ARTICLES Complete genome sequence of the metabolically versatile photosynthetic bacterium Rhodopseudomonas palustris Frank W Larimer1,2,Patrick Chain2,3,Loren Hauser1,2,Jane Lamerdin2,3,7,Stephanie Malfatti2,3,Long Do2,3,7, Miriam L Land1,2, Dale A Pelletier1,2,J Thomas Beatty4,Andrew S Lang4,F Robert Tabita5,Janet L Gibson5, Thomas E Hanson5,7,Cedric Bobst5,Janelle L Torres y Torres6,Caroline Peres6,7,Faith H Harrison6, Jane Gibson6 & Caroline S Harwood6 Rhodopseudomonas palustris is among the most metabolically versatile bacteria known. It uses light, inorganic compounds, or organic compounds, for energy. It acquires carbon from many types of green plant–derived compounds or by carbon dioxide fixation, and it fixes nitrogen. Here we describe the genome sequence of R. palustris, which consists of a 5,459,213-base-pair (bp) circular chromosome with 4,836 predicted genes and a plasmid of 8,427 bp. The sequence reveals genes that confer a remarkably large number of options within a given type of metabolism, including three nitrogenases, five benzene ring cleavage http://www.nature.com/naturebiotechnology pathways and four light harvesting 2 systems. R. palustris encodes 63 signal transduction histidine kinases and 79 response regulator receiver domains. Almost 15% of the genome is devoted to transport. This genome sequence is a starting point to use R. palustris as a model to explore how organisms integrate metabolic modules in response to environmental perturbations. R. palustris is a purple photosynthetic bacterium that belongs to the 5.46 Mb in size. The sequenced strain also harbors a 8.4-kilobase (kb) alpha proteobacteria and is widely distributed in nature as indicated circular plasmid. by its isolation from sources as diverse as swine waste lagoons, earth- worm droppings, marine coastal sediments and pond water. It has RESULTS extraordinary metabolic versatility and grows by any one of the four Major features of the genome modes of metabolism that support life: photoautotrophic or photo- The R. palustris genome has very few repeat nucleotide sequences, synthetic (energy from light and carbon from carbon dioxide), pho- insertion sequence elements or transposons. It has just 16 insertion © 2004 Nature Publishing Group toheterotrophic (energy from light and carbon from organic sequence elements including representatives of the ‘phage’ integrase compounds), chemoheterotrophic (carbon and energy from organic family, four ISR1-like elements and two xerD type elements. No hori- compounds) and chemoautotrophic (energy from inorganic com- zontally transferred islands of DNA are apparent based on anomalous pounds and carbon from carbon dioxide) (Fig. 1). R. palustris enjoys G + C content. R. palustris has 4,836 predicted protein-encoding genes exceptional flexibility within each of these modes of metabolism. It (Table 1 and http://genome.ornl.gov/microbial/rpal/). These include grows with or without oxygen and uses many alternative forms of genes required for the biosynthesis of all its cellular components from inorganic electron donors, carbon and nitrogen. It degrades plant carbon dioxide in keeping with its robust growth in media lacking biomass and chlorinated pollutants and it generates hydrogen as a organic carbon sources. R. palustris has many genes associated with product of nitrogen fixation1,2.ThusR. palustris is a model organism energy metabolism, reflecting its metabolic versatility (Fig. 2). The to probe how the web of metabolic reactions that operates within chromosomal positions and numbered designations of these genes can the confines of a single cell adjusts and reweaves itself in response to be found in Supplementary Table 1 online. There are genes allowing changes in light, carbon, nitrogen and electron sources that are easily oxidation of hydrogen, thiosulfate and carbon monoxide as energy manipulated experimentally. As a critical step in the further develop- and reductant sources. Two homologous NADH dehydrogenase com- ment of this model we have sequenced and annotated the R. palustris plexes that are encoded in the genome likely broker the catabolism of a genome. The genome comprises one circular chromosome that is wide variety of organic compounds, including fatty acids, dicarboxylic 1Genome Analysis and Systems Modeling, Oak Ridge National Laboratory, One Bethel Valley Rd., Oak Ridge, Tennessee 37831, USA. 2Joint Genome Institute, 2800 Mitchell Dr., Walnut Creek, California 94598, USA. 3Lawrence Livermore National Laboratory, 7000 East Ave., Livermore, California 94550, USA. 4Department of Microbiology and Immunology, The University of British Columbia, 6174 University Blvd., Vancouver, British Columbia, Canada V6T 1Z3. 5Department of Microbiology, The Ohio State University, 484 West 12th Ave., Columbus, Ohio 43210, USA. 6Department of Microbiology, 3-432 Bowen Science Bldg., The University of Iowa, Iowa City, Iowa 52242, USA. 7Present addresses: Odyssey Thera, 4550 Norris Canyon Rd., San Ramon, California 94583, USA (J.L.), Department of Biology, University of California San Diego, 9500 Gilman Dr., La Jolla, California 92093, USA (L.D.), Delaware Biotechnology Institute, The University of Delaware, 15 Innovation Way, Newark, Delaware 19711, USA (T.E.H.), Genencor International, 925 Page Mill Rd., Palo Alto, California 94304, USA (C.P.). Correspondence should be addressed to C.S.H. ([email protected]). Published online 14 December 2003; doi:10.1038/nbt923 NATURE BIOTECHNOLOGY VOLUME 22 NUMBER 1 JANUARY 2004 55 ARTICLES H+ Aerobic Anaerobic Chemoheterotrophic Photoheterotrophic growth growth Lignin monomers, ATP other organic ADP compounds H+ Lignin ATP Cell material monomers, Cell other organic material compounds + H 1 H2O H+ ⁄2O2 H+ Chemoautotrophic Photoautotrophic CO2 growth growth Thiosulfate, H2, other inorganic ATP electron donors ADP ATP H+ CO2 Cell Cell material material Thiosulfate, + 1 H2O H + ⁄2O2 H2, other inorganic H electron donors Figure 1 Overview of the physiology of R. palustris. Schematic representations of the four types of metabolism that support its growth are shown. The multicolored circle in each cell represents the enzymatic reactions of central metabolism. http://www.nature.com/naturebiotechnology acids and lignin monomers. The conditions under which these two R. palustris, like other purple phototrophic bacteria, responds to seemingly redundant enzyme systems are expressed have not been lowered light intensity by increasing the amount of light harvesting defined. Terminal oxidase genes should enable R. palustris to use (LH) complexes. These consist of α and β polypeptides bound to bac- nitrite, nitric oxide and nitrous oxide as electron acceptors during teriochlorophyll and a carotenoid, to form a unit that oligomerizes to anaerobic respiration3.There are four sets of genes for terminal oxi- produce complexes that transfer light energy to the reaction center9. ◊ ◊ dases that can function with oxygen: a cytochrome aa3 oxidase, a The pathway of light energy transfer is LH2 LH1 reaction center. cytochrome cbb3 oxidase, a cytochrome d quinol oxidase and a quinol R. palustris differs from other phototrophs in that it has multiple LH2 bd oxidase. Photosynthesis genes enable the use of light as an energy complexes that differ slightly in the wavelengths of light absorbed. source by cyclic photophosphorylation under anaerobic conditions. It tunes its complement of LH2 complexes to harvest light of differing qualities and intensities10.The genome sequence reveals four complete Phototrophy sets of LH2 genes (pucBA) and one incomplete set (Fig. 2 and Genes rpa1505–rpa1554 required for the generation of energy by pho- Supplementary Table 1 online). Two of the four complete sets of tophosphorylation reside in a 55-kb region of the R. palustris chromo- pucBA genes are located near bacteriophytochrome genes rpa3015, © 2004 Nature Publishing Group some. These include genes for bacteriochlorophyll and carotenoid rpa3016 and rpa1490 that may function in the regulation of LH2 com- biosynthesis as well as genes encoding the L, M and H polypeptides plex gene expression. that form the membrane-bound reaction center complex, where light R. palustris has genes (rpa0008 and rpa0009) that are similar to the energy is absorbed to initiate electron transfer reactions. The reaction circadian clock genes, kaiB and kaiC previously identified only in oxy- center genes rpa1527, rpa1528 and rpa1548 are the most highly con- genic photosynthetic bacteria11. R. palustris cells present in anoxic served aspect of this region, sharing from 45 to 60% predicted amino environments generate ample energy by photophosphorylation dur- acid identity with the corresponding genes from Rhodobacter sphaer- ing daylight hours, but may be energy limited at night. Circadian regu- oides,a model organism for the study of anoxygenic photosynthesis4. lation of energy consuming reactions such as nitrogen fixation would However the R. palustris reaction center proteins are most similar (on make sense, but has yet to be shown in R. palustris. the order of 75% amino acid identity) to homologs in the unusual photosynthetic Bradyrhizobium sp. strain ORS278 (ref. 5). This strain Carbon dioxide fixation forms nitrogen-fixing nodules on the stems of the plant Aeschynomene The R. palustris genome encodes two active forms of RubisCO, the key 6 sensitiva,a tropical legume that grows in water logged soils .In addi- enzyme of the Calvin-Benson-Bassham (CBB) pathway of CO2 fixa- tion to a conserved arrangement of photosynthesis genes,