We have been studying the role that protein degradation plays in regulating gene expression and have continued with studies on the linkages between chromosome synthesis and partition of chromosomes during cell division. Turnover of the natural substrates of the Lon ATP-dependent protease, N and SulA, has been shown to be independent of the heat shock protein DnaJ, while the activity of another substrate of Lon, the capsular polysaccharide positive regulator RcsA, has been shown to be dependent on DnaJ activity under most circumstances. These results suggest that the heat shock proteins are not an essential component of Lon-dependent degradation of natural substrates in vivo. The temperature sensitivity of capsule synthesis appears to be due to misfolding of RcsA in vivo; similar misfolding may occur in the absence of DnaJ. Studies on the regulation of rcsA transcription have led to the identification of a small RNA, DsrA RNA. Increased amounts of DsrA RNA lead to increased transcription of rcsA, apparently because the DsrA RNA participates in opening a silenced rcsA promoter. The histone-like protein, HNS, is necessary for silencing. We have continued to investigate the in vivo function of the Clp energy- dependent protease. The specificity of this protease in vivo seems to be dictated by the ATPase subunit. We have found that ClpX, an alternate ATPase subunit, and ClpP mediate degradation of lambda O protein, as well as a number of other substrates. Another possible ATPase subunit has been found by sequence comparisons and its role in in vivo degradation is being examined. We have also been studying the regulatory events of the cell cycle using the mbr mutants, which have an alteration in DNA content per cell. We have been focusing on mbrA, which affects the coupling of DNA replication to cell elongation and have identified several genes required for mbrA to function. In addition, we have cloned mbrA and begun to analyze the physical structure of the gene. Further characterization of mbrA and its suppressors should elucidate the critical role it plays in cell cycle regulation.
