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A study of the effects of a novel rpoA mutation (phs) in Escherichia coli K12Giffard, P. M. January 1986 (has links)
An <i>Escherichia coli</i> strain carrying the <i>phs</i> mutation was isolated by other workers who were interested in determining the role of the Na /H antiport in pH homeostasis. The mutation was observed to cause impaired growth on L-glutamate amd melibiose and was also reported to cause a sensitivity to alkaline conditions due to an impairment in pH homeostasis. Since uptake of both glutamate and melibiose is energised by a Na<sup>+</sup> gradient, these observations were interpreted as evidence that the <i>phs</i> mutation impairs Na<sup>+</sup> /H<sup>+</sup> antiport activity, and so by implication, that the Na<sup>+</sup> /H<sup>+</sup> antiport is involved in pH homeostasis in <i>E. coli</i>. In work for this thesis, evidence was accumulated that the <i>phs</i> mutation does not affect Na<sup>+</sup> /H<sup>+</sup> antiport activity. Proline uptake, which is energised by a Na<sup>+</sup> gradient, was found to be normal in a mutant strain. Also, arabinose uptake via two different transport systems, neither of which is energised by a Na<sup>+</sup> gradient, was found to be impaired by the mutation. K<sup>+</sup> uptake by Na<sup>+</sup> loaded cells, a phenomenon which is thought to give a measure of Na<sup>+</sup> /H<sup>+</sup> antiport activity, was not affected by the mutation. Studies on growth under alkaline conditions indicated that growth is not impaired by the mutation. Evidence was obtained however, that the <i>phs</i> mutation causes filamentation under these conditions. There is a possibility that this may be due to an effect on the penicillin-binding protein content of the cell envelope. Investigations of the effect of the mutation on pH homeostasis suggested that it affects either pH homeostasis, or the validity of the techniques used for determining the cytoplasmic pH. In view of the mapping of the mutation to the gene coding for the α-subunit of RNA polymerase (Rowland <i>et al</i>, 1985), the effects of the mutation on transcription were studied. It was found that the mutation dramatically impairs transcription of genes under the positive regulation of the <i>AraC</i> gene product. An examination of the effect of the mutation on transcription from a variety of different promotors showed that the transcription defect is highly selective. Some evidence was obtained that the mutation impairs the process of positive regulation. To test this, the effect of the mutation on transcription from AraC independent derivatives of an AraC dependent promoter was determined. In the absence of AraC, the mutation had no effect. In the presence of AraC, complex effects were observed. It was concluded that the mutation affects the process of positive regulation or CRP-cAMP mediated derepression of this promoter.
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