Cultivation and Genomic, Nutritional, and Lipid Biomarker ...jb.asm.org/content/192/12/3033.full.pdf · a glass tissue homogenizer in 10 ml Castenholz medium D (10) supplemented with

JOURNAL OF BACTERIOLOGY, June 2010, p. 3033–3042 Vol. 192, No. 120021-9193/10/$12.00 doi:10.1128/JB.01610-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Cultivation and Genomic, Nutritional, and Lipid BiomarkerCharacterization of Roseiflexus Strains Closely Related to

Predominant In Situ Populations Inhabiting YellowstoneHot Spring Microbial Mats�†

Marcel T. J. van der Meer,1,2,3 Christian G. Klatt,2 Jason Wood,2 Donald A. Bryant,4Mary M. Bateson,2 Laurens Lammerts,1 Stefan Schouten,1 Jaap S. Sinninghe Damste,1

Michael T. Madigan,3 and David M. Ward2*NIOZ Royal Netherlands Institute for Sea Research, Department of Marine Organic Biogeochemistry, P.O. Box 59, 1790 AB Den Burg,

Texel, Netherlands1; Montana State University, Department of Land Resources and Environmental Sciences, Bozeman,Montana 597172; Southern Illinois University, Department of Microbiology, Carbondale, Illinois 629013; and

The Pennsylvania State University, Department of Biochemistry and Molecular Biology,University Park, Pennsylvania 168024

Received 10 December 2009/Accepted 19 March 2010

Roseiflexus sp. strains were cultivated from a microbial mat of an alkaline siliceous hot spring in YellowstoneNational Park. These strains are closely related to predominant filamentous anoxygenic phototrophs found inthe mat, as judged by the similarity of small-subunit rRNA, lipid distributions, and genomic and metagenomicsequences. Like a Japanese isolate, R. castenholzii, the Yellowstone isolates contain bacteriochlorophyll a, butnot bacteriochlorophyll c or chlorosomes, and grow photoheterotrophically or chemoheterotrophically underdark aerobic conditions. The genome of one isolate, Roseiflexus sp. strain RS1, contains genes necessary tosupport these metabolisms. This genome also contains genes encoding the 3-hydroxypropionate pathway forCO2 fixation and a hydrogenase, which might enable photoautotrophic metabolism, even though neither isolatecould be grown photoautotrophically with H2 or H2S as a possible electron donor. The isolates exhibittemperature, pH, and sulfide preferences typical of their habitat. Lipids produced by these isolates matchedmuch better with mat lipids than do lipids produced by R. castenholzii or Chloroflexus isolates.

We have investigated laminated cyanobacterial mats in al-kaline siliceous hot springs of Yellowstone National Park (Oc-topus Spring and Mushroom Spring) as models for under-standing principles of microbial community ecology (49, 51,54) and as models of stromatolites (50, 55), which are impor-tant fossilized microbial communities of the Precambrian.These mats are dominated by unicellular cyanobacteria (Syn-echococcus spp.) and filamentous anoxygenic phototrophicbacteria (FAPs) (e.g., Chloroflexus spp. and Roseiflexus spp.);they also contain newly described anoxygenic phototrophicbacteria (8) and many organisms involved in mat decomposi-tion (49). We have studied the composition and structure ofthe mat community using both nucleic acid and lipid biomarkerapproaches (50), as well as the functional contributions of andinteractions among community members (34, 46, 47).

A major finding from molecular analyses was that the pre-dominant Synechococcus spp. of the mat are distantly relatedto readily cultivated strains (15, 49). Careful cultivation ofpredominant strains was necessary before we could observephenotypic properties of Synechococcus sp. isolates that were

useful for understanding the in situ ecology of native matpopulations (2). Genomic analysis of relevant isolates has alsovastly improved our ability to understand metagenomic (5, 25;C. G. Klatt, J. M. Wood, D. B. Rusch, M. M. Bateson, J. F.Heidelberg, A. R. Grossman, D. Bhaya, F. M. Cohan, M. Kuhl,D. A. Bryant, and D. M. Ward, unpublished data) andmetatranscriptomic (Z. Liu, C. G. Klatt, J. M. Wood, D. B.Rusch, M. Ludwig, N. Wittekindt, L. P. Tomsho, S. C. Schus-ter, D. M. Ward, and D. A. Bryant, unpublished data) data-bases, which have the potential to provide a holistic view of thecommunity and to permit observation of how metabolic net-working may occur among its members.

Similarly, based on small-subunit rRNA (SSU rRNA) clon-ing and sequencing and fluorescent in situ hybridization(FISH) probing, the dominant FAPs of the community werefound to be distantly related to readily cultivated Chloroflexusspp. but closely related to Roseiflexus castenholzii (35), a bac-teriochlorophyll a-containing anoxygenic phototroph lackingchlorosomes, which was initially cultivated from a Japanese hotspring (20). Only one SSU rRNA sequence somewhat distantlyrelated to that of Heliothrix oregonensis, the only other FAPknown to possess only bacteriochlorophyll a (37), has beendetected in these mats (35, 56) (Fig. 1). However, SSU rRNAsequences closely related to that of the chlorosome-containingChloroflexus aurantiacus were also detected in the mat at amuch lower abundance than Roseiflexus-like SSU rRNAs, al-though the contribution of Chloroflexus-like organisms wasgreater at 70°C than at 60°C (35). The SSU rRNA sequences of

* Corresponding author. Mailing address: Montana State Univer-sity, Department of Land Resources and Environmental Sciences, P.O.Box 173120, Bozeman, MT 59717. Phone: (406) 994-3401. Fax: (406)994-3933. E-mail: [email protected].

† Supplemental material for this article may be found at http://jb.asm.org/.

� Published ahead of print on 2 April 2010.

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FIG. 1. Neighbor-joining phylogeny based on SSU rRNA sequences of filamentous anoxygenic phototrophic bacteria, showing relatedness ofthe isolates (red text) used in this study to the Chloroflexus (green bracket) and Roseiflexus (red bracket) clades. The bar shows nucleotidesubstitutions per site. All clones in this tree were obtained from Yellowstone hot spring microbial mats, as were Roseiflexus sp. strain RS1,Chloroflexus sp. strain 396-1, and C. aurantiacus strain Y-400-fl. The tree was rooted by a mitochondrial 16S rRNA used as an outgroup.

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R. castenholzii and cultivated Chloroflexus spp. have divergedsubstantially from Roseiflexus- and Chloroflexus-like SSUrRNA sequences detected in mats (35), and thus it is unclearhow representative the phenotypic properties of these isolatesare compared to mat FAPs.

One of the main questions is how the metabolism of culti-vated FAPs observed in laboratory studies and/or inferredfrom genomic analyses relate to the metabolism of in situ FAPpopulations. Both R. castenholzii and C. aurantiacus strainshave been shown to be capable of photoheterotrophic and darkaerobic metabolisms (20, 30, 36), while only some Chloroflexussp. strains have been shown to be photoautotrophic (18, 28).Furthermore, Castenholz and Pierson (11) pointed out thatChloroflexus isolates grew well on inorganic media in dualculture with Synechococcus isolates. These observations sup-port the notion that FAPs are cross-fed small organic com-pounds produced by cyanobacterial photorespiration (3) andfermentation (34) and thus might grow photoheterotrophicallyin the mat. Although a recent pyrosequencing study arguedagainst this producer-consumer relationship (33), we have ob-served evidence that 13CO2 fixed by Synechococcus spp. intopolyglucose is transferred to FAPs via assimilation of [13C]ac-etate derived from cyanobacterial fermentation (46). Com-pound-specific stable carbon isotope studies of FAP biomark-ers also suggested that FAPs may be photoautotrophic (42,45). FAPs that assimilate carbon which has been fixed and thenexcreted by cyanobacteria should have stable carbon isotope

signatures typical of those resulting from the Calvin cycle. Incontrast, the stable carbon isotopic compositions of these FAPbiomarkers showed that they were much heavier than cya-nobacterial lipid biomarkers, suggesting the possibility that insitu FAP populations are photoautotrophic or photomixotro-phic by using the 3-hydroxypropionate pathway. This pathway,which is known to be used by Chloroflexus spp., results inisotopically heavier organic carbon than that fixed in the Calvincycle (21, 22, 40, 43). This photoautotrophic biochemistry ofFAPs in the mat was confirmed in labeling experiments using13C-labeled bicarbonate, which suggested that the 13C labelwas incorporated into FAP biomarkers and FAP biomass (46,47). However, Roseiflexus spp., not Chloroflexus spp., are thedominant mat FAPs, and R. castenholzii has not been shown togrow photoautotrophically (20).

Another concern is the differences in the lipids of FAPisolates compared to lipids that we and others have detected inOctopus Spring and Mushroom Spring mats (14, 39, 41, 42, 55,58, 59). In addition to complex polar lipids (53), these matscontain abundant long-chain (C31 to C37) normal andbranched wax esters (14, 55, 58, 59) (Table 1). C. aurantiacusstrains produce C31 to C37 saturated normal or monounsatu-rated wax esters, as well as long-chain alkenes dominated byhentriacontatriene (C31:3) (26, 39, 43). R. castenholzii producesC37 to C40 normal wax esters and glycosides and fatty glyco-sides consisting of an alkane-1-ol-2-alkanoate (mainlybranched C20 alkane-1,2-diol/C14 fatty acid and branched C20

TABLE 1. Lipid biomarkers in the total lipid extracts of alkaline siliceous hot spring microbial mats and filamentous anoxygenic phototrophicbacteria used in this studya

Compound

Abundance in:

OctopusSpring

MushroomSpring

Roseiflexus sp.strains RS1and RS2b

R. castenholziiHL08 C. aurantiacus

C31:3 alkene tr tr ��n-C31 wax ester �� iso-C31 wax ester � � �n-C32 wax ester �� iso-C32 wax ester � � ��iso/iso-C32 wax ester �C32:1 wax ester �n-C33 wax ester �� iso-C33 wax ester � � �n-C34 wax ester �� iso-C34 wax ester � � ��iso/iso-C34 wax ester �C34:1 wax ester �n-C35 wax ester �� iso-C35 wax ester �n-C36 wax ester � � � ��C36:1 wax ester �n-C37 wax ester � � � �n-C38 wax ester ��n-C39 wax ester �n-C40 wax ester ��C19 alkane-1,2diol tr tr �C20 alkane-1,2-diol tr tr �C21 alkane-1,2-diol tr tr �C34 diol glycoside tr tr ��C36 diol glycoside tr tr ��C37 diol glycoside tr tr tr

a Data are from references 42 to 44 and this study.b Analyzed separately; see Fig. 6.

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alkane-1,2-diol/C16 fatty acid) bonded by glycosidic linkage toa C6 sugar. It does not produce long-chain polyunsaturatedalkenes (44). However, the lipid biomarkers in the mat andthose produced by these cultivated FAPs do not correspondwell (Table 1). The low level of mat alkenes (45) is consistentwith the low relative importance of Chloroflexus spp.; however,the low abundance of long-chain diols and diol glycosides isinconsistent with their abundance in R. castenholzii (44). Al-though the chain lengths of mat wax esters match those ofChloroflexus spp., the mat does not contain unsaturated waxesters typical of Chloroflexus spp., and mat wax ester chainlengths do not match well with those of wax esters produced byR. castenholzii. Furthermore the mat contains branched waxesters, which neither of these FAP isolates produces.

We hypothesized that our ability to understand (i) the func-tional roles that different FAPs play in the mat community and(ii) the microbial sources of major mat lipid biomarkers is onlyas good as the genetic, and thus metabolic and physiological,similarities between the FAPs that have been cultivated andnative FAP populations. Here, we describe the cultivation ofRoseiflexus sp. strains which are genetically relevant comparedto native FAPs of Yellowstone hot spring microbial mats andwhich have heretofore only been described in a very prelimi-nary way (29).

MATERIALS AND METHODS

Samples. Hot spring microbial mats were sampled on 27 July 2002 at anaverage temperature of �60°C from Octopus Spring, Yellowstone NationalPark, Wyoming (7). Mat material was stored in a 50-ml tube completely filledwith spring water; transported to Southern Illinois University, Carbondale, IL,within 3 days; and stored at 4°C until further processing.

Cultivation of FAPs. On 4 September 2002 a subsample was homogenized witha glass tissue homogenizer in 10 ml Castenholz medium D (10) supplementedwith 1 g liter�1 sodium acetate, 1 g liter�1 NaHCO3, 0.2 g liter�1 NH4Cl, 100 mgliter�1 yeast extract, 100 mg liter�1 NaH2SO3, 3 g liter�1 HEPES buffer, andtrace elements (48) and adjusted to a final pH of 8.17 after autoclaving. Bicar-bonate and/or sulfide was added to the mineral medium in some cases (seebelow) from separately sterilized solutions before pH adjustment. A dilutionseries was prepared from 10�2 to 10�12, and each dilution was filtered througha 0.2-�m-pore-size polycarbonate filter. Each filter was incubated as a “floating”filter (12) placed above support filters saturated with approximately 1 ml ofmedium in small petri dishes, and the medium was periodically replenished. Petridishes were sealed in a Gas-Pak jar (Becton-Dickenson) using an H2-plus-CO2

generator to remove O2 and incubated at 58°C with incandescent light of 40�mol photons m�2 s�1. Tiny red colonies on the filters were observed within aweek, but these did not grow following transfer to fresh filters. After 4 months,the original filters had completely dried out but “red-orange growth” was noticedin between the old colonies (10�4 dilution filters); several discrete colonies andthis “red-orange growth” were transferred to a variety of media for cultivation ofanoxygenic phototrophs and incubated under light anoxic or dark oxic condi-tions. Colonies that developed on 0.2% yeast extract agar plates (PE medium[19] with 0.2% yeast extract replacing sodium succinate, acetate, and glutamate)incubated aerobically in darkness at 50°C were transferred regularly to freshmedium incubated under both oxic dark and anoxic light conditions. Tiny singlecolonies were streaked for isolation several times, and the purity of the cultureswas checked by streaking on tryptic soy and nutrient agar plates with oxic andanoxic incubation at 50°C. To grow biomass sufficient for DNA extraction andsequencing, purified cultures were transferred to 0.2% yeast extract liquid me-dium containing approximately 30 �M sulfide and incubated in completely filledtubes anoxically in the light. R. castenholzii strain HL08, which had been kindlyprovided by Satoshi Hanada (Research Institute of Biological Resources, Hi-gashi, Japan), was also grown in 0.2% yeast extract liquid medium and incubatedanoxically in the light.

Analysis of nutrition and growth parameters. Cells pregrown in liquid mediumD containing 0.2% (wt/vol) yeast extract under anoxic light conditions wereinoculated (10%, vol/vol) into fresh medium in which yeast extract was replacedby various single carbon sources or mixtures of carbon sources (0.1%, wt/vol or

vol/vol), as described below. Because of yeast extract carryover in the inoculum,we compared growth on media containing single carbon sources (or the mix) withgrowth in 0.02% yeast extract medium (actually up to 0.04% yeast extract due tocarryover). After incubation in the dark overnight to allow cells to acclimate totheir new culture conditions, these cultures were incubated under anoxic condi-tions in the light or dark, as indicated, and growth was measured turbidometri-cally. In some cases, transfers (10%, vol/vol) were made into media containingsingle carbon sources (or a mixture of carbon sources) to ensure that growth wasnot due to carryover of yeast extract from the initial inoculum. Optimization oftemperature (at pH 8 and without sulfide), pH (at 50°C and without sulfide), andsulfide (at 50°C and pH 8) was investigated in medium D containing 0.2% yeastextract with incubation under anoxic conditions in the light. Except in the case ofsulfide optimization, experiments were done in triplicate.

Absorption spectra. Biomass scraped from growth on 0.2% yeast extract plateswas either (i) resuspended in aqueous 30% bovine serum albumin or (ii) cen-trifuged and then extracted in methanol. Absorption spectra were recorded usinga Hitachi U-2000 spectrophotometer scanning from 400 to 1,200 nm.

Microscopy. Phase-contrast micrographs of cells immobilized on water agarslides were taken with an Olympus B-Max 60 photomicroscope. Transmissionelectron micrographs were taken of cells fixed, stained, and examined as previ-ously described (24).

Nucleic acid analysis. For PCR analysis of FAPs, cell material from growingcolonies was added directly to the PCR mixture and amplification of Chloroflexussp. and Roseiflexus sp. SSU rRNA gene segments was performed according tomethods and using primers reported by Nubel et al. (35).

A neighbor-joining phylogenetic tree was constructed based on 873 nucleotides(nt) between positions 418 and 1291 in the Escherichia coli SSU rRNA sequenceusing the software package ARB (available at http://www.mikro.biologie.tu-muenchen.de) with Jukes-Cantor correction, the pos_var_Bacteria_100 maskingfilter, and bootstrapping with 1,000 replicates. Sequences shorter than this 973-ntalignment were added using the ARB parsimony tool (27).

In genomic and metagenomic analyses, we used metagenomes for Octopus Springand Mushroom Spring microbial mat samples obtained from sites averaging �60°Cand �65°C (Klatt et al., unpublished data) and 20 representative genomes of or-ganisms suspected to be major inhabitants of these mats, including the dominantFAP Roseiflexus sp. strain RS1 described here (accession no. CP000686; http://genome.jgi-psf.org/finished_microbes/ros_r/ros_r.home.html) and the draft ge-nome of Chloroflexus sp. strain 396-1 (9), the Chloroflexus isolate most closely relatedto those in the mat and for which a genome sequence is available (Fig. 1) (52). Weconducted a BLAST analysis (WU-BLASTn, using the settings M � 3, N � �2,wordmask � seg, and default values for all other parameters) to identify the meta-genomic sequences with the best alignments to the 20 genomes. We separatelycreated BLAST databases for the individual genomes of the three FAP isolates wehave used in lipid biomarker analysis: Chloroflexus sp. strain Y-400-fl (accession no.CP001364; http://genome.jgi-psf.org/draft_microbes/chl_y/chl_y.home.html), R. cas-tenholzii (accession no. CP000804; http://img.jgi.doe.gov/cgibin/pub/main.cgi?section�TaxonDetail&taxon_oid�639857015), and Roseiflexus sp. strain RS1. The meta-genomic sequences that could be confidently assigned to mat FAPs from the 20-genome analysis (i.e., �80% nucleotide identity to the Roseiflexus sp. strain RS1 andChloroflexus sp. strain 396-1 genomes) were then searched against each of theRoseiflexus and Chloroflexus genomes individually to identify the percent nucleotideidentity of isolate genes to the homologous FAP genes in the mat.

The Roseiflexus sp. strain RS1 genome data were subjected to the integratedmicrobial genomes (IMG) annotation pipeline of the Joint Genome Institute(JGI) (31). In some cases the functional predictions assigned to genes by IMGwere adjusted after more detailed inspection (see below).

Lipid analysis. Lipids were ultrasonically and sequentially extracted from freeze-dried harvested cells with methanol (three times), dichloromethane (DCM)-meth-anol (1:1) (three times), and DCM (three times) to obtain a total lipid extract (TLE).Part of this TLE fraction was separated into apolar (containing wax esters) and polarfractions by eluting over a small aluminum oxide column in hexane-DCM (1:1) andethyl acetate, respectively. Base hydrolysis of the apolar fraction was performed toanalyze the fatty acids and alcohols that comprise the wax esters (see reference 44 fordetails). The TLE and hydrolyzed apolar fractions were derivatized before analysisusing gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) (see reference 38 for details).

RESULTS

General phenotypic description of new Roseiflexus sp. iso-lates. Discrete red colonies arising early in the attempt tocultivate Roseiflexus sp. strains contained predominantly bac-



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teriochlorophyll c. However, red-orange colonies obtainedfrom the transfer of the red-orange growth that arose later inbetween discrete red colonies on 0.2% yeast extract agar platescontained only bacteriochlorophyll a, indicating that thesecould be Roseiflexus-type organisms. This was confirmed byPCR amplification with Roseiflexus- but not Chloroflexus-spe-cific SSU rRNA primers. Two strains of Roseiflexus sp., RS1and RS2, were isolated, both of which exhibited a filamentousmorphology (Fig. 2A). No evidence of chlorosomes, which arecharacteristically found in green sulfur and green nonsulfurbacteria, was observed in thin sections by transmission electronmicroscopy (Fig. 2B to D). Bright inclusions were common insectioned cells of strain RS1 (Fig. 2D), and these are likely tobe sites of polyhydroxyalkanoate storage. Although intact cellsof both isolates exhibited absorption maxima at 795 nm, theydiffered in absorption maxima at higher wave lengths (i.e., 900nm for strain RS1 and 860 nm for strain RS2); these wave-lengths are indicative of antenna complexes containing bacte-riochlorophyll a (Fig. 3). Absorbance of methanol extracts ofcells at 770 nm confirmed that the cells contained bacterio-chlorophyll a but did not contain detectable amounts of bac-teriochlorophyll c (Fig. 3).

Nutrition. Both strains grew best under photoheterotrophicconditions with 0.2% yeast extract and much more slowly ornot at all with 0.02% yeast extract medium (Table 2; see Fig.S1A and E in the supplemental material). For strain RS1,acetate, pyruvate, a mixture of organic acids (containing ace-tate, glutamate, glycolate, lactate, and malate), glucose, fruc-tose, and possibly malate (but not glutamate, glycolate, bu-tyrate, or lactate) supported growth in light at a lower rate thanthat on 0.2% yeast extract and at a higher rate than thatobserved on 0.02% yeast extract medium (see Fig. S1A to C inthe supplemental material). Continuous growth was not ob-served upon further transfer in medium containing 0.02%yeast extract plus 0.1% acetate or malate (see Fig. S1D in thesupplemental material), suggesting that in addition to acetateor malate, other nutrients or substrates supplied by yeast ex-

tract were required. For strain RS2, acetate, lactate, and theorganic acid mixture (but not glycolate, malate, or glutamate)appeared to stimulate growth in light to a rate that was slightlyhigher than that observed on 0.02% yeast extract medium, butonly after an extended lag phase (see Fig. S1E in the supple-mental material). As with strain RS1, strain RS2 did not growbetter in subculture in medium containing acetate, lactate, ormalate than in medium containing 0.02% yeast extract (seeFig. S1F in the supplemental material).

No growth was observed under photoautotrophic conditionswith 0.02% yeast extract medium supplemented with �1.5 mMsulfide or in aqueous media bubbled with H2 gas and incubatedat 50°C and pH 8 in the light.

Temperature, pH, and sulfide optima. Roseiflexus sp. strainsRS1 and RS2 both grew at temperatures of between 45 and60°C but not at 65°C, with an apparent optimum at 55°C to60°C (Fig. 4, top panel). Strain RS1 grew at pH 7 to 9, andgrowth was optimal at pH 8.1; strain RS2 grew at pH 6 to 9,and growth was optimal at pH 8.1 to 8.5 (Fig. 4, middle panel).For strain RS1, sulfide was optimal at 120 to 240 �M and wasinhibitory at higher levels (Fig. 4, bottom panel).

Genetic relevance of Roseiflexus sp. isolates RS1 and RS2 tomat FAPs. SSU rRNA sequences for strains RS1 and RS2exhibited 99% nucleotide identity to each other. The SSUrRNA of strain RS1 formed a separate clade together with alarge number of Roseiflexus-like clones previously detected at51 to 63°C in the Octopus Spring and Mushroom Spring mi-crobial mats (35), but to the exclusion of the SSU rRNAsequence of R. castenholzii (Fig. 1). Because we have obtainedthe complete genomic sequences of Roseiflexus sp. strain RS1,R. castenholzii, and C. aurantiacus Y-400-fl, as well as meta-genomic data from these microbial mats (9; Klatt et al., un-published data), we could compare the genomic relevance ofthese new Roseiflexus sp. isolates and FAPs that had been usedin prior lipid biomarker studies to native FAP populations. Asshown in Fig. 5, mat metagenomic sequences exhibited thehighest percent nucleotide identity to homologs in the Rosei-flexus sp. strain RS1 genome, with lower percent nucleotideidentity to R. castenholzii homologs and the lowest percentnucleotide identity to C. aurantiacus Y-400-fl homologs.

Features of Roseiflexus sp. strain RS1 and R. castenholziigenomes. Roseiflexus sp. strain RS1 has a single circular chro-mosome of 5,801,598 bp, which is slightly larger than the5,723,298-bp genome of R. castenholzii. Bioinformatics analy-ses provide evidence for a glycolytic pathway and a tricarbox-

FIG. 3. Absorption spectra of Roseiflexus castenholzii and Rosei-flexus sp. strains RS1 and RS2 in aqueous suspension except as noted.

FIG. 2. Phase-contrast (A) and transmission electron microscopy (Bto D) images of Roseiflexus sp. strains RS1 and RS2. The bars indicate 5�m in panel A, 0.5 �m in panel B, and 1 �m in panels C and D.



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ylic acid cycle, which, together with type-1 NADH dehydroge-nase, alternative complex III, and cytochrome oxidase, canaccount for the dark aerobic metabolism observed (see TableS1 in the supplemental material). Likewise, the presence ofgenes encoding type 2 photosynthetic reaction centers, light-harvesting complex I, and bacteriochlorophyll a biosynthesisgenes is consistent with the phototrophic capability and pig-mentation of this organism. The presence of genes encodingthe 3-hydroxypropionate autotrophic pathway may allow thisorganism to acquire at least some of its carbon from carbondioxide via this pathway (25). The Roseiflexus sp. strain RS1genome contains an Ni-Fe hydrogenase encoded by geneshydAB as well as a complete suite of hyp genes potentiallyinvolved in the biosynthesis and maturation of the hydrogenaseenzyme. Collectively, these may confer the ability to oxidizehydrogen as a source of electrons. The genome does not con-

tain homologs of well-characterized genes involved in dissim-ilatory sulfur metabolism, such as dsr, fcc, or sox genes or sqr,which would confer the capability of oxidizing reduced sulfurcompounds. A homolog to a type II sulfide:quinone oxi-doreductase gene (sqr) is found in the genome, but this genemay be involved in sulfide detoxification instead of using sul-fide as a source of electrons for photosynthesis (9). The pres-ence of a putative molybdopterin-containing carbon monoxidedehydrogenase gene in the Roseiflexus sp. strain RS1 genomecould confer the ability to respire CO aerobically as a potentialsource of energy in a mechanism similar to that of carboxy-dotrophic bacteria (23, 32). This capability has recently beenobserved in a nonphototrophic member of the kingdom Chlo-roflexi found in the same microbial mats, Thermomicrobiumroseum, which is distantly related to phototrophic members ofthis kingdom (57).

TABLE 2. Comparison of phenotypic characteristics of new Roseiflexus sp. isolates RS1 and RS2, R. castenholzii, and C. aurantiacus

Characteristic Chloroflexus aurantiacus Roseiflexus castenholziiHLO8 Roseiflexus sp. strain RS1 Roseiflexus sp. strain RS2

Cell diam, �m 0.6–1 0.8–1.9 0.7 0.7

Gliding motility � � NTa NT

Temp range, °C (optimum) 35–70 45–55 (50) 45–60 (55–60) 45–60 (55–60)

pH range (optimum) 7.6–8.4 6–9 (7.5–8) 7–9 (8.1) 6–9 (8.1–8.5)

Sulfide tolerance �2 mM NT Optimum, 100–200 �M NT

MetabolismPhotoautotrophy � NDb ND NDPhotoheterotrophy � � � �Aerobic respiration � � � �

In vivo absorption maxima, nm 740, 808, 868 801, 878 795, 900 795, 860

Bacteriochlorophylls a, c a a a

Chlorosomes � ND ND ND

Carbon nutritionc

0.2% yeast extract �� Fructose NT NT �� NTGlucose (Glc) �� NTLactate �/� � (sl) (�)Glycolate (Gly) NT NT ND NDSuccinate � ND sl NTMalate � ND (�) (sl)Acetate � ND (�) (�)Pyruvate �/�� ND �� NTPropionate NT NT � NTButyrate �� ND ND NTOrganic acid mix NT NT � �

Lipid biomarkersd

C31 alkene � ND ND NDWax esters C31–C36, n- and unsaturated C37–C40 C31–C36, n- and branched C31–C36, n- and branchedDiols ND C34 and C36 glycosides C19–C21 C19–C21

a NT, not tested.b ND, not detected.c Data on photoheterotrophic and photoautotrophic growth of Chloroflexus spp. relative to those of killed cell controls were obtained from references 30 and 28,

respectively. Comparable results for R. castenholzii were obtained from reference 20. Substrates used in other studies but not in this study were not included. ForRoseiflexus sp. strains RS1 and RS2, growth was stimulated above that in 0.02% yeast extract medium by addition of a compound or organic acid mixture (containingacetate, glutamate, glycolate, lactate, and malate). sl, slightly above control value; �/�, some but not all strains grow; �, definitely above control value; ��, significantlyabove control value; parentheses, not above control value upon transfer.

d Lipid data for Chloroflexus aurantiacus are from reference 43, and those for Roseiflexus castenholzii are from reference 44.



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Comparisons of the Roseiflexus sp. strain RS1 genome tothat of R. castenholzii revealed large sets of genes that arecommon to both and some that are unique to each organism.Both Roseiflexus sp. genomes contain a cluster of four colocal-ized genes, with an order of nifHBDK, that are predicted toencode the structural genes of an Mo-nitrogenase. However,both genomes lack any evidence of many other genes typicallyinvolved in biosynthesis and maturation of a functional nitro-genase apoprotein; thus, it is unclear if Roseiflexus spp. havethe ability to fix nitrogen. In addition, both Roseiflexus sp.genomes contain genes potentially allowing for the transport ofboth ferric and ferrous iron and regulation of iron homeostasis.

Because both organisms have genes encoding phosphate ABCtransporters and regulatory enzymes, R. castenholzii and Ro-seiflexus sp. strain RS1 can probably obtain phosphorous in theform of phosphate. Additionally, the Roseiflexus sp. strain RS1genome encodes a phosphonate transporter and associatedregulatory genes; this suggests that this organism may be ableto utilize organophosphates in addition to phosphate as nutri-ent sources. Finally, the Roseiflexus sp. strain RS1 genomeencodes a bacteriorhodopsin, which the R. castenholzii genomelacks. This chromophore-containing protein may be used toharvest light to produce proton motive force for the cell or maybe used as a mechanism to regulate ions in response to chang-ing light conditions (17). A more complete analysis of thegenomes of Roseiflexus sp. strain RS1 and R. castenholzii will bepublished elsewhere.

Lipids. Roseiflexus sp. strains RS1 and RS2 showed nearlyidentical lipid profiles (Fig. 6), which are dominated by C30 toC36 wax esters, including n/iso- and iso/iso-alcohol-fatty acidcombinations, but not unsaturated forms (Table 1). We did notdetect any long-chain alkenes. Both strains also contained C13

to C17 fatty acids, C15 to C18 alcohols, and C19 to C21 diols, butdiol glycosides were not detected.

DISCUSSION

The Roseiflexus sp. strains that we obtained from Yellow-stone hot spring microbial mats closely resemble R. castenhol-zii, which was isolated from microbial mats at Nakabusa hotsprings in Japan. These isolates are similar in filamentousmorphology, the presence of bacteriochlorophyll a, the ab-sence of chlorosomes and bacteriochlorophyll c found in greensulfur bacteria and Chloroflexus spp., and their photohetero-trophic and dark aerobic metabolism (20) (Table 2). However,genomic analyses revealed that despite the inability of micro-biologists to grow R. castenholzii and Roseiflexus sp. strain RS1photoautotrophically, both of these isolates have all of theknown genes for enzymes of the 3-hydroxypropionate pathwayfor CO2 fixation (25) and thus the potential to acquire at leastsome of their cellular carbon from this metabolism. The twogenomic sequences revealed possible shared nutrient acquisi-tion strategies of Roseiflexus spp. (e.g., iron and phosphateuptake and possible nitrogen fixation) but also possible differ-ences, in particular that Roseiflexus sp. strain RS1 may alsoutilize organophosphates as a phosphorous source. The acqui-sition of phosphonates may be important in these alkalinesiliceous hot springs, as there is evidence that Synechococcus

FIG. 4. Growth rates of Roseiflexus sp. strains RS1 and RS2 as afunction of temperature, pH, and sulfide concentration. Error barsrepresent standard errors (n � 3).

FIG. 5. Relationship between Octopus Spring and MushroomSpring microbial mat metagenomic sequences recruited by Roseiflexusand Chloroflexus genomes and homologous sequences in the genomesof Roseiflexus sp. strain RS1 (blue), R. castenholzii (red), and Chlo-roflexus aurantiacus Y-400-fl (green).



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sp. strain B� contains phosphonate uptake and utilization genesas well (1, 5).

The Roseiflexus sp. isolates that we obtained are adapted toslightly higher temperatures (45 to 60°C; optimum, 55°C to60°C) than R. castenholzii (45 to 55°C; optimum, 50°C), con-sistent with the temperatures of the mats from which theycame. The possible existence of temperature-adapted FAPstrains was suggested by Bauld and Brock (4) on the basis ofecophysiological results. Nubel et al. (35) suggested the possi-bility of temperature-adapted strains of Roseiflexus spp. basedon differences in phylogeny and temperature distribution ofSSU rRNA sequences cloned from hot spring mats (6, 16).Differences in light absorption between strains RS1 and RS2may also reflect differential adaptation that might help explainthe cooccurrence of many Roseiflexus-like SSU rRNA se-quences at a single mat location. The Yellowstone and Japa-nese strains of Roseiflexus spp. are also optimally adapted tothe pH of the mats from which they were cultivated. Yellow-stone Roseiflexus sp. strain RS1 was found to be adapted tosulfide levels of 100 to 200 �M, which are typical of these mats(13). Our inability to demonstrate sulfide-associated photoau-totrophic growth might have been due to the use of inhibitoryconcentrations of sulfide; however, genes typically associatedwith sulfide metabolism were not detected in genomic analyses.R. castenholzii was not tested for sulfide tolerance, except that

the isolate was unable to grow photoautotrophically in thepresence of 200 to 400 �M sulfide (20).

Small differences in carbon source nutrition were notedamong R. castenholzii and the two new strains we studied(Table 2). Genetically, however, the Yellowstone isolates werequite divergent from R. castenholzii and were much moreclosely related to the native Roseiflexus species populationsthat predominate in the Yellowstone hot spring mats fromwhich they were cultivated, in terms of both SSU rRNA se-quence similarity (Fig. 1) and the match between genomichomologs (Fig. 5). Surprisingly, this situation is distinctly dif-ferent for two C. aurantiacus strains, J-10-fl from Japan andY-400-fl from Octopus Spring, which differ in gene content byonly four genes and which are also nearly identical (99.98%) inoverall DNA sequence (9).

The mats from which the new Roseiflexus sp. strains wereobtained also contain Roseiflexus-like 3-hydroxypropionategenes (25), consistent with the detection of carbon dioxidefixation activity associated with FAPs on the basis of sulfide-and hydrogen-stimulated incorporation of 13CO2 into wax es-ters and biomass during an early morning low-light period (46,47). However, the association between these lipid biomarkersand specific FAPs was unclear due to the poor match betweenthe lipids of FAP isolates available at the time and mat lipids.As shown in Table 1, the new genetically relevant Roseiflexus

FIG. 6. Lipids detected by gas chromatography-mass spectrometry analysis of total lipid extracts of Roseiflexus sp. strains RS1 and RS2. n,straight; i, � iso; Cx, number of carbon atoms (x); FAme, fatty acid methyl ester (derivatized fatty acid); OTMS, trimethyl silyl derivatized alcohol;1,2 DiOTMS, derivatized alkane-1,2-diol (alcohol groups on the first and second carbon atoms).



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sp. isolates produce wax esters that are nearly identical to thosefound in the mats. In particular, these isolates producebranched-chain wax esters for which no source organism waspreviously known. Furthermore, they do not appear to produceunsaturated wax esters, which are known to be produced byChloroflexus spp. (39, 43) but which are below detection limitsin the mats. Likewise, the new Roseiflexus sp. isolates do notproduce the long-chain, polyunsaturated alkenes typical forChloroflexus spp. or the diol glycosides that are abundant in R.castenholzii. In agreement, both alkenes and diol glycosides arealso only minor compounds in these mats (44, 45).

The results of this study show that our new isolates arerepresentative of predominant FAPs in Yellowstone mats fromwhich they were isolated, and genomic analysis of one isolatesupports the inference from labeling experiments that Rosei-flexus spp. are capable of fixing carbon dioxide during low-lightperiods of the diel cycle via the 3-hydroxypropionate pathway(46). In general, our findings demonstrate the importance ofhaving genetically relevant isolates to understand lipid biomar-kers and complex metabolic networks in microbial communi-ties. In this regard, the Roseiflexus sp. strain RS1 genome hasbeen very useful in analyses of mat metagenomes (Klatt et al.,unpublished data) and metatranscriptomes (Liu et al., unpub-lished data).

ACKNOWLEDGMENTS

We thank W. I. C. Rijpstra for analytical assistance. We thank theU.S. National Park Service for permission to conduct research inYellowstone National Park and for their cooperation in facilitating thework.

This study was supported by U.S. National Aeronautics and SpaceAdministration (NASA) grants NAG5-8824, 13468, and NX09AM87Gand NASA’s support of the Montana State University Thermal BiologyInstitute (NAG5-8807). Genomic sequences were obtained in collab-oration with the Joint Genome Institute (Walnut Creek, CA) and withsupport from Department of Energy grant DE-FG02-94ER20137 andNational Science Foundation grant MCB-0523100 (D.A.B.). The meta-genomics database employed in this work was created under the aus-pices of grant EF-0328698 from the Frontiers in Integrative BiologyProgram for the National Science Foundation to D.M.W.

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