Phylogenetic Analysis of Nitric Oxide Reductase Gene Homologues from Aerobic Ammonia-Oxidizing Bacteria

Home , Nitrosomonas, Nitrosomonas europaea, Paracoccus denitrificans

FEMS Microbiology Ecology 52 (2005) 197–205 www.fems-microbiology.org

Phylogenetic analysis of nitric oxide reductase gene homologues from aerobic ammonia-oxidizing bacteria

Karen L. Casciotti *,1, Bess B. Ward

Department of Geosciences, Princeton University, Princeton, NJ 08540, USA

Received 16 June 2004; received in revised form 15 September 2004; accepted 3 November 2004

First published online 23 November 2004

Abstract

Nitric oxide (NO) and nitrous oxide (N2O) are climatically important trace gases that are produced by both nitrifying and denitrifying bacteria. In the denitrification pathway, N2O is produced from nitric oxide (NO) by the enzyme nitric oxide reductase (NOR). The ammonia-oxidizing bacterium Nitrosomonas europaea also possesses a functional nitric oxide reductase, which was shown recently to serve a unique function. In this study, sequences homologous to the large subunit of nitric oxide reductase (norB) were obtained from eight additional strains of ammonia-oxidizing bacteria, including Nitrosomonas and Nitrosococcus species (i.e., both b- and c-Proteobacterial ammonia oxidizers), showing widespread occurrence of a norB homologue in ammonia-oxidizing bacteria. However, despite efforts to detect norB homologues from Nitrosospira strains, sequences have not yet been obtained. Phylo- genetic analysis placed nitrifier norB homologues in a subcluster, distinct from denitrifier sequences. The similarities and differences of these sequences highlight the need to understand the variety of metabolisms represented within a ‘‘functional group’’ defined by the presence of a single homologous gene. These results expand the database of norB homologue sequences in nitrifying bacteria. Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Ammonia-oxidizing bacteria; Nitriﬁer-denitriﬁcation; Nitric oxide reductase; Nitrite reductase; Nitrous oxide

1. Introduction the flux of nitric oxide (NO) and nitrous oxide (N2O) gases from terrestrial [1–3] and aquatic [4–8] environ- Ammonia-oxidizing nitrifying bacteria play several ments. Because NO and N2O play important roles in important roles in nitrogen cycling in terrestrial, fresh- atmospheric chemistry, global warming, and strato- water, and marine environments. Ammonia-oxidizers spheric ozone depletion, it is important to understand are primarily responsible for the oxidation of ammonia the natural controls on the production of these gases (NH3) to nitrite ðNO2 Þ. This activity is important for [9,10]. þ determining the form of nitrogen ðNH4 ,NO2 ,NO3 Þ Approaches for evaluating the contribution of available for use by primary producers, and it facilitates ammonia-oxidizers to N2O production include inhibi- the loss of nitrogen from ecosystems by denitrification. tion studies [1], chemical and mass balance arguments Ammonia-oxidizing bacteria also contribute directly to [8], environmental constraints (e.g., O2 availability) [11], and stable isotopic analyses of N2O [4,6,12]. The application of genetic tools to study the role of nitrifiers * Corresponding author. Tel.: +1 508 289 3738, fax: +1 508 457 in N2O production has not received much attention (but 2013. E-mail address: [email protected] (K.L. Casciotti). see [13]). In order to develop a genetic approach and 1 Present address: Woods Hole Oceanographic Institution, MS 8, determine appropriate target genes with which to inves- Woods Hole, MA 02543, USA. tigate the role of nitrifiers in N2O production in the

0168-6496/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsec.2004.11.002 198 K.L. Casciotti, B.B. Ward / FEMS Microbiology Ecology 52 (2005) 197–205 environment, it is important to elucidate the pathway(s) cytochrome c oxidases [30]. The nitric oxide reductase and enzymes involved in N2O production by nitrifying found most commonly in denitrifying bacteria is a mem- bacteria. brane-bound dimer of subunits encoded by the genes Evidence exists for two potential pathways for N2O norB and norC [31]. NorB contains the active site, which production in ammonia-oxidizing bacteria. The reduc- consists of a b-type heme prosthetic group as well as a tion of NO2 to N2O is evidenced by stable isotope tracer non-heme iron [32]. Additional heme groups in NorC studies that demonstrate incorporation of NO2 into and NorB are thought to mediate electron transport N2O [14–16] and is supported by biochemical character- from soluble cytochrome c to the catalytic site [33].A ization of a copper-containing nitrite reductase in Nitr- second form of nitric oxide reductase, that also produces osomonas europaea [17–19]. Homologues of nirK and N2O, has been found in several Bacteria and Archaea norB have been fully sequenced in N. europaea [20], [31,34,35,37]. This alternate form of nitric oxide reduc- and partial nirK homologues have also been identified tase accepts electrons from reduced quinol rather than in several marine nitrifiers that are capable of nitrifier- cytochrome c, and it lacks the NorC subunit [31]. In- denitrification [21, Casciotti and Ward, unpublished]. stead, the catalytic subunit (homologous to NorB) has However, mutation of the nirK and norB genes in N. an N-terminal extension that is hypothesized to mediate europaea does not eliminate its ability to produce NO electron transfer from quinol [36]. This alternate form of and N2O. Instead, these mutants have a lowered resis- NOR has been termed qNOR by some authors [31,37] tance to high levels of NO2 and NO [22,23]. While it and has been given the gene designation of norZ in R. is reported that NirK and NorB are active in wild type eutropha [34] or qnorB in other organisms [37]. In this N. europaea and consume nitrite and nitric oxide, paper, NorB will refer to the common cytochrome c-oxi- respectively, the products of these reactions are not iden- dizing NOR and NorZ will refer to the quinol-oxidizing tified. It may be relevant that even though the cyanobac- form, with gene designations norB and norZ, terium Synechocystis PCC 6803 does not gain energy respectively. from nitric oxide reduction and is considered non-deni- Braker and Tiedje [37] explored the distribution of trifying (uses NOR as a detoxification mechanism) the norB in cultured denitrifying strains, and in environmen- product of this enzyme reaction is still N2O [24]. tal samples demonstrated its promise as a functional Regardless of what the specific functions of nirK and gene marker for denitrification. In the current study, norB are in ammonia-oxidizing bacteria the presence of the presence and diversity of norB gene homologues these genetic sequences in ammonia-oxidizing bacteria among several cultured ammonia-oxidizing bacteria and the potential for different uses and controls relative was investigated using the polymerase chain reaction to denitrifying bacteria complicates the interpretation of (PCR) and DNA sequencing. The goal was to examine functional gene diversity in mixed environmental sam- the distribution of norB-like sequences among ammonia ples. Genes encoding nitrite reductase (nirK and nirS) oxidizers and their relationship to denitrifier norB se- have been widely used to study denitrifier diversity due quences. This work is a necessary precursor to studying to the central role of nitrite reductase in the denitrifica- the expression of norB and the role of nitric oxide reduction pathway [13,25–29]. The gene nirS has not been tase in the metabolism of nitrifiers other than N. euro- identified in nitrifying bacteria, but nirK is found in both paea. The results presented here indicate that several nitrifying and denitrifying bacteria [20,21]. The high ammonia-oxidizing bacteria possess a norB homolog diversity of nirK and its ambiguous distinction between similar enough to published denitrifier norB genes that nitrifiers and denitrifiers, however, makes it difficult to they have been detected using degenerate PCR primers distinguish the relative abundance or diversity of these based primarily on denitrifier norB sequences. functional groups on the basis of this gene. In a recent study by Avrahami et al. [13], many of the nirK sequences cloned from a grassland soil in Germany were most closely related to the nirK sequence of the culti- 2. Materials and methods vated strain TA-921i-NH4, an estuarine ammonia oxidizer. The functional designation of these sequences is 2.1. Bacterial culture conditions and DNA extraction therefore somewhat ambiguous and highlights the potential challenges in interpreting the functional signifi- Nitrosomonas europaea and Nitrosomonas eutropha cance of nirK sequences. were grown in Walker medium with deionized water Nitric oxide reductase (NOR) presents an alternative [38]. Nitrosomonas sp. C-113a, Nitrosomonas sp. C-45, target for detection of denitrifiers in environmental stud- Nitrosomonas sp. URW, Nitrosomonas sp. NO3W, and ies that may be simpler to interpret from the standpoint Nitrosococcus oceani were grown in medium with full of functional diversity. The enzyme nitric oxide reduc- strength seawater using the recipe given by Ward [39] tase (NOR), which produces N2O from NO, is a mem- (W medium). Nitrosomonas marina was grown in W ber of the heme-copper oxidase family, which includes medium made up in 50% seawater. Cultures were grown K.L. Casciotti, B.B. Ward / FEMS Microbiology Ecology 52 (2005) 197–205 199 in static 1-L batches at 18 °C in the dark and maintained stituents were the same as for norB2–norB6 reactions. in semi-continuous batch culture with periodic replace- The PCR amplifications were carried out using the fol- ment of 50% of each culture with fresh medium [39]. lowing thermal cycle: 95 °C for 5 min, then 80 °C for Cells (250 ml cultures) were harvested by filtration, 1 min, followed by 25 cycles of 50 °C for 1.5 min, then washed and resuspended in TE buffer (10 mM Tris 72 °C for 2 min, 94 °C for 1 min, then a final annealing HCl, pH 8.5; 1 mM EDTA). Standard procedures for at 50 °C for 1.5 min and extension at 72 °C for 5 min. DNA extraction were followed [40], and DNA was Amplification of norB gene fragments from Nitroso- stored in TE buffer. Nitrosomonas sp. TA-921i-NH4 monas cryotolerans, was achieved using a nested PCR ap- had been lost from the culture collection and archived proach with norB1–norB8 followed by norB2–norB7. genomic DNA was utilized for this study. Purified geno- The PCR protocol for the norB1–norB8 amplification mic DNA from Nitrosomonas cryotolerans was gener- consisted of: 94 °C for 4 min, then 25 cycles of 94 °C ously provided by M.A. Voytek. for 1 min, 55 °C for 1 min, and 72 °C for 1 min. The nested reaction with norB2–norB7 was carried out with 2.2. Primer design and amplification conditions 1 ul of the initial stage in a 50 ll reaction with the follow- ing protocol: 94 °C for 4 min, then 35 cycles of 94 °C for The PCR primers used in this study are given in 1 min, 52 °C for 1 min, and 72 °C for 1 min. A final Table 1. Primers for the norB gene (norB1, norB2, extension at 72 °C was carried out for 7 min. norB6, norB7, and norB8) are degenerate and were designed to amplify a diverse range of norB sequences. 2.3. Cloning and sequencing These primers were based on conserved regions of the norB gene from Pseudomonas fluorescens (AF197467), Amplification products of the expected sizes were ex- Pseudomonas stutzeri (Z28384), Pseudomonas aeruginosa cised from a 1% agarose gel, extracted using the Qia- (AE004489), Paracoccus denitrificans (AB014090), Halo- Quick Gel Extraction Kit (Qiagen), and cloned using monas halodenitrificans (AB010889), Bradyrhizobium the TOPO-TA Cloning Kit (Invitrogen). Transformants japonicum (AJ132911), Rhodobacter sphaeroides were selected and screened for plasmid inserts by PCR (AF000233), Achromobacter cycloclastes (AJ298324), as previously described [21]. Inserts from 7–10 clones and N. europaea (www.jgi.doe.gov). The design of from each cloning experiment were sequenced on both primer norB3 was based only on norB-like sequences strands using the BigDye Kit (Applied Biosystems) with obtained from ammonia-oxidizing bacteria using the vector primers T7 and M13 (reverse). Cycle-sequencing norB2–norB6 primer pair and has several mismatches reactions were precipitated according to the manufac- with known denitrifier norB sequences in the target re- turerÕs instructions and sequenced on an ABI310 genetic gion and is therefore biased towards ammonia oxidizing analyzer. bacteria. Primers norB3 and norB1 were used in a second phase of PCR amplification to extend the length 2.4. Sequence analysis and phylogenetic comparison of nitrifier norB sequences for phylogenetic analysis. The expected norB2–norB6 fragment is 395 bp and the Sequences of norB1–norB3 and norB2–norB6 or expected norB1–norB3 fragment is 236 bp, and the com- norB2–norB7 products from each organism were assem- bined norB1–norB6 sequence is 587 bp. bled into a continuous section using AutoAssembler ver- PCR amplification reactions using the norB2–norB6 sion 1.4.0 (Perkin–Elmer) (10–20 sequences per primer pair contained 50 mM KCl, 10 mM Tris base assembly). Amino acid sequences for NorB were de- (pH 8.0), 2.5 mM MgCl2, 100 lM each dNTP, 100 pmol duced from consensus norB sequences for each organ- each primer, 0.5 ll DNA (10–50 ng), and 1 ll Taq poly- ism, and were aligned using ClustalW [41].A merase in total reaction volume of 50 ll. PCR reactions neighbor-joining tree was constructed for NorB amino for the norB1–norB3 primer pair contained 50 pmol of acid sequences using distance matrix analysis in PAUP* each primer per 50 ll reaction, and the remaining con- 4.0, and 100 bootstrap replicates were performed.

Table 1 PCR primers for norB Primer Positiona Direction Sequence Degeneracy norB1 400–419 Forward 50-CGNGARTTYCTSGARCARCC-30 192-fold norB2 592–610 Forward 50-GACAARHWVTAYTGGTGGT-30 72-fold norB3 616–635 Reverse 50-CCYTCVACCCAGASATGCAC-30 12-fold norB6 967–986 Reverse 50-TGCAKSARRCCCCABACBCC-30 144-fold norB7 1000–1019 Reverse 50-CCRTGGSTRWARWARTTSAC-30 256-fold norB8 1051–1069 Reverse 50-CRTADGCVCCRWAGAAVGC-30 216-fold a Numbering based on complete norB gene sequence from Pseudomonas stutzeri (Z28384). 200 K.L. Casciotti, B.B. Ward / FEMS Microbiology Ecology 52 (2005) 197–205

Parsimony and maximum likelihood analyses were per- 3.2. PCR amplification of norB gene homologues formed on the same alignment using PROTPARS and PROTML from PHYLIP version 3.6b. Sections of the norB gene were successfully amplified from nine ammonia-oxidizing bacteria: N. europaea, N. eutropha, N. marina, Nitrosomonas sp. strains C-113a, 3. Results and discussion URW, NO3W, TA-921i-NH4, N. cryotolerans,and Nitrosococcus oceani. These strains include a variety of 3.1. PCR primer development soil, freshwater, and marine ammonia-oxidizers from both b- and c-subdivisions of the Proteobacteria. Sev- The goal of this study was to compare the diversity eral norB primer combinations were also tested with and relationship of norB gene homologues in ammo- genomic DNA from Nitrosospira briensis, Nitrosospira nia-oxidizing bacteria to those found in denitrifying tenuis,andNitrosospira multiformis. Thus far, while bacteria. In order to accomplish this, norB primers products of the approximately correct size have been ob- were developed prior to the recent publication of a sim- tained from Nitrosospira species, sequence analysis has ilar set of norB primers [37]. The primers described shown them to be non-specific products. here were based on a similar group of full-length norB sequences that are available in GenBank, and they tar- 3.3. NorB phylogenetic analysis get nearly identical conserved regions compared with the cnorB primers described by Braker and Tiedje With the caveat that among nitrifiers, norB has thus [37]. The primers cnorB1F [37] and norB1 (this study) far only been fully sequenced and shown to be func- target nearly identical regions, with one base change in tional in N. europaea [20,23], we have translated the their overlapping region. Primer norB1 extends three PCR products sequenced from nitrifier species into ami- bases longer in the 5Õ direction compared with no acid sequences for phylogenetic analysis. Alignment cnorB1F. Primers cnorB2F and norB2 target identical of the translated nitrifier norB homologues (169 amino regions, with relatively minor differences in degrees of acids) with published NorB sequences and the homolo- degeneracy. The Braker and Tiedje [37] primers gener- gous NorZ protein is relatively unambiguous, with only ally, but not always, allow for greater degeneracy in a few gaps required for the optimal alignment. There are target sequence. Primers cnorB6R and norB6 target 12 predicted membrane-spanning sections in the NorB overlapping regions with some differences in degener- protein [31], and the region amplified by norB2 and acy choices. Primers cnorB7 and norB7 are separated norB6 primers includes the sixth, seventh, and eighth by about 10 bp but do not overlap. NorB8 is farther membrane-spanning sections. The positions of three downstream than any of the primers described previ- conserved histidine residues (H-207, H-258, and H-259 ously. Finally, our internal norB3 primer is specifically in P. stutzeri) that are involved in binding non-heme biased towards nitrifier sequences, having been de- iron at the active site [30], as well as a conserved gluta- signed as an internal primer from alignments of only mate residue (E-211) that is required for enzyme activity the nitrifier norB homologues that were the focus of [42] are conserved in the nitrifier NorB homologues. this study. Overall, the similarity in these independently To include newer database sequences in phylogenetic designed primer sets for norB is noteworthy, and some- analysis, an alignment of 97 AA was used to generate a thing may be learned from the similarities and differ- neighbor-joining tree based on a pairwise distance ma- ences of the two primer sets. For example, we also trix analysis of NorB-like sequences (Fig. 1). The topol- had the greatest success with norB2–norB6 amplifica- ogy of a neighbor-joining tree based on norB/Z nucleic tion, strengthening the recommendation of Braker acid sequences gives the same groupings as in the amino and Tiedje [37] for the use of these target regions for acid-based tree and does not offer any additional phylo- norB amplification. One significant difference in the pri- genetic resolution (not shown). Only NorB-type protein mer design, that may be important for the work de- sequences are included in this comparison, except for the scribed here, is that the norB sequence from N. NorZ-type protein from Synechocystis PCC 6803 [24] europaea, the only nitrifier norB sequence available used as the outgroup. prior to this study, was included in the alignment used The NorB-like sequences have been assigned to six to design primers in this study but apparently not in groups, identified in Fig. 1 and listed in Table 2, to facil- the previous design [37]. This may account for some itate the discussion. Each numbered group is supported of the different choices made in designing the two sets by robust bootstrap values (filled circles in Fig. 1) and of primers. The use of norB primers specifically tar- represents sequences that are >75% identical to each geted for nitrifier norB sequences may provide a meth- other. All of the major groupings discussed below are od of differential amplification and detection of nitrifier also supported by parsimony analysis. Supplemental and denitrifier norB sequences in complex environmen- physiological and phylogenetic information about the tal samples. organisms included in Fig. 1 is given in Table 2. K.L. Casciotti, B.B. Ward / FEMS Microbiology Ecology 52 (2005) 197–205 201

Ochrobactrum anthropi Corynebacterium nephridii Alcaligenes faecalis S-6 Agrobacterium tumefaciens Brucella melitensis Pseudomonas sp. G-179 Achromobacter cycloclastes Sinorhizobium meliloti Bradyrhizobium japonicum Blastobacter denitrificans Rhodopseudomonas palustris cRCR7 Azospirillum brasilense cWM4 cWM31 Rhodobacter sphaeroides Roseobacter denitrificans Paracoccus denitrificans cRCR24 cWM61 cRCR27 cWM29 cWM16 Halomonas halodenitrificans

Nitrosomonas marina* Nitrosomonas sp. C-113a* Nitrosomonas sp. URW* Nitrosomonas sp. NO3W* Nitrosomonas sp. TA-921i-NH4* Nitrosomonas cryotolerans* Nitrosococcus oceani* cRCR1 Nitrosomonas eutropha* Nitrosomonas europaea* Nitrosomonas europaea

Azoarcus tolulyticus Td-3 Azoarcus tolulyticus Td-57 Azoarcus tolulyticus Tol-4 Thauera aromatica cRCR16 cRCR22 cRCR46 41532 Pseudomonas stutzeri Pseudomonas fluorescens Pseudomonas aeruginosa Magnetococcus sp. MC-1 cRCR8 cRCR39 Synechocystis PCC 6803 0.05 changes

Fig. 1. Neighbor-joining tree based on distance matrix analysis of partial NorB amino acid sequences (97 AA), showing the relationship between nitrifier and denitrifier sequences. Bootstrap values greater than 70% are indicated with filled circles, and bootstrap values between 50% and 70% are indicated with open circles. The numbered groupings are based on nodes that are well supported by bootstrapping with both distance and parsimony analysis. Groups 1, 2, 3, 4, and 5 contain NorB sequences from the cNOR family of nitric oxide reductases (16), the outgroup; Synechocystis PCC 6803, possesses the quinol-oxidizing (qNOR), type of nitric oxide reductase (16). The scale bar indicates 0.05 substitutions per site. Nitrifier sequences obtained from this study are indicated by an asterisk (*). Additional sequences were obtained from the GenBank database under accession numbers provided in Table 2.

The sequences from ammonia-oxidizing bacteria form ysis but is unresolved by parsimony. This group contains a coherent cluster (group 1) that is separate from denitri- NorB-like sequences from a diverse range of ammonia- ﬁer sequences (groups 2–6) (Fig. 1). The nitriﬁer cluster is oxidizing bacteria, including Nitrosomonas species from supported by a bootstrap value of 65% by distance anal- the b-subdivision, and N. oceani, a representative of the 202 K.L. Casciotti, B.B. Ward / FEMS Microbiology Ecology 52 (2005) 197–205

Table 2 Properties of organisms included in the NorB phylogenetic comparison Organism Accession No. Taxonomic grouping Nitrite reductasea Commentsa NorB group Ochrobactrum anthropi CAD45406 a-Proteobacteria NirK Denitrifier 2 Corynebacterium nephridii CAD45407 a-Proteobacteria NirK Denitrifier 2 Alcaligenes faecalis S-6 BAA90782 b-Proteobacteria NirK Denitrifier 2 Agrobacterium tumefaciens NP_534866 a-Proteobacteria NirK Denitrifier 2 Brucella melitensis NP_541976 a-Proteobacteria NirK Denitrifier 2 Pseudomonas sp. G-179 AAC79449 a-Proteobacteria NirK Denitrifier 2 Achromobacter cycloclastes CAC27381 b-Proteobacteria NirK Denitrifier 2 Sinorhizobium meliloti NP_435940 a-Proteobacteria NirK N.D. 2 Bradyrhizobium japonicum CAA10810 a-Proteobacteria NirK Denitrifier 2 Blastobacter denitrificans CAD45405 a-Proteobacteria NirK Denitrifier 2 Rhodopseudomonas palustris Unfinished a-Proteobacteria NirK Denitrifier 2 Azospirillum brasilense CAD45408 a-Proteobacteria NirS Denitrifier – Rhodobacter sphaeroides AAC45372 a-Proteobacteria NirK Denitrifier 3 Roseobacter denitrificans BAB84303 a-Proteobacteria NirS Denitrifier 3 Paracoccus denitrificans BAA32546 a-Proteobacteria NirS Denitrifier 3 Halomonas halodenitrificans JE0166 a-Proteobacteria N.D. Denitrifier 5 Nitrosomonas marina AAN34603 b-Proteobacteria NirK Ammonia-oxidizer 1 Nitrosomonas C-113a AAN34601 b-Proteobacteria NirK Ammonia-oxidizer 1 Nitrosomonas URW AAN34607 b-Proteobacteria NirK Ammonia-oxidizer 1 Nitrosomonas NO3W AAN34605 b-Proteobacteria NirK Ammonia-oxidizer 1 Nitrosomonas TA-921i-NH4 AAN34606 b-Proteobacteria NirK Ammonia-oxidizer 1 Nitrosomonas cryotolerans AAV69063 b-Proteobacteria N.D. Ammonia-oxidizer 1 Nitrosococcus oceani AAN34604 c-Proteobacteria N.D. Ammonia-oxidizer 1 Nitrosomonas eutropha AAN34602 b-Proteobacteria N.D. Ammonia-oxidizer 1 Nitrosomonas europaea AAN34608 b-Proteobacteria N.D. Ammonia-oxidizer 1 Nitrosomonas europaea AL954747 b-Proteobacteria NirK Ammonia-oxidizer 1 Azoarcus tolulyticus Td-3 CAD45413 b-Proteobacteria N.D. Denitrifier 4 Azoarcus tolulyticus Td-57 CAD45414 b-Proteobacteria N.D. Denitrifier 4 Azoarcus tolulyticus Tol-4 CAD45415 b-Proteobacteria NirS Denitrifier 4 Thauera aromatica K172 CAD45416 b-Proteobacteria NirS Denitrifier 4 Pseudomonas stutzeri CAA82229 c-Proteobacteria NirS Denitrifier 4 Pseudomonas fluorescens AAG34384 c-Proteobacteria NirS Denitrifier 4 Pseudomonas aeruginosa AAG03913 c-Proteobacteria NirS Denitrifier 4 Magnetococcus sp. MC-1 Unfinished a-Proteobacteria N.D. N.D. 4 Synechocystis PCC 6803 BAA18795 Cyanobacteria None Non-denitrifier – a N.D., Not determined.

c-subdivision nitrifiers. Among group 1 nitrifiers, se- Group 2 contains NorB sequences (89–98% identical) quence identity is high (77–100%) compared to identities from a variety of denitrifying bacteria in the a-andb- between group 1 and group 2 (64–74%), group 3 (61– subdivisions of the Proteobacteria, many of which are 73%), group 4 (59–66%), group 5 (20–30%), and group plant symbionts or pathogens that can also fix N2. All 6 (19–26%). Of interest is the placement of environmen- of the denitrifiers in group 2 possess the copper-type ni- tal clone cRCR1 (CAD45417) from the Red Cedar River trite reductase (NirK) (Table 2). studied by Braker and Tiedje [37]. This environmental Group 3 contains NorB sequences (78–79% identical) clone reproducibly clusters with the nitrifier norB homo- from atypical denitrifiers in the a-subdivision. Both logues and may have originated from a nitrifier host. Roseobacter denitrificans and Rhodobacter sphaeroides Without further information about the genetic back- are capable of phototrophic growth. The NorB from ground of this clone we can not have a definitive answer R. denitrificans is also unusual in that it possesses CuB to this question. The addition of nitrifier sequences to the in the active site (as cytochrome oxidase does), rather norB database, however, expands our knowledge about than FeB as is seen in other known NorB proteins [43]. the physiological diversity of bacteria containing nitric This is surprising considering the high sequence similar- oxide reductase and highlights the possibility that nitrifi- ity of NorB from R. denitrificans to other NorB se- ers may contribute to apparent denitrifier diversity based quences and suggests that relatively few alterations are on partial norB sequences. The next most similar NorB required to change the binding specificity of the active- sequence to group 1 belongs to Halomonas halodenitrifi- site metal. cans, which reproducibly falls outside of that group in its Group 4 consists mainly of denitrifiers with the heme- phylogenetic placement. type nitrite reductase (NirS). Magnetococcus MC-1 is K.L. Casciotti, B.B. Ward / FEMS Microbiology Ecology 52 (2005) 197–205 203 also included in this group, but it does not possess a detected in the environment, is yet to be determined but readily recognizable nirK or nirS gene (as revealed by these are important avenues for future research. The se- searches of its genome). There is no Nir information quences obtained in this study provide some insight into available for Halomonas halodenitrificans nor for the the potential metabolic diversity hidden within denitri- environmental clones that make up group 5. fier diversity based on partial norB gene sequences. These results also provide a good foundation for future 3.4. Comparison to amoA distribution investigation of the role of the denitrification pathway in the metabolism of various marine nitrifying bacteria and The amoA gene, encoding the a subunit of ammonia the role of ammonia-oxidizers in denitrification and monooxygenase has been used widely to study nitrifier N2O production. diversity and phylogenetic relationships. Based on amoA sequences, the nitrifier species from which we obtained 3.6. Sequence Accession Numbers norB sequences are distributed throughout the major groups of Nitrosomonas and Nitrosococcus species. Con- GenBank Accession Nos. AY139082-AY139089 were spicuously absent from the norB dataset, however, are assigned to the norB sequences from C-113a, N. eutro- sequences from the Nitrosospira clade, which are impor- pha, Nitrosomonas marina, N. oceani, NO3W, TA- tant players in many environments [44,45]. The reason 921i-NH4, URW, and N. europaea, respectively. The for our inability to amplify a norB product from Nitros- Nitrosomonas cryotolerans norB sequence has been as- ospira species, whether it is due to primer mismatch or signed the GenBank Accession No. AY654283. absence of the gene is unknown at this time. Nitrosospira species are known to produce N2O with yields similar to Nitrosomonas species [46,47], although production of Acknowledgements N2ObyNitrosospira briensis is less sensitive to C2H2 and O2 levels compared with N. europaea [48]. It is sur- We are grateful to M. Voytek and J. Kirshtein for prising, though, to have successfully amplified a norB- assistance in sequencing Nitrosospira PCR products. like sequence from N. oceani but not from the more Insightful comments from C. Francis, G. OÕMullan, closely related b-subdivision nitrifiers. Full genome M. Voytek, and two anonymous reviewers improved sequencing of Nitrosospira species will provide the most earlier versions of the manuscript. This work was sup- definitive answer to this question, but PCR-based efforts ported by NSF award (CHE-9810248) to B.B.W. and to obtain a norB product are ongoing. Princeton University Harold W. Dodds Fellowship to K.L.C. 3.5. Contrast to NirK phylogeny for nitrifiers and denitrifiers References The phylogenetic relationships based on NorB sequences shown in Fig. 1 provide an interesting compar- [1] Kester, R.A., de Boer, L. and Laanbroek, H.J. (1996) Short ison to phylogenies based on nitrite reductase (NirK) exposure to acetylene to distinguish between nitrifier and denitri- sequences. While NirK-like sequences from some fier nitrous oxide production in soil and sediment samples. FEMS Microbiol. Ecol. 20, 111–120. ammonia-oxidizing bacteria are not phylogenetically [2] Kim, K.-R. and Craig, H. (1993) Nitrogen-15 and oxygen-18 distinguishable from denitrifier sequences [21], the characteristics of nitrous oxide: a global perspective. 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