5268 Nucleic Acids Research, 2008, Vol. 36, No. 16 Our study demonstrated that the initiation sequences of A. aeolicus primase are comprised entirely of cytosines and/or guanines even though its chromosome compo- sition is only 43% GC and there does not appear to be any obvious DNA strand bias for the recognition trinu- cleotides. There is also a disparity in the initiation trinu- cleotide sequences for primases from Firmicutes [d(CTA) and d(TTA) for S. aureus and G. stearothermophilus] and Proteobacteria [d(CTG) for E. coli] despite the presumed proximity of these phyla. In fact, the congruence in pri- mase speciﬁcities for mesophilic S. aureus and thermophi- lic G. stearothermophilus becomes even more striking in this light. Therefore, our ﬁndings suggest that the hyper- thermophilic environment may require a larger number of adaptations than does the thermophilic habitat. Comparisons of the completed genomes from hyper- thermophiles, including A. aeolicus and archaeon Metha- nococcus jannaschii, with bacterial genomes of mesophiles revealed that stable RNAs have a high G+C content even if the genome has a comparatively low G+C content (8). These stable RNAs include 16S RNAs and tRNAs where maintenance of structural stability is particularly impor- tant (38). The extensive hydrogen bonding and modiﬁed bases in these stable RNAs protects them against the deleterious eﬀects of heat by increasing their melting tem- perature (1). Similarly, highly expressed genes have been determined to derive from relatively GC-rich codons more often than genes expressed at a low level in AT-rich micro- bial genomes (41). These ﬁndings suggest that GC base pairing facilitates microbial ﬁtness to thermophily by sta- bilizing various factors of the translation machinery, particularly during cell growth. Our results show that pri- mase initiation speciﬁcity represents another example of thermophilic adaptation, this time for the replication machinery. Primases from bacteriophage T7, S. aureus and E. coli have been shown to have a higher aﬃnity for ssDNA templates that contain the respective initiation sequence than for similar ssDNA not containing the preferred initiation trinucleotide (25,34,42,43). Accordingly, our ﬁndings suggest that the A. aeolicus initiation sequence that is comprised of cytosines and guanines is necessary for stabilizing the interaction among the primase–NTPs– ssDNA complex during initiation, especially due to the geothermal context of this microbe. The additional bond- ing energy between G–C versus A–T base pairs would provide greater thermostability in this complex during the initial steps of primer synthesis. Our results suggest that A. aeolicus primase appears to utilize the thermody- namic strength of GC basepair stacking to help assemble the complex between primase, CTP, GTP and ssDNA. In fact, the stacking free energy of the A. aeolicus primase initiating ribonucleotides with their complements in the template may be nearly twice as favorable ( 2.17 kcal/mol) as the basepair stacking energy that takes place in mesophilic microbial DNA–RNA interac- tions ( 1.00 kcal/mol) (44). Since dinucleotide polymeri- zation is the rate-limiting step during de novo primer synthesis (31), our ﬁndings accentuate the importance of a stable interaction during the initial priming step for subsequent replication of the A. aeolicus genome. We anticipate that primases from other hyperthermophilic bacteria will also utilize initiation trinucleotides that are comprised entirely of cytosines and/or guanines. Our future studies will focus on determining the relationship of template recognition and primase structure. ACKNOWLEDGEMENTS This work was supported in part by a grant from the Department of Defense, Defense Advanced Research Program Agency (award W911NF0510275) to S.H.H. K.S. acknowledges support from the NIH (grant A1068151) and J.M.B. acknowledges support from the NIGMS (GM071747). Funding to pay the Open Access publication charges for this article was provided by the above DARPA award to S.H.H. Conﬂict of interest statement. None declared. REFERENCES 1. Kowalak,J.A., Dalluge,J.J., McCloskey,J.A. and Stetter,K.O. (1994) The role of posttranscriptional modiﬁcation in stabilization of transfer RNA from hyperthermophiles. Biochemistry, 33, 7869–7876. 2. Schlee,S. and Reinstein,J. (2002) The DnaK/ClpB chaperone system from Thermus thermophilus. Cell Mol. Life Sci., 59, 1598–1606. 3. Grogan,D.W. (1998) Hyperthermophiles and the problem of DNA instability. Mol. Microbiol., 28, 1043–1049. 4. Frick,D.N. and Richardson,C.C. (2001) DNA primases. Annu. Rev. Biochem., 70, 39–80. 5. Grompe,M., Versalovic,J., Koeuth,T. and Lupski,J.R. (1991) Mutations in the Escherichia coli dnaG gene suggest coupling between DNA replication and chromosome partitioning. J. Bacteriol., 173, 1268–1278. 6. Liu,J., Dehbi,M., Moeck,G., Arhin,F., Bauda,P., Bergeron,D., Callejo,M., Ferretti,V., Ha,N., Kwan,T. et al. (2004) Antimicrobial drug discovery through bacteriophage genomics. Nat Biotechnol., 22, 185–191. 7. Kobayashi,K., Ehrlich,S.D., Albertini,A., Amati,G., Andersen,K.K., Arnaud,M., Asai,K., Ashikaga,S., Aymerich,S., Bessieres,P. et al. (2003) Essential Bacillus subtilis genes. Proc. Natl Acad. Sci. USA, 100, 4678–4683. 8. Deckert,G., Warren,P.V., Gaasterland,T., Young,W.G., Lenox,A.L., Graham,D.E., Overbeek,R., Snead,M.A., Keller,M., Aujay,M. et al. (1998) The complete genome of the hyperthermophilic bacterium Aquifex aeolicus. Nature, 392, 353–358. 9. Galtier,N. and Lobry,J.R. (1997) Relationships between genomic G+C content, RNA secondary structures, and optimal growth temperature in prokaryotes. J. Mol. Evol., 44, 632–636. 10. Marmur,J. and Doty,P. (1962) Determination of the base compo- sition of deoxyribonucleic acid from its thermal denaturation tem- perature. J. Mol. Biol., 5, 109–118. 11. SantaLucia,J. Jr. (1998) A uniﬁed view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc. Natl Acad. Sci. USA, 95, 1460–1465. 12. De Luchi,D., Gouyette,C. and Subirana,J.A. (2003) The inﬂuence of size on the thermal stability of oligonucleotides: the case of AT sequences. Anal. Biochem., 322, 279–282. 13. Hurst,L.D. and Merchant,A.R. (2001) High guanine-cytosine content is not an adaptation to high temperature: a comparative analysis amongst prokaryotes. Proc. Biol. Sci., 268, 493–497. 14. Burggraf,S., Olsen,G.J., Stetter,K.O. and Woese,C.R. (1992) A phylogenetic analysis of Aquifex pyrophilus. Syst. Appl. Microbiol., 15, 352–356. 15. Wang,H.C. and Hickey,D.A. (2002) Evidence for strong selective constraint acting on the nucleotide composition of 16S ribosomal RNA genes. Nucleic Acids Res., 30, 2501–2507.