1. Genomics and Evolutionary Biology
  2. Microbiology and Infectious Disease
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Insight into the evolution of microbial metabolism from the deep-branching bacterium, Thermovibrio ammonificans

  1. Donato Giovannelli Is a corresponding author
  2. Stefan M Sievert
  3. Michael Hügler
  4. Stephanie Markert
  5. Dörte Becher
  6. Thomas Schweder
  7. Costantino Vetriani Is a corresponding author
  1. Rutgers University, United States
  2. National Research Council of Italy, Italy
  3. Institute for Advanced Studies, United States
  4. Tokyo Institute of Technology, Japan
  5. Woods Hole Oceanographic Institution, United States
  6. DVGW-Technologiezentrum Wasser, Germany
  7. Institute of Pharmacy, Ernst-Moritz-Arndt-University Greifswald, Germany
  8. Ernst-Moritz-Arndt-University Greifswald, Germany
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Cite as: eLife 2017;6:e18990 doi: 10.7554/eLife.18990

Abstract

Anaerobic thermophiles inhabit relic environments that resemble the early Earth. However, the lineage of these modern organisms co-evolved with our planet. Hence, these organisms carry both ancestral and acquired genes and serve as models to reconstruct early metabolism. Based on comparative genomic and proteomic analyses, we identified two distinct groups of genes in Thermovibrio ammonificans: the first codes for enzymes that do not require oxygen and use substrates of geothermal origin; the second appears to be a more recent acquisition, and may reflect adaptations to cope with the rise of oxygen on Earth. We propose that the ancestor of the Aquificae was originally a hydrogen oxidizing, sulfur reducing bacterium that used a hybrid pathway for CO2 fixation. With the gradual rise of oxygen in the atmosphere, more efficient terminal electron acceptors became available and this lineage acquired genes that increased its metabolic flexibility while retaining ancestral metabolic traits.

https://doi.org/10.7554/eLife.18990.001

eLife digest

Life may have arisen on our planet as far back as four billion years ago. Unlike today, the Earth’s atmosphere at the time had no oxygen and an abundance of volcanic emissions including hydrogen, carbon dioxide and sulfur gases. These dramatic differences have led scientists to wonder: how did the ancient microorganisms that inhabited our early planet make a living? And how has microbial life co-evolved with the Earth?

One way to answer these questions is to study bacteria that live today in environments that resemble the early Earth. Deep-sea hydrothermal vents are regions of the deep ocean where active volcanic processes recreate primordial conditions. These habitats support microorganisms that are highly adapted to live off hydrogen, carbon dioxide and sulfur gases, and studying these modern-day microorganisms could give insights into the earliest life on Earth.

Thermovibrio ammonificans is a bacterium that was obtained from an underwater volcanic system in the East Pacific. Giovannelli et al. have now asked if T. ammonificans might have inherited some of its genetic traits from a long-gone ancestor that also thrived off volcanic gases. The genetic makeup of this microorganism was examined for genes that would help it thrive at a deep-sea hydrothermal vent. Next, Giovannelli et al. compared these genes to related copies in other species of bacteria to reconstruct how the metabolism of T. ammonificans might have changed over time.

This approach identified a group of likely ancient genesthat allow a microorganism to use chemicals like hydrogen, carbon dioxide and sulfur to fuel its growth and metabolism. These findings support the hypothesis that an ancestor of T. ammonificans could live off volcanic gases and that the core set of genes involved in those activities had been passed on, through the generations, to this modern-day microorganism. Giovannelli et al. also identified a second group of genes in T. ammonificans that indicate that this bacterium also co-evolved with Earth’s changing conditions, in particular the rise in the concentration of oxygen.

The findings of Giovannelli et al. provide insight into how the metabolism of microbes has co-evolved with the Earth’s changing conditions, and will allow others to formulate new hypotheses that can be tested in laboratory experiments.

https://doi.org/10.7554/eLife.18990.002

Introduction

Deep-branching, anaerobic, thermophilic Bacteria and Archaea inhabit relic environments that resemble the early Earth (Baross and Hoffman, 1985; Martin et al., 2008). Thermophily (Di Giulio, 2003, 2000), anaerobic metabolism (Baross and Hoffman, 1985; Martin et al., 2008; Schopf, 1983) and reliance on substrates of geothermal origin are among the proposed ancestral traits of these microorganisms (Baross and Hoffman, 1985; Di Giulio, 2003, 2000; Lane et al., 2010; Martin et al., 2008; Russell and Martin, 2004; Schopf, 1983). At the same time, their lineages have co-evolved with Earth and their genomes also carry more recently acquired traits. Therefore, these microorganisms can be used as models to reconstruct the evolution of metabolism.

Thermovibrio ammonificans is part of the phylum Aquificae, a deep-branching group of thermophilic bacteria found in geothermal environments (Lebedinsky et al., 2007; Sievert and Vetriani, 2012). Based on phylogenetic analyses of the 16S rRNA gene as well as whole genomes, Aquificae are believed to be the earliest bacterial lineages having emerged on Earth along with the phyla Thermotogae and Thermodesulfobacteria (Di Giulio, 2003; Pitulle et al., 1994; Battistuzzi et al., 2004) (Figure 1, Figure 1—figure supplement 1). All the cultured members of this phylum are chemolithoautotrophs that use hydrogen as an energy source, have optimum growth temperatures between 65˚C and 95°C and rely on the reductive tricarboxylic acid cycle (rTCA) to convert carbon dioxide into biomass (Hügler et al., 2007; Hügler and Sievert, 2011; Sievert and Vetriani, 2012) (Table 1), making them ideal candidates to investigate the evolution of early metabolism.

Figure 1 with 1 supplement see all
Phylogenetic tree of 16S rRNA sequences computed from the current version of the aligned 16S rRNA database obtained from the arb-SILVA project (http://www.arb-silva.de/).
https://doi.org/10.7554/eLife.18990.003
Table 1

Characteristic of representative members of Aquificae phylum.

https://doi.org/10.7554/eLife.18990.005
FamilyOrganismGrowth tempEnergy sourceCarbon sourceTerminal electron acceptorIsolated fromGenome sequence accession numberReferences
AquificaceaeAquifex aeolicus VF595°CH2CO2O2Underwater volcanic vents, Aeolic Islands Sicily, ItalyNC_000918.1(Deckert et al., 1998; Huber et al., 1992)

Hydrogenivirga sp.128–5 R1-1175°CS2O32-, S0CO2NO3-, O2Lau Basin hydrothermal vent area, Pacific OceanNZ_ABHJ00000000(Nakagawa et al., 2004; Reysenbach et al., 2009)

Hydrogenobacter thermophilus TK-675°CH2CO2O2Hot springs in Izu and Kyushu, JapanNC_017161.1(Arai et al., 2010; Kawasumi et al., 1984)

Hydrogenobaculum sp. Y04AAS1*----Marine hydrothermal area, Vulcano Island, ItalyNC_011126.1(Reysenbach et al., 2009; Stohr et al., 2001)

Thermocrinis ruber DSM 1217385°CH2, S2O32-, S0CO2O2Octopus Spring, Yellowstone National Park, Wyoming, USAPRJNA75073(Huber et al., 1998)
DesulfurobacteriaceaeBalnearium lithotrophicum 17S70–75˚CH2CO2S0Deep-sea hydrothermal vent chimney, Suiyo Seamount, JapanNot sequenced(Takai et al., 2003b)

Desulfurobacterium thermolithotrophum DSM 1169970°CH2CO2S0, S2O32-, SO2-Deep-sea hydrothermal chimney, mid-Atlantic ridge, Atlantic OceanNC_015185.1(Göker et al., 2011; L'Haridon et al., 1998)

Phorcysia thermohydrogeniphila HB-875°CH2CO2S0, NO3-Tube of Alvinella pompejana tubeworms, deep-sea hydrothermal vents 13 ˚N, East Pacific Rise, Pacific OceanNot sequenced(Pérez-Rodríguez et al., 2012)

Thermovibrio ammonificans HB-175°CH2CO2S0, NO3-Deep sea hydrothermal vents 9˚N, East Pacific Rise, Pacific OceanNC_014926.1(Giovannelli et al., 2012; Vetriani et al., 2004)
HydrogenothermaceaeHydrogenothermus marinus VM165°CH2CO2O2(1–2%)Deep sea hydrothermal vents 9˚N, East Pacific Rise, Pacific OceanNot sequenced(Stohr et al., 2001)

Persephonella marina EX-H173°CS0CO2O2, NO3-Deep sea hydrothermal vents 9˚N, East Pacific Rise, Pacific OceanNC_012440.1(Götz et al., 2002; Reysenbach et al., 2009)

Sulfurihydrogenibium sp. YO3AOP1170°CS2O32-, S0CO2O2Calcite Hot Springs, Yellowstone National Park, USANC_010730.1(Reysenbach et al., 2009; Takai et al., 2003a)

Venenivibrio stagnispumantis70°CH2CO2O2Terrestrial hot spring Champagne Pool, Waiotapu, New ZealandNot sequenced(Hetzer et al., 2008)
Incertae sedisThermosulfidibacter takai ABI70S6T70°CH2CO2S0deep-sea hydrothermal field at Southern Okinawa Trough, JapanNot sequenced(Nunoura et al., 2008)
  1. *– Strain not formally described whose genome sequence is available. For these strains, the physiological information reported in Table S1 have been collected from MIG associated with the sequencing or from the closest validly published species.

The ability to synthesize new biomass from inorganic precursors, i.e. autotrophic carbon fixation, is a critical step in the global carbon cycle and is considered a key invention during the early evolution of metabolism (Braakman and Smith, 2012; Fuchs, 2011; Hügler and Sievert, 2011). Among the six known pathways of carbon fixation (reviewed in Fuchs, 2011; Hügler and Sievert, 2011), the rTCA cycle (present in the Aquificae, among others) and the reductive acetyl-CoA pathway (present in acetogenic bacteria and methanogens), represent good candidates for the ancestral carbon fixation pathway (Martin and Russell, 2003; Russell and Hall, 2006). The emergence of a reductive acetyl-CoA pathway has been associated with the FeS-rich minerals at alkaline hydrothermal vents (Martin and Russell, 2007, 2003; Wächtershäuser, 1988a) and the presence of homologous core enzymes in both Bacteria and Archaea potentially support its ancestral nature (Fuchs, 2011; Russell and Martin, 2004). The rTCA cycle is considered the modern version of a proposed prebiotic autocatalytic cycle fueled by the formation of the highly insoluble mineral pyrite in sulfur-rich hydrothermal environments (Wächtershäuser, 1988b, 1990). The rTCA cycle is widespread among anaerobic and microaerophilic bacteria including all Aquificae, chemolithoautotrophic Epsilonproteobacteria, Chlorobi, and Nitrospirae (Berg, 2011; Fuchs, 2011; Hügler and Sievert, 2011) in addition to few other bacterial strains. A recent phylometabolic reconstruction hypothesized that all extant pathways can be derived from an ancestral carbon fixation network consisting of a hybrid rTCA cycle / reductive acetyl-CoA pathway (Braakman and Smith, 2012).

Here, we used a comparative genomic approach coupled to proteomic analyses to reconstruct the central metabolism of Thermovibrio ammonificans (Giovannelli et al., 2012; Vetriani et al., 2004), and provide evidence of ancestral and acquired metabolic traits in the genome of this bacterium. We suggest that the last common ancestor within the Aquificae possessed the reductive acetyl-CoA pathway in addition to the previously described rTCA cycle. Furthermore, we show that these two pathways may still coexist within the Desulfurobacteraceae lineage. The simultaneous presence of both pathways of carbon fixation may represent a modern analog of the early carbon fixation phenotype, and suggests that the redundancy of central metabolic pathways could have been common in ancestral microorganisms.

Results

General genome structure and central metabolism of T. ammonificans.

The genome of T. ammonificans HB-1 consists of one chromosome and one plasmid of 1,682,965 bp and 76,561 bp, respectively (Figure 2). The genome encodes for 1890 genes, 1831 of which are protein coding genes and 75.16% of the protein coding genes could be assigned a putative function (Giovannelli et al., 2012). The chromosome contains three ribosomal RNA operons, the first two with a 5 S-23S-16S alignment (coordinates 149,693–152,859 bp and 1,168,776–1,172,141 bp antisense strand, respectively) and the other with a 16 S-23S-5S (coordinates 1,604,527–1,609,585 bp). The second copy of the operon is flanked by the largest CRISPR found in the genome (Figure 2, circle 6). Several other repeats were identified in the chromosome (Figure 2, circle 5). The plasmid contained mainly ORFs with unknown hypothetical functions (Figure 2, circle 9) with the exception of a putative RNA polymerase sigma factor of probable archaeal origin, a DNA Topoisomerase I (43.2% similarity to Hydrogenivirga sp. 128–5 R1-6), a type II secretion protein E (identified also in the preliminary proteome, Figure 3, Figure 3—source data 1) and an ArsR-like regulatory protein, both of which had homologs among the Firmicutes. All other plasmid genes were ORFans with no significant similarities in the database and contained highly repeated DNA (Figure 2, circle 5).

Thermovibrio ammonificans HB-1 genomic map highlighting main genomic features.

The circular chromosome and plasmid are drawn together. Several other repeats were identified in the chromosome (circle 5). From the outer circle: 1 – Dimension of the genome in base pairs (chromosome in violet, plasmid in pink); 2 and 3 – sense and antisense coding sequences colored according to their COGs classification (see legend in the figure); 4 – similarity of each coding sequence with sequences in the non-redundant database; 5 – position of tandem repeat in the genome. A total of 46 tRNA were identified, comprising all of the basic 20 amino acid plus selenocysteine; 6 – position of CRISPRs; 7 – GC mol% content, the red line is the GC mean of the genome (52.1 mol%); 8 – GC skew calculated as G+C/A+T; 9 – localization of coding genes arbitrarily colored according to the metabolic pathways reconstructed (see Figure 1). Colors are consistent trough the entire paper. Total dimension and accession number for the chromosome and plasmid are given.

https://doi.org/10.7554/eLife.18990.006
Figure 3 with 2 supplements see all
Central metabolism of T. ammonificans HB-1.

Enzyme names are reported together with the gene locus number (Theam_number). Primary compounds involved in reactions were also reported, however visualized reactions are not complete. Pathways were arbitrarily color coded according to their reconstructed function and are consistent throughout the paper. Solid shapes represent genes for which the enzyme was found in the proteome, while outlined shapes were only identified in the genome. Circled numbers 1 to 6 represent reaction numbers of the putative reductive acetyl-CoA pathway as described in the text. Abbreviations: NITRATE AMMONIFICATION: NapCMADGH – Periplasmic nitrate reductase complex; NirA – Putative nitrite reductase; AmtB – ammonia transporter; GlnA – L-Glutamine synthetase; GltA – Glutamate synthetase. HYDROGEN OXIDATION: HynABC – Ni-Fe Membrane bound hydrogenase; FrhACB – Cytosolic Ni-Fe hydrogenase/putative coenzyme F420 hydrogenase; HupD – Cyrosolic Ni-Fe hydrogenase maturation protease; HypA-F – Hydrogenases expression/synthesis accessory proteins; EchABCEF – Ech membrane bound hydrogenase complex; HydAB – Cytosolic Ni-Fe hydrogenases potentially involved in ferredoxin reduction; Hdr? – Missing heterodisulfide reductase CoB-CoM; FdhABC – Formate dehydrogenase. ENERGY PRODUCTION: NADH dehydrogenase and ATP synthetase are reported without the names of the single units; Nhe – Sodium/hydrogen symporter. SULFUR REDUCTION: Sqr – Putative sulfate quinone reductase involved in sulfur respiration; Nsr? – FAD/NAD nucleotide-disulphide oxidoreductase; Aps – Sulfate adenylyl transferase and kinase involved in assimilation of sulfur. FLAGELLAR COMPLEX: for simplicity single unit names are not reported. REDUCTIVE ACETYL-CoA: Fhs – Proposed reverse formyl-THF deformylase; PurHT – Formyltransferases (phosphoribosylaminoimidazolecarboxamide and phosphoribosylglycinamide) FolD – Methenyl-THF cyclohydrolase and dehydrogenase; MetVF – Methylene-THF reductase; MetR – Putative methyl-transferase; CodH – Carbon monoxyde dehydrogenase; AcsA – Acetyl-CoA ligase/synthase; AccABCD – Acetyl-CoA carboxylase. REDUCTIVE CITRIC ACID CYCLE: AclAB – ATP-citrate lyase; Mdh – Malate dehydrogenase; FumAB – Fumarate hydratase; FrdAB – Fumarate reductase; SucCD – Succinyl-CoA synthetase; OorABCD – 2-Oxoglutarate synthase; Idh2 – Isocitrate dehydrogenase/2-oxoglutarate carboxylase; AcnB – Aconitate hydratase; PorABDG – Pyruvate synthase; PycAB - Pyruvate carboxylase; PpsA – Phosphoenolpyruvate synthase water dikinase; PyK – Pyruvate:water dikinase. GLUCONEOGENESIS: Eno – Enolase; Pgm – Phosphoglycerate mutase; Pgk – Phosphoglycerate kinase; Gapor – Glyceraldehyde-3-phosphate dehydrogenase; Gapdh – Glyceraldehyde 3-phosphate dehydrogenase; Fba – Predicted fructose-bisphosphate aldolase; Tim – Triosephosphate isomerase; Fbp – Fructose-1,6-bisphosphatase I; Pgi – Phosphoglucose isomerase; Pgm – Phosphoglucomutase.

https://doi.org/10.7554/eLife.18990.007

By combining genome-scale analyses with known physiological traits, we were able to reconstruct the metabolic network of T. ammonificans (Figure 3). As part of the central metabolism of T. ammonificans, we identified the major pathways for carbon fixation, hydrogen oxidation and nitrate reduction to ammonia, and confirmed them by identifying the corresponding expressed enzymes in the proteome (Figure 3, Figure 3—source data 1). Further, we compared the central metabolism of T. ammonificans with closely related species within the Aquificae, and with ecologically similar Epsilonproteobacteria. Comparative genomic analyses with closely related strains provide information about the evolutionary history of the group, while comparison with phylogenetically distant, but ecologically similar species may reveal common adaptive strategies to similar habitats.

The proteome of T. ammonificans

780 proteins, comprising approximately 43% of all predicted ORFs in the database, were identified in cell samples of T. ammonificans grown under nitrate reducing conditions (Figure 3, Figure 3—source data 1), indicating a very high congruence between database entries and sample peptides. Among the most abundant proteins were – besides those involved in general housekeeping functions like the translation elongation factor Tu, ribosomal proteins and chaperones – many rTCA cycle enzymes. The four key enzymes of the rTCA cycle (see next paragraph for details) together yielded a relative abundance of 6% of all identified proteins in the samples. Most of the enzymes putatively involved in the reductive acetyl-CoA pathway (Table 2), including the carbon-monoxide dehydrogenase (CodH), were also detected, although at a lower abundance of 1.25% of all proteins.

Table 2

Enzymes involved in the putative reductive acetyl-CoA pathway in T. ammonificans HB-1, closest relative homolog and homologs within the Aquificae phylum.

https://doi.org/10.7554/eLife.18990.011
Desulfurobac
teriaceae
Hydrogenothe
rmaceae
Aquificaceae
ReactionPutative
enzyme
Putative gene locusClosest relativePutative
origin
D. thermolitho
trophum
Desulfuro
bacterium
sp. TC5-1
P. marinaSulfurihydro
genibium sp. YO3AOP1
H. themophilusHydrogeno
baculum sp. Y04AAS1H
Hydrogeni
virga sp. 128–5 R1-6
T. ruberA. aeolicus
 1Formate
dehydrogenase
Theam_1020Nitratiruptor
sp.
SB155-2 sim.
55% YP_001357016
Methanogensf.e.§ - 30%
YP_00428203#
-**56% YP_002730364f.e.§ - 32% YP_001930236f.e.§ - 33% YP_003433330--f.e.§ - 27% YP_003474076-
 2*5-formyltetra
hydrofolate
cyclo-ligase
Theam_1206Hydrogenivirga
sp. 128–5
R1-1 sim.
41% WP_008285842
Deltaproteo
bacteria / Gram +
41% YP_00428133944% WP_022846876.1f.e.§ - 36% YP_002729868f.e.§ - 38% YP_001931834f.e.§ - 38% YP_003431710f.e.§ - 30% YP_00212153941% WP_00828584240% YP_00347298143% 1SOU_A

formyltetra
hydrofolate
deformylase /
hydrolase
Theam_0826Hydrogenivirga
sp. 128–5
R1-1 sim.
70% WP_008287030
Bacteria82% YP_00428107777% WP_022846479----70% WP_00828703065% NP_214247-

phosphori
bosylglycina
mide formyl
transferase 2
Theam_1211P. marina
sim.
76% YP_002731257
Methanogens
/ Deltaproteo
bacteria
78% YP_00428133564% WP_02284751276% YP_00273125773% YP_001931730f.e.§ - 28% YP_00343307267% YP_00749947974% WP_008288476f.e.§ - 25% YP_003473050f.e.§ - 30% NP_213168

phosphori
bosylaminoi
midazole
carboxamide formyltransferase
/IMP cyclohydrolase
Theam_0328P. marina
sim.
59% YP_002731268
Aquificae / 
Bacteria
86% YP_00428168178% WP_02284735959% YP_00273126856% YP_00193123953% YP_00343327050% YP_00749947357% WP_00828836052% YP_00347433756% NP_214344
 3methylenetetra
hydrofolate
 dehydrogenase / methenyltetra
hydrofolate cyclohydrolase
Theam_1261A. aeolicus sim.
60%
NP_214304
Aquificae /
Gram +
87% YP_00428114774% WP_02284621755% YP_00273147658% YP_00193124059% YP_00551131858% YP_001931240-58% YP_00347310460% NP_214304
 4methylenetetra
hydrofolate reductase
Theam_0919Methanosrcina barkeri sim. 48%
YP_305816
Methanogens87% YP_00428114774% WP_02284621755% YP_00273147658% YP_00193124059% YP_00551131858% YP_002121558f.e.§ - 24% WP_00828678358% YP_00347310460% NP_214304
 55-methyltetra
hydrofolate
corrinoid/iron sulfurprotein
 methyltransferase
Theam_1184Clostridium glycolicum
sim.
42% WP_018589788
Gram +82% YP_00428148472% WP_022846603f.e.§ - 31% YP_002729885f.e.§ - 38% YP_001930381f.e.§ - 37% YP_003432344f.e.§ - 34% YP_002122031f.e.§ - 38% WP_008287326f.e.§ - 36% YP_003473438f.e.§ - 34% NP_213375
 6Carbon-monoxide
 dehydrogenase
Theam_1337Candidatus Methano
peredens
sp. BLZ1 sim.
56% KPQ43483
Archaea / Gram +87% WP_013638427
38%
WP_013638030
76% WP_02284714341% YP_002729904------
  1. * – Reactions are numbered according to Figure 1. For reaction two we reported the possible enzymes that could substitute the missing 10-fomyl-THF synthetase (Fhs). The enzymes are listed in order of decreasing likelihood of their involvement in the reaction based on putative substrate affinity.

  2. † – Reaction six is catalyzed by the Acs/CodH complex, reported here separately.

  3. ‡ – Putative origin of the gene was calculated as consensus taxonomic assignment of the first 100 bastp hits against the nr database. Double assignment implies equal number of assignments to the two taxonomic groups.

  4. § – f.e. = functional equivalent annotated in the genome with similarity below 40% with T. ammonificans equivalent gene. Not considered a true homolog in the present study.

  5. # – Pairwise similarity to T. ammonificans translated gene and accession number of the homologs.

  6. ** – Missing homolog or functional annotated equivalent in the genome.

Carbon fixation

The genome and proteome of T. ammonificans (Figure 3) supports previous evidence, based on detection of key genes and measurements of enzyme activities, that carbon fixation occurs via the rTCA cycle (Hügler et al., 2007). The rTCA is widespread among anaerobic and microaerophilic bacteria including all Aquificae, chemolithoautotrophic Epsilonproteobacteria, Chlorobi, Nitrospina aaaand Nitrospirae, in addition to few other bacterial strains (e.g. Desulfobacter hydrogenophilus, Magnetococcus marinus MC-1 and the endosymbiont of the vent tubeworm, Riftia pachyptila; Hügler and Sievert, 2011). We identified all the genes responsible for the functioning of the rTCA in the genome and proteome of T. ammonificans (Figure 3), including the key enzymes fumarate reductase (frdAB, gene loci Theam_1270–1275), ATP-citrate (pro-S)-lyase (aclAB, 1021–1022), 2-oxoglutarate synthase (oorABCD, 1410–1413) and isocitrate dehydrogenase (idh2, 1023). T. ammonificans synthesizes 2-oxoglutarate from succinyl-CoA rather than via citrate, a feature in common with methanogenic Archaea and Desulfurococcales, and present in numerous strict anaerobes that use the rTCA cycle (Fuchs, 2011). Comparative analyses showed that several genes associated with the rTCA cycle and the gluconeogenesis pathway were highly conserved in all Aquificae, with the exception of the genes coding for the ATP-citrate lyase and the isocitrate-dehydrogenase, which are absent in the Aquificaceae (see details below) (Hügler et al., 2007). In general, T. ammonificans genes had a high percent identity to the genes of D. thermolithotrophum, its closest relative whose genome sequence is available.

Two separate variants of the rTCA cycle are known (Braakman and Smith, 2012; Hügler et al., 2007). In the two-step variant – also known as symmetric variant, with respect to the enzymatic reaction catalyzed in the two arcs of the rTCA cycle – citrate cleavage is accomplished by the combined action of the enzymes citryl-CoA synthetase (CCS) and citryl-CoA lyase (CCL) and the carboxylation of 2-oxoglutarate is catalyzed by 2-oxoglutarate carboxylase and oxalosuccinate reductase (Aoshima, 2007; Aoshima and Igarashi, 2008; Hügler and Sievert, 2011). This variant of the rTCA cycle was previously proposed as ancestral (Braakman and Smith, 2012; Hügler et al., 2007). By contrast, in the one-step variant of the rTCA cycle, also known as asymmetric variant and more widely distributed, the cleavage of citrate and the carboxylation of 2-oxoglutarate are performed in a one-step fashion (Braakman and Smith, 2012; Hügler et al., 2007)(Figure 3, Figure 3—figure supplement 1). This variant is found in T. ammonificans.

The genome of T. ammonificans also codes for an incomplete reductive acetyl-CoA pathway (Figure 3 and Table 2). The key enzyme of this carbon fixation pathway, which is found in acetogens, methanogens, sulfate-reducers and anaerobic ammonium oxidizers (anammox; Berg et al., 2010), is the bifunctional heterotetramer enzyme carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS). The reductive acetyl-CoA pathway is believed to be the most ancient carbon fixation pathway on Earth (Fuchs, 2011). In T. ammonificans, the genes coding for the CO-dehydrogenase (codh catalytic subunit Theam_1337; Table 2 and Figure 3) is present and also expressed. However, rather than coding for the classical bifunctional type II CodH, T. ammonificans codes for the type V, which is present in numerous deep-branching organisms (Figure 4) and whose function is unknown (Techtmann et al., 2012). Sequence analysis of the T. ammonificans CodH protein sequence revealed the presence of conserved residues necessary for the interaction with Acs and for the coordination of the NiFeS cluster. We hypothesize that the type V CodH, which, based on structural and size considerations may represent the most ancestral version of this class of enzymes (Frank Robb, personal communication), may catalyze the reduction of CO2 to CO. The codh gene of T. ammonificans shared high similarity with that of D. thermolithotrophum (87 similarity), while the similarity with the next closest homologous genes dropped below 54%. The genome of D. thermolithotrophum also codes for a classical bifunctional Type II CodH (Dester_0417 and Dester_0418; Figure 4) in addition to the type V CodH. The two genomes also share a CodH iron-sulfur accessory protein (Theam_0999 and Dester_0418, 81% similarity). Additional laboratory analyses will be required to confirm the role of the type V CodH in carbon fixation.

Neighbor-joining tree showing the position of the carbon monoxide dehydrogenase, CodH, of T. ammonificans (in bold).

Bootstrap values based on 1000 replications are shown at branch nodes. Only bootstrap values above 50% are reported. Bar, 10% estimated substitutions. The metabolism of each organism is reported on the side.

https://doi.org/10.7554/eLife.18990.012

Gluconeogenesis

T. ammonificans uses the Embden-Meyerhof-Parnas pathway to synthesize pentose and hexose monosaccharides. We identified in the genome the key genes of this pathway, fructose-1,6-biphosphatase (fbp, Theam_1323; identified also in the proteome, Figure 3) and all other genes necessary for the functioning of the pathway (Figure 3). While the fbp gene is present in Desulfurobacterium thermolithotrophum, and in the genome of Sulfurohydrogenibium sp. YO3AOP1, Persephonella marina and Hydrogenivirga sp. 128–5 R1-6, it is absent from the genome of A. aeolicus (Deckert et al., 1998) and H. thermophilus, suggesting that in those species an alternative unidentified pathway may be active.

Hydrogen oxidation

Hydrogen is the only electron donor used by T. ammonificans and its importance in the metabolism of this bacterium is reflected by the diversity of hydrogenases found in the genome, whose encoding genes are organized in two large clusters (Figure 2 circle 9). The first cluster is composed of eight genes encoding for the complete Group 4 Ech hydrogenases (echABCDEFG, Theam_0476–0482) and the alpha, beta, gamma and delta subunits of the Group 3b multimeric cytoplasmic Hyd sulfhydrogenase (hydABCD, Theam_0484–0487). The second hydrogenase cluster in the genome codes for the maturation protein HypF (hypF, Theam_1116), the three subunit of the Group 3a cytoplasmic F420-reducing hydrogenase Frh (frhACB, Theam_1117–1119) and the Group 1 hydrogenases Hyn (hynABC, Theam_1121–1123) with associated maturation factors/chaperones Hyp (Theam_1124–1128). We also found other hydrogenase maturation factors (Theam_0922–0924) and the formate dehydrogenase Fdh (fdhABC, Theam_1020 and Theam_0966–0967 respectively) scattered in the genome.

We reconstructed the phylogeny of T. ammonificans [NiFe]-hydrogenases (Figure 5), and we assigned them to known classes of hydrogenases (Groups 1 through 4) according to their phylogenetic position in the tree and the presence of known conserved amino acid motifs typical of each group (Vignais and Billoud, 2007). Group 1 [NiFe]-hydrogenases are normally coupled to anaerobic respiration through electron transfer to the membrane quinone and menaquinone pool. Comparative analyses of the hydrogenases revealed that the Group 1 cluster in T. ammonificans (Hyn) is more similar to the hydrogenases found in Deltaproteobacteria than to homologs in other Aquificae (Figure 5). This finding raises interesting questions about the origin of the hydrogen oxidation Group 1 enzymes in T. ammonificans and suggests that an event of lateral gene transfer occurred between T. ammonificans and the Deltaproteobacteria. Further, the periplasmic nitrate reductase (napA) and the nap operon structure in T. ammonificans also appears to be closely related to that of Deltaproteobacteria, suggesting that a horizontal gene transfer event involved the entire hydrogen oxidation (Hyn)/nitrate reduction respiratory pathway (see discussion).

Neighbor-Joining tree showing the position and classification of the [NiFe]-hydrogenases found in the genome of T.ammonificans.

Bootstrap values based on 1000 replication are shown at branch node. Bar, 20% estimated substitution.

https://doi.org/10.7554/eLife.18990.013

Contrarily to members of the Aquificaceae and Hydrogenothermaceae, T. ammonificans and D. thermolithotrophum genomes code for both Group 3 cytoplasmic hydrogenases, Group 3a F420-reducing hydrogenases (frhACB) and Group 3b multimeric cytoplasmic sulfhydrogenases (hydABCD; Figure 5; Jeon et al., 2015). Comparative analyses of these two clusters revealed that other members of the Desulfurobacteriaceae family were the closest relatives, while the next closest enzymes were those found in hyperthermophilic Euryarchaeota (i.e. Pyrococcus abyssi) and in methanogens (Figure 5). Overall, these results imply a polyphyletic origin for the group 3 cytoplasmic hydrogenases. Comparative analyses revealed similarities with hyperthermophilic Euryarchaeota and methanogens (Figure 5), suggesting a possible common ancestry for the group 3 cytoplasmic hydrogenases of Archaea and Desulfurobacteriaceae. Together with Group 3b sulfhydrogenases and Group 4 Ech, F420-reducing hydrogenases are in fact typically found in methanogens in which the F420-cofactor is used as an electron carrier in the reduction of CO2 or other C1 compounds to methane (Vignais and Billoud, 2007). Cultures of T. ammonificans grown with nitrate as the terminal electron acceptor did not present the typical autofluorescence of methanogens due to F420-cofactor fluorescence when observed under UV in epifluorescence microscopy and the genome lacks the gene involved in the biosynthetic pathway of the F420-coenzyme. Due to these observations, we suggest that these hydrogenases may work in conjunction with ferredoxin in T. ammonificans. Group 3b Hyd sulfhydrogenases appear to be of mixed origins despite being present in a single operon. Three of the subunits share similarities with the Thermococcales (Euryarchaeota; hydBCD) while the catalytic subunit (HydA) appears to be unique to the Desulfurobacteriacea, not having any other significant homolog in the database (Figure 6). Comparative analyses indicated that Ech shared similarity with Epsilonproteobacteria Group 4 hydrogenases (Figure 5). Interestingly, Group 2 hydrogenases are found in microaerobic members of the Aquificaceae and Hydrogenothermaceae and in microaerobic Epsiloproteobacteria but are not found in anaerobes, suggesting a possible link with oxygen adaptation. In contrast, Group 3a is found in strict anaerobes, including T. ammonificans and methanogens (Figure 5).

Blastp best hit (cut off at 40% similarity) for the T. ammonificans CDS: (a) Desulfurobacterium thermolithotrophum is included in the database; (b) D. thermolithotrophum and Desulfurobacterium sp.

TC5-1 are excluded from the database (best hits are outside of the Desulfurobacteriaceae family). The interactive versions of the Krona plots are available for download at DOI: 10.6084/m9.figshare.3178528.

https://doi.org/10.7554/eLife.18990.014

Finally, Ech hydrogenases are the only Group 4 hydrogenases found in T. ammonificans. Both the reductive TCA cycle and the reductive acetyl-CoA pathway involve reaction steps that are driven by reduced ferredoxin (Fuchs, 2011). In the case of T. ammonificans, the enzymes formate dehydrogenase, CO-dehydrogenase, pyruvate synthase and 2-oxoglutarate synthase require reduced ferredoxin. We hypothesize that T. ammonificans can accomplish ferredoxin reduction in two different ways: the first involves cytosolic hydrogenases (Group 3, Frh and Hyd; Figure 3) commonly found in methanogens, where they reduce ferredoxin via electron bifurcation (Fuchs, 2011); the second involves membrane bound hydrogenases (Group 4, Ech; Figure 3). With the exception of the F420-reducing (Frh) hydrogenases, representatives of the remaining three groups were identified in the proteome of T. ammonificans (Figure 3).

Nitrate ammonification

T. ammonificans HB-1 can use nitrate as a terminal electron acceptor and reduces it to ammonium (Vetriani et al., 2004). Periplasmic nitrate reductases (Nap) have been studied extensively in some Proteobacteria, and are comprised of the catalytic enzymes – typically a heterodimer (NapAB) – associated with various other subunits involved in channeling electrons to the NapA reactive center (Moreno-Vivián et al., 1999; Vetriani et al., 2014). In T. ammonificans, the genes encoding for the Nap complex are organized in a single operon, napCMADGH. Comparative genomic analyses with genomes from Aquificae and Proteobacteria indicate that the nap operon structure of T. ammonificans is unique to this organism, and shares similarity with Desulfovibrio desulfuricans and other Deltaproteobacteria. All other Aquificae whose genomes are available have a Nap operon similar to that of Epsilon- and Gammaproteobacteria (Vetriani et al., 2014). Moreover, unlike other members of the Aquificae, the nitrate reductase of T. ammonificans (encoded by gene Theam_0423, expressed in the proteome; Figure 3) appears to be of the monomeric type (sensu; Jepson et al., 2006), and the NapB subunit is missing (Figure 3). We also identified a putative nitrite reductase-encoding gene possibly involved in the reduction of nitrite to ammonium (NirA; Theam_1000, Figure 3), which was also expressed in the proteome (Figure 3). However, the exact mechanism through which T. ammonificans reduces nitrite to ammonium remains to be experimentally elucidated.

Sulfur reduction

While T. ammonificans is able to reduce elemental sulfur to hydrogen sulfide (Vetriani et al., 2004), the sulfur reduction pathway remains unclear. We identified in the genome a putative polysulfide reductase of the sulfide-quinone reductase family (sqr, Theam_0841, Figure 3). We propose that this membrane-bound complex can utilize polysulfide formed from the reaction of S0 with sulfide (naturally enriched in hydrothermal systems) via quinone oxidation (Figure 3). The putative sqr gene of T. ammonificans shares similarities with other Aquificae and Epsilonproteobacteria (average 56% similarity to both groups). In particular, the sqr gene appears to be conserved also in Caminibacter mediatlanticus (Figure 7; Giovannelli et al., 2011), whose sulfur reduction pathway is yet to be elucidated. Another possibility is that T. ammonificans uses a NAD or FAD-dependent reductase (nsr, Theam_1321) to reduce sulfur, a mechanism previously described in the sulfur-reducing archaeon Pyrococcus furiosus (Schut et al., 2013, 2007). However, the putative polysulfide reductase of T. ammonificans has only about 33% identity to homologous enzymes of sulfur reducing bacteria and archaea identified in the database.

Hives plot presenting the comparative genomic analysis of the T. ammonificans (Tam) genome with the closest relative available genomes of D. thermolithotrophum (Des) and the ecologically similar Epsilonproteobacterium C. mediatlanticus (Cam).

The three axes represent the organism’s linearized genomes with the origin of replication at the center of the figure. Genome size was normalized for visualization purpose. (A) Localization of homologous genes and syntenic regions among the three genomes. Lines colored according to the general pathway code adopted in Figure 3 connect homologous coding sequences. Lines opacity is proportional to similarity between sequences (darker = higher similarity), line width is proportional to the extent of the syntenic area. (B) Hives panel of the homologous genes divided by pathways. Collinear lastZ alignments and interactive dot-plot replicating the analysis are accessible at the GenomeEvolution website with the following permanent addresses: T. ammonificans and D. thermolithotrophum (http://genomevolution.org/r/9vb3); T. ammonificans and C. mediatlanticus (http://genomevolution.org/r/9vb4); D. thermolithotrophum and C. mediatlanticus (http://genomevolution.org/r/9vb8).

https://doi.org/10.7554/eLife.18990.015

Resistance to oxidative stress

T. ammonificans is a strict anaerobe. However, its genome codes for genes involved in the detoxification of oxygen radicals, including a catalase/peroxidase (Theam_0186), whose activity was previously detected (Vetriani et al., 2004), a putative superoxide reductase (Theam_0447), a cytochrome-C peroxidase (Theam_1156) and a cytochrome bd complex (Theam_0494–0496, Figure 3). The latter three enzymes were identified in the proteome (Figure 3). The cythochrome bd complex has been shown to contribute to oxygen tolerance in other anaerobic bacteria (Das et al., 2005), and to contribute to the detoxification of nitrous oxide radicals (Mason et al., 2009).

Energy conversion

The genome of T. ammonificans contains two gene clusters coding for the NADH dehydrogenase (Theam_0731–0745 and Theam_1488–1498). The genes appear to have undergone an inversion during the duplication process and the second copy is missing four genes in the middle of the operon (nuoEFG and a transcriptional regulator). The two clusters share on average 60% gene similarity. Only one copy of the gene for the synthesis of ATP synthetase complex was present (Theam_1605–1606 and Theam_1656–1662). Three different hydrogen-sodium symporters (nhe, Theam_0443, 0413 and 0647) were present with low pairwise similarity. NADH dehydrogenase second cluster and ATP synthetase complex appear to be conserved in the close relative D. thermolithotrophum and in the Epsilonproteobacterium, C. mediatlanticus TB-2 (Figure 3).

Motility, cell sensing and biofilm formation

T. ammonificans posses one to two terminal flagella (Vetriani et al., 2004). The genes involved in flagella formation and assembly are prevalently organized in a single large cluster in the genome (from Theam_1440 to Theam_1468, Figure 7), putatively organized in three distinct operons. The hook-filament junction proteins genes flgK and flgL (Theam_1384 and Theam_1385), the filament and filament cap proteins fliC and fliD (Theam_1160 and Theam_1162) together with few flagellar maturation factors and motor switch genes (motA and motB, Theam_1087 and Theam_1085–1086, respectively) are instead scattered in the genome. The entire cluster of genes shares similarities with D. thermolitotrophum, although the similarity for some of the proteins is as low as 50%. Despite this, comparative analyses revealed a similar organization in D. thermolitotrophum where the cluster appears inverted in the same relative genomic position and constituted one of the main region of synteny (Figure 7b). By contrast, the flagellar genes in the Epsilonproteobacterium, Caminibacter mediatlanticus TB-2, are scattered throughout the genome (Figure 7b). Numerous genes for adhesion and pili were present, generally organized in small clusters (Figure 7b).

Four different putative methyl-accepting chemotaxis sensory transducer proteins were found in the genome (mcp, Theam_0157, 0165, 0845 and 1027). One of those, Theam_0845, is surrounded by receptors cheW and cheA, and by the response regulator cheY. The entire group is organized in a single operon, cheYVBWAZ conserved in D. thermolithotrophum and other Aquificales with the exception of cheW, which shares higher similarities with the Epsilonproteobacterium, C. mediatlanticus TB-2 (Figure 6). We failed to find in the genome of T. ammonificans a homolog of the primary receptor encoding gene cheR (Wadhams and Armitage, 2004). We identified in the genome numerous membrane transporters for molybdenum (modCBA, Theam_0787–0789), iron (fhuDBC, Theam_0192–0194), zinc (znuAB, Theam_0238 and 0689) and cobalt/nickel (cbiOQMN, Theam_1522, 1523, 1525 and 1526) all involved in the uptake of important micro-elements for enzyme and cofactor synthesis. We also identified the complete type II secretion pathway, which is conserved in gram-negative bacteria and responsible for protein and toxin translocation to the extracellular milieu. Proteins secreted by the type II pathway include proteases, cellulases, pectinases, phospholipases, lipases, toxins and in some cases type III and IV pili (Douzi et al., 2012). In some bacteria, the expression of secretion type II pathway genes are regulated either by quorum-sensing or by the environmental factors at the site of colonization (Douzi et al., 2012; Sandkvist, 2001). Biofilm formation in bacteria is often linked to quorum-sensing mechanisms, regulating the settlement of planktonic cells in relationship to environmental sensing, substrate suitability and cell densities. It is likely that T. ammonificans maintains a mostly attached lifestyle in hydrothermal vent environments due to the high turbulence associated with fluid flux and mixing with seawater. We speculate that the type II pathway genes, together with chemotaxis and flagellar/adhesion genes may play a key role in the formation of biofilm. Extracellular secreted proteins may in fact be used to ‘condition’ the colonized substrate and to interact/remodel existing and newly formed biofilms.

Activated methyl cycle

The activated methyl cycle is a conserved pathway in which S-methyl groups from L-methionine are converted into a biochemically reactive form through insertion into an adenosyl group (Vendeville et al., 2005). S-adenosyl-L-methionine provides activated methyl groups for use in the methylation of proteins, RNA, DNA and certain metabolites central to the core cell machinery. The activated methyl cycle exists in two recognized forms, involving respectively luxS or sahH genes (Vendeville et al., 2005). Genome-based reconstruction of the activated methyl cycle indicated that, in T. ammonificans, involves the sahH gene, while a luxS homolog is absent (Figure 3—figure supplement 2). Methionine methyl groups are adenosylated by a methionine adenosyltransferase (samS, Theam_1075). The methyl group is thus activated and readily available for the S-adenosylmethionine-dependent methyltransferase (samT) for methylation of substrate. In T. ammonificans, samT is a pseudogene, as it contains two stop codons (Theam_0630). Homologs of samT are absent in the genome of the other Aquificae and the pathway appears to be incomplete. Only in P. marina and D. themolithotrophum the functionally equivalent DNA-cytosine methyltransferase (dcm) is present. The presence of a gap in such a central pathway in the Aquificae and the retrieval of a samT-like pseudogene in T. ammonificans raises interesting question on the functioning of the activated methyl cycle in those organisms. An interesting working hypothesis is that enzyme thermal stability at the physiological temperature of these bacteria might have driven the loss of samT and the replacement by a not yet identified alternative enzyme in most Aquificae.

Discussion

Evidence for a reductive acetyl-CoA pathway in the Desulfurobacteraceae lineage

The reductive acetyl-CoA pathway is present in acetogenic bacteria, methanogens, sulfate-reducers and anammox bacteria (Berg, 2011; Fuchs, 2011; Hügler and Sievert, 2011). It is believed to be the oldest carbon fixation pathway on Earth, due to the centrality of acetyl-CoA in metabolism, the low energy requirement (~1 ATP required vs 2–3 ATP for the rTCA cycle) and the limited necessity of de novo protein assembly (Berg et al., 2010; Fuchs, 2011). We hypothesize that the reductive acetyl-CoA pathway is used as an additional or alternative pathway to fix CO2 in T. ammonificans.

The finding of a potentially active reductive acetyl-CoA pathway (reactions 1 to 6; Figure 3) in T. ammonificans and other members of the Desulfurobacteraceae is intriguing. The pathway is missing reaction 2, the formyl-THF synthesis for which no obvious 10-formyl-THF synthetase was found (Figure 3). However, the 10-formyl-THF synthetase of the reductive acetyl-CoA pathway may have been replaced by alternative enzymes, such as 5-formyl-THF cycloligase (Theam_1206) or 10-formyl-THF deformylase (Theam_0826) working in reverse (Table 2, Figure 3). Promiscuous enzymes that function in more than one pathway have been long hypothesized and recently described in Escherichia coli (Jensen, 1976; Kim et al., 2010). Assuming low substrate specificity, other candidate enzymes with similar substrate requirements are the phosphoribosylglycinamide formyltransferase (Theam_1211) or phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase (Theam_0328), which could catalyze the synthesis of formyl-THF, albeit with sub-optimal kinetics. While all four enzymes were detected in the proteomic analysis (Figure 3), we think that the possible involvement of a 5-formyl-THF cycloligase in the synthesis of formyl-THF in T. ammonificans is particularly appealing, as this would provide an evolutionary link between the N5-formate uptake of methanogens and N10-formate uptake in acetogens (Braakman and Smith, 2012). Despite the absence of the 10-formyl-THF synthetase, the genome of T. ammonificans encodes and expresses a type V CodH (Figure 3).

We investigated the available genomes of other members of the Aquificae for the presence of genes homologous to the putative reductive acetyl-CoA pathway of T. ammonificans (Table 2). The similarity of the reductive acetyl-CoA pathway-related genes of T. ammonificans to those from organisms outside the Desulfurobacteriaceae is low. The type V codH gene identified in T. ammonificans is found exclusively in members of the family Desulfurobacteriaceae, with exception of a low similarity homolog present in Persephonella marina (41% amino acid identity) (Table 2). Furthermore, most of the genes involved in reaction 3, 4 and 5 (Figure 3) have homologs in other Aquificae or are substituted by functional equivalents with low amino acid identity (<40%). To understand the relationship of the T. ammonificans CodH with the homologous enzymes of methanogens, homoacetogens and sulfate-reducers, we reconstructed the phylogenetic history of these enzymes with a particular focus on type V CodH (Figure 4). Sequences from the Desulfurobacteriaceae appear to be related to those present in the Archaea, Ferroglobus placidus and Archaeoglobus spp., and similar to Methanosarcina spp. and homoacetogenic bacteria. The CodH found in CO-utilizing bacteria is only distantly related to the one found in T. ammonificans (Techtmann et al., 2012), while the CodH sequence of Persephonella marina appears to be one of a kind, with the enzyme of Ferroglobus placidus as its closest relative (44% similarity). Taken together, these findings suggest that, within the Aquificae, the type V CodH is exclusive to the Desulfurobacteriaceae.

The retrieval of the CodH enzyme from the proteome of T. ammonificans (Figure 3, Figure 3—source data 1), along with the catalytic subunit of the formate dehydrogenase (FdhA), suggests that the reductive acetyl-CoA pathway could be operational in T. ammonificans in addition to the rTCA cycle. The simultaneous presence of two distinct carbon fixation pathways has been confirmed so far only in the endosymbiont of the deep-sea vent tubeworm Riftia pachyptila (Markert et al., 2007). Recent findings suggest that the Riftia symbiont can switch from the Calvin-Benson-Bassham (CBB) to the rTCA cycles in response to low-energy supply (e.g., low concentrations of hydrogen sulfide in the vent fluids, being the rTCA cycle more energetically favorable than the CBB cycle; Markert et al., 2007). Genomic evidence of the simultaneous presence of different carbon fixation pathways came also from genome analysis of Ammonifex degensii and Ferroglobus placidus (Berg, 2011; Hügler and Sievert, 2011), where the reductive acetyl-CoA pathway seems to be coupled to an incomplete CBB cycle. The use of different carbon fixation strategies could be advantageous under energy limiting conditions (e.g., deep biosphere), could optimize overall carbon fixation or provide different precursors for biosynthetic pathways, and could be more widespread than originally thought.

Further laboratory analyses will identify the exact role of the type V CodH in T. ammonificans and the potential contribution of the reductive acetyl-CoA to overall carbon fixation.

Comparative genomic of T. ammonificans: ancestral and acquired metabolic traits

The genome of T. ammonificans HB-1 displays a large degree of mosaicism (Figure 6). When comparing the coding sequences (CDS) of T. ammonificans with available genomes, 34% of the CDS shared higher similarity to genes outside the Aquificae, suggesting that these genes were acquired from more distantly related taxa.

We carried out comparative genomic analyses among T. ammonificans and all available Aquificae genomes (Table 3). Desulfurobacterium thermolithotrophum DSM 11699 (its closest relative whose genome was sequenced) and Caminibacter mediatlanticus TB-2 (an Epsilonproteobacterium which, albeit phylogenetically distant, shares the same physiology and occupies a similar ecological niche as T. ammonificans, only at lower temperature; Figure 7) were further used as a direct comparison. We identified areas of synteny between T. ammonificans and the closely related D. thermolithotrophum in the region surrounding the genes encoding for key enzymes responsible for the citrate cleavage in the rTCA cycle (Figure 7b and Figure 8). This region is conserved also within the Hydrogenothermaceae (in particular P. marina) and members of the Epsilonproteobacteria.

Syntheny diagram presenting the genome organization around the rTCA key enzyme ATP citrate lyase.

In T. ammonificans the two subunits of the ATP citrate lyase enzyme are organized in a single operon together with the isocitrate dehydrogenase and the aconitate dehydratase. The numbers inside each gene represent the locus number for the organism. Shaded color connects synthenic regions. The website address below each organism name is a permanent link to the pairwise analysis performed on the Genome Evolution server (http://genomevolution.org/) against T. ammonificans.

https://doi.org/10.7554/eLife.18990.016
Table 3

List of the genomes belonging to the Aquificae phylum used for comparative genomic analyses.

https://doi.org/10.7554/eLife.18990.017
OrganismGenome acc. numberGenome lengthGC contentNum. genes
Aquifex aeolicus VF5NC_0009181,590,79143%1782
Desulfurobacterium sp. TC5-1NZ_ATXC010000011,653,62540%1680
Desulfurobacterium thermolithotrophum DSM 11699NC_0151851,541,96835%1561
Hydrogenivirga sp. 128–5 R1-1NZ_ABHJ010005513,038,24044%3756
Hydrogenobacter thermophilus TK-6NC_0137991,743,13544%1897
Hydrogenobaculum sp. Y04AAS1NC_0111261,559,51435%1631
Persephonella marina EX-H1NC_0124401,983,96637%2067
Persephonella sp. IF05-L8NZ_JNLJ010000011,828,85835%1920
Sulfurihydrogenibium azorense Az-Fu1NC_0124381,640,87733%1722
Sulfurihydrogenibium sp. YO3AOP1NC_0107301,838,44232%1832
Sulfurihydrogenibium subterraneum DSM 15120NZ_JHUV010000011,610,18132%1701
Sulfurihydrogenibium yellowstonense SS-5NZ_ABZS010002281,534,47133%1637
Thermocrinis albus DSM 14484NC_0138941,500,57747%1631
Thermocrinis ruber DSM 23557NZ_CP0070281,521,03745%1625
Thermocrinis sp. GBSNZ_JNIE010000011,315,62541%1417
Thermosulfidibacter takaii ABI70S6NZ_AP0130351,816,67043%1844
Thermovibrio ammonificans HB-1NC_0149261,759,52652%1820

The conserved regions between the genomes of T. ammonificans and D. thermolithotrophum included the two hydrogenase clusters, the flagellum and the NADP dehydrogenase complex. The order and position of the hydrogenase and NADP dehydrogenase clusters were highly conserved also in C. mediatlanticus, while flagellar genes were scattered throughout the genome (Figure 7). These results suggest that the genes encoding hydrogen utilization and functions related to energy conversion are among the oldest genomic regions present in those organisms, and overall are conserved across phyla. When we analyzed the central metabolism of T. ammonificans using comparative genomic approaches, the presence of two distinct groups of genes became evident: the first group was related to early-branching bacterial or archaeal lineages and coded for enzymes involved in several central metabolic pathways, while the second group of genes appeared to have been acquired later. Among the first group of genes, we identified: (I) the cytoplasmic [Ni-Fe]-hydrogenases Group 3 that were related to the enzymes found in methanogens and thermophilic Euryarchaeota, in which they are involved in ferredoxin reduction (Figure 3 and Figure 8); (II) sulfur-reduction related genes (Figure 3); and (III) the genes coding for the enzymes of the reductive acetyl-CoA pathway (Figure 3). These pathways are either present in early branching Archaea, or are directly involved in metabolic reactions that do not require oxygen (or oxygen by-products) and use substrates of geothermal origin that are likely to have been abundant on Early Earth. Moreover, most key reactions in these pathways are catalyzed by enzymes that are extremely sensitive to oxygen. Our findings are consistent with a recent reconstruction of the ancestral genome of the last universal common ancestor (LUCA), which suggest that LUCA was a hydrogen-dependent autotroph capable of S utilization that could fix CO2 via the reductive acetyl-CoA pathway (Weiss et al., 2016). Taken together, these observations suggest that part of the central metabolism of T. ammonificans is ancestral and emerged prior to oxygenic photosynthesis.

By contrast, some genes and associated metabolic pathways appear to have been acquired by the T. ammonificans lineage at a later time. One example is the ability to conserve energy by nitrate reduction. The hypothesis that nitrate respiration is an acquired trait in T. ammonificans is supported by the structure of the nap operon. The monomeric napA gene of T. ammonificans is homologous to that of Desulfovibrio desulfuricans and other Deltaproteobacteria (Figure 6; online interactive version) and apparently has been acquired laterally. Furthermore, it shares high similarity with assimilatory nitrate reductases (Nas) and it could be an evolutionary intermediate form between assimilatory Nas and the dimeric respiratory Nap. In line with this observation, phylogenetic analyses suggest that the membrane bound [Ni-Fe]-hydrogenases of Group 1 and 4 are of delta/epsilonproteobacterial origin (Figure 5). Based on the finding that Group 1 Hyn hydrogenases were expressed when T. ammonificans was grown under nitrate-reducing conditions (Figure 3), we suggest that these enzymes are linked through the membrane quinone pool to nitrate reduction and may have been acquired simultaneously with the nap genes.

A second example is represented by the oxygen radical detoxification enzymes encoded by the genome of T. ammonificans. We propose that such genes are not part of the core, or ancestral genome of T. ammonificans, but that they evolved as a response to exposure to toxic oxygen radicals. In the modern ocean, catalase and peroxidase may provide protection to oxygen-sensitive enzymes during transient exposure to oxygen associated with entrainment of deep seawater in hydrothermal fluids, or when the organism is displaced into the water column. This second scenario may have important implication for the dispersal of vent microorganisms to other vent sites. We hypothesize that T. ammonificans is able to deal with oxygen exposures using two different mechanisms: the first response mechanism would be activated following exposure to oxygen at physiological temperatures (60–80°C). In that case, the oxygen-stress related genes are induced, protecting the cell machinery against damages. The second mechanism would be a response to prolonged exposure to oxygen at temperatures below the physiological threshold. Such prolonged exposure would trigger a metabolic shut-down, enabling survival of the organism in cold seawater. The latter hypothesis is supported by experiments where batch cultures of T. ammonificans were exposed for a prolonged time at 4°C, which revealed the survival of the bacterium despite being exposed to oxygen (data not shown).

In conclusion, the nitrate reduction pathway, as well as oxygen stress-related genes, may represent adaptations acquired by the T. ammonificans lineage to cope with the rise of oxygen on Earth.

Insight into the evolution of carbon fixation

Six different pathways of carbon fixation are known to date (Fuchs, 2011). In the modern biosphere, the Calvin-Benson-Bassham cycle is the dominant mechanism of CO2 fixation. Yet, other anaerobic pathways have been investigated and are proposed to represent the ancestral autotrophic carbon fixation pathway. In particular, the reductive acetyl-CoA pathway is thought to be among the earliest carbon fixation pathways that have emerged on Earth (Fuchs, 2011). This hypothesis is supported by numerous observations: (I) the presence of this pathway in the early branching methanogenic archaea; (II) its low energy requirements and low need of de novo protein synthesis; (III) its capacity to incorporate different one-carbon compounds and carbon monoxide of geothermal origin, and (IV) the extreme oxygen sensitivity of its key enzymes which have common roots in Bacteria and Archaea (reviewed in Berg et al., 2010; Fuchs, 2011; Hügler and Sievert, 2011). Despite differences in their structure, all extant carbon fixation pathways can be theoretically derived from a putative rTCA cycle/reductive acetyl-CoA hybrid pathway, as proposed by a recent phylometabolic reconstruction of the evolution of carbon fixation (Braakman and Smith, 2012). According to this study, both Aquificae and acetogenic bacteria represent the closest living example of the archetypal network, and both diverged from the pre-LUCA pathway under the selective pressure of energy-efficiency (acetogens) and oxygen sensitivity (Aquificae). Further, a recent reconstruction of the genome of LUCA based on gene phyletic pattern reconstruction is consistent with some of these findings and suggests that LUCA’s genomic makeup point to autotrophic acetogenic and methanogenic roots and to the ancestry of the reductive acetyl-CoA pathway (Weiss et al., 2016).

The presence of a CodH type V enzyme in the genome of T. ammonificans and other members of the Desulfurobacteriaceae, and the presence of a type II CodH in Desulfurobacterium thermolithotrophum suggest that the reductive acetyl-CoA pathway could be operational in extant members of the Desulfurobacteriaceae. Furthermore, this finding supports the scenario proposed by Braakman and Smith that a complete and operational reductive acetyl-CoA pathway was present in the ancestor of the phylum Aquificae (Braakman and Smith, 2012). Comparative analyses revealed that the CodH enzyme is conserved only within the Desulfurobacteriaceae, consistent with the strict anaerobic nature of the members of this family and the extreme oxygen sensitivity of CodH. The rise of oxygen has been interpreted as one of the factors responsible for the diversification of carbon fixation from the ancestral pathway, and could explain the subsequent loss of the CodH in the generally facultative microaerobic Hydrogenothermaceae and Aquificaceae within the Aquificae (Table 2).

Comparative genomic analyses of rTCA cycle genes allow the description of a possible evolutionary scenario for the rTCA cycle in Aquificae. Members of the Aquificaceae (e.g., Aquifex aeolicus; Figure 9 and Figure 3—figure supplement 1) have the two-step version of the rTCA cycle (Figure 3—figure supplement 1A), where citrate cleavage is accomplished by the combined action of the enzymes citryl-CoA synthetase and citryl-CoA lyase (encoded by the ccl gene), and the carboxylation of 2-oxoglutarate is catalyzed by the two enzymes 2-oxoglutarate carboxylase and oxalosuccinate reductase (Aoshima et al., 2004; Braakman and Smith, 2012). Since citryl-CoA synthetase and 2-oxoglutarate carboxylase likely evolved by duplication of the genes for the succinyl-CoA synthetase and pyruvate carboxylase (Aoshima, 2007; Braakman and Smith, 2012), a complete rTCA cycle could have evolved in the Aquificaceae from an ancestral, incomplete version of the cycle. In contrast, the two other groups of Aquificae, the Hydrogenothermaceae and the Desulfurobacteriaceae (Figure 1—figure supplement 1), use the one-step – and more recent – version of the rTCA cycle (Figure 10, Figure 9 and Figure 3—figure supplement 1B), involving ATP citrate lyase (ACL, encoded by the acl gene) and isocitrate dehydrogenase, that carry out citrate cleavage and 2-oxoglutarate carboxylation in two single enzyme reactions, respectively (Braakman and Smith, 2012).

Phylogenetic tree of ATP citrate lyase amino acid sequences.

The tree was constructed with Bayesian inference and maximum likelihood methods and presents the phylogenetic relationship among the concatenated subunit of the ATP citrate lyase in different organisms known to use the rTCA cycle. The numbers near the nodes represent the bayesian posterior probability (left number) and the maximum-likelihood confidence values based on 1000 bootstrap replications (right number). Bar, 30% estimated substitutions. Accession numbers are reported in Figure 9—source data 1. Squares – symmetric rTCA cycle variant characterized by the presence of two-step enzyme reactions for the cleavage of citrate and the carboxylation of 2-oxoglutarate. Circles – asymmetric rTCA cycle variant characterized by one-step reaction enzymes catalyzing the above reactions. Triangles – presence of a citryl-CoA lyase in the genome. See Supplementary Materials for details.

https://doi.org/10.7554/eLife.18990.018
Proposed evolution of the carbon fixation pathway in the Aquificae phylum.

Proposed evolution of the carbon fixation pathway in the Aquificae phylum and reconstruction of the ancestral carbon fixation for the last common ancestor (LCA) based on the results of integrated phylogenetic, comparative genomic and phylometabolic analyses.

https://doi.org/10.7554/eLife.18990.020

The enzyme responsible for citrate cleavage, the ATP citrate lyase, likely evolved through gene fusion of the genes of CCS and CCL (Aoshima et al., 2004). Phylogenetic analyses of ACL suggests that this gene fusion event did not happen within the Aquificae, as Nitrospira and Chlorobia have an evolutionary older version of ACL than Hydrogenothermaceae and Desulfurobacteriaceae (Figure 9; Hügler et al., 2007). Hence, it is likely that these two groups acquired ACL through HGT (Figure 9 and Figure 8; Hügler et al., 2007). Furthermore, our comparative analysis showed that: (I) in the Hydrogenothermaceae and in the Desulfurobacteriaceae, the enzymes of the first half of the rTCA cycle, as well as aconitase (acnA), share a common ancestor with the Aquificaceae and can be considered part of the core genome of the phylum Aquificae, while the remaining enzymes are either the result of gene duplication or have been acquired by horizontal gene transfer (Hügler et al., 2007); (II) the two-step citrate cleavage is exclusive to the Aquificacea (Figure 9); (III) the ccl gene is still present in the Hydrogenothermacea (in addition to acl), and one sequenced strain of the Hydrogenothermaceae (S. azorense) still uses the two-step carboxylation of 2-oxoglutarate, suggesting that the two-step version of the rTCA cycle was present in the ancestor of both the Aquificaceae and the Hydrogenothermaceae (Figure 9 and Figure 10). However, neither of the genes for the two-step citrate cleavage or two-step 2-oxoglutrate carboxylation is present in the Desulfurobacteriaceae (Figure 9). Finally, synteny analyses show that the four enzymes necessary to complement the one-step rTCA cycle variant in the Desulfurobacteriaceae from a theoretical ancestral linear rTCA are organized in a single operon (Figure 8).

Taking into consideration evidence from comparative genomics and phylogenetic analyses, the most parsimonious interpretation of the data suggests that the last common ancestor of the Aquificae had an incomplete form of the rTCA that did not proceed past the synthesis of 2-oxoglutarate, and that later on the cycle was closed following two independent evolutionary trajectories (Figure 10): (I) Gene duplication in the lineage that lead to the Aquificaceae and Hydrogenothermaceae; and (II) gene acquisition by horizontal gene transfer in the Desulfurobacteriaceae and Hydrogenothermaceae (the latter replaced the two-step version of the rTCA cycle with the one-step version) (Figure 9 and Figure 10). The reasons behind the presence of two distinct rTCA cycle variants within the extant Aquificae are not known. Braackman and Smith hypothesized that the one-step reactions might have evolved as a way to improve the thermodynamic efficiency of the rTCA cycle (Braakman and Smith, 2012). We hypothesize that temperature may also have played a role in preserving the ancient, more symmetric, two-step citrate cleavage rTCA cycle variant in Aquificaceae (Figure 10). Members of this group have optimum growth temperatures (75–95°C) higher than those of the two other groups (60–75°C; Table 1), and the ‘ancient’ enzymes might be more stable at these high temperatures. In contrast, the ‘newer’ enzymes that catalyze the one-step citrate cleavage might have evolved to function optimally at lower temperatures.

Different members of the Aquificae use either one of the two versions of the rTCA cycle, and all the extant members of this phylum possess the genes encoding for the enzymes of the reductive acetyl-CoA pathway, with the exception of codH (encoding for the CO-dehydrogenase), which is only found in the Desulfurobacteriaceae. Therefore, we hypothesize that the last common ancestor of the Aquificae possessed the complete reductive acetyl-CoA pathway (Figure 10). While members of the Desulfurobacteriaceae kept the CodH due to their obligate anaerobic lifestyle, microaerophilic Aquificae (Hydrogenothermaceae and Aquificaceae) lost this extremely oxygen-sensitive enzyme (Figures 4 and 10). The presence of the gene encoding CodH in P. marina (Hydrogenothermaceae) is the only exception, and suggests that codH was lost independently in the two lineages (Figure 10).

Alltogether, our results suggest that the last common ancestor of the Aquificae combined the reductive acetyl-CoA pathway with an incomplete form of the rTCA that did not proceed past the synthesis of 2-oxoglutarate (Figure 10). A similar incomplete version of the rTCA pathway, consisting only of the reactions from acetyl-CoA to 2-oxoglutarate, is present in extant methanogens (Berg, 2011). Thus, our phylometabolic reconstruction of the ancestral state of carbon fixation in the Aquificae (Figure 10) is conceptually consistent with chemoautotrophic processes of extant Bacteria and Archaea, and may represent the earliest carbon fixation pathway.

Conclusions

We propose that the ancestor of Thermovibrio ammonificans was originally a hydrogen oxidizing, sulfur reducing bacterium that used a hybrid carbon fixation pathway for CO2 fixation. The simultaneous presence of the rTCA cycle and of the reductive acetyl-CoA pathway of carbon fixation in T. ammonificans may represent a modern analog of the early carbon fixation phenotype, and suggests that the redundancy of central metabolic pathways was common in ancestral microorganisms. With the gradual rise of oxygen in the atmosphere, more efficient terminal electron acceptors became available and this lineage acquired genes that increased its metabolic flexibility - e.g., the capacity to respire nitrate - along with enzymes involved in the detoxification of oxygen radicals. However, this lineage also retained its core, or ancestral, metabolic traits. Given the early branching nature of the phylum Aquificae and the ability of T. ammonificans and the Desulfurobacteriaceae to thrive in hydrothermal environments relying on energy sources of geothermal origins (namely carbon dioxide, hydrogen and elemental sulfur), we argue that these microorganisms represent excellent models to investigate how metabolism co-evolved with Earth’s changing environmental conditions.

Materials and methods

Strain isolation was described in (Vetriani et al., 2004). Growth condition, DNA extraction, sequencing strategy and automatic annotation were published in (Giovannelli et al., 2012).

Manual curation of the genome

Manual curation of the genome was performed using blastn and blastp (McGinnis and Madden, 2004) against the non-redundant database (Pruitt et al., 2007), the conserved domain database (Marchler-Bauer et al., 2005), the Kyoto Encyclopedia of Genes and Genomes (Kanehisa and Goto, 2000) and the PFAM database (Sonnhammer et al., 1998). Coding sequence similarities were compared using translated protein sequence. Metabolic pathways were reconstructed on the basis of available genomic, physiologic and biochemical information and using Kyoto Encyclopedia of Genes and Genomes (Kanehisa and Goto, 2000) and SEED (Overbeek et al., 2014) pathways as template.

Comparative genomics

Genome maps were drawn using Circos (Krzywinski et al., 2009), parsing blast results with ad hoc bash scripting. Comparative analyses between the genomes of T. ammonificans, those of representative members of the Aquificae (reported in Table 3), Desulfurobacterium thermolithotrophum (Göker et al., 2011) and Caminibacter mediatlanticus (Giovannelli et al., 2011) were performed using the GenomeEvolution pipeline CoGe (Lyons et al., 2008). Aquificae genomes were selected among all available genome within this phylum to maximize diversity while minimizing redundancy. All the available genomes of validly published Aquificae species were selected for analysis. To this set we added the reference genomes for the genera Hydrogenivirga and Hydrogenobaculum, as no genome sequence is available for validly published species of these genera. We also selected two additional genomes belonging to the genera Desulfurobacterium (the closest relative to the genus Thermovibrio) and Persephonella, respectively. Excluded genomes include either alternative assemblies of selected genomes or closely related genomes with a gapped genome similarity above 90%. LastZ pairwise alignments of selected genomes were used to draw three-way plots using the Hive Plot software (Krzywinski et al., 2012). Gene context and operonic structures were reconstructed using BioCyc (Karp et al., 2005) and FgenesB. Operons were manually screened and their structure selected based on gene context and available literature on the specific gene. When it was not possible to discriminate between the two alternative operonic predictions, both were reported in the text. Similarities between T. ammonificans genes and other prokaryotic genomes were found performing blastp analyses against the nr database. The top three blast results were retained and further analyzed, ranking the genes for their best hits. The procedure was repeated removing from the database the genome of D. thermolithotrophum and Desulfurobacterium sp. TC5-1, thus searching for the best hit outside of the Desulfurobacteriaceae family. The results were analyzed and interactive Krona plots (Ondov et al., 2011) drawn linking the T. ammonificans genes with its closest match in the database. The interactive plots are accessible at DOI: 10.6084/m9.figshare.3178528. Images were drawn or edited using the open source vector drawing program Inkscape (http://inkscape.org/).

Phylogenetic analyses

Phylogenetic analyses were performed using the approaches defined below for each tree. The phylogenetic tree presented in Figure 1 was computed from the current 16S rRNA database alignment available from the ARB-SILVA project (http://www.arb-silva.de). The maximum likelihood three was computed from the ARB-SILVA alignment using PHYLML (Guindon and Gascuel, 2003) and the GTR model. The phylogenetic tree in Figure 1—figure supplement 1 was constructed by aligning complete or near complete 16S rRNA sequences obtained from NCBI and representing the phylum Aquificae. The 16S rRNA sequences were aligned with ClustalO (Thompson et al., 1997) and Gblocks (Castresana, 2000) and the alignment was manually refined using SEAVIEW (Galtier et al., 1996). The maximum likelihood phylogeny was inferred from the alignment of 1455 sites using PHYML (Guindon and Gascuel, 2003), the GRT model and 1000 bootstrap replications. The CODH tree presented in Figure 4 was computed using a selected set of amino acid sequences and the neighbor-joining method. Alignments were obtained using Muscle (Edgar, 2004) and Gblocks (Castresana, 2000), manually refined using SEAVIEW, and phylogenetic distances calculated using 255 sites and the Observed Divergence matrix. The neighbor-joining method was used to evaluate tree topologies using Phylo_win (Galtier et al., 1996) and their robustness was tested by bootstrap analysis with 1000 resamplings. The tree for the catalytic subunit of the [NiFe]-hydrogenases presented in Figure 5 was computed using a selected set of amino acid sequences and the same approach described above for the CODH tree and was based on 539 sites. The accession number for the sequences used in trees presented in Figure 1—figure supplement 1, Figures 4 and 5 are reported within each tree. The ATP citrate lyase phylogenetic tree was reconstructed with Bayesian inference and maximum likelihood methods. Both subunit of the ATP citrate lyase (AclB and AclA) were aligned individually to retrieved homologs using Muscle (Edgar, 2004) and SEAVIEW (Galtier et al., 1996). The alignments were concatenated and refined using Gblock (Castresana, 2000). A hypothetical ancestral ATP citrate lyase enzyme was manually constructed by concatenating the citryl-CoA synthetase and citryl-CoA lyase of H. thermophilus and A. aeolicus, respectively, and used as the outgroup (Hügler et al., 2007). The maximum likelihood phylogeny was inferred from the alignment using PHYML with the Wag substitution model (Whelan and Goldman, 2001) and 1000 bootstrap resamplings. The substitution model was selected based on AIC values using ProTest3 (Darriba et al., 2011). Bayesian phylogeny was inferred using MrBayes (Ronquist and Huelsenbeck, 2003) performing 500,000 generations with two parallel searches with the Wag amino acid matrix model (selected using forward selection among all possible substitution models) and a burn-in of 125,000 generations. Both tree were computed on 1035 identified sites. Accession numbers for the tree presented are reported in Figure 9—source data 1. A combination of phylogenetic analysis and comparative genomic analyses were used to identify lateral gene transfer events.

Phylometabolic reconstruction of the carbon fixation pathway within the aquificae

Phylometabolic analysis (Braakman and Smith, 2012) was used to investigate the carbon fixation pathway in T. ammonificans and its evolutionary relationship to the carbon fixation pathways present in other members of the Aquificae phylum. In phylometabolic analyses, the metabolic pathways of the organism under investigation are compared to those found in related organisms both within and across neighboring clades. By focusing on the pathways, the comparison may reveal variations in multi-enzyme functional units, providing context for the completion of the pathway within the networks of individual organism, while also allowing for the identification of ancestral states and horizontal gene transfer events. The resulting phylometabolic tree includes multiple complete pathways to common essential metabolites, and suggests which evolutionary substitutions are allowed (at either organism or ecosystem levels) among these pathways (see [Braakman and Smith, 2012] and reference therein for a more extensive description of the principles underlying this approach). We reconstructed the carbon fixation pathways in representative genomes of the Aquificae, and compared them. Information regarding the carbon fixation metabolic network was implemented using phylogenetic and comparative genomic information to help reconstruct possible ancestral states of the carbon fixation network based on maximum parsimony principles.

Protein extraction, digestion and identification

T. ammonificans cell pellets were washed in TE buffer (10 mM Tris-HCl pH 7.5, 10 mM EDTA pH 8.0, containing Roche cOmplete protease inhibitor) and soluble proteins were extracted as described by (Heinz et al., 2012). Briefly, cells were disrupted by sonication (2 × 25 s), cell debris was pelleted and protein concentrations in the supernatant were determined according to (Bradford, 1976). 20 µg of protein extract were loaded onto a precast 10% polyacrylamide mini gel in technical triplicates for 1D PAGE (150 V, 45 min). After staining with Coomassie Brilliant Blue, protein-containing gel lanes were excised and cut into 10 equal subsamples each, which were destained (200 mM NH4HCO3, 30% acetonitrile) and digested with trypsin solution (1 µg/ml, Promega, Madison WI, USA) at 37°C over night, before peptides were eluted from the gel pieces in an ultrasonic bath (15 min). As described by (Xing et al., 2015), peptide mixes were subjected to reversed phase C18 column chromatography on a nano-ACQUITY-UPLC (Waters Corporation, Milford, MA, USA). Mass spectrometry (MS) and MS/MS data were recorded with an online-coupled LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). MS data were searched against the forward-decoy T. ammonificans protein database using Sequest (Thermo Fisher Scientific, San Jose, CA, USA; version 27, revision 11) and identifications were filtered and validated in Scaffold (http://www.proteomesoftware.com), as described previously (Heinz et al., 2012). Data for all three technical replicates were merged and exclusive unique peptide count values of all proteins were exported for calculation of normalized spectral abundance factors (NSAF), which are given in Figure 3—source data 1 in %, i.e., as relative abundance of each protein in % of all identified proteins.

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Decision letter

  1. Joerg Bohlmann
    Reviewing Editor; University of British Columbia, Canada

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Insight into the evolution of microbial metabolism from the deep-branching bacterium, Thermovibrio ammonificans" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Richard Losick as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Following below please find the assessments of the three external reviews. As you will see, the paper was overall quite positively received by the reviewers, but there is some substantial additional work in the data analyses and presentation of the paper that needs to be done before a substantially revised submission can be considered for publication in eLife.

Reviewer #1:

The authors use a comparative genomic approach associated to a proteomic analysis to reconstruct the metabolism of Thermovibrio ammonificans and draw an interesting picture of its possible evolution. Particularly interesting is the observation that the genome of T. ammonificans codes for enzymes putatively involved in the reductive acetyl-CoA pathway and in the reductive TCA (rTCA) cycle.

T. ammonificans encodes an unusual CodH (type V), whose function is still unknown. Based on the assumption that the type V CodH works in the rTCA cycle, the authors suggest that in T. ammonificans the reductive acetyl-CoA pathway and the rTCA cycle are both operational and that the bacterium uses one or the other way to fix carbon based on the environmental conditions.

This is a key point of the manuscript and should have some experimental support.

The manuscript is well presented although in some parts the reader needs to have high familiarity with microbial evolution and metabolism.

I have some questions and concerns:

1) Subsection “Carbon Fixation”, second paragraph: why it is not clearly stated the variant used by T. ammonificans?

2) Figures are not numbered in the order of citation in the text. For example, Figure 7 and 8 (subsection “Carbon Fixation”, last paragraph and subsection “Hydrogen oxidation”, second paragraph, respectively) appear in the text right after Figure 3 while Figure 4 is only cited in the third paragraph of the subsection “Insight into the evolution of carbon fixation”. Also Figure 5 appears before Figure 4 (subsection “Comparative genomic of T. ammonificans: ancestral and acquired metabolic traits”, second paragraph)

3) Subsection “Carbon Fixation”, last paragraph: the authors refer to two genes coding for CodH. But Theam_0999 is not listed in Table 2 and it is not clearly explained what it is. Only in the aforementioned paragraph it is stated that it has 81% similarity with a gene of D. thermolithotrophum. Theam_0999 is also not listed in Table 3, then it is presumably not expressed in the tested growth conditions.

4) Subsection “Carbon Fixation”, last paragraph: what are the bases for the hypothesis that type V CodH may represent the most ancient version of these enzymes?

5) Subsection “Sulfur reduction”: if Theam_1321 has no homologs in the database how was its putative function (NAD- or FAD-dependent reductase) assigned?

6) Subsection “Activated methyl cycle”: I missed what is the evidence to conclude that "In T. ammonificans, the activated methyl cycle is of the second type."

Reviewer #2:

Giovanelli et al. performed comparative genomic, proteomic and phylometabolic analyses, of the anaerobic thermophile, Thermovibrio ammonificans and identified two distinct groups of genes. One that codes for enzymes that do not require oxygen and use substrates of geothermal origin and another that may reflect the rise of oxygen on Earth. Based on this they propose the last common ancestor was a hydrogen oxidizing, sulfur reducing bacterium that used a hybrid pathway for CO2 fixation. I find this an interesting manuscript, with potential interest, but have concerns regarding the methods used in the phylogenomic analyses and also the general scope of the paper.

First, the authors start out in the Abstract writing about early earth and evolution and origins of life, but the paper itself is more of a descriptive narrative of the different pathways encoded and expressed by the organism. The links to evolution and origin of life and this organism are buried in the paper behind long stretches of descriptive text that reads more like a genome announcement paper than a manuscript on the evolution of the group. Second, the authors conclusion that Thermovibrio ammonificans encodes ancestral pathways (e.g., hydrogen oxidation) and more recently acquired ones (e.g., nitrate reduction) and a hybrid pathway for CO2 fixation are not novel conclusions as they have already been shown in several of the authors prior works.

The main part of this manuscript, that has potential as a novel contribution, is the genome evolution aspect. However, the authors do not use a strong phlyogenomic approach to support their claim of the original metabolism of the common ancestor. From what I can tell, they base this conclusion on a comparison of the target organism to only two other related strains (in the Methods section “Comparative genomics”). The proper way to test such a hypothesis would be to take all available genomes from the two clades in question and perform a phylogenomic analysis across all members to identify those genes that have been vertically inherited (e.g., shared protein clusters) as opposed to horizontally inherited (Enright et al., 2002 NAS). This can be done using a Markov Cluster algorithm for example (Nelson-Sathi et al., 2015 Nature). Vertically inherited genes can be defined based on two simple criteria: (1) the protein should be present in at least two higher taxa of the two clades in question and (2) its tree should recover monophyly of the two clades (Weiss et al. 2016 Nature Microbiology). Genes meeting both criteria are unlikely to have undergone LGT, and thus were probably present in the last common ancestor. Unfortunately, such an analysis is missing in the authors study, but is needed to support this main conclusion. I cannot support publication until this major concern is addressed.

Specific comments:

Introduction, second paragraph: There is a lot of evidence suggesting that the reductive acetyl CoA was the first carbon fixation pathway, and that the first organism was actually a methanogen, not a bacterium, and that this organism evolved under lower temperature hydrothermal conditions. Please read some of the recent papers from Bill Martins group, for example Weiss et al. 2016 (Nature Microbiology), where they did phylogenomic analyses of all prokaryotic genomes to date.

Introduction, third paragraph: Regarding the reductive acetyl CoA pathway and the origins of life I suggest you should also read the new review by Sojo et al. in Astrobiology (2016).

Introduction, third paragraph, last sentence: This is not a universally accepted concept as the authors imply with this sentence. Please see Weiss et al. 2016 (Nature Microbiology), who show that LUCA was likely a methanogen.

Subsection “Carbon fixation”, last paragraph: If not all the genes in the pathway are encoded, then how do you explain your discovery of all the proteins (stated a few paragraphs above)?

Subsection “Carbon fixation”, last paragraph: Please provide citations for the presence of this pathway in sulfate reducers and anammox bacteria.

“We hypothesize that the type V CodH may represent the most ancestral version of this class of enzymes, and may catalyze the reduction of CO2 to CO”: Do you base this hypothesis on your own data or someone else?

Subsection “Hydrogen oxidation”, end of third paragraph: How is this link suggested here? This is not clearly explained.

Subsection “Comparative genomic of T. ammonificans: ancestral and acquired metabolic traits”, second paragraph: Why did you choose specifically these organisms to compare?

Subsection “Comparative genomic of T. ammonificans: ancestral and acquired metabolic traits”, second paragraph: From what you write here it is not clear how your synteny analysis shows that this horizontal gene transfer occurred.

Subsection “Comparative genomic of T. ammonificans: ancestral and acquired metabolic traits”, third paragraph: Can you discuss the connections to the Weiss et al. paper (2016) which has already analyzed this topic in more greater detail?

Subsection “Comparative genomic of T. ammonificans: ancestral and acquired metabolic traits”, end of third paragraph: It is already well known that reductive acetyl CoA pathway emerged prior to photosynthesis. What do you mean that the central metabolism of T. ammonificans is "ancestral"? This is very general, and not a new idea.

Subsection “Comparative genomic of T. ammonificans: ancestral and acquired metabolic traits”, last two paragraphs: This is an interesting idea, but where is your data supporting this hypothesis? No figures or tables are cited in the text…

Subsection “Insight into the evolution of carbon fixation”, first paragraph: The Brakeman and Smith study is outdated, more updated is Weiss et al. 2016 Nature Microbiology.

Subsection “Insight into the evolution of carbon fixation”, end of first paragraph: I disagree, new analyses of all prokaryotic genomes and biochemical pathways looking at the oldest conserved genes between all organisms suggest LUCA was a methanogen with reductive acetyl CoA pathway (Weiss et al. 2016). Not Aquificae as the authors propose here.

Reviewer #3:

This is a very comprehensive and beautifully written genomic & proteomic analysis of Thermovibrio ammonificans, followed by a comparative analysis of its C fixation pathways with those of the Aquificaceae and the sulfur/hydrogen-oxidizing epsilonproteobacteria. On first reading, I can only find minor details that need attention (see below). After seeing how cursory many genomes are treated, it was a pleasure to read this analysis and its careful exploration of autotrophic pathway evolution.

The underpinnings of this manuscript – dividing biochemical pathways into ancestral and modern, and then dissecting the genome of a bacterium into ancient and modern layers, like different time horizons in an archaeological excavation – require a short comment. The complexity of even "ancient" pathways, genes and enzymes reflects the fact that every "ancient" gene and enzyme that still exists today has a billion-year history of evolutionary finetuning and, often enough, lateral gene transfer and functional repurposing. Nothing has survived from the dawn of life unchanged. This short preface should explain why I take some introductory statements of this article with a grain of salt, for example the big jump from Wachterhauser to the rTCA cycle (see below).

Introduction, second paragraph: If this assessment – the Aquificae as an early lineage – is based on 16S rRNA gene sequences, you should say this explicitly.

Introduction, third paragraph: I don't have access to Waechtershauser 1988 right now, but his article is focusing on surface catalysis of CO2, CO and small organic molecules being assembled into larger molecules, all on a metal-sulfide or pyrite surface; the theme is prebiotic "autotrophic" syntheses catalyzed by a reactive surface. The fully developed rTCA cycle is far away from that.

The last sentence of the subsection “Conclusions” – "we argue that proposed model for the evolution of metabolism presented in this paper may represent the blueprint of the chemistry of early life" – goes too far in the same direction. Most problematically in origin of life scenarios, early probiotic reactions, like covalent bond formations at reactive interfaces or charge separation at mineral surfaces, are separated by a huge gulf from fully developed enzymatic systems, encoded by genes, transcribed by mRNA, translated by ribosomes… you get the idea. Even ancestral enzymes and pathways only provide a view on the evolution of fully differentiated, metabolically active and self-sustaining microorganisms that we would recognize today [with proper genomes, not to forget], but they do not allow a view to the really early stages and origin of life; theories on communal genomes before cell differentiation, and early evolution of protein domains and structural subunits try to fill the gap. This difference should be acknowledged. Again, nothing has survived from the dawn of life unchanged.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Insight into the evolution of microbial metabolism from the deep-branching bacterium, Thermovibrio ammonificans" for further consideration at eLife. Your revised article has been reevaluated by Gisela Storz (Senior Editor), a Reviewing Editor, and two reviewers.

One of the reviews revealed some considerable major concerns. Normally, this would be reason for rejection of the paper. However, some of the issues may be based on a misunderstanding with the original reviews. Therefore, if you can address the remaining major concerns we would consider a further revised submission. The critical review comments follow below.

Following are the key issues that need to be addressed:

Much of the rebuttal consists of straw man arguments regarding the Weiss et al. study and origins of life issue that detract from my main points raised about their analyses. I apologize if the authors got the wrong idea from my original review, which was not to ask them to re-write their manuscript on the origins of life. I was not a co-author on that paper, but it is a good example of extensive taxon sampling and (in principle) a good method for determining vertical inheritance. As the authors note in their rebuttal whether or not Weiss et al. actually followed their method is currently debated in online comment forums, but this is outside the scope of the authors’ manuscript. By focusing on this, I think that the authors may have missed the main point of my review, which was that their methods for phylogenomic analyses were not sufficient (or at least not sufficiently explained in the Methods). It is possible that analyses were done satisfactorily but this is impossible to tell from the information provided. I have re-read the authors Methods carefully a second time in the revised manuscript, yet it is not possible to tell how the authors processed their data in the different analyses.

My main point is that the authors need to explain in their Methods exactly how they did the different phylogenetic analyses, and provide a satisfactory justification for why they chose to treat their data in the way they did (e.g., why no concatenated gene phylogenies?) instead of following other conventional methods for interpreting bacterial phylogenomics. For example, concatenated gene trees possess more phylogenetic signals, and are less susceptible to the stochastic errors than those built from single genes (Wu and Eisen, 2008; Jeffroy et al. 2006). From what I can tell the authors performed single gene phylogenies of a handful of genes picked from the genomes.

The authors write in their rebuttal: "That is incorrect, as members of the Desulfurobacteriaceae, Aquificaceae and Epsilonproteobacteria were included in the analyses and discussed throughout the manuscript. We used all available Aquificae genomes (as indicated in Table 3), and further focused only on the closest relative and the Epsilonproteobacteria as an ecological proxy."

A quick search of all available genomes from the Aquificae in IMG reveals that there are genomes from 29 representatives available for analysis. Table 3 in the manuscript contains 17 of these, which cover the major clades but does not include all available genomes from each clade. Thus, the statement that the authors make that "Comparative analyses between the genomes of T. ammonificans, all available Aquificae genomes…" is incorrect. Taxon sampling (e.g., n=17 vs. n=29) can have an impact on the results of phylogenetic analyses. Usually the addition of more representatives can provide a better resolution. Please justify why you selected only these 17 for analysis.

In the Methods section "Comparative genomics": It is clear from this that you used the genomes in Table 3. But, it is not clear whether these same genomes were used to create the phylogenetic analyses or not.

In the Methods section "Phylogenetic Methods": The authors describe methods for 16S phylogenies and ATP citrate lyase phylogenies. No information is provided regarding the Ni-Fe hydrogenase phylogeny (Figure 5), CO dehydrogenases (Figure 4), or any other phylogenetic analyses that the authors base their conclusions on. At least, not from what I can tell after reading the section. I do not know whether this is intended or a mistake. Since the authors base their conclusions based on these results it seems like a good idea to explain how these analyses were done. For example, please state number of aligned resides for each tree, whether it is amino acids of DNA data, e.g. the standard information that readers deserve to know so that the authors work can be openly evaluated by the scientific community after publication. Another major question remains, which is why have the authors performed only single gene phylogenies as opposed to concatenated gene phylogenies? Please explain. Concatenated gene phylogenies are less susceptible to stochastic errors than those built from single genes.

https://doi.org/10.7554/eLife.18990.021

Author response

Reviewer #1:

The authors use a comparative genomic approach associated to a proteomic analysis to reconstruct the metabolism of Thermovibrio ammonificans and draw an interesting picture of its possible evolution. Particularly interesting is the observation that the genome of T. ammonificans codes for enzymes putatively involved in the reductive acetyl-CoA pathway and in the reductive TCA (rTCA) cycle.

T. ammonificans encodes an unusual CodH (type V), whose function is still unknown. Based on the assumption that the type V CodH works in the rTCA cycle, the authors suggest that in T. ammonificans the reductive acetyl-CoA pathway and the rTCA cycle are both operational and that the bacterium uses one or the other way to fix carbon based on the environmental conditions. This is a key point of the manuscript and should have some experimental support.

Comment on the functionality of the reductive Acetyl-CoA pathway. We agree that the role of the reductive Acetyl-CoA pathway and of the CodH in T. ammonificans is controversial. To begin to understand the role of the reductive Acetyl-CoA pathway in T. ammonificans we carried out experiments aimed to assess the C isotopic fractionation during autotrophic growth of the bacterium under different electron accepting conditions (nitrate and sulfur). Our hypothesis was that under less efficient electron accepting conditions (i.e., sulfur reduction), T. ammonificans might preferentially use the reductive acetyl-CoA pathway, which requires ~1 ATP vs 2-3 ATP for the rTCA cycle. Briefly, we analyzed the biomass 13C fractionation with respect to the CO2 used as substrate by the organisms in triplicated independent experiments. T. ammonificans biomass δ 13 C isotopic fractionation ranged between -6 ‰ and -8 ‰. The values obtained are in the range of values reported in the literature for the rTCA cycle (Berg et al. 2010), confirming the activity of rTCA as main strategy of carbon fixation under the condition tested. Cells of T. ammonificans grown on sulfur as terminal electron acceptor consistently had isotopic value more negative than population grown in nitrate reducing condition of about 2 ‰. While this difference is small and does not support the activity of the reductive acetyl-CoA pathway, it raises interesting questions regarding the differential isotopic fractionation of carbon while growing with alternative electron acceptors. Because results were inconclusive we decided against including these new experiments in our manuscript. Further experiments that go beyond the scope of this manuscript, whose original objective was to put forth a number of hypotheses based on genomic reconstruction, will need to be designed to shed light on the function of the reductive Acetyl-CoA pathway in T. ammonificans.

The manuscript is well presented although in some parts the reader needs to have high familiarity with microbial evolution and metabolism.

I have some questions and concerns:

1) Subsection “Carbon Fixation”, second paragraph: why it is not clearly stated the variant used by T. ammonificans?

A sentence stating that the asymmetric variant of the rTCA cycle is found in T. ammonificans was added (subsection “Carbon Fixation”, second paragraph).

2) Figures are not numbered in the order of citation in the text. For example, Figure 7 and 8 (subsection “Carbon Fixation”, last paragraph and subsection “Hydrogen oxidation”, second paragraph, respectively) appear in the text right after Figure 3 while Figure 4 is only cited in the third paragraph of the subsection “Insight into the evolution of carbon fixation”. Also Figure 5 appears before Figure 4 (subsection “Comparative genomic of T. ammonificans: ancestral and acquired metabolic traits”, second paragraph)

The figure order and numbering has been corrected throughout the manuscript. Thank you for pointing out this inconsistency!

3) Subsection “Carbon Fixation”, last paragraph: the authors refer to two genes coding for CodH. But Theam_0999 is not listed in Table 2 and it is not clearly explained what it is. Only in the aforementioned paragraph it is stated that it has 81% similarity with a gene of D. thermolithotrophum. Theam_0999 is also not listed in Table 3, then it is presumably not expressed in the tested growth conditions.

Theam_0999 codes for an iron-sulfur cluster accessory protein to CodH (coded by the gene Theam_1337). Reference to Theam_0999 is now present only in the last paragraph of the subsection “Carbon Fixation”.

4) Subsection “Carbon Fixation”, last paragraph: what are the bases for the hypothesis that type V CodH may represent the most ancient version of these enzymes?

The possibilty that type V CodH constitute the ancestral form of the enzyme was suggested by Frank Robb while discussing the Techtmann et al., 2012 paper. Briefly, the structure of the protein retains all the catalytic and binding residuals of a canonical CodH while presenting a simpler (and smaller) overall structure. A clarifying sentence was added to the last paragraph of the subsection “Carbon Fixation”.

5) Subsection “Sulfur reduction”: if Theam_1321 has no homologs in the database how was its putative function (NAD- or FAD-dependent reductase) assigned?

Subsection “Sulfur reduction”: The statement of Theam_1321 being unique to T. ammonificans was revised.

6) Subsection “Activated methyl cycle”: I missed what is the evidence to conclude that "In T. ammonificans, the activated methyl cycle is of the second type."

Comment on the activated methyl cycle: In T. ammonificans the activated methyl cycle involves the sahH gene, while a luxS homolog is absent. We clarified this in the subsection “Activated methyl cycle”. A figure depicting the cycle described in the text was also added a diagram of the activated methyl cycle in T. ammonificans as Figure 3—figure supplement 2.

Reviewer #2:

Giovanelli et al. performed comparative genomic, proteomic and phylometabolic analyses, of the anaerobic thermophile, Thermovibrio ammonificans and identified two distinct groups of genes. One that codes for enzymes that do not require oxygen and use substrates of geothermal origin and another that may reflect the rise of oxygen on Earth. Based on this they propose the last common ancestor was a hydrogen oxidizing, sulfur reducing bacterium that used a hybrid pathway for CO2 fixation. I find this an interesting manuscript, with potential interest, but have concerns regarding the methods used in the phylogenomic analyses and also the general scope of the paper.

First, the authors start out in the Abstract writing about early earth and evolution and origins of life, but the paper itself is more of a descriptive narrative of the different pathways encoded and expressed by the organism. The links to evolution and origin of life and this organism are buried in the paper behind long stretches of descriptive text that reads more like a genome announcement paper than a manuscript on the evolution of the group.

The first general comment was about the evolution of early metabolism, which is the main focus of the manuscript. Reviewer 2 stated that the manuscript was too focused on the central metabolism of T. ammonificans, and that such focus is a distraction from the evolution theme. Our answer to that comment is that, to draw any general conclusion on the evolution of metabolism in the Desufurobacteraceae, it was critical to investigate in depth the central metabolic pathways in our model organism. We tried to strike a balance between the two topics by reporting our genome-derived findings on the central metabolic pathways in T. ammonificans (i.e., our data) in the Results section, while discussing the evolutionary implications of our findings in the Discussion section. Reviewers 1 and 3 praised the presentation style of the manuscript.

Second, the authors conclusion that Thermovibrio ammonificans encodes ancestral pathways (e.g., hydrogen oxidation) and more recently acquired ones (e.g., nitrate reduction) and a hybrid pathway for CO2 fixation are not novel conclusions as they have already been shown in several of the authors prior works.

The second general comment of reviewer 2 stated that our conclusions on the ancestral and acquired genome of T. ammonificans, including the hybrid carbon fixation pathway, are not novel and that we had shown these data previously. That is absolutely incorrect, as the data presented here, along with our interpretation, is original work never published before. We did not publish other papers linking the evolution and metabolism of the Desulfurobacteraceae family and, to our knowledge, neither did others.

The main part of this manuscript, that has potential as a novel contribution, is the genome evolution aspect. However, the authors do not use a strong phlyogenomic approach to support their claim of the original metabolism of the common ancestor. From what I can tell, they base this conclusion on a comparison of the target organism to only two other related strains (in the Methods section “Comparative genomics”). The proper way to test such a hypothesis would be to take all available genomes from the two clades in question and perform a phylogenomic analysis across all members to identify those genes that have been vertically inherited (e.g., shared protein clusters) as opposed to horizontally inherited (Enright et al., 2002 NAS). This can be done using a Markov Cluster algorithm for example (Nelson-Sathi et al., 2015 Nature). Vertically inherited genes can be defined based on two simple criteria: (1) the protein should be present in at least two higher taxa of the two clades in question and (2) its tree should recover monophyly of the two clades (Weiss et al. 2016 Nature Microbiology). Genes meeting both criteria are unlikely to have undergone LGT, and thus were probably present in the last common ancestor. Unfortunately, such an analysis is missing in the authors study, but is needed to support this main conclusion. I cannot support publication until this major concern is addressed.

The third general comment was related to our comparative genomics and phylometabolic analyses. Reviewer 2 states that the genome of T. ammonificans was compared only to two closely related species. That is incorrect, as members of the Desulfurobacteriaceae, Aquificaceae and Epsilonproteobacteria were included in the analyses and discussed throughout the manuscript. We used all available Aquificae genomes (as indicated in Table 3), and further focused only on the closest relative and the Epsilonproteobacteria as an ecological proxy.

Specific comments:

Introduction, second paragraph: There is a lot of evidence suggesting that the reductive acetyl CoA was the first carbon fixation pathway, and that the first organism was actually a methanogen, not a bacterium, and that this organism evolved under lower temperature hydrothermal conditions. Please read some of the recent papers from Bill Martins group, for example Weiss et al. 2016 (Nature Microbiology), where they did phylogenomic analyses of all prokaryotic genomes to date.

Comments on the “first organism being a methanogen”. Reviewer 2 refers to Weiss et al., 2016 (DOI: 10.1038/NMICROBIOL.2016.116), which was published after our manuscript was submitted to eLife. The main message of the Weiss et al. paper is that the reconstruction of the most ancestral microbial genome (the authors refer to the genome of LUCA – Last Universal Common Ancestor) suggests that the earliest microorganisms were acetogens (bacteria) and/or methanogens (archaea). Based on their genome reconstruction the authors conclude that LUCA was an anaerobic, thermophilic autotroph that fixed CO2 via the reductive Acetyl-CoA pathway and could live on H2, CO2 and N2 in an environment similar to deep-sea hydrothermal vents. Hence, the statement from reviewer 2 that Weiss et al. concluded that LUCA was a methanogen is incorrect, as it could have been an acetogen as well. That LUCA might have been an acetogen is in agreement with the hypothesis put forward by Braakman and Smith, 2012, who took a completely different approach to reconstruct the origin of microbial metabolism. Having said that, we tend to agree with that scenario proposed by Weiss et al. (and by Braakman and Smith), which, with the exception of nitrogen fixation, fits very nicely with the metabolism of T. ammonificans. It needs to be noted that the presence of a nitrogenase-encoding gene in the genome of LUCA proposed by Weiss et al. is currently a subject of debate on PubMed Commons(https://www.ncbi.nlm.nih.gov/pubmed/27562259). However, while basically our findings are in agreement with the Weiss et al. paper, we want to stress that our study was not designed to identify the earliest form of microbial metabolism. We demonstrate that T. ammonificans is a good model to reconstruct the evolutionary history of early metabolism but we did not suggest that the lineage that led to the present-day Desulfurobacteriaceae and to T. ammonificans had the same metabolism of LUCA. What happened prior to the emergence of the Desulfurobacteriaceae lineage cannot be gleaned by our analyses, and it is not an objective of our study.

Introduction, third paragraph: Regarding the reductive acetyl CoA pathway and the origins of life I suggest you should also read the new review by Sojo et al. in Astrobiology (2016).

Comment on the reductive Acetyl-CoA pathway and the origin of life. Reviewer 2 refers to the recent work by Sojo et al., 2015 (DOI: 10.1089/ast.2015.1406), which proposes three interesting – but so far untested – hypotheses for CO2 reduction in a very specific environment – alkaline vents. The first hypothesis is that the synthesis of Acetyl-CoA was originally driven by the geochemistry of alkaline vents and it was not genetically encoded. The second hypothesis is that the synthesis of Acetyl-CoA as we know it in extant methanogens and acetogens was predated by a “lost” pathway not found in modern cells. The authors themselves refer to this hypothesis as quite “radical” as, among other assumptions, requires that the Haedean oceans were fairly oxidizing. The third hypothesis implies that the redox gradient at alkaline vents was sufficient to reduce CO2. Hypotheses 1 and 3 rely essentially on prebiotic chemistry, while hypothesis 2 assumes a “lost” pathway of which there is no trace in extant microorganisms. We apologize if we conveyed the wrong message, but our manuscript is not about prebiotic chemistry and the origin of life. Our manuscript is about the reconstruction of the central metabolic pathways in an early-branching – but modern – bacterium, T. ammonificans. From that reconstruction we made an effort to tease apart “early” and “acquired” traits to reconstruct the evolution of metabolism in this bacterium. But by “early” or “ancestral” we do not imply “at the origin of life”. Again, what happened prior to the emergence of the Desulfurobacteriaceae lineage cannot be gleaned by our analyses, and it is not an objective of our study. By stating that, among the six known carbon fixation pathways, the rTCA cycle and the reductive Acetyl-CoA pathway are “good candidates for the ancestral carbon fixation pathway”, we refer to a fairly extensive body of work published by others (including work by Bill Martin’s group, to whom reviewer 2 often refers) and we mean “good candidates” – we do not imply that such pathways are the original pathways.

Introduction, third paragraph, last sentence: This is not a universally accepted concept as the authors imply with this sentence. Please see Weiss et al. 2016 (Nature Microbiology), who show that LUCA was likely a methanogen.

See our first response to specific comments.

Subsection “Carbon fixation”, last paragraph: If not all the genes in the pathway are encoded, then how do you explain your discovery of all the proteins (stated a few paragraphs above)?

Comment on the discrepancy between gene and protein detection. We changed “All” to “Most” (subsection “The proteome of T. ammonificans”).

Subsection “Carbon fixation”, last paragraph: Please provide citations for the presence of this pathway in sulfate reducers and anammox bacteria.

Request to add a reference to substantiate the claim that the CodH is also found in sulfate reducers and anammox. The Berg et al., 2010 reference was added to the last paragraph of the subsection “Carbon Fixation”.

“We hypothesize that the type V CodH may represent the most ancestral version of this class of enzymes, and may catalyze the reduction of CO2 to CO”: Do you base this hypothesis on your own data or someone else?

Comment of the hypothesis that type V CodH is the most ancestral group. Please see related answer to reviewer 1.

Subsection “Hydrogen oxidation”, end of third paragraph: How is this link suggested here? This is not clearly explained.

Comment on the link between the distribution of hydrogenases and oxygen adaptation. Type 2 hydrogenases are found in microaerobic members of the Aquificacea and Hydrothermaceae, while type 3a hydrogenases are found in the strictly anaerobic Desulfurobacteriaceae and in methanoges. We clarified this statement in the text (subsection “Hydrogen oxidation”, third paragraph).

Subsection “Comparative genomic of T. ammonificans: ancestral and acquired metabolic traits”, second paragraph: Why did you choose specifically these organisms to compare?

Subsection “Comparative genomic of T. ammonificans: ancestral and acquired metabolic traits”, second paragraph: Comment on the choice of organisms for direct comparative genomic analyses. As explained in the text, we choose D. thermolithotrophum because it is the closest relative to T. ammonificans whose genome is available, and C. mediatlanticus because, while being phylogetically distant from T. ammonificans, occupies a similar ecological niche and has a similar central metabolism (it is a hydrogenotroph that conserves energy by nitrate or sulfur reduction and fixes CO2 via the rTCA cycle).

Subsection “Comparative genomic of T. ammonificans: ancestral and acquired metabolic traits”, second paragraph: From what you write here it is not clear how your synteny analysis shows that this horizontal gene transfer occurred.

Comment on the possible horizontal gene transfer (HGT) of the ATP citrate lyase between Epsilonproteobacteria and Desulfurobacteraceae. This HGT event was previously suggested by Hügler et al., 2007, Env. Microbiol 9:81. However, we agree with reviewer 2 that the synteny diagram in Figure 8 does not indicate such HGT event, and we removed the last sentence of the paragraph.

Subsection “Comparative genomic of T. ammonificans: ancestral and acquired metabolic traits”, third paragraph: Can you discuss the connections to the Weiss et al. paper (2016) which has already analyzed this topic in more greater detail?

Subsection “Comparative genomic of T. ammonificans: ancestral and acquired metabolic traits”, end of third paragraph: It is already well known that reductive acetyl CoA pathway emerged prior to photosynthesis. What do you mean that the central metabolism of T. ammonificans is "ancestral"? This is very general, and not a new idea.

Comments on relating our analysis of the genome of T. ammonificans with the work of Weiss et al., 2016, and on the ancestry of the central metabolism of T. ammonificans. Our genome reconstruction and phylogenetic analyses indicate that the core or ancestral groups of genes involved in the central metabolism of T. ammonificans include genes coding for Group 3 hydrogenases, genes coding for enzymes of the reductive Acetyl-CoA pathway and genes involved in sulfur reduction. These pathways are catalyzed by enzymes that are extremely sensitive to oxygen which are either present in early branching Archaea, or are directly involved in metabolic reactions that do not require oxygen (or oxygen by-products) and use substrates of geothermal origin. Weiss et al. reconstruction of the LUCA genome is surprisingly consistent with our findings: the authors conclude that “FeS and transition metals are relics of ancient metabolism, that life arose at hydrothermal vents and that sulfur was involved in ancient metabolism”. Similarly to T. ammonificans, LUCA was an H2-dependent autotroph capable of S utilization that could fix CO2 via the reductive Acetyl-CoA pathway. What this means is that part of the genome of T. ammonificans, a modern bacterium that lives today at deep-sea vents, bears some striking similarities to the putative genome of LUCA as reported by Weiss et al. Therefore, part of the central metabolism of T. ammonificans is ancestral. We added a sentence to the third paragraph of the subsection “Comparative genomic of T. ammonificans: ancestral and acquired metabolic traits” with a reference to the Weiss et al. paper.

Subsection “Comparative genomic of T. ammonificans: ancestral and acquired metabolic traits”, last two paragraphs: This is an interesting idea, but where is your data supporting this hypothesis? No figures or tables are cited in the text.

Comment on the oxygen radical detoxification enzymes being acquired as a response to oxygen exposure. This is a hypothesis generated on the widely-accepted assumption that prior to the onset of oxygenic photosynthesis the Earth was anoxic. Hence, it is reasonable to posit that the oxygen radical detoxification machinery is a relatively “late” acquisition in the lineage that led to T. ammonificans. We are currently carrying out experiments to test the function of these enzymes in T. ammonificans. Such experiments go beyond the scope of this paper.

Subsection “Insight into the evolution of carbon fixation”, first paragraph: The Brakeman and Smith study is outdated, more updated is Weiss et al. 2016 Nature Microbiology.

Comment on the Braakman and Smith study being “outdated”, and the underlying suggestion by reviewer 2 to refer exclusively to the Weiss et al. study. We disagree. First, the Weiss et al. paper was published after our manuscript was submitted to eLife. Second, the Weiss et al. paper does not refute the Braakman and Smith findings. Rather, despite the radically different approaches used in the two studies – the Braakman and Smith is based on phylometabolic reconstruction while the Weiss et al. is based on gene phyletic pattern reconstruction – both point to the basal lineage being autotrophic and using the reductive Acetyl-CoA pathway. We included a reference to the Weiss paper in the first paragraph of the subsection “Insight into the evolution of carbon fixation”.

Subsection “Insight into the evolution of carbon fixation”, end of first paragraph: I disagree, new analyses of all prokaryotic genomes and biochemical pathways looking at the oldest conserved genes between all organisms suggest LUCA was a methanogen with reductive acetyl CoA pathway (Weiss et al. 2016). Not Aquificae as the authors propose here.

Reviewer 2 disagrees with the conclusion of the Braakman and Smith study, states that Weiss et al. demonstrate that LUCA was a methanogen, and implies that we propose that LUCA was an Aquificae. First, Weiss at al. suggest that LUCA’s genomic makeup points to an autotroph that fixed carbon via the reductive acetyl-CoA pathway, and suggest acetogenic and/or methanogenic roots. They do not unequivocally show that LUCA was a methanogen. Second, we are not proposing that LUCA was an Aquificae. Instead, we are reporting findings from Braakman and Smith, which are partly in agreement with Weiss et al. (please see the paragraph above), and we discuss that our finding of the reductive acetyl-CoA pathway in T. ammonificans is in line with the hypothesis that this pathway of autotrophic carbon fixation was present in the basal lineage. Again, this is in agreement with findings from BOTH Braakman and Smith and Weiss et al.

Reviewer #3:

This is a very comprehensive and beautifully written genomic & proteomic analysis of Thermovibrio ammonificans, followed by a comparative analysis of its C fixation pathways with those of the Aquificaceae and the sulfur/hydrogen-oxidizing epsilonproteobacteria. On first reading, I can only find minor details that need attention (see below). After seeing how cursory many genomes are treated, it was a pleasure to read this analysis and its careful exploration of autotrophic pathway evolution.

The underpinnings of this manuscript – dividing biochemical pathways into ancestral and modern, and then dissecting the genome of a bacterium into ancient and modern layers, like different time horizons in an archaeological excavation – require a short comment. The complexity of even "ancient" pathways, genes and enzymes reflects the fact that every "ancient" gene and enzyme that still exists today has a billion-year history of evolutionary finetuning and, often enough, lateral gene transfer and functional repurposing. Nothing has survived from the dawn of life unchanged. This short preface should explain why I take some introductory statements of this article with a grain of salt, for example the big jump from Wachterhauser to the rTCA cycle (see below).

Introduction, second paragraph: If this assessment – the Aquificae as an early lineage – is based on 16S rRNA gene sequences, you should say this explicitly.

Introduction, second paragraph: The assessment that Aquificae are an early-branching lineage is based on phylogenetic analyses of both the 16S rRNA gene and whole genomes. We added a sentence stating that.

Introduction, third paragraph: I don't have access to Waechtershauser 1988 right now, but his article is focusing on surface catalysis of CO2, CO and small organic molecules being assembled into larger molecules, all on a metal-sulfide or pyrite surface; the theme is prebiotic "autotrophic" syntheses catalyzed by a reactive surface. The fully developed rTCA cycle is far away from that.

The comment about Wächtershäuser’s iron-sulfur-based prebiotic synthesis model being far away from a functional rTCA cycle is correct. To avoid ambiguities, we removed the references to Wächtershäuser’s work associated to this specific statement. However, the idea that Wächtershäuser’s proposed autocatalytic “surface” metabolism eventually transitioned to a primitive, sulfur dependent version of modern rTCA cycle is embedded in his work and is presented further down the paragraph (Introduction, third paragraph).

The last sentence of the subsection “Conclusions” – "we argue that proposed model for the evolution of metabolism presented in this paper may represent the blueprint of the chemistry of early life" – goes too far in the same direction. Most problematically in origin of life scenarios, early probiotic reactions, like covalent bond formations at reactive interfaces or charge separation at mineral surfaces, are separated by a huge gulf from fully developed enzymatic systems, encoded by genes, transcribed by mRNA, translated by ribosomes… you get the idea. Even ancestral enzymes and pathways only provide a view on the evolution of fully differentiated, metabolically active and self-sustaining microorganisms that we would recognize today [with proper genomes, not to forget], but they do not allow a view to the really early stages and origin of life; theories on communal genomes before cell differentiation, and early evolution of protein domains and structural subunits try to fill the gap. This difference should be acknowledged. Again, nothing has survived from the dawn of life unchanged.

We removed the reference to the chemistry of early life from the concluding sentence and emphasized the importance of using early branching microorganisms to investigate the evolution of metabolism (subsection “Conclusions”).

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Following are the key issues that need to be addressed:

Much of the rebuttal consists of straw man arguments regarding the Weiss et al. study and origins of life issue that detract from my main points raised about their analyses. I apologize if the authors got the wrong idea from my original review, which was not to ask them to re-write their manuscript on the origins of life. I was not a co-author on that paper, but it is a good example of extensive taxon sampling and (in principle) a good method for determining vertical inheritance. As the authors note in their rebuttal whether or not Weiss et al. actually followed their method is currently debated in online comment forums, but this is outside the scope of the authors’ manuscript. By focusing on this, I think that the authors may have missed the main point of my review, which was that their methods for phylogenomic analyses were not sufficient (or at least not sufficiently explained in the Methods). It is possible that analyses were done satisfactorily but this is impossible to tell from the information provided. I have re-read the authors Methods carefully a second time in the revised manuscript, yet it is not possible to tell how the authors processed their data in the different analyses.

My main point is that the authors need to explain in their Methods exactly how they did the different phylogenetic analyses, and provide a satisfactory justification for why they chose to treat their data in the way they did (e.g., why no concatenated gene phylogenies?) instead of following other conventional methods for interpreting bacterial phylogenomics. For example, concatenated gene trees possess more phylogenetic signals, and are less susceptible to the stochastic errors than those built from single genes (Wu and Eisen, 2008; Jeffroy et al. 2006). From what I can tell the authors performed single gene phylogenies of a handful of genes picked from the genomes.

In this second revision, we have significantly amended our Methods section (see the Methods section in the revised manuscript) and taken further step to ensure reproducibility of our analyses. We have also expanded the phylogenetic section by adding additional information requested by reviewer 2, including the number of sites used for the reconstruction. We strongly believe that reproducibility in science is a very important issue, and we strive to make our methods and data publicly available. All comparative genomic analysis and alignments carried out were already directly accessible as permanent url in the public genomeevolution.org server.

In order to identify the ancestral and acquired portion of the central metabolism of T. ammonificans, we used a phylometabolic – rather than a phylogenomic – approach. In phylometabolic analyses, the metabolic pathways of the organism under investigation are compared to those found in related organisms both within and across neighboring clades. By focusing on the pathways, the comparison may reveal variations in multi-enzyme functional units, providing context for the completion of the pathway within the networks of individual organism, while also allowing for the identification of ancestral states and horizontal gene transfer events. The resulting phylometabolic tree includes multiple complete pathways to common essential metabolites, and suggests which evolutionary substitutions are allowed (at either organism or ecosystem levels) among these pathways (see Braakman and Smith, 2012 and reference therein for a more extensive description of the principles underlying this approach and more recently Braakman et al., 2017 for ecological application of this principles). In this second revision we included a section in the Methods explaining the rationale underlying phylometabolic approaches.

The authors write in their rebuttal: "That is incorrect, as members of the Desulfurobacteriaceae, Aquificaceae and Epsilonproteobacteria were included in the analyses and discussed throughout the manuscript. We used all available Aquificae genomes (as indicated in Table 3), and further focused only on the closest relative and the Epsilonproteobacteria as an ecological proxy."

A quick search of all available genomes from the Aquificae in IMG reveals that there are genomes from 29 representatives available for analysis. Table 3 in the manuscript contains 17 of these, which cover the major clades but does not include all available genomes from each clade. Thus, the statement that the authors make that "Comparative analyses between the genomes of T. ammonificans, all available Aquificae genomes…" is incorrect. Taxon sampling (e.g., n=17 vs. n=29) can have an impact on the results of phylogenetic analyses. Usually the addition of more representatives can provide a better resolution. Please justify why you selected only these 17 for analysis.

In the Methods section "Comparative genomics": It is clear from this that you used the genomes in Table 3. But, it is not clear whether these same genomes were used to create the phylogenetic analyses or not.

At the time of writing, a quick search on the NCBI genome page will confirm the availability of 23 genomes divided in 17 Aquificae species. However, the number of available genomes is constantly increasing (the genome of Thermocrinis minervae was released in 2016/12/02 while Desulfurobacterium indicum was released in 2017/01/23; please see the list at the end of this letter).

In our analysis we included all the available genomes of validly published Aquificae species – that is, species that have been physiologically characterized and are recognized by the International Committee on Systematics of Prokaryotes – and for which the type strain is available from a cell culture collection (e.g., ACCT or DSMZ). This will allow for future studies based on our findings to go beyond comparative genomics and test our hypotheses experimentally, using comparative physiological, transcriptomic and proteomic analyses. However, in order to maximize the diversity of our genome dataset, we made two exceptions to this rule. The first exception was to include in the analysis one genome to represent the genus Hydrogenivirga and one to represent the genus Hydrogenobaculum (for which we selected the current reference genome, see NCBI page), as no genome sequence is available for validly published species of these genera. The second exception was to select two additional genomes belonging to the genera Desulfurobacterium (the closest relative to the genus Thermovibrio) and Persephonella, respectively.

The genomes excluded from our analysis are:

Four closely related genomes of the genus Hydrogenobaculum (average gapped genome similarity above 94% to the included Hydrogenobaculum sp. Y04AAS1 genomehttps://www.ncbi.nlm.nih.gov/genome/neighbors/13671?genome_assembly_id=301047)

An alternative assembly of Hydrogenobacter thermophilus TK-6 already included in the analysis

One genome of the genus Persephonella (gapped genome similarity above 92% to Persephonella sp. IF05-L8 included in the analysis,https://www.ncbi.nlm.nih.gov/genome/neighbors/32114?genome_assembly_id=202711).

In the Methods section "Phylogenetic Methods": The authors describe methods for 16S phylogenies and ATP citrate lyase phylogenies. No information is provided regarding the Ni-Fe hydrogenase phylogeny (Figure 5), CO dehydrogenases (Figure 4), or any other phylogenetic analyses that the authors base their conclusions on. At least, not from what I can tell after reading the section. I do not know whether this is intended or a mistake. Since the authors base their conclusions based on these results it seems like a good idea to explain how these analyses were done. For example, please state number of aligned resides for each tree, whether it is amino acids of DNA data, e.g. the standard information that readers deserve to know so that the authors work can be openly evaluated by the scientific community after publication. Another major question remains, which is why have the authors performed only single gene phylogenies as opposed to concatenated gene phylogenies? Please explain. Concatenated gene phylogenies are less susceptible to stochastic errors than those built from single genes.

We agree with reviewer 2 about the usefulness of concatenated phylogenies. However, the use of concatenated vs. single protein phylogenies depends on the objective of the study. Concatenated phylogenies are a powerful tool to resolve the phylogenetic relationships among organisms, and undoubtedly are less susceptible to stochastic errors (Wu and Eisen, 2008). Concatenated phylogenies are thus the appropriate mode of analysis when the objective is to resolve organismal relationships. However, concatenated phylogenies cannot be used to reconstruct the phylogenetic history of a gene (unless the concatenated phylogeny is performed exclusively on the multiple subunit of the same protein complex, as we have performed in the present study for the ATP-citrate lyase). Phylogenies of single enzyme (that can include multiple subunits) are necessary to reconstruct the phylogenetic history of the metabolic pathways they represent. In this context, the use of concatenated phylogenies, which include different genes or enzymes, can mask lateral gene transfer events, which need to be investigated singularly (Boussau et al., 2008; Philippe and Douady, 2003; Bapteste et al., 2004). Our objective was to identify the ancestral and acquired complement of the genome of T. ammonificans, and compare it with the genomes of other members of the Aquificae. A revision of the overall phylogenetic relationship among the different species and families of the Aquificae was not the object of this study. Rather, we aimed at reconstructing the history of key enzymes diagnostic of specific biochemical pathways, and at comparing the reconstructed histories of such key enzymes among members of Aquificae.

Therefore, we believe that the use of single protein phylogenies is appropriate to the objectives of our study.

https://doi.org/10.7554/eLife.18990.022

Article and author information

Author details

  1. Donato Giovannelli

    1. Institute of Earth, Ocean and Atmospheric Sciences, Rutgers University, New Brunswick, United States
    2. Institute of Marine Science, National Research Council of Italy, Ancona, Italy
    3. Program in Interdisciplinary Studies, Institute for Advanced Studies, Princeton, United States
    4. Earth-Life Science Institute, Tokyo Institute of Technology, Tokyo, Japan
    Contribution
    DG, Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing—original draft, Writing—review and editing
    For correspondence
    giovannelli@marine.rutgers.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon 0000-0001-7182-8233
  2. Stefan M Sievert

    Biology Department, Woods Hole Oceanographic Institution, Woods Hole, United States
    Contribution
    SMS, Conceptualization, Formal analysis, Funding acquisition, Methodology, Writing—review and editing
    Competing interests
    The authors declare that no competing interests exist.
  3. Michael Hügler

    DVGW-Technologiezentrum Wasser, Karlsruhe, Germany
    Contribution
    MH, Conceptualization, Formal analysis, Investigation, Writing—review and editing
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon 0000-0002-2820-0333
  4. Stephanie Markert

    Pharmaceutical Biotechnology, Institute of Pharmacy, Institute of Pharmacy, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany
    Contribution
    SM, Data curation, Formal analysis, Methodology, Writing—review and editing
    Competing interests
    The authors declare that no competing interests exist.
  5. Dörte Becher

    Institute for Microbiology, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany
    Contribution
    DB, Formal analysis, Methodology, Writing—review and editing
    Competing interests
    The authors declare that no competing interests exist.
  6. Thomas Schweder

    Pharmaceutical Biotechnology, Institute of Pharmacy, Institute of Pharmacy, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany
    Contribution
    TS, Conceptualization, Data curation, Methodology, Writing—review and editing
    Competing interests
    The authors declare that no competing interests exist.
  7. Costantino Vetriani

    1. Institute of Earth, Ocean and Atmospheric Sciences, Rutgers University, New Brunswick, United States
    2. Department of Biochemistry and Microbiology, Rutgers University, New Brunswick, United States
    Contribution
    CV, Conceptualization, Formal analysis, Supervision, Funding acquisition, Writing—original draft, Project administration, Writing—review and editing
    For correspondence
    vetriani@marine.rutgers.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon 0000-0002-8141-8438

Funding

National Science Foundation (MCB 04-56676)

  • Costantino Vetriani

National Aeronautics and Space Administration (NNX15AM18G)

  • Costantino Vetriani

National Science Foundation (OCE 03-27353)

  • Costantino Vetriani

National Science Foundation (MCB 08-43678)

  • Costantino Vetriani

National Science Foundation (OCE 09-37371)

  • Costantino Vetriani

National Science Foundation (OCE 11-24141)

  • Costantino Vetriani

National Science Foundation (MCB 15-17567)

  • Donato Giovannelli
  • Costantino Vetriani

National Science Foundation (OCE-1136727)

  • Stefan M Sievert

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

The authors gratefully acknowledge the support of the Deep Carbon Observatory. Thanks to Sandra Beyer for assistance in the lab and to Sebastian Grund for MS measurements. The genome of Thermovibrio ammonificans was sequenced under the auspices of the US Department of Energy. Work on T. ammonificans was supported, in part, by NSF grants MCB 04–56676, OCE 03–27353, MCB 08–43678, OCE 09–37371, OCE 11–24141, OCE11-36451, and NASA grant NNX15AM18G to CV, NSF grant MCB 15–17567 to CV and DG, and by the New Jersey Agricultural Experiment Station. DG was supported by a Postdoctoral Fellowship from the Institute of Marine and Coastal Sciences and a Postdoctoral Fellowship from the Center for Dark Energy Biosphere Investigations (C-DEBI). Funding to SMS was provided by NSF grant OCE-1136727 and a senior fellowship by the Alfried Krupp Wissenschaftskolleg Greifswald, Germany. This publication was in part supported by the ELSI Origins Network (EON), which is supported by a grant from the John Templeton Foundation. The opinions expressed in this publication are those of the authors and do not necessarily reflect the views of the John Templeton Foundation. This paper is C-DEBI contribution 368.

Reviewing Editor

  1. Joerg Bohlmann, Reviewing Editor, University of British Columbia, Canada

Publication history

  1. Received: June 21, 2016
  2. Accepted: April 23, 2017
  3. Accepted Manuscript published: April 24, 2017 (version 1)
  4. Version of Record published: May 23, 2017 (version 2)

Copyright

© 2017, Giovannelli et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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