Pyrococcus abyssi

Supplementary material

An integrated analysis of the genome of the hyperthermophilic archaeon Pyrococcus abyssi

Georges Cohen1, Valérie Barbe2, Didier Flament3, Michael Galperin4, Roland Heilig2, Odile Lecompte5, 
Olivier Poch5, Daniel Prieur6, Joël Quérellou3, Raymond Ripp5, Jean-Claude Thierry5, John Van der Oost7,
Jean Weissenbach2, Yvan Zivanovic8 and Patrick Forterre8.
1-Institut Pasteur, 25,28 rue du Docteur Roux, 75724 Paris CEDEX 15, France. 2-Genoscope, CNS, 2, rue Gaston Crémieux, CP 5706, 91057 EVRY cedex, France. 3-Ifremer, Centre de Brest, DRV/VP/LMBE, BP 70, 29280 Plouzané, France. 4-National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD 20894, USA. 5-CNRS-IGBMC, BP 163, 1 rue Laurent Fries, 67404 Illkirch, France. 6-Université de Bretagne Occidentale, IUEM, Place Nicolas Copernic, TBI, 29280 Plouzané, France. 7-Wageningen University, Hesselink van Suchtelenweg 4, 6703 CT Wageningen, the Netherlands. 8-Université Paris-Sud, Centre Universitaire d'Orsay, Institut de Génétique et Microbiologie, UMR, CNRS, 8621, Bat 409, 91405 Orsay Cedex, France. Mol Microbiol. 47:1495-512 (2003).

DNA replication, chromosome segregation and cell division:

The other eukaryal-like replication proteins than can be readily identified in the P. abyssi proteome are the MCM helicase (PAB2373), DNA pol I (PAB1128), RNAse H II (PAB0352), the two subunits of DNA primase (PAB2235, PAB2236), the three subunits of RP-A (RP-A39: PAB2163, RP-A14: PAB2164 and RP-A21: PAB2165), the PCNA clamp (PAB1465), the flap-endonuclease FEN 1 (PAB1877) and DNA ligase (PAB2002).


In general, promoter regions are A/T rich to facilitate local unwinding of the DNA helix upon transcription initiation. In addition, the A/T enrichment of archaeal intergenic regions can be attributed to the presence of TATA box and transcription factor B recognition elements (BRE): the consensus sequence of 19 mapped promoters of Pyrococcus furiosus has a similar A/T bias (at position -36/-23 relative to the transcription start site: [G/A],A,A,A,N,N,T,T,[A/T],[A/T],[A/T],[A/T],A) (Verhees 2002). The predicted non coding regions in the Pyrococcus genome indeed display a strong over-representation of 6 trinucleotides (AAA, AAT, ATT, TAA, TTA and TTT) whereas coding regions do not exhibit any clear-cut bias in overall trinucleotides composition (see figure).

Click to enlarge

Frequencies of all 64 trinucleotides have been computed for complete genome, coding and intergenic regions. The ratio of observed (%Obs) to full genome frequencies (%total) is displayed in the histogram.


The membrane potential is used to drive the flagellar motor. Like other Pyrococci, P. abyssi has typical archaeal flagella. The flagellar operon contains 3 copies of the flagellin gene flaB (PAB1378-PAB1380), followed by flagellar accessory genes flaCDEFGHIJ (PAB1381-PAB1387).

Although archaeal flagella differ from bacterial ones in both structure and composition (Thomas et al., 2001), several flagellar assembly proteins such as the ATPase FlaI (PAB1386) and the signal peptidase FlaK (PAB1309) show similarity to proteins involved in the biogenesis of type IV pili in bacteria (Bardy and Jarrell, 2002). A set of methyl-accepting chemotaxis proteins has been identified in the genome (PAB1026-PAB1027, PAB1330-PAB1336).

The fact that this operon is also present in P. horikoshii but not in P. furiosus, has been suggested to correlate with the wider range of biosynthetic pathways encoded by the latter genome, which might obviate the need for the chemotactic response (Maeder et al., 1999).

Amino acid biosynthesis

The entire set of enzymes involved in the 10-step tryptophan biosynthesis pathway is encoded in the P. abyssi GE5 genome. The first steps of this pathway, leading from phosphoenolpyruvate and erythrose-4-phosphate to chorismate, are encoded in a single aroGBDEKP---AC putative operon (PAB0297-PAB0307), which additionally includes genes for an ABC-type transport system of unknown substrate specificity. In P. abyssi the genes encoding chorismate mutase and prephenate dehydrogenase, required for Phe and Tyr synthesis are missing (Table ).

The enzymes that constitute the classical bacterial lysine biosynthesis pathway are not encoded by the P. abyssi genome. However, the gene cluster encoding an alternative route, the alpha-aminoadipic acid (AAA) pathway that was recently characterised in the thermophilic bacterium Thermus thermophilus (Nishida et al., 1999), appears to be conserved in P. abyssi, as well as in P. horikoshii and P. furiosus. The lys operon encodes enzymes that resemble their counterparts in leucine and arginine synthesis: leuACDB, argCBDE, a small ORF with similarity to a zinc ribbon, and a RimK-like gene which has previously been proposed to code for an ATP-dependent carboxylate-amine/thiol ligase (Koonin et al., 1997). In P. furiosus it has recently been proposed that these genes are also involved in the synthesis of ornithine, which is converted in 3 steps to arginine by ArgFGH (Brinkman et al., 2002). Unlike P. furiosus, however, P. abyssi appears not to possess argFGH, in agreement with its arginine auxotrophy (Table).

All the enzymes necessary for the synthesis of threonine are present. Aspartate kinase is represented by two genes (PAB1674 and PAB1675) indicating a possible different regulation of threonine and methionine biosynthesis, since the product of aspartokinase, aspartylphosphate is a common precursor of these two amino acids. Genes for three other enzymes of threonine biosynthesis, aspartate semialdehyde dehydrogenase (PAB1678), homoserine kinase (PAB1676) and threonine synthase (PAB1677), are clustered with the aspartate kinase genes, forming a probable operon, whereas the homoserine dehydrogenase gene (PAB0610) is located next to several genes of methionine biosynthesis (PAB0605-PAB0608). The enzymes of the branch leading from threonine to isoleucine are found, with the exception of the small regulatory subunit of aceto-hydroxyacid synthase. The enzymes common to valine and isoleucine biosynthesis are present in P. abyssi as well as the threonine dehydratase, specific to isoleucine synthesis. The genes encoding the common isoleucine/valine biosynthesis enzymes are clustered with the genes of leucine biosynthesis (PAB0888 to PAB0895, including PAB2424) (Table ).

All the enzymes required for serine, glycine and cysteine biosynthesis are present in P. abyssi, with the exception of PAPS phosphotransferase and sulphite reductase. Of the two subunits of bacterial NADPH-dependent glutamate synthase, the large subunit (equivalent to the E. coli GltB and B. subtilis GltA) is missing in P. abyssi, while the small subunit (GltD in E. coli) is encoded in two copies (PAB1738 and PAB1214). In Pyrococcus kodakaraensis (Jongsareejit et al., 1997), a homotetramer of this subunit was found to be capable of both glutamine-dependent and ammonia-dependent synthesis of glutamate without the presence of an equivalent of the E. coli large subunit. The glutamine synthetase gene (PAB1292) is present in P. abyssi. Although no protein catalysing a covalent modification has been detected, it should be noted that the tyrosine residue on which the adenylylation occurs in E. coli as well as six surrounding residues are conserved in the P. abyssi sequence. Several aminotransferases have been detected, catalysing the final steps of valine, leucine and isoleucine synthases, as well as aspartate and glutamate synthesis from oxaloacetate and 2-oxoglutarate. In addition P. abyssi possesses an aromatic amino acid aminotransferase, several omega amino acid aminotransferases and a serine-glyoxylate aminotransferase.

Although P. abyssi has been found to be methionine auxothroph, several orthologs of bacterial enzymes involved in methionine biosynthesis are present, with the notable exception of homoserine acyltransferase and methyltetrahydrofolate reductase. The B12-dependent variant of methionine synthetase is not encoded in P. abyssi, but B12-independent enzyme, methylating homocysteine into methionine is found in two copies (PAB0608 and PAB2361). Also found are the archaeal type S-adenosylmethionine synthetase (PAB2094) and a S-adenosylhomocysteinase (PAB1372), which is probably involved in a methionine salvage pathway. Despite the reported proline prototrophy, none of the classical proteins responsible for proline biosynthesis has been detected in P. abyssi GE5 genome, raising the possibility of an unique proline synthesis route for Pyrococci (Fig. 1, text).

Amino acid biosynthesis

Gene name

(predicted) PAB

Phe, Tyr biosynthesis







Trp biosynthesis





His biosynthesis

his operon


Ser biosynthesis



Gly biosynthesis



Thr biosynthesis*



Cys biosynthesis



Leu biosynthesis*



Ile, Val biosynthesis*



Met biosynthesis*



Pro biosynthesis**

novel type ?


Lys biosynthesis (AAA-type)



Arg biosynthesis



Ala biosynthesis



Asp biosynthesis


Several ATs

Glu biosynthesis



Gln biosynthesis



Asn biosynthesis




Nucleotides & cofactors



Purine biosynthesis*



Pyrimidine biosynthesis



NAD biosynthesis



Heme biosynthesis



Cobalamin biosynthesis



Folate biosynthesis



Pyridoxal biosynthesis



Biotin biosynthesis



Coenzyme A biosynthesis*



Heme biosynthesis



Anabolic capacity of P. abyssi as deduced from genome analysis.
Predicted genes/operons involved in amino acid biosynthesis are indicated by PAB identifier; when no gene has been identified it is indicated (No). In some cases there is a discrepancy with experimentally-determined autotrophy (*) or prototrophy (**).

Vitamin biosynthesis

P. abyssi does not encode enzymes of the biotin biosynthesis and has to import it through a still uncharacterised transport system. It encodes, however, two copies of biotin-(acetyl-CoA carboxylase) ligase, which links biotin to the biotin-carboxyl carrier protein. One of these two copies is fused in a bi-functional protein BirA (PAB0104) to the biotin-dependent transcriptional regulator, as is the case in many bacteria. In contrast of P. furiosus, P. abyssi and P.horikoshii do not encode enzymes of riboflavin biosynthesis and we were not able to identify transporters involved in flavin uptake.

P. abyssi encodes transketolase and acetolactate synthase, the thiamine diphosphate-dependent enzymes, and apparently can both synthesise thiamine and acquire it from the outside. Indeed, P. abyssi encodes a putative ABC-type transport system, consisting of adjacent genes for a thiamine-binding periplasmic protein (PAB1835), an ATPase (PAB0545), and a permease (PAB0543). In addition, P. abyssi encodes homologs of most (but not all) bacterial enzymes of thiamine biosynthesis (ThiC, PAB1930; ThiD, PAB1646; ThiE, PAB1645; ThiL, PAB2358). In contrast, the enzymes involved in the synthesis of the thiazole moiety in bacteria are only partly represented in P. abyssi. This could be due to the fact that bacteria produce thiazole ring from 1-deoxy-D-xylulose 5-phosphate, which appears to be a bacteria-specific sugar. The sugar that serves as thiazole precursor in archaea remains unknown, but the proteins responsible for the introduction of sulphur into the molecule (ThiS, PABs5591; ThiF, PAB2302; and ThiI, PAB0226 and PAB0561) seem to be the same. It should be noted that as compared to bacterial ThiD, PAB1646 and all other archaeal homologs of ThiD contain an additional 180-aa C-terminal domain of unknown function that is most likely involved in thiamine biosynthesis.

P. abyssi encodes a complete set of enzymes of pyridine nucleotide biosynthesis (NadA, PAB2345; NadB, PAB2343; NadC, PAB2347). The apparent absence of 1-deoxy-D-xylulose 5-phosphate in archaea (see above) suggests that pyridoxine ring is formed from some other sugar by products of the PDX1 and PDX2 (formerly SNZ/SNO) genes, described in Cercospora nicotianae and in yeast (Ehrenshaft and Daub, 2001). These two genes (PAB0537 and PAB0538) are the only pyridoxine biosynthesis genes found in P. abyssi.

P. abyssi genome does not contain heme biosynthesis related genes. However, genes encoding enzymes for the last steps of adenosylcobalamin biosynthesis have been detected: cobalamin biosynthesis protein CbiB (PAB0025), cobalamin-5-phosphate synthase(CobS, PAB2320), ATP:corrinoid adenosyltransferase (BtuR, PAB2289), and nicotinic acid mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT, PAB2326). It appears that P. abyssi would have to import a corrinoid precursor of the cobalamine.


Isoprenoid biosynthesis and utilization

Acetyl CoA C-acetyltransferase (PAB0907), 3-hydroxy-3-methylglutaryl CoA synthase (PAB0906) and reductase (PAB2106) as well as mevalonate kinase (PAB0372) are found in P.abyssi, in accordance with all Archaea investigated to date. The orthologs of the proteins, accounting for the route from mevalonate to pyrophosphomevalonate are missing, as are the enzymes of the alternative deoxy-D-xylulose phosphate synthase pathway (Smit and Mushegian, 2000). Whatever is the source of pyrophosphomevalonate, an isopentenyldiphosphate isomerase is synthesised by P. abyssi (PAB1662) as well as a multifunctional isoprenyldiphosphate synthase (PAB2389), a homolog of an enzyme from Archaeoglobus fulgidus that catalyses the synthesis of geranylgeranyldiphosphate (Wang et al., 1999). A polyprenyldiphosphate synthase (PAB0394) has been detected. as well as a geranylgeranyl hydrogenase (PAB01O9), known to be a precursor of phytol, component of chlorophylls.


Cited References

Bardy, S.L., and Jarrell, K.F. (2002)
FlaK of the archaeon Methanococcus maripaludis possesses preflagellin peptidase activity.
FEMS Microbiol Lett 208: 53-59.

Brinkman, A.B., Bell, S.D., Lebbink, R.J., De Vos, W.M., and Van der Oost, J.
The Sulfolobus solfataricus Lrp-like protein LysM regulates lysine biosynthesis in response to lysine avalability.
J Biol Chem (in press).

Ehrenshaft, M., and Daub, M.E. (2001)
Isolation of PDX2, a second novel gene in the pyridoxine biosynthesis pathway of eukaryotes, archaebacteria, and a subset of eubacteria.
J Bacteriol 183: 3383-3390.

Jongsareejit, B., Rahman, R.N., Fujiwara, S., and Imanaka, T. (1997)
Gene cloning, sequencing and enzymatic properties of glutamate synthase from the hyperthermophilic archaeon Pyrococcus sp. KOD1.
Mol Gen Genet 254: 635-642.

Koonin, E.V., Mushegian, A.R., Galperin, M.Y., and Walker, D.R. (1997)
Comparison of archaeal and bacterial genomes: computer analysis of protein sequences predicts novel functions and suggests a chimeric origin for archaea.
Mol Microbiol 25: 619-637.

Maeder, D.L., Weiss, R.B., Dunn, D.M., Cherry, J.L., Gonzalez, J.M., DiRuggiero, J., and Robb, F.T. (1999)
Divergence of the hyperthermophilic archaea Pyrococcus furiosus and P. horikoshii inferred from complete genomic sequences.
Genetics 152: 1299-1305.

Nishida H, Nishiyama M, Kobashi N, Kosuge T, Hoshino T, Yamane H.
A prokaryotic gene cluster involved in synthesis of lysine through the amino adipate pathway: a key to the evolution of amino acid biosynthesis.
Genome Res. 1999 Dec;9(12):1175-83.

Smit, A., and Mushegian, A. (2000)
Biosynthesis of isoprenoids via mevalonate in Archaea: the lost pathway.
Genome Res 10: 1468-1484.

Thomas, N.A., Bardy, S.L., and Jarrell, K.F. (2001)
The archaeal flagellum: a different kind of prokaryotic motility structure.
FEMS Microbiol Rev 25: 147-174.

Verhees, C.H. (2002)
Molecular characterization of glycolysis in Pyrococcus furiosus.
Thesis Wageningen University.

Wang, C.W., Oh, M.K., and Liao, J.C. (1999)
Engineered isoprenoid pathway enhances astaxanthin production in Escherichia coli.
Biotechnol Bioeng 62: 235-241.