APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 2006, p. 6299–6315 Vol. 72, No. 9 0099-2240/06/$08.00ϩ0 doi:10.1128/AEM.00463-06 Copyright © 2006, American Society for Microbiology. All Rights Reserved. Complete Genome Sequence of the Marine, Chemolithoautotrophic, Ammonia-Oxidizing Bacterium Nitrosococcus oceani ATCC 19707† Martin G. Klotz,1* Daniel J. Arp,2 Patrick S. G. Chain,3,4 Amal F. El-Sheikh,1 Loren J. Hauser,5 Norman G. Hommes,2 Frank W. Larimer,5 Stephanie A. Malfatti,3,4 Jeanette M. Norton,6 Amisha T. Poret-Peterson,1 Lisa M. Vergez,3,4 and Bess B. Ward7 University of Louisville, Louisville, Kentucky 402921; Oregon State University, Corvallis, Oregon 973312; Lawrence Livermore National Laboratory, Livermore, California 945503; Joint Genome Institute, Walnut Creek, California 945984; Oak Ridge National Laboratory, Oak Ridge, Tennessee 378315; Utah State University, Logan, Utah 843226; and Princeton University, Princeton, New Jersey 085447 Received 26 February 2006/Accepted 16 June 2006 The gammaproteobacterium Nitrosococcus oceani (ATCC 19707) is a gram-negative obligate chemolithoau- totroph capable of extracting energy and reducing power from the oxidation of ammonia to nitrite. Sequencing (and annotation of the genome revealed a single circular chromosome (3,481,691 bp; G؉C content of 50.4% and a plasmid (40,420 bp) that contain 3,052 and 41 candidate protein-encoding genes, respectively. The genes encoding proteins necessary for the function of known modes of lithotrophy and autotrophy were identified. Contrary to betaproteobacterial nitrifier genomes, the N. oceani genome contained two complete rrn operons. In contrast, only one copy of the genes needed to synthesize functional ammonia monooxygenase and hydroxyl- amine oxidoreductase, as well as the proteins that relay the extracted electrons to a terminal electron acceptor, were identified. The N. oceani genome contained genes for 13 complete two-component systems. The genome also contained all the genes needed to reconstruct complete central pathways, the tricarboxylic acid cycle, and the Embden-Meyerhof-Parnass and pentose phosphate pathways. The N. oceani genome contains the genes required to store and utilize energy from glycogen inclusion bodies and sucrose. Polyphosphate and pyrophos- phate appear to be integrated in this bacterium’s energy metabolism, stress tolerance, and ability to assimilate carbon via gluconeogenesis. One set of genes for type I ribulose-1,5-bisphosphate carboxylase/oxygenase was identified, while genes necessary for methanotrophy and for carboxysome formation were not identified. The N. oceani genome contains two copies each of the genes or operons necessary to assemble functional complexes ؉ ؉ I and IV as well as ATP synthase (one H -dependent F0F1 type, one Na -dependent V type). The ammonia-oxidizing bacterium Nitrosococcus oceani tracted from natural seawater (61, 74). In addition to the truly ATTC 19707 (Fig. 1) (Bacteria, Proteobacteria, Gammapro- marine environment, N. oceani was detected by immunofluo- teobacteria, Chromatiales, Chromatiaceae, Nitrosococcus, Ni- rescence and fluorescent in situ hybridization in the saline trosococcus oceani) was the first ammonia-oxidizing bacterium waters of Lake Bonney, a permanently ice-covered lake in isolated by enrichment culture from seawater (Nitrosocystis Antarctica (70). Nitrosococcus halophilus has been isolated oceanus) (53), and it resembles the original type strain, Nitroso- only from saline ponds (39) and has not been detected in other coccus winogradskyi 1892, which was lost. As a member of the environments by using molecular probes. order Chromatiales, the purple sulfur bacteria, N. oceani is a The general role of nitrifying bacteria in marine systems is to member of the evolutionarily oldest taxonomic group capable link the oxidizing and reducing processes of the nitrogen cycle of lithotrophic ammonia catabolism. To date, N. oceani and N. by converting ammonium to nitrate. This conversion is respon- halophilus are the only recognized species of gammaproteobac- sible for maintaining nitrate, the major component of the fixed terial ammonia-oxidizing bacteria (AOB). All other cultivated nitrogen pool in the oceans, which is present almost every- aerobic AOB are Betaproteobacteria, and their members have where below a few hundred meters at concentrations ap- been detected in soils, freshwater, and sediments as well as proaching 40 ␮M. The deep nitrate reservoir of the oceans, marine environments (41). In contrast, gammaproteobacterial believed to have come about by abiotic processes on the pri- AOB have only been found in marine or saline environments. mordial Earth (48), is a huge pool of nitrogen whose availabil- Nitrosococcus oceani has been detected in many marine envi- ity to primary producers in the surface layer is still controlled ronments using immunofluorescence (72, 78) and more re- largely by physical processes (27). The nitrification process cently on the basis of cloned gene sequences from DNA ex- produces oxidized forms of nitrogen that are lost via denitri- fication and anaerobic ammonia oxidation (anammox) (34, 65). By converting nitrogenous compounds released as waste * Corresponding author. Mailing address: Department of Biology, products of metabolism into NOx intermediates that can act University of Louisville, 139 Life Science Building, Louisville, KY both as oxidants and reductants for the fixed N removal pro- 40292. Phone: (502) 852-7779. Fax: (502) 852-0725. E-mail: aem @mgklotz.com. cesses (14, 73), nitrification closes the global nitrogen cycle. † Supplemental material for this article may be found at http://aem Nitrification occurs in both the water column and in sediments .asm.org/. of marine environments. In sediments, nitrification is often 6299 6300 KLOTZ ET AL. APPL.ENVIRON.MICROBIOL. FIG. 1. Transmission electron micrograph of Nitrosococcus oceani ATCC 19707. Cultures of N. oceani were grown until slowing readjustment of the pH with potassium carbonate indicated the beginning of transition of the culture into stationary growth phase. Cells were harvested by centrifugation and sent to the University of Wisconsin—Madison Electron Microscopy facility for further processing and electron microscopy. The scale bar at the lower right (500 nm) indicates the average cell size of 1.5 ␮m in diameter. tightly coupled with denitrification and can account for a sig- contained numerous genes that encode putative copper blue nificant fraction of the total oxygen consumption in sediments proteins (54), including one (CENSYa_1582) with significant (80). It has been discovered only recently that aerobic ammo- sequence similarity to the NcgA and NirK protein family, nia oxidation is also carried out by some Crenarchaeota (58 and which is implicated in nitrite reduction by AOB (6). Since references therein). Ko¨nneke et al. (38) reported the isolation AMO activity is also copper dependent, it appears that ammo- of a marine crenarchaeote, Candidatus Nitrosopumilus mari- nia oxidation in Archaea is entirely based on the function of tima, that was able to grow chemolithoautotrophically by aer- copper-containing proteins. Because copper is known to be obically consuming ammonia and producing nitrite. This phys- redox active only under oxic conditions and because the cyto- iological observation has been supported by the identification chrome-based core module of bacterial ammonia catabolism of DNA sequences similar to the genes encoding the three (encoded by the hao gene cluster) has evolved from bacterial subunits of ammonia monooxygenase (AMO) from AOB (38). inventory involved in (anaerobic) denitrification (7), it is highly On the other hand, the recently completed genome sequence likely that ammonia catabolism in the Archaea has evolved of the crenarchaeote Cenarchaeum symbiosum, a symbiont of fairly late by incorporating an AMO-like function into an am- marine sponges (28) predicted to be an ammonia-oxidizing monia-independent metabolism. Recent phylogenetic analyses archaeon (AOA), lacked all open reading frames (ORFs) with of known Amo subunit protein sequences (28, 58, and M. G. sequence similarity to genes known to be essential to ammonia Klotz, unpublished results) suggest, indeed, that the AOA oxidation in all AOB such as hydroxylamine oxidoreductase likely obtained their amo genes via lateral transfer from AOB. (HAO) and cytochromes c554 and cm552 (7). Furthermore, the It will thus be interesting to identify genetically and biochem- C. symbiosum genome lacked the genes for other cytochrome ically how the AOA resolved the tasks of aerobic ammonia proteins known to control nitrosating stress in AOB (31), ex- oxidation and detoxification of the resulting NOx compounds. cept for one (norQ) of the four required genes for functional The susceptibility of ammonia oxidizers to inhibition by sun- NO reductase (28). In contrast, the C. symbiosum genome light (due to the light sensitivity of ammonia monooxygenase VOL. 72, 2006 N. OCEANI GENOME SEQUENCE 6301 [AMO]) is probably responsible for the characteristic distribu- assess function. Manual functional assignments were assessed on an individual tion of nitrification in the water column; maximal rates occur in gene-by-gene basis as needed. Nucleotide sequence accession numbers. surface waters near the bottom of the euphotic zone (74). The sequence and annotation of the complete N. oceani strain ATCC 19707 genome is available at GenBank/EMBL/ Nitrification rates decrease with increasing depth as the rate of DDBJ accession numbers CP000127