The product of the Escherichia coli lexA gene has been identified, and the regulation of lexA gene expression in vivo has been examined. A series of specialized transducing phages carring lexA⁺ and 3 different amber lexA alleles was constructed by in vivo recombination between λlexA3 and host lexA alleles. These phages were characterized extensively to confirm that they carried the appropriate lexA allele. The lexA gene product was identified by comparison of the polypeptides encoded by λlexA3 and the amber lexA phages. A 24,000 dalton polypeptide, synthesized after infection of both amber-suppressor and non-suppressor hosts by λlexA3 was not synthesized following amber lexA phage infection of non-suppressor hosts. Synthesis of this polypeptide following amber lexA phage infection was restored by the presence of an amber suppressor mutation in the host. On the basis of these data, the 24,000 dalton polypeptide was identified as the lexA gene product. Regulation of lexA gene expression in vivo was examined by hybridization experiments to measure lexA mRNA levels. The basla level of lexA mRNA in wild type E. coli was found to be .006% of total mRNA. Treatment of the bacteria with 100 erglmm² ultraviolet irradiation (UV) led to an eight-fold increase in lexA mRNA levels within 10 minutes, the lexA mRNA remained elevated until 70 minutes after irradiation, then slowly declined. By comparison, the level of recA mRNA increased from .05% to .51% of total mRNA within 10 minutes following UV irradiation, then declined. Both lexA and recA genes were induced by nalidixic acid treatment; the induction was not as rapid as UV induction and different relative induction kinetics of the two genes were seen. The levels of lexA and recA mRNAS were measured in several mutant strains following UV-irradiation.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

No description available.

Under conditions of phosphate starvation Escherichia coli can solubilize mineral phosphates, such as dicalcium phosphate, to orthophosphate which is then available for uptake and cell growth processes. lac operon fusions were created using MudX phage, and mineral phosphate solubilization (Mps) mutants were identified by their inability to solubilize mineral phosphate on plate assays. Four of these mutants have been mapped on the E. coli chromosome via Hfr matings and are located at two distinct portions of the chromosome; between 23 and 50 minutes and between 60 and 90 minutes. One mutant in each region has phosphate starvation inducible (Psi) promoter activity. One of these mutants (DB1047) was mapped to between 69 and 75 minutes via F' matings, and fine structure mapped to 75 minutes by hybridization with λ clones from a genomic library of Escherichia coli. DB1047 was characterized more closely and found to exhibit pleiotropy with regard to several membrane related traits. Evidence that this is a single insertional event comes from the simultaneous loss of all traits tested in spontaneous revertants. Additionally, a Tn5 mutant was identified that was identical for these traits. Our data strongly support the hypothesis that the mutation carried by DB1047 is in the ompB locus. This locus consists of the two regulatory cotranscribed genes, ompR and envZ. This locus is involved in regulation of transcription of the ompC and ompF genes for outer membrane porin proteins, and is located at 75 minutes on the chromosome as is the DB1047 mutation. DB1047 lacks the outer membrane porin OmpF, a phenotype previously attributed to envZ mutants. However, the ompR321 mutant resembles DB1047 in reduced ability to solubilize phosphate. Additional supporting evidence for the DB1047 mutation belonging to the ompB locus comes from the most recent report that mutations in the himA gene, which I found to be deficient in the ability to solubilize phosphate, also affect the regulation of production of the outer membrane porin OmpF.

No description available.

No description available.

No description available.

No description available.

No description available.

No description available.