ited in the DDBJ/EMBL/GenBank databases under the fol- cloning with the pGEM-T-vector system (Promega). Plasmid lowing accession numbers: DQ777727–DQ777739 (16S inserts were amplified and sequenced with vector-specific rRNA gene sequences), DQ844792–DQ844810 (gyrB gene primers M13-forward and M13-reverse (Promega). To sequences), DQ679936–DQ679946 (bphA1 and ditA1 account for potential PCR and sequencing errors, at least gene sequences amplified with primer set bphAf371B/ eight clones from each ligation reaction were screened for bphAr1153-2) and DQ789330–DQ789350, DQ844811– similar sequence types and consensus sequences [deduced DQ844910, DQ844726–DQ844791, DQ852290–DQ852307 from at least three identical or nearly identical ( 1% differ- (ditA1 gene sequences amplified with primer set ditAf543/ ence) clones] were generated for further analysis. Raw ditAr1186). 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 per- Acknowledgements formed using BLASTN and BLASTP programs from the National Centre for Biotechnology Information website. Nucleotide The authors wish to thank Professor W.W. Mohn for kindly sequences of ditA1 and gyrB genes were translated and providing the resin acid-degrading reference strains used in aligned at the protein level using CLUSTALW implemented in this work. We also thank Julia Bötel, Silke Kahl, Christel MEGA software version 3.1 (Kumar et al., 2004), and back- Kakoschke and Beate Jaschok-Kentner for excellent techni- translated to obtain the corresponding DNA alignments. ISPa cal assistance, and Heinrich Steinmetz for performing the © 2007 The Authors Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 1202–1218