FEMS Microbiology Ecology 43 (2003) 349^357 www.fems-microbiology.org

Nitrite reductase genes in halobenzoate degrading denitrifying

Bongkeun Song Ã, 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 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 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. ß 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 reasons. In the environment, nitrite reduction can remove toxic nitrite and prevent the production of carcinogens Denitri¢cation is de¢ned as dissimilatory transforma- generated by nitrite and amines. In bacterial metabolism, tion of nitrate or nitrite to gas concomitant with energy nitrite reduction is the key process in denitri¢cation, which conservation. This process has been thoroughly examined converts nitrite to the gaseous product, nitric oxide. This because of its importance in the global nitrogen cycle, the reaction serves a respiratory function in denitrifying bac- production of greenhouse gases and the removal of con- teria and is an e¡ective process for removal of nitrogen taminants in the environment. Several enzymes are in- from the pool of ¢xed nitrogen, making it unavailable for volved in the complete denitri¢cation process. Nitrate is most other organisms. Nitrite reductase, the enzyme that converted to dinitrogen through the sequential action of catalyzes this process, is located in the periplasmic space the enzymes nitrate reductase, nitrite reductase, nitric ox- [2,3] and occurs in two major types: cytochrome cd1-type ide reductase and nitrous oxide reductase (for reviews see nitrite reductase (NirS) and Cu-type nitrite reductase [1^3]). (NirK). Both appear to perform the same physiological 3 Nitrite reduction is ecologically important for several reaction producing NO from NO2 (for reviews see [1,2]). The capability for dissimilatory nitrite reduction cannot be easily detected by relying on taxonomic a⁄nities iden-

* Corresponding author. Tel.: +1 (609) 258-6294; ti¢ed by 16S rRNA gene probes because nitrite reducing Fax: +1 (609) 258-0796. bacteria are distributed in many genera of the prokaryotes E-mail address: [email protected] (B. Song). [4] and many of these genera also contain non-denitrifying

0168-6496 / 02 / $22.00 ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S0168-6496(02)00433-6

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 Bertani broth. All other strains were grown on M-R2A to investigate denitrifying organisms in the environment. medium as described previously [20]. Stable bacterial con- Nitrite reductase genes have been successfully detected by sortia were obtained as described previously [18]. All the gene probe analysis and direct polymerase chain reaction cultures growing in denitrifying liquid media [19] were (PCR) ampli¢cation with gene-speci¢c primers [5^12]. monitored periodically for the loss of nitrate and nitrite More than 200 complete or partial sequences of nitrite using an ion chromatography system (Dionex DX-100, reductase genes from pure cultures and environmental Sunnyvale, CA, USA) with conductivity detector as pre- samples have already been deposited in the GenBank da- viously described [15]. tabase. Removal of halogenated aromatic contaminants has 2.2. Genomic bacterial DNA isolation also been linked to denitri¢cation [13^15] and haloben- zoate degrading denitrifying bacteria have been isolated Chromosomal DNA of the strains in the genera Azoar- from various geographic sites with di¡ering ecological cus and Thauera was isolated as previously described [21]. site characteristics [16]. Taxonomic diversity and function- DNA from other strains and bacterial consortia was ex- al diversity of denitrifying halobenzoate degrading bacte- tracted using a modi¢ed phenol:chloroform method [22]. ria were described previously from studies in pure cultures The purity of the DNA was determined by measuring ab- and bacterial consortia [16^18]. The isolates were classi¢ed sorbance at 230, 260 and 280 nm. into nine genera belonging to the K-, L-, and Q-subdivisions of the [16]. The isolates belonging in the 2.3. PCR ampli¢cation of nitrite reductase genes genera Azoarcus and Thauera were further characterized in terms of their taxonomic and metabolic diversity related to The primers for nitrite reductase genes (nirS and nirK) aromatic compound degradation [17]. Denitrifying bacte- have been described previously [11,12,23]. For direct PCR rial consortia capable of degrading 4-chlorobenzoate were ampli¢cation of the Cu-type nitrite reductase gene (nirK), characterized with terminal restriction fragment length primers Cunir3^4 were used with a touchdown PCR pro- polymorphism (T-RFLP) analysis of 16S rRNA genes gram as described previously [12]. Three di¡erent sets of and the nucleotide sequence responsible for each terminal primers were used for PCR ampli¢cation of cytochrome restriction fragment (T-RF) was determined by 16S rRNA cd1-type nitrite reductase gene (nirS). Primers Nir1^2, spe- gene cloning and sequencing [18]. Two major populations, ci¢c for a central region (721 bp) of the nirS gene of one assigned to the genus Thauera, and another related to Pseudomonas stutzeri [11], and degenerate primers nirS1F the genera Ralstonia and Limnobacter, were associated and nirS6R [7] were used for nirS gene ampli¢cation from with 4-chlorobenzoate degradation under denitrifying con- halobenzoate degrading isolates and bacterial consortia ditions [18]. However, the genetic diversity of functional following the previously described conditions [7,11].In genes in these halobenzoate degrading denitrifying bacte- addition, degenerate primers Nir3^4 (Nir3-AAYGT- ria and their relationship to other denitrifying groups has NAARGARACBGG and Nir4-ACRTTRAAYTTNCC- not been investigated previously. The genes encoding ni- NGT) designed by Krishtein and Ward [23] were used to trite reductase were thus investigated in this study using amplify the 3P end sequences of nirS genes (730 bp). PCR direct PCR ampli¢cation methods with gene-speci¢c prim- ampli¢cation was performed with Nir3^4 primers in a to- ers for the ¢rst time to determine the type of nitrite reduc- tal volume of 50 Wl containing 5 Wlof10UPCR bu¡er tase present and to examine the diversity of nitrite reduc- (500 mM KCl, 200 mM Tris^HCl [pH 8.4]), 1.5 mM tase genes in halobenzoate degrading denitrifying bacteria MgCl2,20WM of each deoxyribonucleoside triphosphate, and some related species. 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 dena- 2. Materials and methods turation of 30 s at 95‡C, primer annealing for 2 min at 47‡C, and followed by 2 min extension at 72‡C. The am- 2.1. Bacterial strains and growth conditions pli¢ed products were examined on 1.0% agarose gels by electrophoresis and then were puri¢ed using a Qiaquick1 Twenty halobenzoate degrading denitrifying strains and gel extraction kit (Qiagen, Bothell, WA, USA) according 15 additional denitrifying strains in the Proteobacteria, as to the manufacturer’s instructions. well as two denitrifying bacterial consortia capable of de- grading 4-chlorobenzoate were examined in this study (Ta- 2.4. Cloning and sequencing PCR products ble 1). All the strains in the genus Thauera were cultivated anaerobically in a minimal salts medium [19] with succi- The cleaned PCR products were used for direct cloning nate as a carbon source and nitrate as an electron accep- with the TOPO-TA1 cloning system (Invitrogen, Carls- tor, with the exception of Thauera mechernichensis TL1T bad, CA, USA) following the manufacturer’s instruction. (DSM 12266), which was cultured aerobically in Luria^ The ligated plasmids were transformed in high transform-

FEMSEC 1463 24-2-03 B. Song, B.B. Ward / FEMS Microbiology Ecology 43 (2003) 349^357 351 ing e⁄ciency Escherichia coli TOP10FP1 (Invitrogen) fol- yielded the ampli¢ed nir gene products (Table 1) although lowing the manufacturer’s instructions. The transformed all 35 strains were capable of nitrite reduction, which was cells were plated on Luria^Bertani agar plates containing veri¢ed by the loss of nitrite during anaerobic growth 50 Wgml31 kanamycin. One strand of the inserts was (data not shown). Complete denitri¢cation was not as- initially sequenced with the primers M13 reverse or T7 sayed, but disappearance of nitrite from the medium was using Big-dye1 terminator chemistry (Applied Biosystems, taken as evidence for the capability of nitrite reduction. Foster City, CA, USA) and ABI model 310 automated nirK genes were found in eight isolates of the genera DNA sequencer (Applied Biosystems). In addition, the Ochrobactrum, Ensifer and Mesorhizobium, as well as inserts were sequenced again with the PCR primers for Pseudomonas mendocina CH91. nirS genes were success- nirS or nirK genes. fully ampli¢ed from seven halobenzoate degrading isolates and 11 additional strains of the genera Acidovorax, Azoar- 2.5. Phylogenetic analysis of nitrite reductase genes cus and Thauera with the various nirS primer sets (Nir1^2, Nir3^4 and NirS1F^6R). Interestingly, nirS genes could The amino acid sequences of the nirS and nirK genes in be ampli¢ed from most of the Thauera strains (except the reference species were obtained from the GenBank and Thauera mechernichensis) and database. The GenBank accession numbers of reference Azoarcus evansii with the Nir1^2 primers, even though nirS and nirK genes used in this study are given in Figs. the Nir1^2 primers were designed with 100% identity to 1 and 2. The partial amino acid sequences of NirS (240 the nirS gene of P. stutzeri. The nirS genes were also amino acids) and NirK (140 amino acids) were aligned ampli¢ed with the degenerate Nir3^4 and NirS1F^6R using the ClustalW program (http://www.ebi.ac.uk/clus- primers except from AR-1, which was talw/). The phylogenetic analyses were performed with ampli¢ed only with the Nir1^2 primers. In addition, two the PHYLIP 3.5 program [24] according to the Dayho¡ denitrifying bacterial consortia were determined to contain PAM matrix method [25]. Phylogenetic trees were recon- nirS genes by successful PCR ampli¢cation with the structed using the neighbor-joining method [26]. The SEQ- NirS1F^6R primers, but nirK genes were not detected in BOOT program was used to obtain the con¢dence level the consortia by PCR ampli¢cation with Cunir3^4 prim- for neighbor-joining analysis using 100 bootstrapped data ers. sets [27]. 3.2. Phylogenetic analysis of Cu-type nitrite reductase 2.6. T-RFLP analysis of nitrite reductase genes sequences (nirK) in halobenzoate degrading denitrifying isolates For T-RFLP analysis, primer nirS1F 5P end-labeled with 6-FAM (5-[6]-carboxy-£uorescein, Operon Technol- The nirK gene fragments obtained by PCR ampli¢cation ogy, Alameda, CA, USA) and unlabeled nirS6R primers with the Cunir3^4 primers were sequenced and translated were used to amplify nirS genes from the extracted DNA to amino acid sequences for phylogenetic analysis. The samples of bacterial consortia. PCR products were puri- NirK sequences from halobenzoate degrading isolates in ¢ed using a Qiaquick1 gel extraction kit (Qiagen). The the genus Ochrobactrum clustered with the NirK sequence concentration of puri¢ed PCR products were determined of the type strain of Ochrobactrum anthropi (Fig. 1), which by image analysis of the agarose gel and quanti¢ed by was consistent with the 16S rRNA gene phylogeny [16]. comparison to the concentration of standards. The puri- The NirK sequence of Mesorhizobium sp. 4FB11 was more ¢ed PCR products (40 ng) were digested with 1 U of MspI closely related to the NirK sequences of Alcaligenes faeca- restriction endonuclease (New England Biolabs, Beverly, lis ATCC 8750 and Nitrosomonas sp. TA-921i-NH4 than MA, USA) for 6 h at 37‡C. The digested samples were those of Rhizobium, Sinorhizobium and Bradyrhizobium analyzed on an ABI 310 automated sequencer. The sizes strains (Fig. 1), although strain 4FB11 was closely related of fragments were compared with internal standards and to the genera Bradyrhizobium and Sinorhizobium on the determined by the GeneScan software (Applied Biosys- basis of 16S rRNA sequence analysis [16]. The NirK se- tems). quences from halobenzoate degrading isolates in the genus Ensifer had more than 98.5% identity to NirK sequences from Achromobacter cycloclastes (Fig. 1). P. mendocina 3. Results CH91 had a NirK, which is closely related to those of Pseudomonas aureofaciens, Achromobacter xylosoxidans 3.1. PCR ampli¢cation of nirS and nirK genes and Alcaligenes spp. with about 81% identities (Fig. 1).

Twenty halobenzoate degrading denitrifying isolates 3.3. Phylogenetic analysis of cytochrome cd1-type nitrite and 15 additional denitrifying bacterial strains were exam- reductase sequences (nirS) in denitrifying isolates ined for the presence of nir genes with speci¢c primers for ampli¢cation of nirS and nirK genes. Twenty-seven strains The nirS gene fragments ampli¢ed with the Nir1^2,

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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 NirS12 NirS34 NirS16 NirK L subdivision of the Proteobacteria Azoarcus tolulyticus Tol4T 3 ++3 Aquifer sediment Mo¡et ¢eld, California AY078271 2FB2 3333Estuarine sediment Arthur Kill, New Jersey n/a 2FB6 33+ 3 Agricultural soil Wyoming AY078272 4FB10 3333Estuarine sediment Shihwa, Korea n/a Azoarcus toluvorans Td21T 3 ++3 Muck soil Clinton, Michigan AY078270 Azoarcus toluclasticus MF63T 3333Aquifer sediment Mo¡et ¢eld, California n/a MF58 3333Aquifer sediment Mo¡et ¢eld, California n/a Azoarcus evansii KB740T +++3 Creek sediment USA AY078269 AXT +++3 Selenium contaminated California AY078264 water Thauera aromatica K172T +++3 Anaerobic sludge Konstanz, Germany AY078256 T1 + + + 3 Petroleum contaminated California AY078257 soil AR-1 + 333Activated sludge Tu«bingen-Lustnau, Germany AY078260 3CB2 + 3 + 3 Estuarine sediment Arthur Kill, New Jersey AY078258 3CB3 + + + 3 Agricultural soil Wyoming AY078259 Thauera chlorobenzoica 3CB-1T +++3 River sediment Albany, New York AY078261 4FB1 + + + 3 Estuarine sediment Arthur Kill, New Jersey AY078262 4FB2 + + + 3 Estuarine sediment Arthur Kill, New Jersey AY078263 47LolT +++3 Activated sludge Germany AY078265 Thauera terpenica 58EuT 3 ++3 Activated sludge Germany AY078266 21Mol 3 ++3 Activated sludge Germany AY078267 Thauera mechernichensis TL1T 3 ++3 Land¢ll leachate Mechernich, Germany AY078268 Acidovorax sp. 2FB4 3333River sediment Kyungan, Korea n/a 2FB5 3333River sediment Kyungan, Korea n/a 2FB7 33+ 3 River sediment Kyungan, Korea AY078273

K subdivision of the Proteobacteria Ochrobactrum sp. 3CB4 333+ Estuarine sediment Arthur Kill, New Jersey AY078250 3CB5 333+ Agricultural soil Wyoming AY078251 2FB10 333+ Agricultural soil Viikki, Finland AY078249 4FB13 333+ Agricultural soil Viikki, Finland AY078252 4FB14 333+ River sediment Kyungan, Korea AY078253 Ensifer sp. 2FB8 333+ River sediment Kyungan, Korea AY078247 4FB6 333+ Agricultural soil Wyoming AY078248 Mesorhizobium sp. 4FB11 333+ Marine sediment Hopewell Rock, Canada AY078254 Bradyrhizobium sp. 2FB3 3333Pond sediment New Brunswick, New Jersey n/a Q subdivision of the Proteobacteria Pseudomonas mendocina ATCC 3333Agricultural soil Mendoca, Argentina n/a 25411T CH91 333+ Agricultural soil Mendoca, Argentina AY078255 Bacterial consortium 4-chlorobenzoate degrading denitrifying 33+ 3 Estuarine sediment Arthur Kill, New Jersey AY078274 consortium cultures 33+ 3 Agricultural soil Wyoming to AY078278 +: ampli¢ed and veri¢ed by sequencing; 3: no ampli¢cation;n/a: not available.

Nir3^4 or NirS1F^6R primers were sequenced and trans- used here to illustrate the phylogenetic relationships lated to amino acid sequences to compare phylogenies. among the strains because most of the nirS genes from Phylogenetic analysis of the NirS sequences obtained the genera Acidovorax, Azoarcus and Thauera were ampli- with the Nir1^2 primers showed that all the NirS sequen- ¢ed with the Nir3^4 and NirS1F^6R primers and because ces from the Thauera strains and A. evansii were closely more NirS sequences covering this region are available in related to the NirS sequence of P. stutzeri with more than the GenBank database. The NirS sequences in all the 80% identity, as expected based on the speci¢city of the Acidovorax, Azoarcus and Thauera strains, except Azoar- primers (data not shown). cus tolulyticus and T. mechernichensis, clustered with the The translated amino acid sequences of the nirS genes NirS sequences of P. stutzeri and A. faecalis with more obtained with the Nir3^4 and NirS1F^6R primers were than 85% amino acid sequence identities (Fig. 2). How-

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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 trifying conditions was examined with T-RFLP analysis T. mechernichensis TL1T clustered together regardless of and cloning of nirS genes from PCR-ampli¢ed products their and were distantly related to the NirS using the NirS1F^6R primers. T-RFLP analysis detected sequences of Pseudomonas £uorescens and Pseudomonas relatively few peaks in both consortia, but showed that the aeruginosa. The NirS sequences of these two species shared consortium culture established with Arthur Kill sediments 97.8% amino acid sequence identity and about 66% iden- had greater nirS gene diversity than that from Wyoming tity to the NirS sequences from P. £uorescens and P. aeru- soils (Fig. 3). Increasing the T-RFLP signal strength by ginosa. Interestingly, the NirS sequence from A. tolulyticus using more template DNA did not yield a greater number 2FB6 did not cluster with that of A. tolulyticus Tol4T, of fragments. Further e¡ort for cloning of nirS genes was although they were designated as the same species. The focused on the Arthur Kill consortium culture. RFLP NirS sequences from T. aromatica, Thauera chlorobenzoica analysis with HaeIII endonuclease digestion was per- and T. terpenica clustered according to the taxonomic clas- formed to ¢nd distinct RF patterns from the nirS gene si¢cation of the strains [17]. The NirS sequences from the clones. Five distinct RFs (clones nirSAK1 to nirSAK5) species Azoarcus toluvorans and A. evansii were grouped were found in 96 clones (data not shown). Clones corre- together and separated from the cluster of Thauera NirS sponding to ¢ve di¡erent RFs were sequenced and trans- sequences, although NirS sequences from two genera share lated to amino acid sequences to compare phylogenies about 86% identity. However, the NirS sequence from with known NirS sequences. Phylogenetic analysis showed Acidovorax sp. 2FB7 was closely related to those of that clones nirSAK1, nirSAK2 and nirSAK3 were closely T. chlorobenzoica regardless of their taxonomic di¡erence. related to the NirS sequences from T. chlorobenzoica, A. tolulyticus and P. aeruginosa, respectively (Fig. 2). 3.4. Diversity of nirS genes in bacterial consortium cultures However, NirS sequences of clones nirSAK4 and nirSAK5 were not associated with any known NirS sequences (Fig. Diversity of nirS genes in two di¡erent bacterial con- 2). T-RFLP analysis of nirS genes in the Arthur Kill con- sortia capable of degrading 4-chlorobenzoate under deni- sortium showed that at least three major T-RFs were

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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 compari- son. 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 showed low relatedness with less than 68% identity, T-RFs of 97 bp and 136 bp length were matched with although one clone (U14) shared 85% identity with the clones nirSAK5 and nirSAK1, respectively. The T-RF of NirK sequence of Mesorhizobium sp. 4FB11 (data not 166 bp length was matched with clones nirSAK2 and nir- shown). The NirS sequences from Acidovorax, Azoarcus SAK3, which were not di¡erentiated with MspI restriction and Thauera, as well as from denitrifying bacterial con- endonuclease digestion (Fig. 3) but were di¡erentiated sortium culture did not show high a⁄nities to any of the with RFLP analysis with HaeIII endonuclease digestion environmental clones from the previous studies [10,28] (data not shown). Clone nirSAK4 yielded a 367-bp (data not shown). T-RF, which was observed as a small peak on T-RFLP analysis (Fig. 3). 4. Discussion 3.5. Comparison of Nir sequences with environmental clones 4.1. Distribution of nir genes in the halobenzoate degrading All the nir genes cloned from bacterial strains and con- denitrifying bacteria and related strains sortium in this study were compared with nir genes cloned from environmental samples in other studies [10,28]. Pair- Halobenzoate degrading denitrifying isolates were wise comparison of NirK sequences from this study and studied here for the ¢rst time to examine functional gene 56 di¡erent environmental NirK sequences from marine diversity related to dissimilatory nitrite reduction. Both sediments [10] and forested upland and marsh soils [28] types of nir genes were found in these isolates although

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97 Arthur Kill sediment

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 endonu- clease MspI was used for digestion. they were originally enriched on the basis of their capa- undetected and that the nir gene diversity in the consortia bilities for halobenzoate degradation under denitrifying could have been underestimated. In addition, nitrite reduc- conditions. Di¡erent halobenzoates, such as 2-£uoroben- tion by the isolates cannot be considered as proof of ca- zoate, 4-£uorobenzoate and 3-chlorobenzoate, used as a pacity for complete denitri¢cation to N2. Their capabilities sole energy and carbon source did not appear to be related for complete denitri¢cation can only be veri¢ed with the to the presence of di¡erent types of nir genes. In addition, production of N2OorN2 during anaerobic growth. The neither gene type was correlated with a particular kind of isolates might not contain nirK or nirS genes although ecosystem or geographical distribution. Halobenzoate de- they reduced nitrite;nitrite reduction to ammonium, for grading denitrifying isolates and related strains were de- example, involves non-homologous genes. Thus, further rived from sources ranging from agricultural soils to ma- characterization of their denitri¢cation potential and fur- rine sediments and from North America to Asia and ther primer development for nir genes will both be neces- Scandinavia (Table 1). nirS genes were retrieved from sary in order to investigate the genetic diversity of nitrite strains re£ecting this entire range of ecological settings reductase genes in both pure cultures and environmental and geographical origins. Although nirK was detected in samples. fewer total strains, it was represented in more di¡erent genera, in isolates from around the globe. 4.2. Genetic diversity of Cu-type nitrite reductase An interesting correlation was observed between the type of nitrite reductase gene and 16S rRNA gene based The copper-type nitrite reductase has been found in taxonomy of the strains in this study. The nirS genes various denitrifying bacteria, nitrifying bacteria [12], and were found only in the closely related genera Acidovorax, archaea [30]. Most nirK genes have been described in three Azoarcus and Thauera. nirK genes, however, were detected subdivisions (K, L, and Q) of the Proteobacteria [7,9,10,12]. in the more phylogenetically diverse genera Ensifer, Ochro- For the most part, the phylogeny of NirK sequences in bactrum, Pseudomonas and Mesorhizobium. The nirS and this study correlated with the phylogeny of 16S rRNA nirK genes were not both detected within a single genus in genes. The NirK sequences from Ochrobactrum strains this study. However, some large genera, such as Alcali- were grouped together with the NirK sequence of the genes and Pseudomonas, with many denitrifying members, type strain. In addition, the NirK sequence from P. men- are reported to have both types of nir gene. Further com- docina grouped with the one of P. aureofaciens, again, parisons between functional gene and 16S rRNA phylog- consistent with their 16S rRNA gene-based taxonomy. enies will be necessary to evaluate the congruence in the However, the NirK sequences from Ensifer and Mesorhi- genera investigated here. Failure to detect nir genes in zobium strains did not correlate with 16S rRNA gene- denitrifying strains closely related to those in which nir based taxonomy but were more closely related to the genes were detected may imply greater diversity of nir NirK sequences from the genera Alcaligenes and Achromo- genes in denitrifying bacteria. Pairwise comparison of bacter in the L subdivision of the Proteobacteria. NirK sequences used in this study and a new class of copper-containing nitrite reductase (AniA and related 4.3. Genetic diversity of cytochrome cd1-type nitrite NirK) showed high variation in their amino acid sequen- reductase ces [29]. The nirK genes belonging to the new class would not have been detected with the primers used in this study. Cytochrome cd1-type nitrite reductase genes (nirS) have Thus it is possible that some genes in the isolates went 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 the presence of the other nirS gene clones (nirSAK2, nir- known nirS genes were sequenced from members of the SAK3 and nirSAK4) showed that functional gene diver- K and Q subdivisions of the Proteobacteria, except the ones sity is higher than taxonomic diversity in this bacterial from A. faecalis and Ralstonia eutropha. This study ex- consortium culture. tended 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 Acknowledgements because of their involvement in halobenzoate and aro- matic compound degradation under denitrifying condi- This work was funded in part by the Department of tions. Most of the NirS sequences from these genera Energy and National Science Foundation awards to were closely related to the NirS sequences of P. stutzeri, B.B.W. in the Q-Proteobacteria, although the strains are very dif- ferent in taxonomy. This may provide evidence for hori- zontal gene transfer of nirS among the genera Pseudomo- References nas, Acidovorax, Azoarcus and Thauera. However, novel NirS sequences were found in A. tolulyticus Tol4T and [1] Zumft, W.G. (1997) Cell biology and molecular basis of denitri¢ca- T. mechernichensis TL1T, which are closely related to tion. Microbiol. Mol. Biol. Rev. 61, 533^616. each other in spite of their speciation, and cluster sepa- [2] Berks, B.C., Ferguson, S.J., Moir, J.W.B. and Richardson, D.J. (1995) Enzymes and associated electron transport systems that cata- rately from the genera Pseudomonas and Ralstonia (Fig. lyze the respiratory reduction of nitrogen oxides and oxyanions. Bio- 2). These clusters may re£ect di¡erent physiological chim. Biophys. Acta 1232, 97^173. characteristics for nitrite reduction from the other strains, [3] Knowles, R. (1982) Denitri¢cation. Microbiol. Rev. 46, 43^70. such as aerobic denitri¢cation, which has been reported in [4] Zumft, W.G. (1992) The denitrifying prokaryotes. In: The Prokary- T. mechernichensis [31]. In addition, A. tolulyticus 2FB6 otes (Balows, A., Tru«per, H.G., Dworkin, M., Harder, W. and T Schleifer, K.-H., Eds.), pp. 554^582. Springer Verlag, New York. has a di¡erent nirS gene from A. tolulyticus Tol4 al- [5] Ye, R.W., Fries, M.R., Bezborodnikov, S.G., Averill, B.A. and though they share 79.9% similarity of whole genomic Tiedje, J.M. (1993) Characterization of the structural gene encoding DNAs [17]. a copper-containing nitrite reductase and homology of this gene to The di¡erence between NirS sequence phylogeny and DNA of other denitri¢ers. Appl. Environ. Microbiol. 59, 250^254. speciation of Acidovorax, Azoarcus and Thauera strains [6] Ward, B. (1995) Diversity of culturable denitrifying bacteria;limits of rDNA RFLP analysis and probes for the functional gene, nitrite (Fig. 2) implies the independent evolution of genes in- reductase. Arch. Microbiol. 163, 167^175. volved in metabolism. It is desirable to examine the genet- [7] Braker, G., Fesefeldt, A. and Witzel, K.-P. (1998) Development of ic diversity of other metabolic genes, such as the genes PCR primer systems for ampli¢cation of nitrite reductase genes (nirK encoding aromatic compound degradation under denitri- and nirS) to detect denitrifying bacteria in environmental samples. fying conditions, in order to understand the evolutionary Appl. Environ. Microbiol. 64, 3769^3775. [8] Smith, G.B. and Tiedje, J.M. (1992) Isolation and characterization of history of metabolic pathways. a nitrite reductase gene and its use as a probe for denitrifying bac- The denitrifying consortia examined in this study have teria. Appl. Environ. Microbiol. 58, 376^384. been characterized for 4-chlorobenzoate degradation and [9] Hallin, S. and Lindgren, P.-E. (1999) PCR detection of genes encod- taxonomic diversity previously [18]. Two major 16S rRNA ing nitrite reductase in denitrifying bacteria. Appl. Environ. Micro- gene clones were found to be involved in 4-chlorobenzoate biol. 65, 1652^1657. [10] Braker, G., Zhou, J., Wu, L., Devol, A.H. and Tiedje, J.M. (2000) degradation. One 16S rRNA gene clone (4CB1) was Nitrite reductase genes (nirK and nirS) as functional markers to in- closely related to the genus Thauera and the other vestigate diversity of denitrifying bacteria in paci¢c northwest marine (4CB2) was distantly related to the genera Ralstonia and sediment communities. Appl. Environ. Microbiol. 66, 2096^2104. Limnobacter [18]. Phylogenetic analysis of NirS sequences [11] Ward, B., Cockcroft, A. and Kilpatrick, K. (1993) Antibody and from the bacterial consortium derived from the Arthur DNA probes for detection of nitrite reductase in seawater. J. Gen. Microbiol. 139, 2285^2293. Kill sediment sample showed that clone nirSAK1 was [12] Casciotti, K.L. and Ward, B.B. (2001) Dissimilatory nitrite reductase very similar to the NirS sequences from the genus genes from autotrophic ammonia-oxidizing bacteria. Appl. Environ. Thauera. Thus, clone nirSAK1 is probably derived from Microbiol. 67, 2213^2221. the same organism represented by the 16S rRNA gene [13] Ha«ggblom, M.M., Rivera, M.D. and Young, L.Y. (1996) Anaerobic clone 4CB1 in this consortium, although isolation of a degradation of halogenated benzoic acids coupled to denitri¢cation observed in a variety of sediment and soil samples. FEMS Microbiol. pure culture is required to verify the connection between Lett. 144, 213^219. nirS and 16S rRNA genes. In addition, clone nirSAK5, [14] Ha«ggblom, M.M., Rivera, M. and Young, L.Y. (1993) In£uence of generating a T-RF of 97 bp length, could be derived from alternative electron acceptors on the anaerobic biodegradability of the same organism containing the 16S rRNA gene clone chlorinated phenols and benzoic acids. Appl. Environ. Microbiol. 4CB2 on the basis of comparison of T-RFLP patterns of 59, 1162^1167. [15] Vargas, C., Song, B., Camps, M. and Ha«ggblom, M.M. (2000) An- 16S rRNA genes and nirS genes (data not shown). Fur- aerobic degradation of £uorinated aromatic compounds. Appl. Mi- thermore, even though T-RFLP may still underestimate crobiol. Biotechnol. 53, 342^347. the number of di¡erent sequences present in a sample, [16] Song, B., Palleroni, N.J. and Ha«ggblom, M.M. (2000) Isolation and

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