<I>Escherichia coli</I> mutants impaired in growth and survival at low external pH in minimal medium were selected and attempts made to identify the disrupted genes. This study suggested that <I>clpX</I>, encoding a heat-shock induced protease and molecular chaperone, was functional in survival of <I>E. coli </I>at pH 3.3. Promoter probe plasmid libraries of <I>Salmonella typhimurium </I>LT2 DNA were created in <I>E. coli </I>and screened for acid-inducible transcriptional elements, and transcriptionally active fragments of degradative amino-acid decarboxylase genes recovered. Chromosomal gene fusions to the reporter gene <I>lacZ </I>in <I>E. coli</I> generated by Mu DII 1734 insertion were screened in a similar way and suggested that the gene encoding adenylate cyclase (<I>cya</I>) could be induced by mild cytoplasmic acidification. The sequence of a gene known to be inducible by cytoplasmic acidification, <I>inaA, </I>became available during the course of this study. The 5' region of this gene was used to generate a set of plasmids carrying fragments of the acid-inducible promoter transcriptionally fused to a luciferase based reporter system. Elements of the sequence required for induction by cytoplasmic acidification were identified. One of these reporter constructs was used to screen an <I>E. coli </I>Tn<I>10</I> chromosomal insertional mutant library for genes involved in the regulation of <I>inaA. </I>One such mutant had a multiple antibiotic resistant (<I>mar</I>) phenotype. The disrupted loci in 2 other mutants were identified by inverse PCR, sequence analysis and database searches. Both were known only as open reading frames (ORFs) discovered during the sequencing of the entire <I>E. coli</I> genome, and were tentatively identified as <I>yddB </I>(closely linked to <I>gadB </I>and <I>gadC</I>; required for glutamate dependent acid resistance) and <I>f300</I> (closely linked to <I>pldA; </I>required for detergent resistance). The promoter of <I>f300</I> was shown to be sensitive to cytoplasmic acidification. The <I>inaA</I> promoter was also demonstrated to be induced at the onset of stationary phase, and to be independent of the stationary phase and weak-acid inducible σ factor RpoS and also of cAMP levels.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:245982 |
| Date | January 1997 |
| Creators | Stephen, John R. |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=130919 |
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