| The emergence of bacterial resistance to virtually all known antibiotics poses a serious threat to human health. As pharmaceutical companies retreat from the drug discovery field, it falls to academia to identify and validate novel antibacterial targets. Teichoic acids are a ubiquitous feature of Gram-positive cell walls that may represent such a target. This thesis focuses on the dispensability and function of the teichoic acid polymerase from the model organism Bacillus subtilis.;Conditional complementation of a thermosensitive mutant and a tagF deletion strain confirmed that polymerase activity is required for growth. In addition, cell wall analysis and the inability of teichuronic acid to compensate for a tagF lesion showed that the importance of teichoic acid biosynthesis involves more than simply providing an anionic determinant in the cell wall.;The TagF enzyme was purified for the first time for in vitro biochemical analysis. A novel polymerase assay was developed that allowed for the determination of steady-state kinetic parameters while preserving the reaction product for further characterization. Thus, polymerase activity was conclusively attributed to TagF for the first time. The dependence of TagF activity on pH, combined with mutational analysis, identified several critical residues. In vivo complementation experiments showed that equivalent residues were also critical for the function of the related TagB enzyme. This analysis showed that TagF serves as a model for other poorly characterized teichoic acid synthetic enzymes and gave the first clues about the mechanism of polymerization.;Teichoic acid polymerization was also studied in the absence of membrane. This work demonstrated that TagF did not require membrane association for activity, but that this association was involved in regulating polymer length. Further analysis showed that length regulation likely did not require accessory proteins, a feature that could distinguish teichoic acid polymerization from similar processes in Gram-negative bacteria. |
