Studies on the effects of temperature, pH, and anaerobiosis on gene expression in E. coli K-12
Auger, Elizabeth Ann
Bennett, George N.
Doctor of Philosophy
This research investigated the effects of temperature, pH, and anaerobiosis on gene expression in Escherichia coli. In the first series of experiments, the effects of FirA, a histone-like protein, and two temperature sensitive mutants on chromosomal lac operon expression were examined. The mutant Fir proteins have no effect on the transcription of the lac operon at 30$\sp\circ$C or upon a temperature shift to 40$\sp\circ$C. The effects of temperature on in vivo expression of E. coli promoters were also examined. The tet and anti-tet promoters of pBR322, lacUV5, trp, several hybrid promoters, and spacing variants of the tet promoter followed the general trend of increasing expression level between 20$\sp\circ$C and 37$\sp\circ$C with a leveling or some decrease in expression above 37$\sp\circ$C. Only the tet::trp promoter and the consensus sequence synthetic tet promoters deviated from this pattern, increasing in activity above 40$\sp\circ$C. No effects of temperature were observed on the expression profiles of trp promoters with altered flanking sequences. The effects of temperature on the induction of the trp promoter by 3-$\beta$-indole acrylic acid were examined. Five $\mu$g/ml was sufficient to derepress the trp promoter at the same induction ratio from 20$\sp\circ$C to 45$\sp\circ$C. The biodegradative lysine decarboxylase (LDC) of E. coli is induced by low pH, anaerobiosis, and excess lysine. A series of isogenic E. coli strains were constructed to determine the effects of CadR, Fnr, Cya, CRP, and Pgi on LDC expression. These strains were grown in a rich medium similar to Falkow decarboxylase medium at pH 5.5, pH 6.8, and pH 8.0 under aerobic and anaerobic conditions. The pH and air responses were retained in these mutants demonstrating that these regulatory processes do not mediate LDC regulation. To further study the regulation of biodegradative LDC, as well as biodegradative arginine decarboxylase, a Mu lac fusion library was constructed. Stable LDC deficient lac fusions which are regulated by low pH or anaerobic conditions were identified. Two stable arginine decarboxylase deficient lac fusions were observed to be regulated by low pH and anaerobic conditions. Three LDC deficient lac fusion strains regulated by low pH or anaerobiosis have been mapped to the cadA gene.