Zhang, Limei2024-08-152024-08-1520002000https://hdl.handle.net/1828/20267The lytB gene is located at about 0.5 min on Escherichia coli genetic linkage map. It is part of an operon, designated ileS-lsp, which consists of 5 genes: ribF, ileS, lsp, slpA and lytB (5' to 3'). Previous studies on the lytB gene suggested that it may be involved in the regulation of the starvation stress response, known as the stringent response, and in mediating tolerance to penicillin. However, recent studies indicate that the original lytB mutant actually carried multiple mutations, and the major phenotypic characteristics were due to a temperature-sensitive mutation in the ileS gene. A second unusual and unidentified dominant "temperature-sensitive mutation lies downstream of the lytB. Importantly, no mutation was found in lytB, and the function of this gene therefore currently unknown. The 5 genes in the ileS-lsp operon share no obvious relationship to each other, and there are no clues here regarding a possible function for lytB. The main objective of this thesis was to clone and express the E. coli lytB gene as a first step toward determining the biological function of LytB. The lytB gene was amplified from a previously cloned copy of ileS-lsp operon by polymerase chain reaction. It was cloned expressed from three different expression vectors. It was most efficiently expressed as a glutathione S-tranferase fusion protein from the cloning vector, pGEX-5X-1, and was purified in this form in high yield. The purified LytB protein was >95% pure as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Western blot analysis. The lytB gene is predicted to encode a 35.5-kDa protein, and this was confirmed in this study. Although the protein has not been quantified, preliminary experiments involving a Western blot analysis of crude cell extracts from wild type bacteria with a polyclonal anti-LytB antiserum indicate that LytB is a low abundance protein. The protein appeared to be unstable, and this characteristic may be worth further study. Several attempts were made to disrupt the chromosomal copy of the lytB gene. These attempts involved the in vitro insertion of a genetic element known as the n interposon into the lytB gene cloned into a co1E1 vector. The Ω interposon encodes spectinomycin resistance which offers the advantage of a direct selection for constructs. It also contains efficient transcriptional and translational terminators on its 3'-end and is consequently an effective gene disruptor. Three independent attempts were made to transform the constructed plasmid into a po1A (DNA polymerase 1-deficient) mutant strain. Co1E1 plasmids are unstable in Po1A mutants, and this method is used to force the insertion of cloned genes into the chromosome of the host by homologous recombination. All 3 attempts were unsuccessful. About 97% of the spectinomycin-resistant transformants still carried the plasmid in a stable form and presumably had developed a way of stabilizing Co1E1 plasmids, possibly through spontaneous suppressor mutations. The remaining 3% of the spectinomycin-resistant transformants carried the Ω interposon in their chromosomes, but they still produced LytB as determined by Western blot analysis. The disrupted lytB gene apparently entered the chromosome of these strains by illegitimate recombination. Two other approaches to disrupt lytB were also unsuccessful. An attempt to identify prtoeins that interact with LytB by the yeast two-hybrid technology was also unsuccessful. A major conclusion of this study is that the lytB gene in E. coli appears to be indispensable.77 pagesAvailable to the World Wide WebThesis