The long term objectives of this research are to understand the molecular basis of cell division (septum formation) and its inhibition by DNA-damaging agents (ultraviolet light, nalidixic acid, nitrofurantoin) that induce a distress response (the SOS response) in Escherichia coli. Other closely related objectives are to understand the details of intracellular protein breakdown and overproduction of capsular polysaccharide. Cell division, ultraviolet radiation, and proteolysis studies in bacteria may provide basic answers that are applicable to solving human cancer which involves the heritable loss of control of cell division. Use of antibiotics that interfere with cell division in these studies (penicillins, cephalosporins) may also provide important basic information useful in designing new antibiotics. Capsular polysaccharides are important determinants of virulence for a number of bacteria that are pathogenic for humans.
The specific aims are: 1. Study in vivo proteolysis and enzymes of capsular polysaccharide synthesis regulated by the CapR (Lon) ATP hydrolysis-dependent protease. This will be done by recloning the capR gene so that it is under lac operator control. The alteration of enzymes of capsular polysaccharide synthesis and degradation or synthesis of proteins after switching the CapR ATP hydrolysis-dependent protease on or off will be determined by measuring the enzymes spectrophotometrically and the polypeptides by two dimensional gels with autoradiography. Polypeptides detected as unstable will be tested directly using the pure CapR ATP hydrolysis dependent protease. 2. Clone the equivalent of the E. coli K-12 1on (capR) gene from Salmonella, Shigella and Caulobacter and purify and measure the enzymatic and DNA binding activities of the corresponding proteins by techniques previously developed. 3. Identify, localize, purify and determine the activity of proteins specified by genes involved in cell division. The genes known as ftsA, ftsQ, sulB (ftsZ, sfiB), and sulA (sfiA) and ftsI (pbp3, sep) are those being studied. Recloning, membrane fractionation and protein purification techniques will be applied. 4. Purify the major outer membrane-localized Ompt protein and its precursor and determine the N-terminal amino acid sequence of both. Determine the proteolytic specificity of OmpT protein which is thought to be a trypsin-like enzyme that processes pro-OmpT protein to OmpT protein.
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| Storts, D R; Aparicio, O M; Schoemaker, J M et al. (1989) Overproduction and identification of the ftsQ gene product, an essential cell division protein in Escherichia coli K-12. J Bacteriol 171:4290-7 |
| Storts, D R; Markovitz, A (1988) Construction and characterization of mutations in hupB, the gene encoding HU-beta (HU-1) in Escherichia coli K-12. J Bacteriol 170:1541-7 |
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