Multiple genome sequences reveal adaptations of a phototrophic bacterium to sediment microenvironments Yasuhiro Odaa, Frank W. Larimerb, Patrick S. G. Chainc,d,e, Stephanie Malfattic,d, Maria V. Shinc,d, Lisa M. Vergezc,d, Loren Hauserb, Miriam L. Landb, Stephan Braatschf, J. Thomas Beattyf, Dale A. Pelletierb, Amy L. Schaefera, and Caroline S. Harwooda,1 aDepartment of Microbiology, University of Washington, Seattle, WA 98195; bGenome Analysis and Systems Modeling, Oak Ridge National Laboratory, Oak Ridge, TN 37831; cJoint Genome Institute, Walnut Creek, CA 94598; dLawrence Livermore National Laboratory, Livermore, CA 94550; eDepartment of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824; and fDepartment of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada Edited by Robert Haselkorn, University of Chicago, Chicago, IL, and approved October 14, 2008 (received for review September 13, 2008) The bacterial genus Rhodopseudomonas is comprised of photo- exist in soils and sediments, but on a microscale that is generally synthetic bacteria found widely distributed in aquatic sediments. too small for human observation. The genus Rhodopseudomonas Members of the genus catalyze hydrogen gas production, carbon consists of photosynthetic Alphaproteobacteria of extreme met- dioxide sequestration, and biomass turnover. The genome se- abolic versatility. Members of the genus are ubiquitous in quence of Rhodopseudomonas palustris CGA009 revealed a sur- temperate aquatic sediments (7–9), and isolates classified as prising richness of metabolic versatility that would seem to explain Rhodopseudomonas spp. can grow with or without light or its ability to live in a heterogeneous environment like sediment. oxygen, fix nitrogen, and have highly developed biodegradation However, there is considerable genotypic diversity among Rhodo- abilities. The sequenced genome of Rhodopseudomonas palustris pseudomonas isolates. Here we report the complete genome strain CGA009 revealed much of the genetic basis for this sequences of four additional members of the genus isolated from versatility and, we presumed, its ability to grow in heterogeneous a restricted geographical area. The sequences confirm that the environments typical of sediments (10). However, expanding isolates belong to a coherent taxonomic unit, but they also have beyond the analysis of this single strain, a genotypic character- significant differences. Whole genome alignments show that the ization of 75 isolates of Rhodopseudomonas from sediment circular chromosomes of the isolates consist of a collinear back- samples at three different sites revealed significant strain-to- bone with a moderate number of genomic rearrangements that strain differences (11). That study showed that the genus Rho- impact local gene order and orientation. There are 3,319 genes, dopseudomonas consists of distinct populations and raised the 70% of the genes in each genome, shared by four or more strains. possibility that each population has distinctive physiological Between 10% and 18% of the genes in each genome are strain characteristics. To investigate this possibility we sequenced the specific. Some of these genes suggest specialized physiological genomes of four genotypically distinct isolates of Rhodopseudo- traits, which we verified experimentally, that include expanded monas and analyzed selected physiological traits. Our findings light harvesting, oxygen respiration, and nitrogen fixation capa- show that although the isolates share many characteristics in bilities, as well as anaerobic fermentation. Strain-specific adapta- common, each strain has a unique set of genes for physiologies tions include traits that may be useful in bioenergy applications. that define them as distinct ecotypes. The ecotypes have likely This work suggests that against a backdrop of metabolic versatility evolved to take advantage of microenvironments in sediments. that is a defining characteristic of Rhodopseudomonas, different Strain-specific adaptations that allow anaerobic fermentation, ecotypes have evolved to take advantage of physical and chemical expanded biodegradation, or expanded light-harvesting capabil- MICROBIOLOGY conditions in sediment microenvironments that are too small for ities are also potentially useful in applications for biohydrogen human observation. production by Rhodopseudomonas. alphaproteobacteria ͉ ecotype ͉ genomes ͉ photosynthesis ͉ Results rhodopseudomonas Selection of Rhodopseudomonas Strains for Genome Sequencing. Rhodopseudomonas strains isolated by direct plating from three atural populations of closely related bacteria are commonly freshwater sediment samples from the Netherlands belonged to Ncomprised of physiologically and genetically distinct vari- several distinct clades based on 16S rRNA analysis (Fig. 1). We ants, referred to as ecotypes (1). Ecotypes are thought to have selected three strains (BisB18, BisB5, and BisA53) isolated from evolved by adapting to environmental conditions in the natural the top 0.5 cm of claylike sediment that was present 1–2 cm below habitats from which they derive. Some have suggested that ecotypes are the fundamental biological units, rather than species, which have no generally agreed upon theoretical basis in Author contributions: Y.O., P.S.G.C., and C.S.H. designed research; Y.O., S.M., M.V.S., L.M.V., S.B., and A.L.S. performed research; Y.O., F.W.L., P.S.G.C., L.H., M.L.L., J.T.B., D.A.P., microbiology (2, 3). Perhaps the best example of the analysis of and C.S.H. analyzed data; and Y.O. and C.S.H. wrote the paper. ecotypes at the genome level comes from studies of the marine The authors declare no conflict of interest. cyanobacterial genus Prochlorococcus. Prochlorococcus isolates This article is a PNAS Direct Submission. from various depths in ocean waters vary in growth responses to Data deposition: The sequence and annotations of the complete Rhodopseudomonas light intensity and have been classified as high-light- or low- chromosomes have been deposited in GenBank/EMBL/DDBJ [accession nos. CP000250 light-adapted ecotypes. The genome sequences of a collection of (strain HaA2), CP000283 (strain BisB5), CP000301 (strain BisB18), and CP000463 (strain these isolates revealed the molecular basis for the high-light and BisA53)]. low-light ecological differentiation of natural populations (4–6). 1To whom correspondence should be addressed. E-mail: [email protected]. In contrast to open ocean environments, which tend to be This article contains supporting information online at www.pnas.org/cgi/content/full/ homogeneous on a large scale, soils and sediments are hetero- 0809160105/DCSupplemental. geneous on a large scale. Homogeneous environments likely © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0809160105 PNAS ͉ November 25, 2008 ͉ vol. 105 ͉ no. 47 ͉ 18543–18548 Downloaded by guest on September 25, 2021 0.01 Clade TIE-1 100 NCIB8288 585 514 420 859 794 AP1 316 40 437 438 BIS3 80 405 488 59 tnecreP fo seneg 492 CGA009 515 462 73 KD1 355 510 335 95 529 DCP3 60 544 429 507 92 WS17 92 BIS6 40 85 90 BIS10 DX-1 BisB18 2,752 2,740 2,751 2,746 2,760 84 BisA53 20 55 HaA2 100 BisB5 0 100 96 NCIMB8252 CGA009 HaA2 BisB18 BisB5 BisA53 BIS23 (4,833) (4,683) (4,886) (4,397) (4,884) 86 BIS18 BIS17 Fig. 2. Comparative gene inventories of five strains of Rhodopseudomonas. BIS14 BIS11 Ortholog categories were determined using OrthoMCL (37). Each category is color coded as follows: genes shared by all five genomes including paralogs 97 Bradyrhizobium japonicum USDA 110 92 Bradyrhizobium sp. ORS278 (black bars); genes shared by four genomes including paralogs (red); genes Bradyrhizobium sp. BTAi1 shared by three genomes including paralogs (green); genes shared by two Nitrobacter winogradskyi Nb-255 genomes including paralogs (yellow); and strain-specific genes including in- paralogs (light blue). Numbers in the bars represent number of genes in each Fig. 1. Phylogenetic relationships of Rhodopseudomonas and Bradyrhizo- category. bium strains based on partial (1,256 bp) 16S rRNA gene sequences. Bootstrap values (100 replicates) are given at branch points. Bar represents substitutions per site. Nitrobacter winogradskyi Nb-255 was used to root the tree. Se- which encode phagelike elements, and CGA009 has a vanadium quenced strains are indicated in red. nitrogenase gene cluster. In general, the genomes are so similar that we used only CGA009 in our comparisons. When the the surface of a river along its bank. Strains BisB18 and BisB5 genomes of pairs of strains are aligned, some pairs, such as were isolated from the same 0.5 g of sediment sample, whereas BisB18 and BisA53, have similar genome architectures. Other BisA53 was from a sample taken about 5 m away. The river was pairs, such as the comparison of CGA009 with HaA2, have large not obviously polluted but it was near a small industrial area. A inversions of DNA relative to each other. In most pairwise fourth strain (HaA2) came from a site roughly 240 km from the comparisons, many rearrangements and sequence inversions are first two locations. It was obtained from a 1–2 mm-thick patch observed even though the general gene order and overall of leaf litter, roots, and sediment present Ϸ2 cm below the genome architecture is preserved (supporting information (SI) surface of a shallow pond that was formed by the accumulation Fig. S1). All of the genomes exhibit homogenized GC skews (Fig. of rainwater