microorganisms Article Genome Sequence of Rhodoferax antarcticus ANT.BRT; A Psychrophilic Purple Nonsulfur Bacterium from an Antarctic Microbial Mat Jennifer M. Baker 1, Carli J. Riester 1, Blair M. Skinner 1, Austin W. Newell 1, Wesley D. Swingley 2, Michael T. Madigan 3, Deborah O. Jung 3, Marie Asao 4, Min Chen 5, Patrick C. Loughlin 5, Hao Pan 5, Yuankui Lin 5, Yaqiong Li 5, Jacob Shaw 6, Mindy Prado 6, Chris Sherman 6, Joseph Kuo-Hsiang Tang 6,†, Robert E. Blankenship 6, Tingting Zhao 7,‡, Jeffrey W. Touchman 7,§ and W. Matthew Sattley 1,* 1 Division of Natural Sciences, Indiana Wesleyan University, Marion, IN 46953, USA; [email protected] (J.M.B.); [email protected] (C.J.R.); [email protected] (B.M.S.); [email protected] (A.W.N.); 2 Department of Biological Sciences, Northern Illinois University, DeKalb, IL 60115, USA; [email protected] 3 Department of Microbiology, Southern Illinois University, Carbondale, IL 62901, USA; [email protected] (M.T.M.); [email protected] (D.O.J.) 4 Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA; [email protected] 5 School of Life and Environmental Sciences, The University of Sydney, New South Wales 2006, Australia; [email protected] (M.C.); [email protected] (P.C.L.); [email protected] (H.P.); [email protected] (Yu.L.); [email protected] (Ya.L.) 6 Departments of Biology and Chemistry, Washington University in Saint Louis, St. Louis, MO 63130, USA; [email protected] (J.S.); [email protected] (M.P.); [email protected] (C.S.); [email protected] (J.K.-H.T.); [email protected] (R.E.B.) 7 School of Life Sciences, Arizona State University, Tempe, AZ 85287, USA; [email protected] (T.Z.); [email protected] (J.W.T.) * Correspondence: [email protected]; Tel.: +1-765-677-2128 † Present address: Materials and Manufacturing Directories, Air Force Research Laboratory, WPAFB, Ohio, OH 45433, USA ‡ Present address: PPD Pharmaceuticals, Austin, TX 78744, USA § Present address: Monsanto Company, Woodland, CA 95695, USA Academic Editor: Martin von Bergen Received: 11 January 2017; Accepted: 18 February 2017; Published: 21 February 2017 Abstract: Rhodoferax antarcticus is an Antarctic purple nonsulfur bacterium and the only characterized anoxygenic phototroph that grows best below 20 ◦C. We present here a high-quality draft genome of Rfx. antarcticus strain ANT.BRT, isolated from an Antarctic microbial mat. The circular chromosome (3.8 Mbp) of Rfx. antarcticus has a 59.1% guanine + cytosine (GC) content and contains 4036 open reading frames. In addition, the bacterium contains a sizable plasmid (198.6 kbp, 48.4% GC with 226 open reading frames) that comprises about 5% of the total genetic content. Surprisingly, genes encoding light-harvesting complexes 1 and 3 (LH1 and LH3), but not light-harvesting complex 2 (LH2), were identified in the photosynthesis gene cluster of the Rfx. antarcticus genome, a feature that is unique among purple phototrophs. Consistent with physiological studies that showed a strong capacity for nitrogen fixation in Rfx. antarcticus, a nitrogen fixation gene cluster encoding a molybdenum-type nitrogenase was present, but no alternative nitrogenases were identified despite the cold-active phenotype of this phototroph. Genes encoding two forms of ribulose 1,5-bisphosphate carboxylase/oxygenase were present in the Rfx. antarcticus genome, a feature that likely provides autotrophic flexibility under varying environmental conditions. Lastly, genes for assembly of both type IV pili and flagella are present, with the latter showing an unusual degree of clustering. This report represents the first genomic analysis of a psychrophilic anoxygenic phototroph and Microorganisms 2017, 5, 8; doi:10.3390/microorganisms5010008 www.mdpi.com/journal/microorganisms Microorganisms 2017, 5, 8 2 of 16 Microorganisms 2017, 5, 8 2 of 16 with the latter showing an unusual degree of clustering. This report represents the first genomic analysis of a psychrophilic anoxygenic phototroph and provides a glimpse of the genetic basis for maintainingprovides a glimpse a phototrophic of the genetic lifestyle basis in fora perman maintainingently cold, a phototrophic yet highly lifestylevariable, in environment. a permanently cold, yet highly variable, environment. Keywords: purple anoxygenic phototroph; photosynthesis gene cluster; light-harvesting complex; psychrophile;Keywords: purple Antarctica; anoxygenic Rhodoferax phototroph; antarcticus photosynthesis; nitrogen fixation; gene cluster;nitrogenase light-harvesting complex; psychrophile; Antarctica; Rhodoferax antarcticus; nitrogen fixation; nitrogenase 1. Introduction 1. Introduction Anoxygenic phototrophic bacteria are widespread in nature, and within this group, the purple nonsulfurAnoxygenic bacteria phototrophic(PNB) are by bacteriafar the most are widespread metabolically in nature,diverse. and Species within of PNB thisgroup, are either the Alpha purple- Alpha ornonsulfur Betaproteobacteria bacteria (PNB) and are are noted by far for the their most capacity metabolically to grow diverse. both phototrophically Species of PNB are(anoxic/light) either and- or Betaproteobacteria chemotrophically and(oxic/dark). are noted This for their broad capacity metabolic to grow diversity both phototrophically allows PNBs (anoxic/light)to adjust their and metabolismchemotrophically to fit available (oxic/dark). conditions This broad and metabolicresources diversityin a wide allowsvariety PNBs of habitats to adjust [1]. their metabolism to fitPNBs available have conditions been isolated and resources from several in a wide extrem varietye environments, of habitats [1 ].including hot, cold, acidic, alkaline,PNBs and have hypersaline been isolated [1,2]. The from success several of extremePNBs in environments,these harsh habitats including infers hot, that cold, they acidic,have evolvedalkaline, important and hypersaline biochemical [1,2]. modifications The success ofto support PNBs in photosynthesis these harsh habitats under infersstressful that conditions. they have Rhodoferaxevolved important antarcticus biochemical, one of four modifications species of the togenus support and photosynthesisa member of the under Betaproteobacteria stressful conditions. (Figure Rhodoferax antarcticus Betaproteobacteria 1), is the first purple ,bacterium one of four isolated species from of the a genus permanently and a member cold environment, of the a microbial(Figure mat on1), Rossis the Island, first purpleMcMurdo, bacterium Antarctica isolated [3] (Table from 1). a permanentlyThe organism coldis a small environment, curved rod, a microbial highly motile mat by on flagellarRoss Island, means, McMurdo, and contains Antarctica bacteriochlorophyll [3] (Table1). The (Bchl) organism a. Rfx. is a smallantarcticus curved grows rod, highlyat 0 °C motile and by flagellar means, and contains bacteriochlorophyll (Bchl) a. Rfx. antarcticus grows at 0 ◦C and optimally near 15 °C,◦ major tenets of psychrophiles [4]. A second and phenotypically distinct strain ofoptimally Rfx. antarcticus near 15, strainC, major Fryx1 tenets (Figure of psychrophiles 1), was isolated [4]. from A second the water and phenotypically column of the distinctpermanently strain ice-coveredof Rfx. antarcticus Lake Fryxell,, strain McMurdo Fryx1 (Figure Dry1 Valleys), was isolated [5]. from the water column of the permanently ice-covered Lake Fryxell, McMurdo Dry Valleys [5]. Figure 1. The 16S rRNA gene phylogenetic tree of Rfx. antarcticus strain ANT.BRT and related T FigureBetaproteobacteria 1. The 16Swith rRNAEscherichia gene phylogenetic coli (Gammaproteobacteria tree of Rfx. )antarcticus as the outgroup. strain ANT.BR rRNA gene and sequences related Betaproteobacteriawere obtained from with the Escherichia National Centercoli (Gammaproteobacteria for Biotechnology) Information as the outgroup. (NCBI) rRNA GenBank gene database sequences [6] wereand alignedobtained with from ClustalW the National using Center Mega7 for [ 7Biot], withechnology a final Information data set of 1380 (NCBI) nucleotides. GenBank Phylogeneticdatabase [6] andanalysis aligned was with conducted ClustalW using using the Mega7 maximum [7], likelihoodwith a final method data set in of conjunction 1380 nucleotides. with the Phylogenetic Jukes–Cantor analysiscorrection was [8 conducted]. Bootstrap using values the above maximum 50 (100 likelihood replicates) method are shown in conjunction at their respective with thenodes. Jukes–Cantor correction [8]. Bootstrap values above 50 (100 replicates) are shown at their respective nodes. Microorganisms 2017, 5, 8 3 of 16 Table 1. Classification and general features of Rhodoferax antarcticus str. ANT.BRT *. Property Term Classification Domain: Bacteria Phylum: Proteobacteria Class: Betaproteobacteria Order: Burkholderiales Family: Comamonadaceae Genus: Rhodoferax Species: Rhodoferax antarcticus Type strain: ANT.BRT (ATCC 700587; DSMZ 24876) Gram stain Negative Cell shape Vibrio to spirillum Motility Highly motile Endosporulation Non-endospore forming Temperature range 0–25 ◦C Optimum temperature 15–18 ◦C pH range; Optimum 6–8; 7 Acetate, pyruvate, lactate, succinate, malate, fumarate, glucose, Carbon sources fructose, sucrose, citrate, aspartate Habitat Algal–bacterial microbial mat Salinity 0%–2% NaCl (w/v) Oxygen requirement Facultative aerobe Biotic relationship Free-living Pathogenicity Non-pathogenic Geographic location Cape Royds, Ross Island, Antarctica Sample collection December, 1993