The complete genome sequence of Staphylothermus marinus reveals differences in sulfur metabolism among heterotrophic Crenarchaeota
- Iain J Anderson1Email author,
- Lakshmi Dharmarajan2,
- Jason Rodriguez2, 3,
- Sean Hooper1,
- Iris Porat4, 13,
- Luke E Ulrich5,
- James G Elkins6,
- Kostas Mavromatis1,
- Hui Sun7,
- Miriam Land6,
- Alla Lapidus7,
- Susan Lucas7,
- Kerrie Barry8,
- Harald Huber9,
- Igor B Zhulin5,
- William B Whitman4,
- Biswarup Mukhopadhyay2, 3, 10,
- Carl Woese11,
- James Bristow12 and
- Nikos Kyrpides1
© Anderson et al; licensee BioMed Central Ltd. 2009
Received: 05 September 2008
Accepted: 02 April 2009
Published: 02 April 2009
Staphylothermus marinus is an anaerobic, sulfur-reducing peptide fermenter of the archaeal phylum Crenarchaeota. It is the third heterotrophic, obligate sulfur reducing crenarchaeote to be sequenced and provides an opportunity for comparative analysis of the three genomes.
The 1.57 Mbp genome of the hyperthermophilic crenarchaeote Staphylothermus marinus has been completely sequenced. The main energy generating pathways likely involve 2-oxoacid:ferredoxin oxidoreductases and ADP-forming acetyl-CoA synthases. S. marinus possesses several enzymes not present in other crenarchaeotes including a sodium ion-translocating decarboxylase likely to be involved in amino acid degradation. S. marinus lacks sulfur-reducing enzymes present in the other two sulfur-reducing crenarchaeotes that have been sequenced – Thermofilum pendens and Hyperthermus butylicus. Instead it has three operons similar to the mbh and mbx operons of Pyrococcus furiosus, which may play a role in sulfur reduction and/or hydrogen production. The two marine organisms, S. marinus and H. butylicus, possess more sodium-dependent transporters than T. pendens and use symporters for potassium uptake while T. pendens uses an ATP-dependent potassium transporter. T. pendens has adapted to a nutrient-rich environment while H. butylicus is adapted to a nutrient-poor environment, and S. marinus lies between these two extremes.
The three heterotrophic sulfur-reducing crenarchaeotes have adapted to their habitats, terrestrial vs. marine, via their transporter content, and they have also adapted to environments with differing levels of nutrients. Despite the fact that they all use sulfur as an electron acceptor, they are likely to have different pathways for sulfur reduction.
Crenarchaeota is one of the two major phyla of the domain Archaea. Many crenarchaeotes are heterotrophic, anaerobic, sulfur-reducing hyperthermophiles, but the crenarchaeotes with completely sequenced genomes are primarily aerobes. Of the archaea with published genomes, only Hyperthermus butylicus and Thermofilum pendens are heterotrophic, obligate sulfur-reducing anaerobes [1, 2]. More genomes are needed from anaerobic crenarchaeotes in order to determine if their phenotypic similarities are reflected in their genomes.
Staphylothermus marinus was isolated from a black smoker and from volcanically heated sediments . It is a hyperthermophile, with a maximum growth temperature of 98°C. Its name reflects its proclivity to form clusters of up to 100 cells. At high concentrations of yeast extract it forms large cells up to 15 μm in diameter. It is a strict anaerobe and grows heterotrophically on complex media. H2S, CO2, acetate and isovalerate are metabolic products, suggesting a metabolism similar to that of the Thermococcales of the phylum Euryarchaeota. Dark granules observed within the cytoplasm may consist of glycogen. While S. marinus can survive in the absence of sulfur and produce hydrogen rather than H2S, it requires sulfur for growth . An unusual cell surface protein named tetrabrachion has been characterized from S. marinus , and a 24-subunit phosphoenolpyruvate-utilizing enzyme with a unique structure has also been studied .
Here we report the complete genome of the anaerobic, sulfur-reducing archaeon S. marinus and a comparative analysis with other sulfur-reducing heterotrophic crenarchaeotes. While some features in S. marinus are similar to H. butylicus  and T. pendens , including peptide fermentation enzymes, there are also major differences, particularly in the electron transport machinery.
The genome of S. marinus F1 consists of a circular chromosome of 1.57 Mbp. There is one copy each of 5S, 16S, and 23S ribosomal RNA. About 59% of protein-coding genes begin with an AUG codon, 8% with GUG, and 33% with UUG. The low number of GUG start codons reflects the low GC content of this genome (35.7% GC). The ribosomal protein L12ae gene (Smar_1096) does not have a valid start codon, but this is likely to be an essential gene. Based on alignment with L12ae proteins from other archaea, it appears that the S. marinus gene begins with an ATC start codon. S. marinus has 12 regions of CRISPR repeats containing between 5 and 17 repeats. Twelve CRISPR-associated proteins are found in the vicinity of three of the repeats, between coordinates 323,400 and 345,500 (Smar_0308-Smar_0325), and one other CRISPR-associated protein is found at a different location not close to any repeats (Smar_1195).
Genome size (bp)
Coding region (bp)
G+C content (bp)
Genes with function prediction
Genes in ortholog clusters
Genes in paralog clusters
Genes assigned to COGs
Genes assigned Pfam domains
Genes with signal peptides
Genes with transmembrane helices
Comparison of COG categories among the three sulfur-reducing crenarchaeotes.
Amino acid transport and metabolism
Carbohydrate transport and metabolism
Cell cycle control, cell division, chromosome partitioning
Cell wall/membrane/envelope biogenesis
Chromatin structure and dynamics
Coenzyme transport and metabolism
Energy production and conversion
General function prediction only
Inorganic ion transport and metabolism
Intracellular trafficking, secretion, and vesicular transport
Lipid transport and metabolism
Nucleotide transport and metabolism
Posttranslational modification, protein turnover, chaperones
RNA processing and modification
Replication, recombination and repair
Secondary metabolites biosynthesis, transport, and catabolism
Signal transduction mechanisms
The S. marinus genome contains several protein families not found before in crenarchaeotes, and these are discussed below. S. marinus is the first crenarchaeote found to have an arginine decarboxylase belonging to COG1166 (Smar_0204), which includes the speA gene of E. coli. This protein family is also found in one euryarchaeote, Methanosaeta thermophila. Most euryarchaeota have a pyruvoyl-dependent arginine decarboxylase . T. pendens and Cenarchaeum symbiosum also contain this type of enzyme. No arginine decarboxylase has been identified in other crenarchaeote genomes. Phylogenetic analysis of the S. marinus arginine decarboxylase (not shown) does not indicate a clear case of lateral gene transfer, and this enzyme was not identified during the search for laterally transferred genes (see below).
S. marinus contains a probable cell surface protein (Smar_0566) with 4 copies of the pfam03640 repeat, which has not been found in any other crenarchaeal genome. This repeat is present in two methanogens, Candidatus Methanoregula boonei and Candidatus Methanosphaerula palustris. It is also found in a wide variety of bacteria, but its function is unknown.
S. marinus is unique among crenarchaeotes in having a sodium ion-translocating decarboxylase for energy generation (Smar_1503-1504). It also has three putative operons containing subunits of multisubunit cation/proton antiporters, although these are likely to belong to large membrane-bound ion-translocating enzyme complexes rather than acting as cation antiporters (see below). S. marinus is the first crenarchaeote found to have a type I restriction-modification system (Smar_0761-0763).
Twenty-one probable laterally transferred genes were identified using the program SIGI-HMM . One gene is by itself (Smar_0375), there are three pairs of genes (Smar_0568-0569, Smar_0846-0847, and Smar_1144-1145) and there is one cluster of 17 genes (Smar_1525-1541) in which 14 of the genes are predicted to be laterally transferred. Twelve of the laterally transferred genes are predicted to have come from other Crenarchaeota, six from Euryarchaeota, and the remaining three have unknown donors. Six of the 17 genes are likely to be pseudogenes, suggesting that they were transferred but then are degrading. From these findings we conclude that lateral transfer has not played a large role in shaping S. marinus gene content, and most if not all gene transfers have come from other archaea.
The presence of transporters for peptides and carbohydrates suggests that both types of compounds can serve as carbon and energy sources. S. marinus has four ABC transporters for carbohydrates (Smar_0088-0091, Smar_0108-0111, Smar_0299-0302, Smar_1146-1149) and two for peptides (Smar_0270-0274, Smar_0342-0346). It has a carbohydrate secondary transporter of the glycoside-pentoside-hexuronide (GPH) family (Smar_0710), and it is the first crenarchaeote found to have a peptide transporter of the oligopeptide transporter (OPT) family (Smar_1400). There are no ABC transporters for amino acids, but a probable amino acid transporter of the neurotransmitter:sodium symporter (NSS) family is present (Smar_0285). The presence of secondary transporters (GPH, OPT, and NSS), which have low affinity and high capacity, suggests that there are times when S. marinus is exposed to high levels of nutrients, and it can conserve energy by using secondary transporters instead of ATP-dependent transporters.
S. marinus has a glycolysis pathway similar to Aeropyrum pernix, with ATP-dependent glucokinase (Smar_1514) and phosphofructokinase (Smar_0007). Glycogen synthase (Smar_1393) and phosphorylase (Smar_0246) are present, suggesting that the dark granules observed in S. marinus cells are composed of glycogen. Similar to other crenarchaeotes and thermococci, S. marinus has pyruvate:ferredoxin oxidoreductase (Smar_1447-1450) and ADP-forming acetyl-CoA synthase (Smar_0449, Smar_1241-1242) for ATP synthesis from pyruvate. Three other 2-ketoacid:ferredoxin oxidoreductases are present (Smar_0291-292, Smar_0997-1000, Smar_1443-1444) that are probably involved in amino acid degradation.
S. marinus, like the other heterotrophic crenarchaeotes H. butylicus and T. pendens, has lost almost all amino acid biosynthetic enzymes, although it has retained a few pathways for specific physiological reasons. For instance, glutamine is needed for its function as a nitrogen donor. Like the other heterotrophic crenarchaeotes, S. marinus can make pyrimidines but not purines. Enzymes for synthesis of several cofactors are present in S. marinus, in contrast to T. pendens, which lacks many cofactor synthesis pathways. S. marinus can likely synthesize riboflavin, pyridoxine, and coenzyme A, but it probably must acquire heme from the environment.
Electron transport/sulfur reduction
Sulfur reduction enzymes and their presence in the three sulfur-reducing heterotrophic crenarchaeotes.
Sulfur/polysulfide reductase (molybdoenzyme)
Smar_0018-0030, Smar_0645-0657, Smar_1057-1071
Subunit composition of multisubunit membrane-bound complexes from Pyrococcus species and S. marinus.
S. marinus produces hydrogen when sulfur is limiting . Two of the multisubunit complexes are potentially involved in hydrogen production. One set of S. marinus proteins (Smar_1060-Smar_1063) clusters strongly with E. coli hydrogenases 3 and 4 in phylogenetic trees, and may form a membrane-bound hydrogenase. Smar_0018 and Smar_0020 have similarity (61% and 39%, respectively) to subunits of Methanosarcina mazei ech hydrogenase subunits, and hydrogenase accessory proteins are found in their vicinity (Smar_0012-0013, Smar_0015). It is likely that at least one of these clusters is involved in hydrogen production.
The other complexes may be involved in sulfur reduction either directly or indirectly. One of the clusters (Smar_1057-1071) is close on the chromosome to a pyridine nucleotide-disulfide oxidoreductase (Smar_1055). It is possible that this cluster is involved in sulfur respiration, where Smar_1055 acts as a NAD(P)H-dependent polysulfide reductase and the other ORFs are involved in the generation of NAD(P)H through a membrane-based electron transport system that oxidizes reduced ferredoxin and translocates protons across the membrane. The system would allow energy generation from an overall sulfur-dependent oxidation of peptides and amino acids and it would be similar to the mbx-NAD(P)H elemental sulfur oxidoreductase (NSR) system that has been described for P. furiosus .
Comparison of the three sulfur-reducing crenarchaeotes
The major difference in habitat between these three organisms is that S. marinus and H. butylicus were isolated from marine environments [1, 3] while T. pendens was isolated from a terrestrial solfatara . Marine environments have relatively high concentrations of sodium and potassium compared to terrestrial springs, and this influences the complement of transporters encoded by the three genomes. For example, S. marinus and H. butylicus use the Trk type of potassium transporter (COG0168), which is a proton or sodium symporter, while T. pendens uses the more energy-intensive ATP-dependent kdp-type potassium transporter (COG2060, COG2216, COG2156). Also, S. marinus and H. butylicus have a greater number and variety of sodium symporters than T. pendens. They both have sodium-dependent multidrug efflux pumps of the MATE family (COG0534) and amino acid transporters of the neurotransmitter:sodium symporter family (pfam00209), while only S. marinus has a transporter of the sodium:solute symporter family (pfam00474).
Both T. pendens and H. butylicus have formate dehydrogenases while S. marinus lacks this enzyme. Formate can be used as an electron donor with sulfur as electron acceptor to generate energy. S. marinus also lacks the FdhE protein, which is involved in formate dehydrogenase formation, while the other two have it.
There are also differences in the ability to utilize carbohydrates among the three organisms. As discussed above, T. pendens has a greater number of carbohydrate transporters than the other two. According to the CAZy database http://www.cazy.org, H. butylicus has no glycosyl hydrolases, while S. marinus has ten and T. pendens has fifteen. Also H. butylicus apparently does not store glycogen as it lacks glycogen synthase and phosphorylase, but the other two have these. H. butylicus also lacks enzymes for utilization of galactose and N-acetylglucosamine. Surprisingly while S. marinus and T. pendens have probable glucokinases related to the characterized Aeropyrum pernix enzyme , H. butylicus has a protein related to the broad-specificity hexokinase from Sulfolobus tokodaii . This suggests that, while it may not be able to break down polysaccharides, it may be able to utilize simple sugars.
There are similarities and differences among the three genomes in the genes involved in biosynthesis. Many of the genes shared by S. marinus and H. butylicus but missing from T. pendens are involved in cofactor metabolism. T. pendens appears to be unable to make riboflavin, coenzyme A, pyridoxine, and possibly other cofactors, and it has transporters for biotin and riboflavin that are not found in the other two. Among the three organisms only H. butylicus has a heme biosynthesis pathway. On the other hand, all three organisms are unable to make most amino acids and purines, although they do have the pyrimidine biosynthetic pathway. S. marinus and H. butylicus have ABC transporters of the basic membrane protein family (pfam02608) that probably transport nucleosides , but T. pendens lacks this type of transporter. In fact T. pendens does not have any identifiable nucleoside or nucleobase transporters, so it likely has undiscovered families to transport these compounds.
There are other differences between these three organisms that do not directly reflect the habitats they live in. H. butylicus is surprisingly lacking some enzymes of central metabolism. It has no identifiable fructose-bisphosphate aldolase and no phosphoenolpyruvate synthase or pyruvate phosphate dikinase. Since fructose-bisphosphate aldolase is essential for hexose and pentose synthesis, it likely has a new version of this enzyme. H. butylicus also does not have an asparaginyl-tRNA synthetase; however, it is the only one of the three to have an Asp-tRNA(Asn)/Glu-tRNA(Gln) amidotransferase, but the A subunit of this enzyme (Hbut_0594) has a frameshift. Since this appears to be an essential enzyme for H. butylicus, the gene may still be functional.
The crenarchaeotes H. butylicus, S. marinus, and T. pendens are similar phenotypically in that they all degrade peptides and/or carbohydrates using 2-oxoacid:ferredoxin oxidoreductases, they are anaerobes, and they are dependent on sulfur reduction to dispose of electrons. They all have genomes in the size range of 1.6–1.8 Mbp. Having these three genome sequences allows comparative studies to determine whether their phenotypic similarity is reflected in their genome sequences.
While the central metabolic pathways for generation of ATP from peptides appear to be similar between the three, there are also differences. For instance there are different sets of transporters used by the marine organisms versus the terrestrial one. However the biggest differences between the three relate to the availability of nutrients. On one extreme is H. butylicus, which has no glycosidases and is capable of synthesizing most if not all cofactors it needs. This organism appears to be more specialized than the other two in that it is restricted to the use of peptides and amino acids as energy sources, although formate can also be utilized. On the other extreme is T. pendens which has many glycosidases and relies on its environment for most cofactors, thus it is used to being in a nutrient-rich environment. Probably a terrestrial solfatara environment allows nutrients to be concentrated as compared to marine environments in which nutrients may be quickly dispersed. S. marinus falls in the middle ground as it has several glycosidases like T. pendens but it encodes most cofactor biosynthesis pathways like H. butylicus. Its use of secondary transporters and ABC transporters suggests that at least at some times it is exposed to high levels of nutrients. It is adapted to an environment that contains carbohydrates as well as proteinaceous substrates, but in which cofactors are not present at high levels.
Characterized membrane-bound sulfur and polysulfide reductases have three subunits [16–19]. The A and B subunits are related in sequence, but the C subunits belong to different protein families. T. pendens and H. butylicus have putative three-subunit sulfur reductases in which all subunits are adjacent on the chromosome. These complexes have the standard A and B subunits, but they differ in their C subunits. The T. pendens C subunit belongs to the same family as the W. succinogenes psrC subunit, while the H. butylicus C subunit is related to sreC of Acidianus ambivalens. S. marinus lacks this type of sulfur or polysulfide reductase, and it is the only crenarchaeote other than C. symbiosum to lack this family of molybdopterin oxidoreductases (COG0243).
T. pendens and H. butylicus also have putative NADPH:sulfur oxidoreductases similar to the P. furiosus enzyme , which is also absent in S. marinus. However, S. marinus has three mbh/mbx-related multisubunit complexes, which are not found in the other two genomes. The overall picture of sulfur reduction shows that T. pendens and H. butylicus may use similar pathways, while S. marinus uses different ones. This is in contrast to the phylogenetic positions of these organisms: S. marinus and H. butylicus belong to the order Desulfurococcales, while T. pendens belongs to the order Thermoproteales. The molybdoenzymes are widespread within Crenarchaeota, missing only in S. marinus, and may represent the ancestral path for sulfur reduction in Crenarchaeota. This analysis, however, rests on comparison to sulfur reduction enzymes characterized in other organisms, and new sulfur reduction pathways may be identified in the future.
The three heterotrophic sulfur-reducing crenarchaeotes have adapted to their habitats, terrestrial vs. marine, via their transporter content, and they have also adapted to environments with differing levels of nutrients, with T. pendens being adapted to a nutrient-rich environment and H. butylicus adapted to an environment in which only peptides are present. S. marinus appears to have different electron transport pathways compared to the phenotypically similar organisms T. pendens and H. butylicus, showing that this phenotype is not encoded by the same genotype in these organisms.
S. marinus strain F1 is available from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) as DSM 3639T and from the American Type Culture Collection (ATCC) as ATCC 43588. T. pendens Hrk5 is available from DSMZ as DSM 2475, and H. butylicus is available from DSMZ as DSM 5456. S. marinus F1 cells were grown in a 300 liter fermenter at 85°C in SME medium with 0.1% yeast extract, 0.1% peptone, and 0.7% elemental sulfur under a 200 kPa N2 atmosphere. Cells grew to a density of 3 × 108 cells/ml in 3 days. Cell pellets were stored at -85°C. DNA was extracted based on the method of Zhou et al. . One gram of cells was dissolved in 4.5 ml extraction buffer (100 mM Tris, pH 8.0, 100 mM EDTA, 100 mM sodium phosphate, and 1.5 M NaCl). After 200 micrograms of proteinase K were added, cells were incubated for 30 minutes at 37°C. A solution of 0.5 ml 20% SDS was added, and then the mixture was incubated at 65°C for 2 hours. Proteins were removed by extraction with 5 ml phenol. The sample was centrifuged for 30 minutes at 19000 rpm in a Sorvall SS34 rotor at 10°C, and the upper phase was discarded. The sample was then extracted twice with chloroform and isoamyl alcohol (24:1) to remove phenol. DNA was precipitated with 3 ml isopropanol at room temperature overnight. The sample was then centrifuged for 30 minutes. The pellet was washed with 5 ml 70% ethanol and recentrifuged. The pellet was dried and then dissolved in 1 ml LiChrosolv (Merck, Darmstadt, Germany). RNA was removed by addition of 20 μg DNAse-free RNAse and incubation for 4 hours at 37°C.
The genome of S. marinus was sequenced at the Joint Genome Institute (JGI) using a combination of 3 kb, 8–10 kb and 40 kb (fosmid) DNA libraries. For all three libraries, shearing is followed by blunt end repair; then the DNA is isolated on an agarose gel and the appropriate section of the gel is cut out. For fosmid libraries, DNA is separated on a pulsed-field gel. DNA is extracted from the gel and then cloned into the pUC18 vector for 3 kb libraries, the pMCL200 vector for 8–10 kb libraries, or the pCC1FOS vector for 40 kb fosmid libraries. Sequencing is carried out from both ends of the inserts using BigDye Terminators and ABI3730XL DNA sequencers. More detailed information about library construction and sequencing, including protocols and reagents, is available at http://www.jgi.doe.gov/sequencing/protocols/prots_production.html. Draft assemblies were based on 23766 total reads. All three libraries provided 13.3× coverage of the genome. The Phred/Phrap/Consed software package http://www.phrap.com was used for sequence assembly and quality assessment [28–30]. All mis-assemblies were corrected and all gaps between contigs were closed by custom primer walk using subclones or PCR products as templates. A total of 657 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. The Phred quality score for this genome is Q50, which corresponds to one miscalled base per 100,000 bases. The genome sequence of S. marinus can be accessed in GenBank [GenBank: CP000575]. The Genomes On Line Database (GOLD) accession number is http://genomesonline.org/GOLD_CARDS/Gc00511.html. Genes were identified using a combination of Critica  and Glimmer  followed by a round of manual curation.
Analysis of the S. marinus genome was carried out with the Integrated Microbial Genomes (IMG) system . Proteins unique to S. marinus or missing from S. marinus but present in other crenarchaeotes were identified with the phylogenetic profiler in IMG. Transposable elements were identified by BLAST against the ISFinder database . CRISPR repeats were identified with the CRISPR Recognition Tool .
Laterally transferred genes were identified with SIGI-HMM . DNA and protein alignments were generated with CLUSTAL W . The phylogenetic tree was generated with MrBayes 3.1.2  with 1,000,000 generations sampled every 100 generations. The first 250,000 generations were discarded as burn-in. The tree was viewed and manipulated with njplot .
To generate clusters for comparative genomics, we retrieved all amino acid sequences for S. marinus, H. butylicus, and T. pendens along with their blastp  (e-value < 10-6) similarity scores, from the Integrated Microbial Genomes database . Thereafter, we divided the resulting network of protein similarities into distinct similarity matrices. Each matrix (cluster of proteins) was then successively partitioned into two child clusters using a spectral clustering procedure [40, 41]. This procedure is analogous to a random walk of a particle moving over the proteins of the network. At each transition, the particle moves to an adjacent protein with probabilities corresponding to the similarity between proteins. The amount of time the particle spends in a given sub-network will determine whether this is indeed a cluster of its own or not. The magnitude of the second eigenvalue of the similarity matrix for a network will determine how fast the particle approaches its stationary distribution . Here, we chose to partition the network if the second eigenvalue was 0.8 or more. This approach resulted in 1041 clusters of a total of 2653 proteins with homologs within two or more of the organisms.
This work was performed under the auspices of the US Department of Energy's Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231, Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344, and Los Alamos National Laboratory under contract No. DE-AC02-06NA25396. L. D., J. R., and B. M. were supported by a NASA Astrobiology: Exobiology and Evolutionary Biology grant NNG05GP24G to B. M. I. P. and W. B. W. were supported by DOE contract number DE-FG02-97ER20269. L. E. U. and I. B. Z. were supported by grant number GM72285 from the National Institutes of Health. J. G. E. was supported by the DOE Genomes to Life program. M. L. was supported by the Department of Energy under contract DE-AC05-000R22725.
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