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 À (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 À bacteria). Aerobic chemolithotrophic oxidation of ammonia oxidoreductase (HAO, haoA): NH2OH1H2O ! NO21 by bacteria proceeds in two consecutive steps. First, ammo- 5H114eÀ (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. c 2011 Federation of European Microbiological Societies FEMS Microbiol Ecol 76 (2011) 39–48 Published by Blackwell Publishing Ltd. All rights reserved Nitrosococcus watsonii 41 microscopy, the ammonia-containing growth medium elsewhere, together with the results of their analysis and [12.5 mM (NH4)2SO4] was amended with hydroxylamine characterization. and adjusted weekly to a final concentration of