JOURNAL OF BACTERIOLOGY, May 2003, p. 2759–2773 Vol. 185, No. 9 0021-9193/03/$08.00ϩ0 DOI: 10.1128/JB.185.9.2759–2773.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved. Complete Genome Sequence of the Ammonia-Oxidizing Bacterium and Obligate Chemolithoautotroph Nitrosomonas europaea† Patrick Chain,1,2 Jane Lamerdin,1,2 Frank Larimer,1,3 Warren Regala,1,2 Victoria Lao,1,2 Miriam Land,1,3 Loren Hauser,1,3 Alan Hooper,4 Martin Klotz,5 Jeanette Norton,6 Luis Sayavedra-Soto,7 Dave Arciero,4 Norman Hommes,7 Mark Whittaker,4 and Daniel Arp7* Joint Genome Institute, Walnut Creek, California 945981; Lawrence Livermore National Laboratory, Livermore, California 945502; Oak Ridge National Laboratory, Oak Ridge, Tennessee 378313; University of Minnesota, Minneapolis, Minnesota 554554; University of Louisville, Louisville, Kentucky 402085; Utah State University, Logan, Utah 843226; and Oregon State University, Corvallis, Oregon 973317 Received 21 November 2002/Accepted 20 February 2003 Nitrosomonas europaea (ATCC 19718) is a gram-negative obligate chemolithoautotroph that can derive all its energy and reductant for growth from the oxidation of ammonia to nitrite. Nitrosomonas europaea participates in the biogeochemical N cycle in the process of nitrification. Its genome consists of a single circular chromo- some of 2,812,094 bp. The GC skew analysis indicates that the genome is divided into two unequal replichores. Genes are distributed evenly around the genome, with ϳ47% transcribed from one strand and ϳ53% tran- scribed from the complementary strand. A total of 2,460 protein-encoding genes emerged from the modeling Downloaded from effort, averaging 1,011 bp in length, with intergenic regions averaging 117 bp. Genes necessary for the catabolism of ammonia, energy and reductant generation, biosynthesis, and CO2 and NH3 assimilation were identified. In contrast, genes for catabolism of organic compounds are limited. Genes encoding transporters for inorganic ions were plentiful, whereas genes encoding transporters for organic molecules were scant. Complex repetitive elements constitute ca. 5% of the genome. Among these are 85 predicted insertion sequence elements in eight different families. The strategy of N. europaea to accumulate Fe from the environment involves several jb.asm.org classes of Fe receptors with more than 20 genes devoted to these receptors. However, genes for the synthesis of only one siderophore, citrate, were identified in the genome. This genome has provided new insights into the growth and metabolism of ammonia-oxidizing bacteria. by on May 18, 2008 Nitrosomonas europaea is a bacterium that can derive all its sites contaminated with chlorinated aliphatic hydrocarbons energy and reductant for growth from the oxidation of ammo- (44, 45, 61, 76). nia to nitrite. The cell’s demand for carbon has to be met Ammonia-oxidizing bacteria such as Nitrosomonas convert Ϫ almost entirely by the fixation of carbon dioxide. Additional NH3 to NO2 by the successive action of ammonia monooxy- mineral salts complete the cell’s nutritional needs. Although genase (AMO) and hydroxylamine oxidoreductase (HAO): ϩ ϩ ϩϩ Ϫ 3 ϩ 3 Ϫ ϩ ϩ this bacterium can incorporate small amounts of organic com- NH3 O2 2H 2e NH2OH H2O NO2 5H Ϫ pounds into cellular biomass (19, 20, 59, 60), there is an obli- ϩ 4e . Two of the four electrons return to the AMO reaction, gate requirement for oxidation of ammonia and assimilation of and two are either reductant for biosynthesis or pass to a inorganic nutrients to support growth. As such, this bacterium terminal electron acceptor (41, 83). is a member of a small group of bacteria known as obligate N. europaea is a member of the proteobacteria, a large group chemolithoautotrophs. of eubacteria of presumed photosynthetic ancestry (73). All N. europaea and other ammonia-oxidizing bacteria partici- terrestrial ammonia-oxidizing bacteria currently identified be- pate in the biogeochemical N cycle in the process of nitrifica- long to the ␤-subdivision of the proteobacteria, including the tion (the biological conversion of reduced nitrogen in the form Nitrosomonas and Nitrosospira genera, whereas marine strains ϩ ␥ of ammonia [NH3] or ammonium [NH4 ] to oxidized N in the are found in both the -subdivision Nitrosococcus genus and Ϫ Ϫ ␤ form of nitrite [NO2 ], nitrate [NO3 ], or gaseous forms [NO, the -subdivision genera (34, 82). N2O]). The gaseous products of nitrification, NO and N2O, are N. europaea is the best studied of the ammonia-oxidizing “greenhouse gases,” whereas the soluble forms are readily bacteria at the molecular level. Previous work has focused leached into ground and surface waters. In contrast to these primarily on unique aspects of this bacterium’s growth, namely, detrimental consequences of ammonia oxidation, nitrifiers in- its ability to utilize NH3, and the bioenergetics associated with crease N availability to plants, are important to the treatment this process. Thus, the pathway for NH3 oxidation has received of wastewater, and have potential for the bioremediation of considerable attention at both the biochemical and the genetic levels. AMO is a heteromultimeric enzyme encoded by the amoCAB operon that exists in two strikingly similar copies in * Corresponding author. Mailing address: Department of Botany the N. europaea genome (9, 37, 47, 54, 55). HAO is a homotri- and Plant Pathology, Oregon State University, Corvallis, OR 97331- 2902. Phone: (541) 737-1294. Fax: (541) 737-5310. E-mail: arpd@bcc mer in which each subunit harbors eight c-type hemes, one of .orst.edu. which binds substrate. Electrons pass from HAO through two † For a commentary on this article, see page 2690 in this issue. tetraheme c cytochromes (cytochromes c554 and cm552)to 2759 2760 CHAIN ET AL. J. BACTERIOL. ubiquinone (79). Hence, one theoretical HAO-electron accep- TABLE 1. Summary of N. europaea genome tor complex includes 48 c-hemes. Similar to the multiple copies Parameter Value of the amo operon, the hao gene is present in three nearly No. of chromosomes........................................................................ 1 identical copies (55). Gene sequences for three cytochromes No. of bp ...........................................................................................2,812,094 that participate in NH3 oxidation have also been determined GC content (%)................................................................................ 50.7 (7, 39), and some of these exist as multiple copies in the Coding density (%).......................................................................... 88.4 genome. In contrast to NH oxidation, other cellular processes No. of predicted protein coding genes.......................................... 2,460 3 No. of predicted proteins with putative function......................... 1,863 have received only limited attention. Analysis of the complete No. of unknown proteins with matches to other genome sequence has allowed new insights into all aspects of proteins of unknown function..................................................... 285 the growth and environmental responses of this ammonia- No. of unknown proteins unique to N. europaea......................... 312 No. of predicted RNA coding genes ............................................. 47 oxidizing bacterium. No. of tRNAs.................................................................................... 41 No. of 16S-Ala tRNATGC-Ile tRNAGAT-23S-5S tRNAs............. 1 MATERIALS AND METHODS No. of genes in protein categorya: Energy............................................................................................ 103 Construction, isolation, and sequencing of small-insert and large-insert li- Transport....................................................................................... 285 braries. Genomic DNA, isolated from the N. europaea (ATCC 19718), was DNA replication........................................................................... 212 sequenced by using a conventional whole-genome shotgun strategy (30). Briefly, Transcription................................................................................. 36 random 2- to 3-kb DNA fragments were isolated after mechanical shearing. Translation .................................................................................... 136 These gel-extracted fragments were concentrated, end repaired, and cloned into Regulation and signaling............................................................. 187 pUC18. Double-ended plasmid sequencing reactions were carried out by using Amino acid metabolism............................................................... 120 PE BigDye Terminator chemistry (Perkin-Elmer, Foster City, Calif.) and se- Carbohydrate metabolism ........................................................... 55 quencing ladders were resolved on PE 377 automated DNA sequencers. Two Nucleotide metabolism................................................................ 48 rounds (near 55,000 reads each) of small-insert library sequencing were per- Cofactor metabolism.................................................................... 80 formed, generating 17-fold redundancy. Lipid metabolism.......................................................................... 48 Downloaded from A large-insert (ϳ30 kb) fosmid library was also constructed by Sau3AI partial Secondary metabolites ................................................................. 48 digestion of genomic DNA and cloning into the pFos1 cloning vector (46).