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
10 figures and 3 tables


Figure 1 with 1 supplement
Phylogenetic tree of 16S rRNA sequences computed from the current version of the aligned 16S rRNA database obtained from the arb-SILVA project (
Figure 1—figure supplement 1
Maximum likelihood tree showing the 16S rRNA relationship of the Aquificae phylum.

Thermotoga spp. were used as outgroup. Bootstrap values based on 1000 replication are shown at branch node. Bar, 5% estimated substitution.
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.
Figure 3 with 2 supplements
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.
Figure 3—source data 1

List of the proteins identified in the proteome of T. ammonificans grown under nitrate reducing conditions.
Figure 3—figure supplement 1
Reductive TCA cycle variants found in extant bacterial lineages.

(A) two-step variant of the rTCA cycle, also known as symmetric variants, were the carboxylation of 2-oxoglutarate is performed in two steps by the enzymes 2-oxoglutarate carboxylase and oxalosuccinate reductase, and the cleavage of citrate is performed in two step by the citryl-CoA synthetase and citryl-CoA lyase enzymes; (B) one-step variant of the rTCA cycle, also known as asymmetric variants, were the carboxylation of 2-oxoglutarate is performed in a single step by the enzymes isocitrate dehydrogenase, and the cleavage of citrate to acetyl-CoA is performed in a single reaction by the ATP citrate lyase enzyme.
Figure 3—figure supplement 2
The activated methyl cycle of T. ammonificans reconstructed from the genome.
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.
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.
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.
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 (; T. ammonificans and C. mediatlanticus (; D. thermolithotrophum and C. mediatlanticus (
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 ( against T. ammonificans.
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.
Figure 9—source data 1

Accession number of the ATP citrate lyase sequences used for the reconstruction of the phylogenetic history of the enzyme presented in Figure 2 in the main text.
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.


Table 1

Characteristic of representative members of Aquificae phylum.
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.

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.
Putative gene locusClosest relativePutative
D. thermolitho
sp. TC5-1
P. marinaSulfurihydro
genibium sp. YO3AOP1
H. themophilusHydrogeno
baculum sp. Y04AAS1H
virga sp. 128–5 R1-6
T. ruberA. aeolicus
SB155-2 sim.
55% YP_001357016
Methanogensf.e.§ - 30%
-**56% YP_002730364f.e.§ - 32% YP_001930236f.e.§ - 33% YP_003433330--f.e.§ - 27% YP_003474076-
sp. 128–5
R1-1 sim.
41% WP_008285842
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

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

mide formyl
transferase 2
Theam_1211P. marina
76% YP_002731257
/ Deltaproteo
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

carboxamide formyltransferase
/IMP cyclohydrolase
Theam_0328P. marina
59% YP_002731268
Aquificae / 
86% YP_00428168178% WP_02284735959% YP_00273126856% YP_00193123953% YP_00343327050% YP_00749947357% WP_00828836052% YP_00347433756% NP_214344
 dehydrogenase / methenyltetra
hydrofolate cyclohydrolase
Theam_1261A. aeolicus sim.
Aquificae /
Gram +
87% YP_00428114774% WP_02284621755% YP_00273147658% YP_00193124059% YP_00551131858% YP_001931240-58% YP_00347310460% NP_214304
hydrofolate reductase
Theam_0919Methanosrcina barkeri sim. 48%
Methanogens87% YP_00428114774% WP_02284621755% YP_00273147658% YP_00193124059% YP_00551131858% YP_002121558f.e.§ - 24% WP_00828678358% YP_00347310460% NP_214304
corrinoid/iron sulfurprotein
Theam_1184Clostridium glycolicum
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
Theam_1337Candidatus Methano
sp. BLZ1 sim.
56% KPQ43483
Archaea / Gram +87% WP_013638427
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.

Table 3

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

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  1. Donato Giovannelli
  2. Stefan M Sievert
  3. Michael Hügler
  4. Stephanie Markert
  5. Dörte Becher
  6. Thomas Schweder
  7. Costantino Vetriani
Insight into the evolution of microbial metabolism from the deep-branching bacterium, Thermovibrio ammonificans
eLife 6:e18990.