microorganisms Article Analysis of the Complete Genome of the Alkaliphilic and Phototrophic Firmicute Heliorestis convoluta Strain HHT 1 1 1 1, Emma D. Dewey , Lynn M. Stokes , Brad M. Burchell , Kathryn N. Shaffer y, 1 1, 2 2 Austin M. Huntington , Jennifer M. Baker z , Suvarna Nadendla , Michelle G. Giglio , 3 4,§ 5, 3 Kelly S. Bender , Jeffrey W. Touchman , Robert E. Blankenship k, Michael T. Madigan and W. Matthew Sattley 1,* 1 Division of Natural Sciences, Indiana Wesleyan University, Marion, IN 46953, USA; [email protected] (E.D.D.); [email protected] (L.M.S.); [email protected] (B.M.B.); kshaff[email protected] (K.N.S.); [email protected] (A.M.H.); [email protected] (J.M.B.) 2 Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD 21201, USA; [email protected] (S.N.); [email protected] (M.G.G.) 3 Department of Microbiology, Southern Illinois University, Carbondale, IL 62901, USA; [email protected] (K.S.B.); [email protected] (M.T.M.) 4 School of Life Sciences, Arizona State University, Tempe, AZ 85287, USA; jeff[email protected] 5 Departments of Biology and Chemistry, Washington University in Saint Louis, St. Louis, MO 63130, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-765-677-2128 Present address: School of Pharmacy, Cedarville University, Cedarville, OH 45314, USA. y Present address: Department of Microbiology and Immunology, University of Michigan, Ann Arbor, z MI 48109, USA. § Present address: Intrexon Corporation, 1910 Fifth Street, Davis, CA 95616, USA. Present address: 3536 South Kachina Drive, Tempe, AZ 85282, USA. k Received: 26 January 2020; Accepted: 22 February 2020; Published: 25 February 2020 Abstract: Despite significant interest and past work to elucidate the phylogeny and photochemistry of species of the Heliobacteriaceae, genomic analyses of heliobacteria to date have been limited to just one published genome, that of the thermophilic species Heliobacterium (Hbt.) modesticaldum str. Ice1T. Here we present an analysis of the complete genome of a second heliobacterium, Heliorestis (Hrs.) convoluta str. HHT, an alkaliphilic, mesophilic, and morphologically distinct heliobacterium isolated from an Egyptian soda lake. The genome of Hrs. convoluta is a single circular chromosome of 3.22 Mb with a GC content of 43.1% and 3263 protein-encoding genes. In addition to culture-based observations and insights gleaned from the Hbt. modesticaldum genome, an analysis of enzyme-encoding genes from key metabolic pathways supports an obligately photoheterotrophic lifestyle for Hrs. convoluta. A complete set of genes encoding enzymes for propionate and butyrate catabolism and the absence of a gene encoding lactate dehydrogenase distinguishes the carbon metabolism of Hrs. convoluta from its close relatives. Comparative analyses of key proteins in Hrs. convoluta, including cytochrome c553 and the Fo alpha subunit of ATP synthase, with those of related species reveal variations in specific amino acid residues that likely contribute to the success of Hrs. convoluta in its highly alkaline environment. Keywords: heliobacteria; Heliorestis convoluta; alkaliphilic bacteria; soda lake; anoxygenic phototroph; bacteriochlorophyll g Microorganisms 2020, 8, 313; doi:10.3390/microorganisms8030313 www.mdpi.com/journal/microorganisms Microorganisms 2020, 8, 313 2 of 24 1. IntroductionMicroorganisms 2020, 8, 313 2 of 23 1.Heliobacteria Introduction comprise a unique group of strictly anaerobic, anoxygenic phototrophs that have been isolated from a wide diversity of soil and aquatic habitats [1–4]. Unlike all other phototrophic Heliobacteria comprise a unique group of strictly anaerobic, anoxygenic phototrophs that have bacteria, heliobacteria use bacteriochlorophyll (Bchl) g as the chief chlorophyll pigment for phototrophic been isolated from a wide diversity of soil and aquatic habitats [1–4]. Unlike all other phototrophic growth [5], but despite their ability to use light as an energy source, heliobacteria are apparently bacteria, heliobacteria use bacteriochlorophyll (Bchl) g as the chief chlorophyll pigment for incapablephototrophic of autotrophic growth [5], growth but despite and, thus, their areability obligate to use heterotrophslight as an energy [4,6]. source, Heliobacteria heliobacteria are are the onlyapparently phototrophs incapable of the of large autotrophic bacterial growth phylum and, Firmicutesthus, are obligate[4,7,8], heterotrophs and although [4,6]. they Heliobacteria typically stainare Gram-negatively, the only phototrophs thin of sections the large of bacterial cells of phylum heliobacteria Firmicutes exhibit [4,7,8], aand Gram-positive although they celltypically wall morphologystain Gram-negatively, [9,10]. In addition thin tosections these of distinctive cells of heliobacteria properties, cellsexhibit of a heliobacteria Gram-positive are cell able wall to differentiatemorphology into [9,10]. heat-resistant In addition endospores to these [4distinctiv,11], ande someproperties, heliobacteria cells of haveheliobacteria also demonstrated are able to the abilitydifferentiate to reduce into heat-resistant toxic metals, en suchdospores as Hg [4,11],2+, and and thereforesome heliobact mayeria be have useful also for demonstrated applications the in bioremediationability to reduce [12,13 ].toxic metals, such as Hg2+, and therefore may be useful for applications in bioremediationSpecies of Heliobacteriaceae [12,13]. can be divided into two physiological groups—neutrophiles and alkaliphiles—thatSpecies of track Heliobacteriaceae closely with can their be divided phylogeny into [7two] (Figure physiological1). Included groups—neutrophiles in the neutrophilic and clade,alkaliphiles—that the moderate thermophile track closelyHbt. with modesticaldum their phylogenywas [7] the(Figure first 1). heliobacterium Included in the to neutrophilic have its genome clade, sequencedthe moderate and, with thermophile its simple phototrophicHbt. modesticaldum machinery was consistingthe first heliobacterium of a type I reaction to have center its (RC)genome and no peripheralsequenced antenna and, with photocomplex, its simple phototrophic has been amachinery model organism consisting for of studies a type of I photosynthesisreaction center (RC) and relatedand photochemistry no peripheral antenna [6,14]. Likephotoc otheromplex, neutrophilic has been heliobacteria,a model organismHbt. for modesticaldum studies of photosynthesisexhibits both phototrophicand related growth photochemistry in the light [6,14]. and chemotrophic Like other neutrophilic growth in heliobacteria, the dark [3,15 Hbt.–17 modesticaldum]. exhibits both phototrophic growth in the light and chemotrophic growth in the dark [3,15–17]. FigureFigure 1. Phylogenetic 1. Phylogenetic (16S (16S rRNA) rRNA) tree tree of Heliorestisof Heliorestis convoluta convolutaand and related relatedFirmicutes Firmicutes.. Heliobacteria,Heliobacteria, the onlythe only phototrophic phototrophicFirmicutes Firmicutes, are, are divided divided into into alkaliphilic alkaliphilic and and neutrophilic neutrophilic species. species.Heliobacterium Heliobacterium modesticaldummodesticaldum(boxed) (boxed) is theis the model model organism organism for for physiological physiological and and biochemical biochemical studiesstudies ofof the heliobacteria;heliobacteria;Hrs. Hrs. convoluta convoluta(boxed) (boxed) is is the the first first alkaliphilic alkaliphilic heliobacterium heliobacterium toto havehave aa describeddescribed genome.genome. Note Note that that the the branching branching pattern pattern shown shown here here suggests suggests a a possible possible alkaliphilic alkaliphilic originorigin to the heliobacteria,heliobacteria, as previously as previously discussed discussed by by Sattley Sattley and an Swingleyd Swingley [7 ].[7]. The The weighted weighted neighbor-joiningneighbor-joining methodmethod [18] and[18] and Jukes-Cantor Jukes-Cantor corrected corrected distance distance model model were were used used for for tree tree construction. construction. NodesNodes representrepresent bootstrap bootstrap values values ( 50%) (≥50%) based based on 100 on replicates,100 replicates, and andEscherichia Escherichia coli wascoli was used used to root to root the tree.the ≥ GenBanktree. accessionGenBank accession numbers fornumbers eachsequence for each sequence used in the used analysis in the areanalysis shown are in shown parentheses, in parentheses, adapted fromadapted Sattley andfrom Swingley Sattley and [7], SwingleyAdv. Bot. [7], Res. Adv.2013 Bot., 66 Res., 67–97, 2013, Copyright66, 67–97, Copyright 2013 Elsevier 2013 Ltd. Elsevier Ltd. Microorganisms 2020, 8, 313 3 of 24 Microorganisms 2020, 8, 313 3 of 23 Species of alkaliphilicalkaliphilic heliobacteria grow optimally between pH 8–9.5 and, unlikeunlike neutrophilicneutrophilic heliobacteria, are obligate photoheterotrophs, us usinging light and organic compounds for growth but incapableincapable ofof chemotrophic chemotrophic growth growth in darkness in darkness [19–22]. [19–22]. Consistent Consistent with other with alkaliphilic other heliobacteriaalkaliphilic originatingheliobacteria from originating the soils andfrom waters the soils of soda and lakes waters [19 ,of20 soda,22], Hrs. lakes convoluta [19,20,22],str. Hrs. HHT convolutawas isolated str. fromHHT thewas shore isolated of the from alkaline the shore (pH 10)of the Lake alkaline El Hamra (pH (Figure10) Lake2A), El locatedHamra in(Figure the Wadi