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FEMS Microbiology Ecology 43 (2003) 349^357 www.fems-microbiology.org Nitrite reductase genes in halobenzoate degrading denitrifying bacteria Bongkeun Song , Bess B. Ward Department of Geosciences, Princeton University, Princeton, NJ 08544-1003, USA Received 5 July 2002; received in revised form 9 October 2002 ; accepted 18 October 2002 First published online 14 November 2002 Abstract Diversity of the functional genes encoding dissimilatory nitrite reductase was investigated for the first time in denitrifying halobenzoate degrading bacteria and in two 4-chlorobenzoate degrading denitrifying consortia. Nitrite reductase genes were PCR-amplified with degenerate primers (specific to the two different types of respiratory nitrite reductase, nirS and nirK), cloned and sequenced to determine which type of nitrite reductase was present in each isolate and consortium. Halobenzoate degrading isolates belonging to the genera Ochrobactrum, Ensifer and Mesorhizobium, as well as Pseudomonas mendocina CH91 were found to have nirK genes, which were closely related to the previously published nirK genes of Ochrobactrum anthropi, Achromobacter cycloclastes, Alcaligenes faecalis and Pseudomonas aureofaciens, respectively. The isolates assigned to the genera Acidovorax, Azoarcus and Thauera as well as all other species in the genera Thauera and Azoarcus contained nirS genes, which were closely related to the nirS genes from Pseudomonas stutzeri with some exceptions. In addition, only nirS genes were found in 4-chlorobenzoate degrading denitrifying consortia. Three different major terminal restriction fragments from the nirS genes were detected by terminal restriction fragment length polymorphism analysis of the consortia, and five different nirS genes were cloned from one consortium. Three nirS gene clones were closely related to nirS genes from Thauera chlorobenzoica, Azoarcus tolulyticus and Pseudomonas aeruginosa, respectively. The phylogeny of nir genes was not entirely congruent with the 16S rRNA phylogeny of the genera nor was it correlated with the ecological and geographical origins or isolation substrates used for isolation and enrichment of consortia. 8 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Denitri¢cation ; Nitrite reductase; nirS; nirK; Halobenzoate 1. Introduction Denitri¢cation is de¢ned as dissimilatory transformation of nitrate or nitrite to gas concomitant with energy conservation. This process has been thoroughly examined because of its importance in the global nitrogen cycle, the production of greenhouse gases and the removal of contaminants in the environment. Several enzymes are involved in the complete denitri¢cation process. Nitrate is converted to dinitrogen through the sequential action of the enzymes nitrate reductase, nitrite reductase, nitric oxide reductase and nitrous oxide reductase (for reviews see [1^3]). Nitrite reduction is ecologically important for several * Corresponding author. Tel. : +1 (609) 258-6294; Fax : +1 (609) 258-0796. E-mail address : [email protected] (B. Song). reasons. In the environment, nitrite reduction can remove toxic nitrite and prevent the production of carcinogens generated by nitrite and amines. In bacterial metabolism, nitrite reduction is the key process in denitri¢cation, which converts nitrite to the gaseous product, nitric oxide. This reaction serves a respiratory function in denitrifying bacteria and is an e¡ective process for removal of nitrogen from the pool of ¢xed nitrogen, making it unavailable for most other organisms. Nitrite reductase, the enzyme that catalyzes this process, is located in the periplasmic space [2,3] and occurs in two major types : cytochrome cd1 -type nitrite reductase (NirS) and Cu-type nitrite reductase (NirK). Both appear to perform the same physiological reaction producing NO from NO3 2 (for reviews see [1,2]). The capability for dissimilatory nitrite reduction cannot be easily detected by relying on taxonomic a⁄nities identi¢ed by 16S rRNA gene probes because nitrite reducing bacteria are distributed in many genera of the prokaryotes [4] and many of these genera also contain non-denitrifying 0168-6496 / 02 / $22.00 8 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 1 6 8 - 6 4 9 6 ( 0 2 ) 0 0 4 3 3 - 6 FEMSEC 1463 24-2-03 350 B. Song, B.B. Ward / FEMS Microbiology Ecology 43 (2003) 349^357 strains. Using functional genes is an alternative approach to investigate denitrifying organisms in the environment. Nitrite reductase genes have been successfully detected by gene probe analysis and direct polymerase chain reaction (PCR) ampli¢cation with gene-speci¢c primers [5^12]. More than 200 complete or partial sequences of nitrite reductase genes from pure cultures and environmental samples have already been deposited in the GenBank database. Removal of halogenated aromatic contaminants has also been linked to denitri¢cation [13^15] and halobenzoate degrading denitrifying bacteria have been isolated from various geographic sites with di¡ering ecological site characteristics [16]. Taxonomic diversity and functional diversity of denitrifying halobenzoate degrading bacteria were described previously from studies in pure cultures and bacterial consortia [16^18]. The isolates were classi¢ed into nine genera belonging to the K-, L-, and Q-subdivisions of the Proteobacteria [16]. The isolates belonging in the genera Azoarcus and Thauera were further characterized in terms of their taxonomic and metabolic diversity related to aromatic compound degradation [17]. Denitrifying bacterial consortia capable of degrading 4-chlorobenzoate were characterized with terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes and the nucleotide sequence responsible for each terminal restriction fragment (T-RF) was determined by 16S rRNA gene cloning and sequencing [18]. Two major populations, one assigned to the genus Thauera, and another related to the genera Ralstonia and Limnobacter, were associated with 4-chlorobenzoate degradation under denitrifying conditions [18]. However, the genetic diversity of functional genes in these halobenzoate degrading denitrifying bacteria and their relationship to other denitrifying groups has not been investigated previously. The genes encoding nitrite reductase were thus investigated in this study using direct PCR ampli¢cation methods with gene-speci¢c primers for the ¢rst time to determine the type of nitrite reductase present and to examine the diversity of nitrite reductase genes in halobenzoate degrading denitrifying bacteria and some related species. 2. Materials and methods 2.1. Bacterial strains and growth conditions Twenty halobenzoate degrading denitrifying strains and 15 additional denitrifying strains in the Proteobacteria, as well as two denitrifying bacterial consortia capable of degrading 4-chlorobenzoate were examined in this study (Table 1). All the strains in the genus Thauera were cultivated anaerobically in a minimal salts medium [19] with succinate as a carbon source and nitrate as an electron acceptor, with the exception of Thauera mechernichensis TL1T (DSM 12266), which was cultured aerobically in Luria^ Bertani broth. All other strains were grown on M-R2A medium as described previously [20]. Stable bacterial consortia were obtained as described previously [18]. All the cultures growing in denitrifying liquid media [19] were monitored periodically for the loss of nitrate and nitrite using an ion chromatography system (Dionex DX-100, Sunnyvale, CA, USA) with conductivity detector as previously described [15]. 2.2. Genomic bacterial DNA isolation Chromosomal DNA of the strains in the genera Azoarcus and Thauera was isolated as previously described [21]. DNA from other strains and bacterial consortia was extracted using a modi¢ed phenol:chloroform method [22]. The purity of the DNA was determined by measuring absorbance at 230, 260 and 280 nm. 2.3. PCR ampli¢cation of nitrite reductase genes The primers for nitrite reductase genes (nirS and nirK) have been described previously [11,12,23]. For direct PCR ampli¢cation of the Cu-type nitrite reductase gene (nirK), primers Cunir3^4 were used with a touchdown PCR program as described previously [12]. Three di¡erent sets of primers were used for PCR ampli¢cation of cytochrome cd1 -type nitrite reductase gene (nirS). Primers Nir1^2, speci¢c for a central region (721 bp) of the nirS gene of Pseudomonas stutzeri [11], and degenerate primers nirS1F and nirS6R [7] were used for nirS gene ampli¢cation from halobenzoate degrading isolates and bacterial consortia following the previously described conditions [7,11]. In addition, degenerate primers Nir3^4 (Nir3-AAYGTNAARGARACBGG and Nir4-ACRTTRAAYTTNCCNGT) designed by Krishtein and Ward [23] were used to amplify the 3P end sequences of nirS genes (730 bp). PCR ampli¢cation was performed with Nir3^4 primers in a total volume of 50 Wl containing 5 Wl of 10UPCR bu¡er (500 mM KCl, 200 mM Tris^HCl [pH 8.4]), 1.5 mM MgCl2 , 20 WM of each deoxyribonucleoside triphosphate, 1 WM of each primer, 1 U Taq polymerase, and V100 ng of genomic DNA. The PCR cycle was started with a 5 min denaturation step at 95‡C, followed by 30 cycles of denaturation of 30 s at 95‡C, primer annealing for 2 min at 47‡C, and followed by 2 min extension at 72‡C. The ampli¢ed products were examined on 1.0% agarose gels by electrophoresis and then were puri¢ed using a Qiaquick1 gel extraction kit (Qiagen, Bothell, WA, USA) according to the manufacturer’s instructions. 2.4. Cloning and sequencing PCR products The cleaned PCR products were used for direct cloning with the TOPO-TA1 cloning system (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instruction. The ligated plasmids were transformed in high transform- FEMSEC 1463 24-2-03 B. Song, B.B. Ward / FEMS Microbiology Ecology 43 (2003) 349^357 ing e⁄ciency Escherichia coli TOP10FP1 (Invitrogen) following the manufacturer’s instructions. The transformed cells were plated on Luria^Bertani agar plates containing 50 Wg ml31 kanamycin. One strand of the inserts was initially sequenced with the primers M13 reverse or T7 using Big-dye1 terminator chemistry (Applied Biosystems, Foster City, CA, USA) and ABI model 310 automated DNA sequencer (Applied Biosystems). In addition, the inserts were sequenced again with the PCR primers for nirS or nirK genes. 2.5. Phylogenetic analysis of nitrite reductase genes The amino acid sequences of the nirS and nirK genes in the reference species were obtained from the GenBank database. The GenBank accession numbers of reference nirS and nirK genes used in this study are given in Figs. 1 and 2. The partial amino acid sequences of NirS (240 amino acids) and NirK (140 amino acids) were aligned using the ClustalW program (http://www.ebi.ac.uk/clustalw/). The phylogenetic analyses were performed with the PHYLIP 3.5 program [24] according to the Dayho¡ PAM matrix method [25]. Phylogenetic trees were reconstructed using the neighbor-joining method [26]. The SEQBOOT program was used to obtain the con¢dence level for neighbor-joining analysis using 100 bootstrapped data sets [27]. 351 yielded the ampli¢ed nir gene products (Table 1) although all 35 strains were capable of nitrite reduction, which was veri¢ed by the loss of nitrite during anaerobic growth (data not shown). Complete denitri¢cation was not assayed, but disappearance of nitrite from the medium was taken as evidence for the capability of nitrite reduction. nirK genes were found in eight isolates of the genera Ochrobactrum, Ensifer and Mesorhizobium, as well as Pseudomonas mendocina CH91. nirS genes were successfully ampli¢ed from seven halobenzoate degrading isolates and 11 additional strains of the genera Acidovorax, Azoarcus and Thauera with the various nirS primer sets (Nir1^2, Nir3^4 and NirS1F^6R). Interestingly, nirS genes could be ampli¢ed from most of the Thauera strains (except Thauera terpenica and Thauera mechernichensis) and Azoarcus evansii with the Nir1^2 primers, even though the Nir1^2 primers were designed with 100% identity to the nirS gene of P. stutzeri. The nirS genes were also ampli¢ed with the degenerate Nir3^4 and NirS1F^6R primers except from Thauera aromatica AR-1, which was ampli¢ed only with the Nir1^2 primers. In addition, two denitrifying bacterial consortia were determined to contain nirS genes by successful PCR ampli¢cation with the NirS1F^6R primers, but nirK genes were not detected in the consortia by PCR ampli¢cation with Cunir3^4 primers. 3.2. Phylogenetic analysis of Cu-type nitrite reductase sequences (nirK) in halobenzoate degrading denitrifying isolates 2.6. T-RFLP analysis of nitrite reductase genes For T-RFLP analysis, primer nirS1F 5P end-labeled with 6-FAM (5-[6]-carboxy-£uorescein, Operon Technology, Alameda, CA, USA) and unlabeled nirS6R primers were used to amplify nirS genes from the extracted DNA samples of bacterial consortia. PCR products were puri¢ed using a Qiaquick1 gel extraction kit (Qiagen). The concentration of puri¢ed PCR products were determined by image analysis of the agarose gel and quanti¢ed by comparison to the concentration of standards. The puri¢ed PCR products (40 ng) were digested with 1 U of MspI restriction endonuclease (New England Biolabs, Beverly, MA, USA) for 6 h at 37‡C. The digested samples were analyzed on an ABI 310 automated sequencer. The sizes of fragments were compared with internal standards and determined by the GeneScan software (Applied Biosystems). 3. Results 3.1. PCR ampli¢cation of nirS and nirK genes Twenty halobenzoate degrading denitrifying isolates and 15 additional denitrifying bacterial strains were examined for the presence of nir genes with speci¢c primers for ampli¢cation of nirS and nirK genes. Twenty-seven strains The nirK gene fragments obtained by PCR ampli¢cation with the Cunir3^4 primers were sequenced and translated to amino acid sequences for phylogenetic analysis. The NirK sequences from halobenzoate degrading isolates in the genus Ochrobactrum clustered with the NirK sequence of the type strain of Ochrobactrum anthropi (Fig. 1), which was consistent with the 16S rRNA gene phylogeny [16]. The NirK sequence of Mesorhizobium sp. 4FB11 was more closely related to the NirK sequences of Alcaligenes faecalis ATCC 8750 and Nitrosomonas sp. TA-921i-NH4 than those of Rhizobium, Sinorhizobium and Bradyrhizobium strains (Fig. 1), although strain 4FB11 was closely related to the genera Bradyrhizobium and Sinorhizobium on the basis of 16S rRNA sequence analysis [16]. The NirK sequences from halobenzoate degrading isolates in the genus Ensifer had more than 98.5% identity to NirK sequences from Achromobacter cycloclastes (Fig. 1). P. mendocina CH91 had a NirK, which is closely related to those of Pseudomonas aureofaciens, Achromobacter xylosoxidans and Alcaligenes spp. with about 81% identities (Fig. 1). 3.3. Phylogenetic analysis of cytochrome cd1 -type nitrite reductase sequences (nirS) in denitrifying isolates The nirS gene fragments ampli¢ed with the Nir1^2, FEMSEC 1463 24-2-03 352 B. Song, B.B. Ward / FEMS Microbiology Ecology 43 (2003) 349^357 Table 1 Determination of nitrite reductase genes and sources of bacterial strains Genus and species Strain PCR ampli¢cation Ecological source Geographical source GenBank number of nir gene Mo¡et ¢eld, California Arthur Kill, New Jersey Wyoming Shihwa, Korea Clinton, Michigan Mo¡et ¢eld, California Mo¡et ¢eld, California USA California AY078271 n/a AY078272 n/a AY078270 n/a n/a AY078269 AY078264 Konstanz, Germany California AY078256 AY078257 Tu«bingen-Lustnau, Germany Arthur Kill, New Jersey Wyoming Albany, New York Arthur Kill, New Jersey Arthur Kill, New Jersey Germany Germany Germany Mechernich, Germany Kyungan, Korea Kyungan, Korea Kyungan, Korea AY078260 AY078258 AY078259 AY078261 AY078262 AY078263 AY078265 AY078266 AY078267 AY078268 n/a n/a AY078273 NirS12 NirS34 NirS16 NirK L subdivision of the Proteobacteria Azoarcus tolulyticus Tol4T 2FB2 2FB6 4FB10 Azoarcus toluvorans Td21T Azoarcus toluclasticus MF63T MF58 Azoarcus evansii KB740T Thauera selenatis AXT Thauera aromatica Thauera chlorobenzoica Thauera linaloolentis Thauera terpenica Thauera mechernichensis Acidovorax sp. 3 3 3 3 3 3 3 + + + 3 3 3 + 3 3 + + + 3 + 3 + 3 3 + + 3 3 3 3 3 3 3 3 3 K172T T1 + + + + + + 3 3 AR-1 3CB2 3CB3 3CB-1T 4FB1 4FB2 47LolT 58EuT 21Mol TL1T 2FB4 2FB5 2FB7 + + + + + + + 3 3 3 3 3 3 3 3 + + + + + + + + 3 3 3 3 + + + + + + + + + 3 3 + 3 3 3 3 3 3 3 3 3 3 3 3 3 Aquifer sediment Estuarine sediment Agricultural soil Estuarine sediment Muck soil Aquifer sediment Aquifer sediment Creek sediment Selenium contaminated water Anaerobic sludge Petroleum contaminated soil Activated sludge Estuarine sediment Agricultural soil River sediment Estuarine sediment Estuarine sediment Activated sludge Activated sludge Activated sludge Land¢ll leachate River sediment River sediment River sediment 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 + + + + + + + + 3 Estuarine sediment Agricultural soil Agricultural soil Agricultural soil River sediment River sediment Agricultural soil Marine sediment Pond sediment Arthur Kill, New Jersey Wyoming Viikki, Finland Viikki, Finland Kyungan, Korea Kyungan, Korea Wyoming Hopewell Rock, Canada New Brunswick, New Jersey AY078250 AY078251 AY078249 AY078252 AY078253 AY078247 AY078248 AY078254 n/a 3 3 3 3 Agricultural soil Mendoca, Argentina n/a 3 3 3 + Agricultural soil Mendoca, Argentina AY078255 3 3 3 3 + + 3 3 Estuarine sediment Agricultural soil Arthur Kill, New Jersey Wyoming AY078274 to AY078278 K subdivision of the Proteobacteria Ochrobactrum sp. 3CB4 3CB5 2FB10 4FB13 4FB14 Ensifer sp. 2FB8 4FB6 Mesorhizobium sp. 4FB11 Bradyrhizobium sp. 2FB3 Q subdivision of the Proteobacteria Pseudomonas mendocina ATCC 25411T CH91 Bacterial consortium 4-chlorobenzoate degrading denitrifying consortium cultures +: ampli¢ed and veri¢ed by sequencing ; 3: no ampli¢cation; n/a: not available. Nir3^4 or NirS1F^6R primers were sequenced and translated to amino acid sequences to compare phylogenies. Phylogenetic analysis of the NirS sequences obtained with the Nir1^2 primers showed that all the NirS sequences from the Thauera strains and A. evansii were closely related to the NirS sequence of P. stutzeri with more than 80% identity, as expected based on the speci¢city of the primers (data not shown). The translated amino acid sequences of the nirS genes obtained with the Nir3^4 and NirS1F^6R primers were used here to illustrate the phylogenetic relationships among the strains because most of the nirS genes from the genera Acidovorax, Azoarcus and Thauera were ampli¢ed with the Nir3^4 and NirS1F^6R primers and because more NirS sequences covering this region are available in the GenBank database. The NirS sequences in all the Acidovorax, Azoarcus and Thauera strains, except Azoarcus tolulyticus and T. mechernichensis, clustered with the NirS sequences of P. stutzeri and A. faecalis with more than 85% amino acid sequence identities (Fig. 2). How- FEMSEC 1463 24-2-03 B. Song, B.B. Ward / FEMS Microbiology Ecology 43 (2003) 349^357 353 Fig. 1. Phylogenetic tree of nirK sequences on the basis of 140 amino acids. Scale bar represents 10 amino acids di¡erence in 100. Bootstrap values greater than 50 are reported above. GenBank accession numbers for the nirK genes are listed in Table 1 and given in parentheses. The nirK genes cloned in this study are reported in bold type. ever, the NirS sequences from A. tolulyticus Tol4T and T. mechernichensis TL1T clustered together regardless of their taxonomy and were distantly related to the NirS sequences of Pseudomonas £uorescens and Pseudomonas aeruginosa. The NirS sequences of these two species shared 97.8% amino acid sequence identity and about 66% identity to the NirS sequences from P. £uorescens and P. aeruginosa. Interestingly, the NirS sequence from A. tolulyticus 2FB6 did not cluster with that of A. tolulyticus Tol4T , although they were designated as the same species. The NirS sequences from T. aromatica, Thauera chlorobenzoica and T. terpenica clustered according to the taxonomic classi¢cation of the strains [17]. The NirS sequences from the species Azoarcus toluvorans and A. evansii were grouped together and separated from the cluster of Thauera NirS sequences, although NirS sequences from two genera share about 86% identity. However, the NirS sequence from Acidovorax sp. 2FB7 was closely related to those of T. chlorobenzoica regardless of their taxonomic di¡erence. 3.4. Diversity of nirS genes in bacterial consortium cultures Diversity of nirS genes in two di¡erent bacterial consortia capable of degrading 4-chlorobenzoate under deni- trifying conditions was examined with T-RFLP analysis and cloning of nirS genes from PCR-ampli¢ed products using the NirS1F^6R primers. T-RFLP analysis detected relatively few peaks in both consortia, but showed that the consortium culture established with Arthur Kill sediments had greater nirS gene diversity than that from Wyoming soils (Fig. 3). Increasing the T-RFLP signal strength by using more template DNA did not yield a greater number of fragments. Further e¡ort for cloning of nirS genes was focused on the Arthur Kill consortium culture. RFLP analysis with HaeIII endonuclease digestion was performed to ¢nd distinct RF patterns from the nirS gene clones. Five distinct RFs (clones nirSAK1 to nirSAK5) were found in 96 clones (data not shown). Clones corresponding to ¢ve di¡erent RFs were sequenced and translated to amino acid sequences to compare phylogenies with known NirS sequences. Phylogenetic analysis showed that clones nirSAK1, nirSAK2 and nirSAK3 were closely related to the NirS sequences from T. chlorobenzoica, A. tolulyticus and P. aeruginosa, respectively (Fig. 2). However, NirS sequences of clones nirSAK4 and nirSAK5 were not associated with any known NirS sequences (Fig. 2). T-RFLP analysis of nirS genes in the Arthur Kill consortium showed that at least three major T-RFs were FEMSEC 1463 24-2-03 354 B. Song, B.B. Ward / FEMS Microbiology Ecology 43 (2003) 349^357 Fig. 2. Phylogenetic tree of nirS sequences obtained with the Nir3^4 and NirS1F^6R primers. 240 amino acids were used in each sequence for comparison. Scale bar represents 10 amino acids di¡erence in 100. Bootstrap values greater than 50 are reported above. GenBank accession numbers for the nirS genes are listed in Table 1 and given in parentheses. The nirS genes cloned in this study are reported in bold type. present of 97 bp, 136 bp and 166 bp length (Fig. 3). The T-RFs of 97 bp and 136 bp length were matched with clones nirSAK5 and nirSAK1, respectively. The T-RF of 166 bp length was matched with clones nirSAK2 and nirSAK3, which were not di¡erentiated with MspI restriction endonuclease digestion (Fig. 3) but were di¡erentiated with RFLP analysis with HaeIII endonuclease digestion (data not shown). Clone nirSAK4 yielded a 367-bp T-RF, which was observed as a small peak on T-RFLP analysis (Fig. 3). showed low relatedness with less than 68% identity, although one clone (U14) shared 85% identity with the NirK sequence of Mesorhizobium sp. 4FB11 (data not shown). The NirS sequences from Acidovorax, Azoarcus and Thauera, as well as from denitrifying bacterial consortium culture did not show high a⁄nities to any of the environmental clones from the previous studies [10,28] (data not shown). 4. Discussion 3.5. Comparison of Nir sequences with environmental clones All the nir genes cloned from bacterial strains and consortium in this study were compared with nir genes cloned from environmental samples in other studies [10,28]. Pairwise comparison of NirK sequences from this study and 56 di¡erent environmental NirK sequences from marine sediments [10] and forested upland and marsh soils [28] 4.1. Distribution of nir genes in the halobenzoate degrading denitrifying bacteria and related strains Halobenzoate degrading denitrifying isolates were studied here for the ¢rst time to examine functional gene diversity related to dissimilatory nitrite reduction. Both types of nir genes were found in these isolates although FEMSEC 1463 24-2-03 B. Song, B.B. Ward / FEMS Microbiology Ecology 43 (2003) 349^357 355 Arthur Kill sediment 97 136 166 367 Wyoming soil Fig. 3. T-RFLP analysis of nirS genes in bacterial consortia capable of degrading 4-chlorobenzoate under denitrifying conditions. Restriction endonuclease MspI was used for digestion. they were originally enriched on the basis of their capabilities for halobenzoate degradation under denitrifying conditions. Di¡erent halobenzoates, such as 2-£uorobenzoate, 4-£uorobenzoate and 3-chlorobenzoate, used as a sole energy and carbon source did not appear to be related to the presence of di¡erent types of nir genes. In addition, neither gene type was correlated with a particular kind of ecosystem or geographical distribution. Halobenzoate degrading denitrifying isolates and related strains were derived from sources ranging from agricultural soils to marine sediments and from North America to Asia and Scandinavia (Table 1). nirS genes were retrieved from strains re£ecting this entire range of ecological settings and geographical origins. Although nirK was detected in fewer total strains, it was represented in more di¡erent genera, in isolates from around the globe. An interesting correlation was observed between the type of nitrite reductase gene and 16S rRNA gene based taxonomy of the strains in this study. The nirS genes were found only in the closely related genera Acidovorax, Azoarcus and Thauera. nirK genes, however, were detected in the more phylogenetically diverse genera Ensifer, Ochrobactrum, Pseudomonas and Mesorhizobium. The nirS and nirK genes were not both detected within a single genus in this study. However, some large genera, such as Alcaligenes and Pseudomonas, with many denitrifying members, are reported to have both types of nir gene. Further comparisons between functional gene and 16S rRNA phylogenies will be necessary to evaluate the congruence in the genera investigated here. Failure to detect nir genes in denitrifying strains closely related to those in which nir genes were detected may imply greater diversity of nir genes in denitrifying bacteria. Pairwise comparison of NirK sequences used in this study and a new class of copper-containing nitrite reductase (AniA and related NirK) showed high variation in their amino acid sequences [29]. The nirK genes belonging to the new class would not have been detected with the primers used in this study. Thus it is possible that some genes in the isolates went undetected and that the nir gene diversity in the consortia could have been underestimated. In addition, nitrite reduction by the isolates cannot be considered as proof of capacity for complete denitri¢cation to N2 . Their capabilities for complete denitri¢cation can only be veri¢ed with the production of N2 O or N2 during anaerobic growth. The isolates might not contain nirK or nirS genes although they reduced nitrite; nitrite reduction to ammonium, for example, involves non-homologous genes. Thus, further characterization of their denitri¢cation potential and further primer development for nir genes will both be necessary in order to investigate the genetic diversity of nitrite reductase genes in both pure cultures and environmental samples. 4.2. Genetic diversity of Cu-type nitrite reductase The copper-type nitrite reductase has been found in various denitrifying bacteria, nitrifying bacteria [12], and archaea [30]. Most nirK genes have been described in three subdivisions (K, L, and Q) of the Proteobacteria [7,9,10,12]. For the most part, the phylogeny of NirK sequences in this study correlated with the phylogeny of 16S rRNA genes. The NirK sequences from Ochrobactrum strains were grouped together with the NirK sequence of the type strain. In addition, the NirK sequence from P. mendocina grouped with the one of P. aureofaciens, again, consistent with their 16S rRNA gene-based taxonomy. However, the NirK sequences from Ensifer and Mesorhizobium strains did not correlate with 16S rRNA genebased taxonomy but were more closely related to the NirK sequences from the genera Alcaligenes and Achromobacter in the L subdivision of the Proteobacteria. 4.3. Genetic diversity of cytochrome cd1 -type nitrite reductase Cytochrome cd1 -type nitrite reductase genes (nirS) have been sequenced from various bacteria in the K, L and Q FEMSEC 1463 24-2-03 356 B. Song, B.B. Ward / FEMS Microbiology Ecology 43 (2003) 349^357 subdivisions of the Proteobacteria [7^10]. Most of the known nirS genes were sequenced from members of the K and Q subdivisions of the Proteobacteria, except the ones from A. faecalis and Ralstonia eutropha. This study extended the diversity of nirS genes in denitrifying bacteria in the L subdivision of the Proteobacteria. The genera Acidovorax, Azoarcus and Thauera were of special interest because of their involvement in halobenzoate and aromatic compound degradation under denitrifying conditions. Most of the NirS sequences from these genera were closely related to the NirS sequences of P. stutzeri, in the Q-Proteobacteria, although the strains are very different in taxonomy. This may provide evidence for horizontal gene transfer of nirS among the genera Pseudomonas, Acidovorax, Azoarcus and Thauera. However, novel NirS sequences were found in A. tolulyticus Tol4T and T. mechernichensis TL1T , which are closely related to each other in spite of their speciation, and cluster separately from the genera Pseudomonas and Ralstonia (Fig. 2). These clusters may re£ect di¡erent physiological characteristics for nitrite reduction from the other strains, such as aerobic denitri¢cation, which has been reported in T. mechernichensis [31]. In addition, A. tolulyticus 2FB6 has a di¡erent nirS gene from A. tolulyticus Tol4T although they share 79.9% similarity of whole genomic DNAs [17]. The di¡erence between NirS sequence phylogeny and speciation of Acidovorax, Azoarcus and Thauera strains (Fig. 2) implies the independent evolution of genes involved in metabolism. It is desirable to examine the genetic diversity of other metabolic genes, such as the genes encoding aromatic compound degradation under denitrifying conditions, in order to understand the evolutionary history of metabolic pathways. The denitrifying consortia examined in this study have been characterized for 4-chlorobenzoate degradation and taxonomic diversity previously [18]. Two major 16S rRNA gene clones were found to be involved in 4-chlorobenzoate degradation. One 16S rRNA gene clone (4CB1) was closely related to the genus Thauera and the other (4CB2) was distantly related to the genera Ralstonia and Limnobacter [18]. Phylogenetic analysis of NirS sequences from the bacterial consortium derived from the Arthur Kill sediment sample showed that clone nirSAK1 was very similar to the NirS sequences from the genus Thauera. Thus, clone nirSAK1 is probably derived from the same organism represented by the 16S rRNA gene clone 4CB1 in this consortium, although isolation of a pure culture is required to verify the connection between nirS and 16S rRNA genes. In addition, clone nirSAK5, generating a T-RF of 97 bp length, could be derived from the same organism containing the 16S rRNA gene clone 4CB2 on the basis of comparison of T-RFLP patterns of 16S rRNA genes and nirS genes (data not shown). Furthermore, even though T-RFLP may still underestimate the number of di¡erent sequences present in a sample, the presence of the other nirS gene clones (nirSAK2, nirSAK3 and nirSAK4) showed that functional gene diversity is higher than taxonomic diversity in this bacterial consortium culture. 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