Nitrosococcus Watsonii Sp. Nov., a New Species of Marine Obligate

RESEARCH ARTICLE Nitrosococcus watsonii sp. nov., a new species of marine obligate ammonia-oxidizing bacteria that is not omnipresent in the world’s oceans: calls tovalidate the names ‘Nitrosococcus halophilus’and ‘Nitrosomonas mobilis’ Mark A. Campbell1, Patrick S.G. Chain2,3,4, Hongyue Dang5, Amal F. El Sheikh1, Jeanette M. Norton6, Naomi L. Ward7, Bess B. Ward8 & Martin G. Klotz1,5

1Evolutionary and Genomic Microbiology Laboratory, Department of Biology, University of Louisville, Louisville, KY, USA; 2Los Alamos National Laboratory, Genome Science Group, Bioscience Division, Los Alamos, NM, USA; 3Metagenomics Program, Joint Genome Institute, Walnut Creek, CA, USA; 4Center for Microbial Ecology, Michigan State University, East Lansing, MI, USA; 5Centre for Bioengineering and Biotechnology & State Key Laboratory of Heavy Oil Processing, China University of Petroleum (East China), Qingdao, China; 6Department of Plants, Soils and Climate, Utah State University, Logan, UT, USA; 7Department of Molecular Biology, University of Wyoming, Laramie, WY, USA; and 8Department of Geosciences, Princeton University, Princeton, NJ, USA

Correspondence: Martin G. Klotz, Abstract Evolutionary and Genomic Microbiology Laboratory, Department of Biology, University Local associations between anammox bacteria and obligate aerobic bacteria in the of Louisville, 139 Life Sciences Building, genus Nitrosococcus appear to be signiﬁcant for ammonia oxidation in oxygen Louisville, KY 40292, USA. Tel.: 11 502 852 minimum zones. The literature on the genus Nitrosococcus in the Chromatiaceae 7779; fax: 11 502 852 0725; e-mail: family of purple sulfur bacteria (Gammaproteobacteria, Chromatiales) contains [email protected] reports on four described species, Nitrosococcus nitrosus, Nitrosococcus oceani, ‘Nitrosococcus halophilus’ and ‘Nitrosomonas mobilis’, of which only N. nitrosus and Previous addresses: Patrick S.G. Chain, N. oceani are validly published names and only N. oceani is omnipresent in the Lawrence Livermore National Laboratory, world’s oceans. The species ‘N. halophilus’ with Nc4T as the type strain was Livermore, CA 94550, USA. Naomi L. Ward, The Institute for Genomic proposed in 1990, but the species is not validly published. Phylogenetic analyses of Research, Rockville, MD 20850, USA. signature genes, growth-physiological studies and an average nucleotide identity analysis between N. oceani ATCC19707T (C-107, Nc9), ‘N. halophilus’ strain Nc4T Received 12 August 2010; revised 27 November and Nitrosococcus sp. strain C-113 revealed that a proposal for a new species is 2010; accepted 29 November 2010. warranted. Therefore, the provisional taxonomic assignment Nitrosococcus watso- Final version published online 11 January 2011. nii is proposed for Nitrosococcus sp. strain C-113T. Sequence analysis of Nitroso- coccus haoAB signature genes detected in cultures enriched from Jiaozhou Bay DOI:10.1111/j.1574-6941.2010.01027.x sediments (China) identiﬁed only N. oceani-type sequences, suggesting that different patterns of distribution in the environment correlate with speciation in Editor: Michael Wagner the genus Nitrosococcus.

Keywords ammonia oxidation; Nitrosococcus; Nitrosococcus watsonii C-113; ‘Nitrosococcus halophilus’ Nc4; Nitrosomonas mobilis Nc2.

membrane-bound enzyme, ammonia monooxygenase Introduction (AMO, amoCAB), in the following reaction: NH312e 1 1 Members of the genus Nitrosococcus are marine aerobic O212H ! NH2OH1H2O (Hooper et al., 2005). The ammonia-oxidizing bacteria (AOB) that belong to the class subsequent oxidation of hydroxylamine to nitrite is facili-

MICROBIOLOGY ECOLOGY MICROBIOLOGY Gammaproteobacteria, order Chromatiales (the purple sulfur tated by the soluble periplasmic enzyme, hydroxylamine bacteria). Aerobic chemolithotrophic oxidation of ammonia oxidoreductase (HAO, haoA): NH2OH1H2O ! NO21 by bacteria proceeds in two consecutive steps. First, ammo- 5H114e (Hooper et al., 2005). The oxidation of hydro- nia is converted to hydroxylamine via a multisubunit xylamine to nitrite yields four electrons, two of which are

FEMS Microbiol Ecol 76 (2011) 39–48 c 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 40 M.A. Campbell et al. returned to the upstream monooxygenase reaction, and the 1990; Koops & Pommerening-Roser,¨ 2001; Koper et al., remaining two electrons are the sole source for generating 2004)]. However, distinction of an additional strain (Nitro- useable energy and reductant. HAO is the key enzyme in sococcus sp. C-113, not yet formally described) from bacterial ammonia catabolism due to its dual role as an N. oceani has been marginal based on comparison of their extractor of reductant and as a detoxicant of the highly 16S rRNA gene sequences (Ward & O’Mullan, 2002) and mutagenic intermediate hydroxylamine. For these reasons, this might have prevented its recognition in reported use of its encoding gene, haoA, as a functional marker gene environmental samples. The complete genome sequence of has been proposed (Klotz & Stein, 2008) and successfully N. oceani ATCC19707 has been published recently (Klotz tested in molecular ecological studies (Schmid et al., 2008). et al., 2006). Here we describe generation of additional In contrast to the omnipresent and diverse betaproteobac- genome sequences for Nitrosococcus sp. strain C-113 and terial AOB within the two genera Nitrosospira and Nitroso- ‘N. halophilus’ strain Nc4, and use of comparative genome monas, Nitrosococcus species are restricted to marine analysis as a basis for proposed description of a new species environments and salt lakes. Extensive sampling indicated of Nitrosococcus (Nitrosococcus watsonii) with its type strain that the species Nitrosococcus oceani is distributed in oceans C-113T. We also propose solutions to the current taxonomic worldwide (Ward & O’Mullan, 2002). Further, Nitrosococcus disarray within the genus Nitrosococcus. Ecological support has recently been identified to cooperate as the dominant for the distinctiveness of N. oceani and ‘N. watsonii’ C-113 nitrifier with anammox bacteria in hypoxic layers of several was obtained by investigating the molecular diversity of the oxygen minimum zones (OMZ), in which anammox dom- gammaproteobacterial haoAB gene in an environment for inates the process of N removal from the system (Lam et al., which a highly diverse presence of betaproteobacterial AOB 2007). has been recently established (Dang et al., 2010). Our The genus Nitrosococcus was originally established with enrichment cultures from Jiaozhou Bay (China) contained Nitrosococcus nitrosus (Buchanan, 1925) as its type species. exclusively haoAB genes with the highest sequence similarity The type strain of this species has been lost from culture to published sequences from N. oceani, but not ‘N. watsonii’ (agreement among attending scientists at the First Interna- or ‘N. halophilus.’ tional Conference on Nitrification, July 4–9, 2009, Louis- ville, KY), rendering N. nitrosus a type species with no validly described type strain. The only remaining validly Materials and methods described species with an assigned type strain in active culture is N. oceani (Watson, 1971; Skerman et al., 1980). Bacterial strains, culture maintenance and DNA While Koops et al. (1990) isolated and first described isolation another species of Nitrosococcus,‘Nitrosococcus halophilus’, Nitrosococcus sp. strain C-113 was first described in 1965 by this species name has not yet been validated despite being Stanley Watson (Fig. 1; Watson, 1965) and has been main- listed in Bergey’s Manual of Systematic Bacteriology with tained since then as a nonaxenic enrichment culture with a strain Nc4 as the proposed type strain (Garrity & Holt, predominantly coccus cell type. To purge the enrichment 2001). The taxonomic confusion within the genus Nitroso- culture of heterotrophic bacterial contaminants visible by coccus is further compounded by the description of ‘Nitro- sococcus mobilis’ (Koops et al., 1976); this species name has never been validated. Twenty years later, analysis of 16S rRNA gene sequences from ‘N. mobilis’ strains Nc2 and Nm93 indicated that ‘N. mobilis’ and recognized Nitrosomo- nas species constitute a monophyletic assemblage within the Betaproteobacteria (Pommerening-Roser¨ et al., 1996), fol- lowed by Purkhold et al. (2000), Aakra et al. (2001) and others. The affilation of N. mobilis with the Betaproteobac- teria was initially demonstrated by Woese et al. (1984) and later confirmed by Head et al. (1993) and Teske et al. (1994). Designation of Nc2 as the type strain of ‘N. mobilis’ has been suggested (Koops & Pommerening-Roser,¨ 2005; Garrity et al., 2007); however, the binomial has never been validated. Nitrosococcus oceani strains and ‘N. halophilus’ strain Nc4 can be distinguished on the basis of both phylogenetic Fig. 1. Electronmicrographs added in 1969 to the note of collection on analysis (16S rRNA gene sequences) and physiological November 13, 1967, on pages 112 and 113 in the culture book for AOB criteria [i.e. N. halophilus is not ureolytic (Koops et al., of Dr Stanley W. Watson.

[12.5 mM (NH4)2SO4] was amended with hydroxylamine characterization. and adjusted weekly to a final concentration of 0.2 mM. Medium acidification and nitrite production were moni- Environmental sample collection, enrichments tored as described before for the growth of N. oceani and DNA isolation (Alzerreca et al., 1999; Klotz et al., 2006). After 4 weeks of Sediment samples were collected in October 2008 at speci- continuous exposure to hydroxylamine at 0.2 mM, inspec- fied locations (i.e. A3, B2, J1 or P1) in Jiaozhou Bay near tion of the culture under the light microscope revealed only Qingdao (China) and prepared as described recently (Dang one cell type (Fig. 1). The presence of an axenic culture was et al., 2010). Enrichment cultures were started by mixing also confirmed by the fact that only 16S rRNA gene 0.5 g of frozen sediment into 200 mL of hydroxylamine- sequences identical to the previously determined Nitroso- amended artificial seawater and maintained as standing coccus sp. strain C-113 (GenBank accession AF153343) were batch cultures. Enrichments were propagated by transfer- retrieved. ring 5 mL of culture into 200 mL of fresh culture media after In experiments following Koops et al. (1990) to deter- the phenyl-red pH indicator showed a change in color at mine the tolerance of Nitrosococcus sp. strain C-113 to its least five times. own substrate, 100 mL (in a 250-mL Erlenmeyer flask) of For DNA isolation, 200 mL of J1, B2, P3, D5, A3, A5 and marine AOB medium with 12.5 mM (NH ) SO were in- 4 2 4 P1 enrichment cultures were filtered (0.20 mm pore size, oculated with Nitrosococcus sp. strain C-113 and incubated Fisher Scientific), the filters were cut in half and placed at 30 1C in the dark; the pH was adjusted daily with K CO . 2 3 separately into two 1.5-mL tubes. One tube was stored at After 2 weeks, the culture was partitioned in 10-mL aliquots 80 1C and the other was used for subsequent DNA to new 100-mL Erlenmeyer flasks following adjustment of isolation and analysis. DNA was extracted using the Ultra- the (NH ) SO concentration in duplicate flasks to 12.5, 4 2 4 CleanTM Microbial DNA Isolation Kit and a Fastpreps 100, 200, 400, 600 and 800 mM with a pH of 8. Like centrifuge as reported earlier (Poret-Peterson et al., 2008). ‘N. halophilus’ (Koops et al., 1990), Nitrosococcus sp. strain The presence of sufficient DNA was verified for each C-113 does not grow at ammonium sulfate concentrations sediment sample using the PCR and universal primers of 1 M or higher. Nitrite was measured immediately after targeting the 16S rRNA gene. inoculation and once daily as reported by Koops et al. (1990) for N. oceani and ‘N. halophilus’. Ammonium shift PCR-cloning, sequencing and analysis of experiments were performed to identify the optimum con- gammaproteobacterial haoAB gene fragments centration: 12.5 and 400 mM ammonium-grown cells were inoculated into fresh medium containing 100 mM ammo- PCR was performed using the degenerate primer pair nium sulfate and nitrite concentration was measured daily. HaoAF (50-YTGYCAYAAYGGRGYNGAYCAYAAYGAGT) To prepare cells for genome sequencing, Nitrosococcus sp. and HaoBRev1 (50-ANNYGMYGRGMASYRTCCCACCA), strain C-113 was grown as 200–400 mL batch cultures, in designed for the detection of gammaproteobacterial haoAB 2-L Erlenmeyer flasks, for 3 weeks, at 30 1C, in the dark. genes, and the isolated enrichment community DNA as a Cultures were grown without shaking, in artificial seawater template. The primers have been successfully tested using as described previously for N. oceani ATCC19707 (Alzerreca genomic DNA isolated from pure cultures of Gammapro- et al., 1999), with the modification of an increased teobacteria including N. oceani strain AFC27, ‘N. halophilus’, sodium concentration (600 mM) and the presence of hydro- Nitrosococcus sp. strain C-113, Methylococcus capsulatus or xylamine (0.2 mM). The cultures were routinely checked for Methylomicrobium album BG8 (data not shown). Genomic contamination. DNA from N. oceani ATCC19707 was used as a positive For genomic DNA preparations, cells were harvested by control. Amplicons, obtained only from the J1 enrichment centrifugation at late stationary growth phase as determined culture and N. oceani ATCC19707, were cloned into by spectrophotometric estimation of cell density and pCR2.1-TOPO plasmids (Invitrogen, Carlsbad, CA). After observation of decreased acid production. Genomic DNA transformation of the recombinant plasmids into chemically was isolated using the Wizard genomic DNA purification competent Escherichia coli TOP10TM cells, insert-positive kit (Promega, Madison, WI) according to the manufac- transformants were selected on Luria–Bertani medium agar turer’s recommendations, and stored at 20 1C until supplemented with 50 mgmL1 each of ampicillin and further use. Genomic DNA was also obtained from cultures kanamycin and 40 mg mL1 X-gal and transferred to a of ‘N. halophilus’ Nc4 using protocols described above for master plate. Ten colonies were randomly selected from the Nitrosococcus sp. strain C-113. The experimental details master plate for plasmid isolation (Wizard Plasmid Prep, of sequencing the entire genomes of Nitrososoccus sp. Promega) and insert sequencing (University of Louisville strain C-113 and ‘N. halophilus’ Nc4 will be described DNA core facility). Insert nucleotide sequences were

analyzed using SEQUENCHER (v4.6, Genecodes, Madison, WI) Chain (10 000 000 generations) in three independent runs. and seven (out of 10) nonidentical insert sequences and The searches were conducted assuming an equal or a gamma available Nitrosococcus haoAB gene sequences (Klotz et al., distribution of rates across sites, sampling every 1000th 2008; Schmid et al., 2008) were used to produce multiple generation and using strict molecular clock and HKY sequence alignments with CLUSTALX v.1.83 (Thompson et al., substitution models (Hasegawa et al., 1985) for analyses of 1997) (IUB DNA weight matrix with gap opening and gap the 16S rRNA, gyrB and rpoD genes and the WAG empirical extension penalties of 35/15 and 0.75/0.35, respectively, for amino acid substitution model (Whelan & Goldman, 2001) the pairwise/multiple sequence alignments). The DNA for analyses of the AmoA and HaoA proteins. Unrooted alignments were subjected to Bayesian inference of phylo- 50% majority rule consensus phylograms were constructed geny using the BEAST package [BEAUTI v1.5.3, BEAST v1.5.3, for 16S rRNA (1428 nucleotides, Fig. 2a) and concatenated TREEANNOTATOR v1.5.3, FIGTREE v.1.3 (Drummond & Rambaut, gyrB and rpoD genes (4363 nucleotides, Fig. 2b), for which 2007)]. Tree likelihoods (ignoring ambiguities) were deter- only posterior probability values o 1.0 are shown. Sources mined for unique sites within the alignment by creating a for the sequences used in the alignments are indicated in the Monte–Carlo Markov Chain (10 000 000 generations) in respective phylogenetic trees. The mean branch lengths are three independent runs. The searches were conducted characterized by a scale bar indicating the evolutionary assuming an equal distribution of rates across sites, sam- distance (percent changes). pling every 1000th generation and using strict molecular clock and HKY substitution models (Hasegawa et al., 1985). Average nucleotide identity (ANI) between An unrooted 50% majority rule consensus phylogram was purple sulfur bacterial genomes constructed. In order to unequivocally determine the species status of strain C-113, the ANI between the whole genome sequence of Analysis of gammaproteobacterial rrsA, gyrB Nitrosococcus sp. strain C-113 (one chromosome and two and rpoD gene sequences plasmids) and genome sequences of N. oceani ATCC19707 (CP000127; plasmid: CP000126) as well as those of other For the AOB-specific proteins AmoA and HaoA, and for purple sulfur bacteria, including N. oceani strain AFC-27 highly conserved rrsA, gyrB and rpoD genes, sequence (ABSG00000000) and ‘N. halophilus’strainNc4(1chromo- similarities were investigated initially using the NCBI BLAST some and 1 plasmid; under embargo), Allochromatium program (Altschul et al., 1990). Protein sequences deduced vinosum strain DSM 180 (CP001896; plasmids: CP001897, from experimentally determined nucleotide sequences were CP001898), Halorhodospira halophila strain SL1 (CP000544), analyzed with the PSORT (Nakai & Kanehisa, 1991), and Alkalilimnicola ehrlichei strain MLHE-1 (CP000453), Thioalk- TMHMM (http://www.cbs.dtu.dk/services/TMHMM/) pro- alivibrio sp. strains HL-EbGR7 (CP001339) and K90mix grams to identify hydrophobic domains that could serve as (CP001905; plasmid: CP00196) and Halothiobacillus neopolita- signal peptides for export into the periplasm or constitute nus strain c2 (CP001801) was calculated. The ANI calculations membrane-spanning domains. The various deduced full- were performed using the in silico DNA–DNA hybridization length protein sequences from Nitrososoccus sp. strain C-113 method (Konstantinidis & Tiedje, 2005; Goris et al., 2007) and ‘N. halophilus’ Nc4, respective BLAST hits retrieved from implemented in the JSPECIES software (http://www.imedea.uib-c the GenBank/EMBL database and homologs found in sic.es/jspecies/about.html; Richter & Rossello-Mora, 2009). unpublished genome sequences were used to produce multi- BLAST optionsusedwereasfollows;drop-offvalueforgapped ple sequence alignments using CLUSTALX v.1.83 (Thompson alignment, x = 150; penalty for a nucleotide mismatch, q = 1, et al., 1997) (Gonnet 250 protein weight matrix with gap filter query sequence, F; expectation value, e =1e 15; and opening and gap extension penalties of 35/15 and 0.75/0.35, number of processors to use, a = 2. ANI calculation settings respectively, for the pairwise/multiple sequence alignments). were 30% for identity (%), 70% for alignment and 1020 bp for A distance neighbor-joining guide tree was constructed Ã length. using the BIONJ function in PAUP v. 4.10b (Swofford, 1999) and observed differences between the resulting tree topology Strain availability and known phylogenetic relationships were used for manual refinement of the CLUSTALX alignments. The GenBank/EMBL/DDBJ accession numbers for the 16S The refined protein and gene alignments were subjected rRNA gene sequences are as follows: AF152243 for strain C- to Bayesian inference of phylogeny using the BEAST package 113; AF287298, AJ298748, Nhal_R0018 and Nhal_R0025 for T [BEAUTI v1.5.3, BEAST v1.5.3, TREEANNOTATOR v1.5.3, FIGTREE strain Nc4 ; and M96403, AF287297 and AJ298728 for v.1.3 (Drummond & Rambaut, 2007)]. Tree likelihoods strain Nc2 T. Prior attempts to maintain these strains in pure (ignoring ambiguities) were determined for unique sites culture in major type culture collections have failed. The within the alignment by creating a Monte–Carlo Markov strains above are presently in pure culture in the following

Fig. 2. Unrooted 50% majority rule consensus phylograms constructed after Bayesian inference of phylogeny for 16S rRNA (1428 nucleotides, Fig. 2a) and concatenated gyrB and rpoD genes (4363 nucleotides, Fig. 2b). Only posterior probability values smaller than 1.0 are shown at the respective nodes. Sources for the sequences used in the alignments are indicated in the respective phylogenetic trees. Mean branch lengths are characterized by a scale bar indicating the evolutionary distance (percent changes). research laboratories: N. watsonii strain C-113T, M.G. Klotz sococcus haoAB gene sequences in cultures enriched from (University of Louisville), J.M. Norton (Utah State Univer- Jiaozhou Bay sediments (China) identiﬁed only N. oceani- sity) and the WHOI culture collection; ‘N. halophilus’ strain type sequences (Fig. 3). This and the fact that ‘N. halophilus’ Nc4T, M.G. Klotz (University of Louisville), Youn Hwan (strains Nc7 and Nc4 were isolated from a salt lake in Saudi (California State University at Fresno) and A. Pommeren- Arabia and a salt lagoon off Sardinia in the Mediterranean ing-Roser¨ (Universtat¨ Hamburg); ‘N. mobilis’ strain Nc2T,A. Sea, respectively) and Nitrosococcus sp. strain C-113 (iso- Pommerening-Roser¨ (Universtat¨ Hamburg) and M. Wagner lated in the Mediterranean Sea near Port Said) were, so far, (Universitat¨ Wien). retrieved in isolated locations whereas strains of N. oceani are found worldwide (Ward & O’Mullan, 2002) suggests that different patterns of distribution in the environment corre- Results and discussion late with speciation in the genus Nitrosococcus. Phylogenetic analyses of the Nitrosococcus sp. strain C-113 Unequivocal evidence for taxonomic delineation at the AmoA and HaoA proteins generated results identical to species level was obtained by calculating the ANI of repre- phylogenetic analyses of AmoA (Tavormina et al., 2010) and sentative genome sequences. The ANI values of the Nitroso- HaoA (Klotz et al., 2008) proteins published recently. These coccus strain C-113 with N. oceani ATCC19707 were results and the phylograms constructed for the 16S rRNA ANIb = 89.29% and and ANIm = 89.87% (chromosome (Fig. 2a and Norton, 2010) and concatenated gyrB and rpoD only, Fig. 4) as well as ANIb = 89.34% and ANIm = 89.85% genes (Fig. 2b) suggest that Nitrosococcus sp. strain C-113, (chromosome and plasmids), which is well below the 94% ‘N. halophilus’ strain Nc4 and strains in the N. oceani species threshold for species delineation that corresponds to the are different phylotypes. Interestingly, our analysis of Nitro- recommended cut-off of 70% similarity in DNA–DNA

Fig. 4. Plot of the calculated ANI as a function of BLASTN-based similarities of the 16S rRNA gene sequences for pair-wise comparison between known, genome-sequenced purple sulfur bacteria and Nitrosococcus watsonii strain C-113. ANIb, ANI using BLAST alignments; ANIm, ANI using MUMMER alignments. Triangles, pairwise calculations including N. watsonii genome data; open circles, all other purple sulfur bacterial genome comparisons. 1, Noce ATCC19707/Noce AFC27; 2, Nwat C-113/Noce ATCC19707; Nwat C-113/Noce AFC27; 3, Nhal Nc4/Nwat C-113; Nhal Nc4/Noce ATCC19707; Nwat Nc4/Noce AFC27; 4, Hhal SL1/Noce ATCC19707; Hhal SL1/Noce AFC27; 5, circled data points: Noce or Nwat or Nhal/Alvin or Tgr7 or TK90.

ammonium salt, as indicated by a maximum of nitrite Fig. 3. Unrooted 50% majority rule consensus phylogram constructed production at this concentration (Fig. 5 and Koops et al., after Bayesian inference of phylogeny for a 1266-nucleotide-long alignment of fragments of haoAB gene sequences obtained from Jiaozhou 1990). This is also supported for Nitrosococcus sp. strain C- Bay sediment enrichment cultures, pure cultures of Nitrosococcus oceani 113 by ammonium shift experiments to the optimum ATCC19707 and AFC27, Nitrosococcus halophilus Nc4, Nitrosococcus concentration (inset to Fig. 5). watsonii C-113 and Methylomicrobium album BG8 (GQ471938) as the Based on the data presented here, we propose and call for outgroup. Posterior probability values are shown at the nodes. Mean the validation of the provisional taxonomic assignment ‘N. branch lengths are characterized by a scale bar indicating the evolu- watsonii’ with strain C-113 as the type strain. Furthermore, tionary distance (percent changes). we call for validation of strain Nc4 as the type strain for ‘N. halophilus’ (Koops et al., 1990), thereby justifying inclusion of both species among the validly described species of the hybridization experiments (Konstantinidis & Tiedje, 2005; genus Nitrosococcus. Goris et al., 2007). This result and preliminary identification The original isolate of N. nitrosus was not a marine strain of massive differences in the arrangement of genes and gene (Winogradsky, 1892; Koops et al., 1976), and no isolate or clusters in these two Nitrosococcus genomes suggest that DNA that is not a phylotype (ribotype) similar to N. oceani Nitrosococcus sp. strain C-113 is the representative of a or ‘N. halophilus’ (Fig. 2; Ward & O’Mullan, 2002) has since species distinct from N. oceani and also from ‘N. halophilus’ been retrieved from the open ocean. Koops et al. (1976) were (Fig. 3). This is also supported by significant differences in not able to unambiguously classify the isolated coccus- physiological properties between N. oceani and Nitrosococ- shaped strain Nc2 either as Nitrosomonas or Nitrosococcus cus sp. strain C-113 as summarized in Table 1. In experi- according to the existing key (Watson, 1974); however, to ments designed to determine ammonium salt tolerance avoid the creation of a new genus and because of its (Koops et al., 1990), Nitrosococcus sp. strain C-113, like the morphological similarities to the type species N. nitrosus, more distantly related ‘N. halophilus’, was significantly less they classified strain Nc2 in the genus Nitrosococcus (Koops tolerant to higher ammonium concentrations than N. oceani et al., 1976). (Fig. 5). In contrast to N. oceani,‘N. halophilus’ and Although coccoid forms are not currently found within Nitrosococcus sp. strain C-113 do not grow at ammonium Nitrosomonas, the order Nitrosomonadales [in which Nitro- salts concentrations of 1 M or higher, but the optimum somonas is located (Skerman et al., 1980)] also includes concentration for all three species is at around 100 mM Nitrosospira (Skerman et al., 1980; Head et al., 1993;

Table 1. Selected ecological, physiological, cellular and genetic properties of cultured Nitrosococcus species Species Nitrosococcus oceani ‘Nitrosococcus halophilus’‘Nitrosococcus watsonii’ (type strains) ATCC19707 (C-107, Nc10) Nc4 (not validated) C-113 (not validated) Preferred habitat Marine Marine and salt lakes Marine Points of isolation Omnipresent Salt lagoon or lake Mediterranean Sea, Port Said Salt requirements Obligately halophilic Obligately halophilic Obligately halophilic NaCl optimum Around 500 mM Around 700 mM Around 600 mM Temperature optimum 28–32 1C 28–32 1C 28–32 1C pH optimum 7.6–8 7.6–8 7.6–8 Ammonium tolerance o 1200 mM up to 600 mM o 1600 mM Ammonium optimum 100 mM 100 mM 100–200 mM Urease genes/ureolytic Positive/positive Negative/negative Positive/negative Cell shape Coccus or short rod Coccus or short rod Coccus or short rod Membrane structure PM and intracytoplasmic PM and intra-cytoplasmic PM and intra-cytoplasmic membrane stack membrane stack membrane stack Flagellation Polar or lophotrichous Lophotrichous Polar (tufts not observed) Genome G1C content 50.3% 51.6% 50.1% Nucleoid 3 481 691 bp 4 079 427 bp 3 328 570 bp Plasmids 1 (40 420 bp) 1 (65 833 bp) 2 (39 105 bp, 5611 bp)

Inclusion bodies Glycogen/starch; poly-Pi Glycogen/starch; poly-Pi Glycogen/starch; poly-Pi

Data presented in this table are a compilation of results from unpublished work in the Klotz laboratory and the following references: Fiencke & Bock (2006), Koops et al. (1990), Koops & Pommerening-Roser¨ (2001), Koper et al. (2004) and Ward & O’Mullan (2002).

Pommerening-Roser¨ et al., 1996; Purkhold et al., 2000; Aakra et al., 2001; Koops & Pommerening-Roser,¨ 2005; Garrity et al., 2007). Therefore, this publication shall also serve as a call for validation of the new species ‘N. mobilis’ with strain Nc2 as the type strain. Recent literature contains numerous reports on the varied contributions of Thaumarchaea and Proteobacteria to aerobic ammonia oxidation in both aquatic and terrestrial environments. However, only a few papers included experimental data on the expression of the amoA marker gene and/or nitrification rates in addition to the commonly measured abundance of amoA gene copies in the isolated community DNA. Of these more comprehensive papers, two reported on Fig. 5. Plot of relative nitrite production by Nitrosococcus watsonii strain a significant role of gammaproteobacterial AOB highly simi- C-113T, Nitrosococcus oceani strain Nc1 and ‘Nitrosococcus halophilus’ strain Nc4T when grown for 8 days at different concentrations of lar in sequence to N. oceani in the N cycle of OMZ. Based on ammonium. Superscript ‘a’ indicates that the values for N. oceani and molecular ecological (amoA) and isotope fractionation 14 15 ‘N. halophilus’ were obtained from Koops et al. (1990). The inset shows ( N/ N) studies, Lam and colleagues reported that archaea the results of ammonium shift experiments (12.5 and 400 mM ammo- highly similar in sequence to Nitrosopumilus maritimus nium sulfate-grown cells were inoculated into fresh medium containing (Konneke¨ et al., 2005; Walker et al., 2010) as well as N. oceani 100 mM ammonium sulfate and the nitrite concentration was measured were important nitrifiers in the OMZ of the Black Sea, each daily). providing about half of the nitrite required by anaerobically ammonia-oxidizing Planctomycetes, the anammox bacteria Purkhold et al., 2000; Aakra et al., 2001), members of which (Lam et al., 2007). While archaeal and bacterial nitrification are not rod-shaped. Like all described strains of Nitrosomo- and anammox were indirectly coupled in the upper layer, nas europaea and Nitrosomonas eutropha,‘N. mobilis’ strains anammox and nitrification by N. oceani appeared to be can also tolerate high concentrations of ammonia (Juretsch- directly coupled in the suboxic layer of the Black Sea OMZ, ko et al., 1998), providing another unifying phenotypic trait. where archaeal amoA transcripts were scarce (Lam et al., Most importantly, the strains of ‘N. mobilis’ are Betaproteo- 2007). In addition, the recent discovery of a significant bacteria that phylogenetically cluster monophyletically contribution to the N cycle by N. oceani in the lower layer of with N. europaea and N. eutropha (Head et al., 1993; the Peruvian OMZ (Lam et al., 2009) confirmed that extant

AOB in the genus Nitrosococcus are ecologically significant Description of Nitrosococcus watsonii sp. nov. (GenBank and that their ancestors might have played an important role Taxonomy ID: 105559) synonym: Nitrosococcus sp. C-113. in the oceans of the Earth’s proterozoic period (Staley, 2007). Etymology, locality as well as culture history: Nitrosus Given the pioneering role of the anammox process in the (Latin masculine adjective): nitrous; coccus: (Latin mascu- early nitrogen cycle (Klotz & Stein, 2008), detailed studies of line adjective): sphere; watsonii (Latin masculine genitive the inferred direct mutualistic interaction between Nitroso- name): from Watson. The name refers to the collector of the coccus and anammox bacteria in low oxygen environments organism (ammonia oxidizer, marine) motivated by the fact are needed. that, so far, no ammonia-oxidizing bacterium has been Nitrosococcus is a rare obligate aerobic representative of named in honor of Dr Stanley W. Watson. the predominantly anaerobic purple sulfur bacteria (Imhoff, Collected in 1967 at Port Said, Mediterranean Sea, by 2005) and its finding in the lower layers of OMZs is thus Stanley W. Watson. Maintained until 1994 as enrichment puzzling (Lam et al., 2007; Staley, 2007). The genome culture C-113 by Frederica Valois (Woods Hole Oceano- sequences of N. oceani ATCC19707 (Klotz et al., 2006) and graphic Institution). Identified as a gammaproteobacterial unpublished Nitrosococcus genomes contain a richer and AOB in an enrichment culture in 1995 by J.M. Norton (Utah more diverse gene inventory in the quinol-oxidizing branch State University) and later purified using a hydroxylamine of the electron transport chain than is found in the genomes treatment regime by A.F. El Sheikh, M.A. Campbell and of betaproteobacterial AOB (Arp et al., 2007; Stein et al., M.G. Klotz (University of Louisville). 2007; Norton et al., 2008) and amoA gene-encoding archaea Appearing as large cocci or very short rods. Cells contain (Hallam et al., 2006; Walker et al., 2010). Enriched cate- a well-developed intracellular membrane system of an gories include complexes that conserve energy coupled to arrangement that appears as one stack of membrane vesicles the reduction of oxygen and NO (Klotz & Stein, 2010). packed mainly in the center of the cell. Numerous inclusion Knowledge of the taxonomic diversity and spatial distribu- bodies identified by comparison with N. oceani as glycogen/ tion of gammaproteobacterial AOB in the world’s oceans, as starch granules (white color) and polyphosphate granules well as additional molecular and physiological studies, are (dark color). Polar flagella allow for motility, the genotype is thus ecologically relevant and it can be expected that this chemotaxis-positive. Light sensitive; strictly aerobic; mod- research will help us to understand better the functional erately alkaliphilic and mesophilic; grows in the presence of intricacies of the extant N cycle. sodium salts between 200 and 800 mM; optimum growth Emended description of the genus Nitrosococcus (Gen- temperature is 28–32 1C; optimum salt concentration is Bank Taxonomy ID: 1227) synonym: Nitrosococcus Wino- 500–700 mM; the G1C content of the DNA is 50.1%; the gradsky 1892; ex ‘Micrococcus nitrosus’ Migula (1900); ex genome of N. watsonii strain C-113T is presently being ‘Nitrosococcus nitrosus’ Buchanan (Buchanan, 1925; Editor- annotated and analyzed. ial_Board, 1955; Commission, 1958). Name approved by Emended description of the genus Nitrosomonas (Gen- Skerman et al. (1980), but the culture was eventually lost. A Bank Taxonomy ID: 1227) synonym: Nitrosomonas Wino- new isolate ‘Nitrosocystis oceanus strain C-107’ (sic) Watson gradsky 1892 ex Nitrosomonas (Editorial_Board, 1955) ex (1965), Nitrosococcus oceani strain ATCC19707 Watson Nitrosomonas (Commission, 1958), emended by Watson (1971), name approved by Skerman et al. (1980) is now in 1974. Approved Lists 1980 (Skerman et al., 1980). We culture, listed as the type strain and its genome has been propose below the validation of ‘N. mobilis’ as an additional sequenced (Klotz et al., 2006). We propose below the species of the genus Nitrosomonas and Nc2 as its type strain. validation of ‘N. halophilus’ as a species name and an Description of Nitrosomonas mobilis sp. comb. nov. additional species of the genus Nitrosococcus (N. watsonii). synonym: ‘N. mobilis’ (Koops et al., 1976); ex ‘N. mobilis’ The etymology of Nitrosococcus is catalogued using digital (Garrity et al., 2007). optical identifiers as follows: Genus (DOI: 10.1601/ Etymology, locality and culture history as well as diag- nm.2107), Family Chromatiaceae (DOI: 10.1601/tx.2070; nosis as in Koops et al. (1976); phylogenetic assignment to Bavendamm, 1924), Order Chromatiales (DOI: 10.1601/ Nitrosomonas, Nitrosomonadaceae, Nitrosomonadales, Beta- nm.2069; Imhoff, 2005; List_Editor, 2005), Class Gamma- proteobacteria (Head et al., 1993; Pommerening-Roser¨ et al., proteobacteria (DOI: 10.1601/tx.2068), Phylum Proteobac- 1996; Purkhold et al., 2000; Aakra et al., 2001; Koops & teria (DOI: 10.1601/tx.808; Garrity & Holt, 2001). Pommerening-Roser,¨ 2005; Garrity et al., 2007). Description of Nitrosococcus halophilus Nc4 (GenBank Taxonomy ID: 472759): synonym: N. halophilus strain Nc4. Acknowledgements Etymology, locality as well as culture history as in Koops et al. (1990); phylogenetic assignment to the genus Nitroso- We would like to thank Dr Jean P. Euze`by (E´ cole Nationale coccus (Skerman et al., 1980) by this publication with Nc4 as Vet´ erinaire,´ Toulouse, France), Dr George M. Garrity (Mi- the type strain. chigan State University) and anonymous reviewers of a

c 2011 Federation of European Microbiological Societies FEMS Microbiol Ecol 76 (2011) 39–48 Published by Blackwell Publishing Ltd. All rights reserved Nitrosococcus watsonii 47 previous version of this manuscript for invaluable taxo- Boone DR & Castenholz RW, eds), pp. 119–169. Springer, New nomic advice. Pertinent taxonomic information was ac- York. cessed through the ‘NAMES FOR LIFE’ online tool (http:// Garrity GM, Lilburn TG, Cole JR, Harrison SH, Euzeby J & namesforlife.com). Technical assistance by undergraduate Tindall BJ (2007) Part 4. Michigan State University Board of student David Griffith (UofL) is acknowledged. 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