Valerie Mizrahi

The minimal kinetic scheme for DNA polymerization catalyzed by the Klenow fragment of DNA polymerase I (KF) from Escherichia coli has been determined with short DNA oligomers of defined sequence. A key feature of this scheme is a minimal two-step sequence that interconverts the ternary KF.DNAn.dNTP and KF.DNAn+1.PPi complexes. The rate is not limited by the actual polymerization but by a separate step, possibly important in ensuring fidelity [Mizrahi, V., Henrie, R. N., Marlier, J. F., Johnson, K. A., & Benkovic, S. J. (1985) Biochemistry 24, 4010-4018]. Evidence for this sequence is supplied by the observation of biphasic kinetics in single-turnover pyrophosphorolysis experiments (the microscopic reverse of polymerization). Data analysis then provides an estimate of the internal equilibrium constant. The dissociations of DNA, dNTP, and PPi from the various binary and ternary complexes were measured by partitioning (isotope-trapping) experiments. The rate constant for DNA dissociation from KF is sequence dependent and is rate limiting during nonprocessive DNA synthesis. The combination of single-turnover (both directions) and isotope-trapping experiments provides sufficient information to permit a quantitative evaluation of the kinetic scheme for specific DNA sequences.

Long-term survival of nonreplicating Mycobacterium tuberculosis (Mtb) is ensured by the coordinated shutdown of active metabolism through a broad transcriptional program called the stringent response. In Mtb, this response is initiated by the enzymatic action of RelMtb and deletion of relMtb produces a strain (H37RvDeltarelMtb) severely compromised in the maintenance of long-term viability. Although aerosol inoculation of mice with H37RvDeltarelMtb results in normal initial bacterial growth and containment, the ability of this strain to sustain chronic infection is severely impaired. Significant histopathologic differences were noted in lungs and spleens of mice infected with H37RvDeltarelMtb compared with controls throughout the course of the infection. Microarray analysis revealed that H37RvDeltarelMtb suffers from a generalized alteration of the transcriptional apparatus, as well as specific changes in the expression of virulence factors, cell-wall biosynthetic enzymes, heat shock proteins, and secreted antigens that may alter immune recognition of the recombinant organism. Thus, RelMtb is critical for the successful establishment of persistent infection in mice by altering the expression of antigenic and enzymatic factors that may contribute to successful latent infection.

The stringent response utilizes hyperphosphorylated guanine [(p)ppGpp] as a signaling molecule to control bacterial gene expression involved in long-term survival under starvation conditions. In gram-negative bacteria, (p)ppGpp is produced by the activity of the related RelA and SpoT proteins. Mycobacterium tuberculosis contains a single homolog of these proteins (Rel(Mtb)) and responds to nutrient starvation by producing (p)ppGpp. A rel(Mtb) knockout strain was constructed in a virulent strain of M. tuberculosis, H37Rv, by allelic replacement. The rel(Mtb) mutant displayed a significantly slower aerobic growth rate than the wild type in synthetic liquid media, whether rich or minimal. The growth rate of the wild type was equivalent to that of the mutant when citrate or phospholipid was employed as the sole carbon source. These two organisms also showed identical growth rates within a human macrophage-like cell line. These results suggest that the in vivo carbon source does not represent a stressful condition for the bacilli, since it appears to be utilized in a similar Rel(Mtb)-independent manner. In vitro growth in liquid media represents a condition that benefits from Rel(Mtb)-mediated adaptation. Long-term survival of the rel(Mtb) mutant during in vitro starvation or nutrient run out in normal media was significantly impaired compared to that in the wild type. In addition, the mutant was significantly less able to survive extended anaerobic incubation than the wild-type virulent organism. Thus, the Rel(Mtb) protein is required for long-term survival of pathogenic mycobacteria under starvation conditions.