Five new genes apparently involved in the metabolism of cell wall peptidoglycan by Escherichia coli are described. One of these, designated murH, was mapped at 99 min on the E. coli linkage map. The murH1 mutant exhibited temperature- sensitive (ts) growth which was associated with a block in a late step in peptidoglycan synthesis and with peptidoglycan hydrolase-mediated lysis at the restrictive temperature. The murH locus could not be cloned in multicopy vectors but was readily cloned in a single copy phasmid vector derived from phage λ. The instability of murH in multicopy prevented its further characterization. As an alternative approach to characterizing the murH function, extragenic mutations which suppressed the murH1 ts lysis phenotype were isolated. One suppressor mutation, designated smh-A1, (25 min on the genetic linkage map) restored temperature resistance in murH1 mutants but otherwise had no distinguishable phenotype. A second extragenic murH1 suppressor, smhB1 (13 min) conferred a ts lysis phenotype by itself. Interestingly, a combination of murH1 and smhB1 resulted in cosuppression of their lysis phenotypes. The suppressor activities of the smhA1 and smhB1 alleles were relatively specific in that they failed to suppress lysis caused by either mutational (murE or murF) or antibiotic-induced blocks in peptidoglycan synthesis. Two additional ts lysis mutations, lytD1 (mapped at 13 min) and lytE1 (25 min), arose spontaneously in smhB1 and smhA1 backgrounds, respectively. The smhA1 allele suppressed the lysis phenotype of lytE1 but not of lytD1. Furthermore, the combination of smhB1 with either lytD1 or lytE1 resulted in cosuppression of their lysis phenotype. The specificity of the suppressor activities, combined with the similarities in the phenotypes of the mutants representing this collection of loci, suggested functional relationships between the murH, smhA, smhB, lytD, and lytE loci. Four clones which complemented the lytD1 mutation were obtained by screening an E. coli gene library, but it is shown that the complementing activity did not represent the E. coli chromosomal lytD locus. It is shown instead that 2 phage λ genes, identified as cro and cI, accounted for the lytD1 complementing activities in these clones. Evidence is presented which suggests that these clones were derived from phage λ DNA which was fortuitously present as a contaminant in the vector preparation used for construction of the gene library. Since the λ Cro and CI proteins are DNA-binding proteins which bind to identical 17 base-pair recognition sequences (the λ right operator sequences), it is hypothesized that LytD encodes a DNA-binding protein with a similar specificity (i.e., which binds to a λ right operator-like sequence) which regulates, probably negatively, the expression of a gene(s) involved in some way with peptidoglycan hydrolysis. / Graduate
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/9479 |
| Date | 20 June 2018 |
| Creators | Dai, Dexi |
| Contributors | Ishiguro, Edward E. |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | Available to the World Wide Web |
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