The polC gene specifying DNA polymerase III (PolIII) of Staphylococcus aureus (Sa), was cloned with a novel strategy and found to contain a 4305-bp open reading frame (ORF) encoding a polypeptide of approx. 162 kDa. The 1435-codon ORF was engineered into an Escherichia coli (Ec) expression plasmid under the control of the lac promoter and its repressor. Derepression of Ec transformants carrying the recombinant (re-) vector generated high-level synthesis of active re-Sa PolIII. The re-PolIII was purified to > 98% homogeneity and was shown by N-terminal amino acid sequence analysis to be the bona fide product of the Sa polC ORF. The physical and catalytic properties of re-Sa PolIII and its responsiveness to inhibitors of the HPUra type were generally similar to those of Bacillus subtilis (Bs) PolIII. Comparative analysis of the primary structures of Sa PolIII, Bs PolIII and Mycoplasma pulmonis PolIII indicated strong conservation of essential catalytic domains and a novel zinc-finger motif. Comparison of the primary structures of Ec PolIII and these three Gram+ enzymes revealed a region of novel homology and reinforced the likelihood of a specific evolutionary relationship between PolIII of Gram+ and Gram- eubacteria. The polC gene mapped between omega 1074 [Tn551] and recA/ngr on the Sa NCTC 8325 genome.

The major research interest of our laboratory is focused on the replication-specific DNA polymerase III (pol III) family in Gram+ bacteria, and has used Bacillus subtilis (BS) as the primary model enzyme for study. The long range objective of the work of the laboratory is to gain a deeper understanding of the structure and function of Gram+ bacterial DNA polymerase IIIs, a structurally unique class of DNA-dependent DNA polymerase which are uniquely susceptible to inhibition by a specific class of dGTP analogs. The project described in this thesis dissertation deals specifically with the pol III of the Gram+ organism Staphylococcus aureus, and involves the isolation and characterization of DNA pol III from this clinically relevant pathogenic bacterium. A homology-based strategy was devised to clone the structural gene specifying DNA polymerase III of Staphylococcus aureus, SA polC. SA polC was found to contain a 4305-bp open reading frame (ORF) encoding a 162.4 kDa polypeptide, and mapped between Ω1074[Tn551] and recA/ngr on the genome map of S. aureus NCTC 8325. The 1435 codon ORF was engineered into the E. coli expression plasmid pBS(KS) under the control of the lac promoter and its repressor. The translational signals of SA polC were reengineered using expression cassette PCR (ECPCR) to optimize the in vitro expression of SA polC in E. coli. Derepression of E. coli transformants carrying the recombinant vector generated high level expression of active recombinant pol III. The recombinant SA pol III was purified to greater than 98% homogeneity and was shown by N-terminal amino acid analysis to be the bona fide product of the 4305-bp SA polC ORF. The physical and catalytic properties of recombinant SA pol III and its responsiveness to inhibitors of the HPUra type were similar to those of Bacillus subtilis (BS) pol III. Comparative structural analysis of the primary structure of SA pol III and the pol IIIs of B. subtilis and the Gram+ relative Mycoplasma pulmonis indicated strong conservation of essential catalytic domains and a novel zinc-finger motif. Comparison of the primary structures of E. coli pol III and these three Gram+ enzymes suggested a specific evolutionary relationship between the pol IIIs of Gram+ and Gram- bacteria.