temperature of 858C (8). Interestingly, the genomes of these hyperthermophiles are only one-third the size of the Gram-negative E. coli genome and demonstrate few speciﬁc indications of heat-resistance (9). Despite the intuitive expectation that thermophilic adaptations would occur, such as a higher G+C genome content with a higher melting temperature (10 12), the A. aeolicus chromosome has a G+C content of only 43% (8). Therefore, there appears to be little relationship between adaptation of the organism to high growth temperatures and high G+C content in the genome (9,13). Inferences made from the phylogenetic analysis of the 16S ribosomal RNA sequence from A. aeolicus indicate that this microbe is one of the earliest bacteria to have diverged from the evolutionary tree of the eubacteria (14), but other studies suggest that RNA sequence changes were an adaptive response to the elevated temperature of the organism’s environment (9,15). Protein phylogenies have placed A. aeolicus much further from the root, but some studies could not identify a consistent node (8,16). More recent cladistic studies using multiple signature pro- teins indicate that the Aquifex lineage emerged from Gram-positive bacteria, prior to the split of Gram-positive and Gram-negative bacteria (17,18). The proximity of Aquifex to Gram-positive bacteria is consistent with the low GC content of its genome and the encoding of both PolC and DnaE, replicative DNA polymerase III holoen- zymes. In contrast, Aquifex possesses a double membrane structure similar to Gram-negative bacteria. Therefore, questions remain as to whether heat tolerance in A. aeolicus is a legacy of primordial bacterial evolution or a later adaptation to ﬁll a vacant niche in the geothermal envir- onment. We hypothesized that a bacterial hyperthermo- phile might utilize primase initiation sequences that diﬀer from mesophilic microbes. Primases from several viruses, phages and bacteria stu- died to date have required a speciﬁc sequence to initiate synthesis of an RNA polymer de novo (4). The bacterio- phages T4, T7 and SP6 recognize the trinucleotides 50-d(GTT)-30, 50-d(GTC)-30 and 50-d(GCA)-30, respectively (19 21). In E. coli, the primer is most often initiated by the sequence pppApG in vivo (22) and in vitro studies determined that E. coli primase primed single-stranded DNA (ssDNA) templates containing 50-d(CTG)-30 in the absence of the replicative helicase (23). It was unknown whether bacteria from other genera would have diﬀerent recognition sequences until the primases from Geobacillus stearothermophilus and S. aureus were recently shown to recognize 50-d(CTA)-30 and 50-d(TTA)-30 (24,25). Based solely on these three known examples, bacterial primase initiation speciﬁcity appears to be inherited and shared between phylogenetic siblings, but diﬀerent between Gram-negative and Gram-positive bacteria. Nevertheless, the initiation sequences for microbial primases have sev- eral common features: the recognition sequences are three bases in length; primer synthesis initiates complementary to the middle base of the trinucleotide, which is a pyrimi- dine and the third base in the trinucleotide is cryptic, meaning its presence is required to support eﬃcient primer synthesis, but its complement is not incorporated into the RNA primer (4). Nucleic Acids Research, 2008, Vol. 36, No. 16 5261 Although the contiguous structure of A. aeolicus pri- mase zinc-binding and RNA polymerase domains have been resolved by crystallography (26), the activity and the initiation speciﬁcity of this protein had not been previously characterized. The lack of prior biochemical and biological studies on this enzyme and the potential diﬀerence in trinucleotide speciﬁcity presented a chal- lenge for screening. Therefore, a new approach was devel- oped with the capability to not only determine the activity of A. aeolicus primase, but also any other microbial pri- mase that had not been characterized. To test as many trinucleotide sequences as possible, two oligonucleotides were created using a genetic algorithm program such that all 64 trinucleotides were present in one or the other sequence. After the resulting primer lengths were determined, the potential initiation sequences were pre- dicted from the template and then conﬁrmed using trinucleotide-speciﬁc templates. Using this approach, the zinc binding and RNA polymerase domains of A. aeolicus primase were shown to be suﬃcient to synthe- size primers de novo. Further, this replicative enzyme initiated RNA polymer synthesis on trinucleotides not previously known to be recognition sequences for any microbial primase. Aquifex aeolicus primase preferen- tially initiated primer synthesis on 50-d(CCC)-30 and to a lesser degree on 50-d(GCC)-30 and 50-d(CGC)-30. Elucidation of the initiation trinucleotides for A. aeolicus primase from this hyperthermophile has provided the basis for further enzymatic studies. MATERIALS AND METHODS Proteins, reagents and substrates Residues 1 405 of A. aeolicus primase were cloned, expressed and puriﬁed as previously described (26). The C-terminal domain had been deleted to enhance protein solubility. The full-length primases from other bacteria, including S. aureus and E. coli, were used to evaluate the functionality of the two oligonucleotides. Escherichia coli DnaG was isolated as previously described (27) and the other bacterial primases were cloned into pET30 and expressed in E. coli BL21(DE3) according to the manufac- turer’s suggestions (Novagen EMD Biosciences, La Jolla, CA, USA). Protein puriﬁcation was performed using nickel aﬃnity chromatography as recommended by the manufacturer (GE Healthcare, Piscataway, NJ, USA). Enzyme purity and subunit molecular mass were esti- mated by sodium dodecyl sulfate-polyacrylamide gel elec- trophoresis. The concentration of A. aeolicus, S. aureus and E. coli primase were determined using the monomer extinction coeﬃcient of 36 010 M 1 cm 1, 53 890 1 cm 1 and 46 215 M 1 cm 1, respectively, at 280 nm. Ribonucleoside triphosphates (NTPs) were obtained from Promega (Madison, WI, USA). Magnesium acetate, potassium glutamate, HEPES and dithiothreitol (DTT) were from Sigma (St. Louis, MO, USA). The Sephadex G-25 microspin columns were purchased from Amersham (Piscataway, NJ, USA). The WAVE HPLC Nucleic Acid Fragment Analysis System, HPLC Buﬀer A (0.1 M triethylammonium acetate, pH 7.0), HPLC Buﬀer B