pH regulation in enteric bacteria
Escherichia coli 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 clpX, encoding a heat-shock induced protease and molecular chaperone, was functional in survival of E. coli at pH 3.3. Promoter probe plasmid libraries of Salmonella typhimurium LT2 DNA were created in E. coli 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 lacZ in E. coli generated by Mu DII 1734 insertion were screened in a similar way and suggested that the gene encoding adenylate cyclase (cya) could be induced by mild cytoplasmic acidification. The sequence of a gene known to be inducible by cytoplasmic acidification, inaA, 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 E. coli Tn10 chromosomal insertional mutant library for genes involved in the regulation of inaA. One such mutant had a multiple antibiotic resistant (mar) 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 E. coli genome, and were tentatively identified as yddB (closely linked to gadB and gadC; required for glutamate dependent acid resistance) and f300 (closely linked to pldA; required for detergent resistance). The promoter of f300 was shown to be sensitive to cytoplasmic acidification. The inaA 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.