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1.0 INTRODUCTION darkness will ensue each day at sunset in the natural Over the past two decades, there has been an enormous environment! The CO dehydrogenase and associated increase in the number of new thermophilic isolates with hydrogenase components are repressed by light and diverse metabolic strategies, consistent with the diversity induced by CO and darkness, and growth proceeds at of geochemical energy sources associated with continental a slow rate in the absence of light with a relatively low and submerged hydrothermal vents (reviewed in growth yield coefficient of 3.7 g (dry wt.) of cells per mol Reysenbach and Shock 2002). These metabolic pathways of CO oxidized (Champine and Uffen 1987). fuel chemolithoautotrophic primary production in many Thermophilic CO-utilizers produce H , acetate, or hydrothermal ecosystems such as hot springs in Yellowstone 2 methane as their primary end products, and it seems National Park (YNP) where opportunistic microbial reasonable to assume that they are involved in cross- metabolic adaptation is on display, often in brilliant color. feeding other chemolithotrophs such as methanogens,
Carboxydotrophic bacterial thermophiles capable of using acetoclastic methanogens, methanotrophs, H2-oxidizers, or CO(g) as their only source of energy and carbon have S-reducers. This chapter reviews the distribution of several been isolated from diverse geothermal habitats worldwide CO-utilizing thermophiles and describes the isolation of a (Sokolova et al. 2004; Table 1). This unique physiology CO-utilizing organism from Norris Geyser Basin, YNP. is not new to microbiology. First described by Uffen (1976), many phototrophic purple nonsulfur bacteria, 2.0 MATERIALS AND METHODS when cultured anaerobically in the dark, produce H at the 2 2.1 Strains expense of CO, which is converted to CO2. Using tritiated Carboxydothermus hydrogenoformans DSM 6008 water, Uffen showed that H2 was the product in what is and Thermoterrabacterium ferrireducens DSM 11255 now known as the water-gas shift reaction [CO + H2O = were obtained from the Deutsche Sammlung von CO2 + H2]. In these phototrophs, the oxidation of CO appears to be a backup metabolic strategy allowing the Mikroorganismen und Zellkulturen (Braunschweig, cells to maintain a slow growth rate on CO. In effect, the Germany). T. ferrireducens was cultured under strictly capacity for using an alternative energy source represents anaerobic conditions on modified freshwater medium, an important adaptation that, in the case of phototrophs, containing 30 mM glycerol as an electron donor and appears to be justified by the reliable assumption that carbon source, and 20 mM Na-fumarate as an electron
Table 1. CO-oxidizing hydrogenogenic microorganisms and their isolation locales. GenBank Organism Isolation Locale Habitat Reference Accession No. Kunashir, Kuril Islands Mud from hot Svetlichnyi et al. Carboxydothermus hydrogenoformans NC_002972 Kamchatka swamp, 68°C, pH 5.5 1991 Raoul Island, Kermadeck Littoral hydrothermal Svetlichnyi et al. Carboxydothermus restrictus Not Available Archipelago sample 1994 Gezyer Valley, Terrestrial hot spring, Carboxydocella thermautotrophica AY061974 Sokolova et al. 2002 Kamchatka 60°C, pH 8.6 Terrestrial hot spring, Thermosinus carboxydivorans AY519200 Norris Basin, YNP, USA Sokolova et al. 2004 50°C, pH 7.5 Thermococcus sp. AM4 AJ583507 East Pacific Rise (13o N) Hydrothermal vent Sokolova et al. 2004 Caldanaerobacter subterraneus subsp. Marine hydrothermal Sokolova et al. 2001; AF120479 Okinawa Trough pacificus vent, 110-130°C Fardeau et al. 2004 Caldanaerobacter subterraneus Not Available Kunashir, Kuril Islands Terrestrial hot spring Subbotina et al. 2003 strain 2707 166 GEOTHERMAL BIOLOGY AND GEOCHEMISTRY IN YELLOWSTONE NATIONAL PARK
acceptor. C. hydrogenoformans strain Z-2901 (DSM 6008) the cooS gene from C. hydrogenoformans. Phylogenetic was grown as previously described (Svetlichny et al. 1991). analyses were performed by the program Pileup (Genetic T. ferrireducens was mass cultured in a 200 L fermentor Computer Group) and CLUSTAL W. Bootstrap values at the Center of Marine Biotechnology ExSUF facility, were the percentages from a sampling of 1000. The genome o under a gas phase of 100% CO2 at 65 C. Late exponential sequencing project was conducted in collaboration with phase cells were harvested by centrifugation. The cell J. Eisen and colleagues at The Institute for Genomic paste, containing 150 g of cells (wet wt.), was frozen at Research (TIGR), and the primers devised by A. Lebedinski -80oC for purification of the Fe reductase. at the Russian Academy of Sciences in Moscow.
2.2 Sequencing Studies 2.3 Sampling An earlier study presented results of a genome survey Samples were collected from Norris Geyser Basin, within of C. hydrogenoformans strain Z-2901 (Gonzalez and the One Hundred Spring Plain, during June 2000 (Figure Robb 2000). Genomic DNA was extracted from cultures 1). A sample of sediment and geothermal water was of C. hydrogenoformans strain Z-2901 (DSM 60008), collected from a small geothermal pool containing visible and a genomic library was prepared using lambda Zip- organic matter, at ~ neutral pH (7.5) and a temperature Lox, EcoRI arms (Gibco BRL). Gene walking using the of 50oC (lat 44o43ʹ797ʺN, long 110o42ʹ506ʺW). The Vectorette II system (Genosys) and the CooS specific samples were used as inoculum for the isolation of CO- primer 5ʹ-CCA TCG ATA CTT TCA AAC GGC utilizing organisms using cultivation strategies defined GC-3ʹ was performed to complete the sequence of previously (Sokolova et al. 2004).
A B 3.0 RESULTS AND DISCUSSION 3.1 Insights from Sequencing Studies The complete genome sequence of C. hydrogenoformans has provided a gene inventory of a specialized thermophilic carboxydotroph. C. hydrogenoformans was originally described as growing only on CO and pyruvate (Svetlichny et al. 1991). A recent study (Henstra and Stams 2004) C D indicates that C. hydrogenoformans displays more versatile substrate utilization than previously suspected. For example, 9,10- anthraquinone-2,6-disulfonate (AQDS) can act as an alternative to protons as an electron acceptor, where the strain utilizes CO and grows at the normal rate but produces no
H2. One of the most unusual features of the annotated C. hydrogenoformans Z-2901 Figure 1. Sampling locations in Yellowstone National Park used for genome sequence is the presence of five enrichment of anaerobic CO-oxidizing strains. A, B. One Hundred Spring Plain, Norris Geyser Basin, showing the hot spring (80-95oC) containing FeIII oxides from dissimilar and apparently paralogous cooS which Thermosinus carboxydovorans was isolated. Sampling equipment shown genes (Figure 2). The operon encoding in B includes temperature probe, pH meter, and altimeter/barometer. C. Sampling CODH I is homologous and similar in location at Potts Basin. D. High temperature sampling with telescopic probe at full extension. gene order and content to the locus in Primary Energy Metabolism in Geothermal Environments 167
Rhodospirillum rubrum, which is induced # 1 2 3 4 5 by growth in the dark in the presence of 0 0.5 1.0 1.5 2.0 2.5 Mb CO. The occurrence of a cooA gene that cooS encodes a heme-containing transcriptional activator protein with marked similarity to a family of cAMP sensor proteins is of significant interest (Roberts et al. 2004). cooA cooS cooF cooF cooS This operon has been shown to sense CO CODH II and to regulate CODH synthesis and H2 production by R. rubrum and Azotobacter “ Moorella thermacetica ” vinelandii. The cooA homolog from locus “Rhodospirillum rubrum” locus C. hydrogenoformans has been cloned and cooS cooF cooHcooJ cooXcooLcooK cooM cooC cooA expressed in Escherichia coli, and shown to have similar sensing properties in vivo to the cooA from phototrophs (Youn et al. CODH I 2004). Figure 2. Carbon monoxide dehydrogenase (CODH) encoding loci from the full genome sequence of Carboxydothermus hydrogenoformans. The CODH I locus Directional transfer of genetic information corresponds to the Rhodospirillum rubrum dark-and CO-inducible operon that from Archaea to Bacteria is an area of active includes a cooS homolog and a multicomponent-membrane bound hydrogenase. speculation. For instance, in the genome CODH II is a three member putative operon including a cooS homolog with strong sequence similarity to the CODH from the archaeon, Archaeoglobus fulgidus. This sequencing project of C. hydrogenoformans CODH may function in generating reducing potential in the cell for biosynthesis we have found various genes showing (Svetlichnyi et al. 2000). The loci labeled 2, 4, and 5 have no assigned functions at highest homology to members of Archaea present. Locus 5 has a putative five gene operon that is identical in gene order to suggesting the relatively recent transfer the operon from the acetogenic Gram-positive bacterium, Moorella thermoacetica. of genetic material between Archaea and the bacterium C. hydrogenoformans. For physiology of CO-oxidation is widespread, and probably horizontal gene transfer to be feasible, we presume that global. This strain may also be capable of utilizing FeIII anaerobic bacteria and potential archaeal donors occur or selenite as additional electron acceptors, consistent in close physical proximity (Gonzalez and Robb 2000). with the relatively high abundance of Fe, S, and Se in the C. hydrogenoformans is a strictly anaerobic thermophilic Norris Basin environment. Consequently, the Yellowstone bacterium which utilizes CO and produces H2 and CO2 isolate may demonstrate a new mechanism of energy (Svetlichny et al. 1991). Methanogens in hydrothermal conservation, namely CO oxidation with FeIII reduction. vents are known to produce CO under certain conditions, Indeed, FeIII-oxides were present in the hot spring from such as H2 deficiency. These interspecies CO transfers which this isolate was obtained (Figure 1). The isolation of in natural volcanic environments may be examples of a neutrophilic strain from this environment also highlights mutualistic bacterial-archaeal interactions. Evidence the potential variation of hot spring environments. For supporting this hypothesis is forthcoming from the example, although many geothermal springs in Norris genome sequence data of carboxydotrophic bacteria. Basin are acidic, several springs exhibit pH values near 3.2 Isolation and Characterization Studies neutral.
The characterization of Thermosinus carboxidovorans (an The widespread occurrence of anaerobic CO-oxidation extreme thermophile from Norris Basin), which exhibits begs the question as to the significance of these organ- a relatively high growth yield on CO and corresponding isms in the “metabolic milieu.” CO is a key metabolite production of H (Sokolova et al. 2004), confirms that the 2 in methanogenesis and acetogenesis, major pathways in 168 GEOTHERMAL BIOLOGY AND GEOCHEMISTRY IN YELLOWSTONE NATIONAL PARK
Rhodococcus ruber type strain: DSM43338 (X80625) sulfovibrio vulgaris is grown
Ammonifex degensii KC4 (U34975) on pyruvate, it produces CO as a metabolic end product. 85 Thermacetogenium phaeum PB (AB020336) It is possible, therefore, that methanogens and sulfate re- 88 Anaerosinus glycerini DSM 5192(T) (AJ010960) ducers produce CO and, in Rhodospira trueperi (X99671) 83 return, are replenished with 59 H as a result of anaerobic Roseospira mediosalina L1-66 = BN 280 T (AJ000989) 2 99 CO oxidation. Interspecies 99 Rhodospirillum photometricum E-11 (D30777) CO transfers in natural vol- 55 canic environments may be Rhodospirillum rubrum ATCC 11170 (D30778) examples of mutualistic bac- Thermosinus carboxydivorans Nor1 (AY519200) terial-archaeal relationships along with subsequent ge- Selenomonas ruminantium K2 (AF161581) netic exchange, resulting in 100 Moorella thermoacetica ET-5a (AJ242494) a more efficient utilization of 97 available resources. Support- Moorella glycerini YS6 (U82327) 100 ing this assumption, several 84 100 Carboxydothermus hydrogenoformans (NC 002972) associations among anaero- 99 bic bacteria and methano- Thermoterrabacterium ferrireducens (U76364) 98 gens have been reported. Acetonema longum DSM 6540(T) (AJ010964) The reduction of Fe is a trait 93 Dendrosporobacter quercicolus DSM 1736(T) (AJ010962) shared by T. carboxydivorans 67 and T. ferrireducens, possibly Sporomusa acidovorans DSM 3132 Type (AJ279798) a consequence of the Fe-rich Caldanaerobacter subterraneus subsp. pacificus (AF174484) locale from which they were both isolated (One Hundred 96 Thermoanaerobacter ethanolicus 39E (L09164) Spring Plain, Norris Geyser 84 Thermoanaerobacter siderophilus SR4(T) (AF120479) Basin). T. ferrireducens is a 10 thermophilic dissimilatory FeIII-reducing bacterium
Figure 3. Phylogenetic dendogram of carboxydotrophic bacteria and related strains. The belonging to the Firmicutes CO-utilizing strains are Rhodospirillum rubrum, Thermosinus carboxydivorans, Moorella (Slobodkin and Wiegel thermoacetica, Carboxydothermus hydrogenoformans and Caldanaerobacter subterraneus subsp. 1997). We are currently pacificus. The maximum parsimony tree was created in PHYDIT and bootstrapped using 1000 replicates (bootstrap values over 50% are displayed). exploring the mechanism of Fe metabolism in T. ferrireducens and have purified global carbon cycling. There is also evidence that CO is a membrane-bound complex capable of FeIII reduction important in microbial sulfate reduction. Recent studies in vitro. Both T. ferrireducens and T. carboxydivorans are by Voordouw (2002) indicated that, although CO is toxic capable of organotrophic and lithoautotrophic growth with to most sulfate reducing bacteria, metabolic cycling of insoluble and soluble FeIII compounds; have a Gram-type CO is a critical feature for coupling electron transport in positive cell wall surrounded by an s-layer; and contain c- these organisms. Moreover, when the sulfate reducer De- type cytochromes. T. carboxydivorans is capable of growth Primary Energy Metabolism in Geothermal Environments 169
on a similar limited number of organic compounds and is common in hot spring communities. For horizontal gene very similar in growth physiology to C. hydrogenoformans transfer to be feasible, physical proximity and mutualistic and T. ferrireducens (Henstra and Stams 2004). The major or syntrophic relationships among bacteria and archaea exception is the inability of T. ferrireducens to grow on CO, would likely be important factors aiding DNA exchange and to produce H2. Conversely, T. ferrireducens appears to between the domains. Continuous evolutionary pressure be specialized to reduce metals, using FeIII preferentially to gain a dominant position among the anaerobic as a terminal electron acceptor. Since T. ferrireducens has organisms in a geothermal environment appears to no periplasmic space, the mechanism of FeIII reduction be evident. Microorganisms that have obtained a is expected to be different from that in Gram-negative competitive advantage through evolution may have sorted bacteria, and indeed it appears that our preparation is able through combinations of physiological traits either by to carry out cytochrome reduction in response to FeIII, acquiring the required genetic material or by developing unlike Gram-negative bacteria (A. Slobodkin, unpublished associations with microorganisms capable of performing results). complementary functions.
4.0 SUMMARY ACKNOWLEDGMENTS The phylogenetic context of T. carboxydivorans and C. Samples from Yellowstone National Park were taken un- hydrogenoformans is shown relative to several other Gram- der permit number 4056. We gratefully acknowledge the positive bacteria and those related to R. rubrum (Figure help of Steven Miller, who accompanied us with GPS sur- 3). It is evident that CO-dependent hydrogenogenic vey equipment on sampling surveys to Norris Basin, Potts metabolism occurs in a diverse phylogenetic context, Area, and the Glen Africa Basin. Research in the authors’ and is accompanied by a varied repertoire of alternative laboratories is supported by grants from the National Sci- trophic strategies. Chemoautotrophic pathways may ence Foundation (Grant MCB02383387 in the Micro- have been present in ancestral lineages of Archaea and bial Observatories Program); NATO (CLG. 978269); Bacteria (Reysenbach and Shock 2003). An important the Cooperative Research and Development Foundation question that arises in consideration of the phylogenetic (CRDF, Grant No. RB2-2379-MO-02); NASA (with tree is whether carboxydotrophy was widespread and Dr. DiRuggiero); the US Department of Energy (with subsequently lost by most lineages, or whether cooS- The Institute of Genomic Research, [TIGR] on the genome containing gene clusters were subject to widespread lateral sequencing of C. hydrogenoformans); and the ‘Molecu- gene transfer. The closest relative of C. hydrogenoformans lar and Cell Biology’ Program of the Russian Academy of is currently T. ferrireducens, a non-carboxydotroph that Sciences. J. M. G. acknowledges support from the Spanish was isolated from Norris Basin close to the location where Ministry of Science and Technology through a Ramon y T. carboxydivorans was found (Figure 1). The variety Cajal contract and grant REN2002-00041. Publication of alternative chemoautotrophic and organotrophic is contribution number 05-109 from the Center of Marine metabolisms in closely related microorganisms (based on Biotechnology. 16S rDNA) suggests that horizontal gene transfer may be 170 GEOTHERMAL BIOLOGY AND GEOCHEMISTRY IN YELLOWSTONE NATIONAL PARK
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