published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

ompW encodes a widespread outer-membrane porin protein in Gram-negative bacteria. It has been implicated in bacterial responses to various antibiotics and environmental substances such as antibiotics, drugs and mouse mucus etc. Little is known, however, about its regulation and physiological roles during bacterial stress responses. Recently, comparative genomics studies revealed that the ompW gene is a core regulon of the global transcription factor FNR (Fumarate Nitrate Reduction) which mediates the transition from aerobic to anaerobic lifestyle of facultative bacteria. Anaerobiosis represents a predominant challenge encountered by many bacteria in their natural ecological niches and human hosts. This thesis thus aims to elucidate the molecular mechanism of FNR-dependent regulation of ompW expression and its relevance to the anaerobic adaption of the model facultative bacterium E. coli. Regulation of ompW expression by several other key physiological signals related to the anaerobiosis of E. coli, as well as the physiological significance, is also explored systematically. In the first half of the thesis, FNR-dependent regulation of ompW is confirmed by in vivo transcriptional activity assay, and then further confirmed at mRNA and protein level by RT-qPCR and western blotting. EMSA combined with transcriptional activity assay reveals that FNR directly binds with two sites centered at -81.5 and -126.5 bp respectively on ompW promoter (PompW). While binding to the -81.5 site by FNR activates the transcription of ompW, interaction with the -126.5 site represses it, and repression through the -126.5 site is dependent on primary occupancy of the -81.5 site by FNR. Based on these molecular mechanisms, a novel regulatory model of ompW expression during anaerobic adaptation of E. coli is proposed. Growth competition assay further confirmed the physiological significance of this fine-tuned regulation of ompW by FNR in facilitating the fitness and adaptation of E. coli during the transition from aerobic to micro-aerobic and anaerobic lifestyles. In the second half of the thesis, it is demonstrated that two other physiological signals related to the anaerobiosis of E. coli participate in the regulation of ompW, i.e. carbon and electron sources. The molecular mechanisms of how the relevant transcription factors, namely CRP and NarXL, mediate ompW transcription were elucidated: CRP activates the transcription of ompW by binding with the -42.5 site on PompW when glucose is absent; NarL represses the expression of ompW via its binding with the -18.5 site on PompW in the presence of nitrate (the most preferred electron source of E. coli during anaerobic growth). Fumarate is estimated to enter the central channel of OmpW and rescues OmpW-mediated colicin S4 killing of E. coli, suggesting OmpW is a receptor for fumarate and revealing its role in facilitating C4-dicarboxylates utilization. In summary, my study reveals a previously unrecognized, highly co-ordinated and dynamic regulation network for the expression of the widely distributed Gram-negative bacterial minor porin protein OmpW. Given the high conservancy of both the ompW gene and its promoter regions in several pathogenic bacterial species, my study contributes to the understanding of the pathogenicity of these species in the host relevant environment of anaerobiosis. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

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The tryptophan productivity of the genetically-manipulated strain JP4153 was increased 2.5-fold by introducing pMU78, a medium copy-number plasmid carrying a feedback-resistant trp operon. JP4153(pMU78) produced 23.5 g/l of tryptophan at a rate of 0.7 g/l/h when grown at 37 degrees C in a defined glucose and ammonium salts medium in a bench-scale fermentor. / During prolonged cultivation in the presence of antibiotic, the recombinant strain generated faster-growing, production-defective variants, which harboure mutated derivatives of pMU78. Insertion sequences were responsible for the two predominant types of mutation. The plasmid element ISI02 mediated deletions extending into the promoter-proximal region of the plasmid-borne trp operon. ISI0-Right, a chromosomal element, inserted into the promoter/trpE region of the plasmid. Three methods were employed to increase the structural stability of JP4153(pMU78) during the course of the production process. First, the growth of seed cultures was carried out at 30 degrees C, the permissive temperature for the trpS378 mutation carried by the host strain. Second, the seed culture medium was modified by the addition of yeast extract, which appeared to reduce the selective disadvantage conferred by the plasmid. Third, ISI02was deleted from pMU78 to create pMU88. (For complete abstract open document)

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The lexA protein in E. coli is a specific repressor of the recA gene. The lexA protein is cleaved by the recA protein in response to DNA damage. Cleavage derepresses the recA gene resulting in high level synthesis of recA protein and the expression of other DNA damage inducible functions (SOS functions). The lexA3 mutation makes the lexA protein resistant to cleavage and thus inhibits expression of DNA damage inducible functions. A mutant of E. coli has been isolated which exhibits many of the properties expected of a strain carrying an operator-constitutive mutation in the recA gene. The mutation partially suppresses the UV sensitivity of lexA3 strains, maps near the recA structural gene, allows constitutive synthesis of the recA protein and the recA message, and is cis-acting. Strains carrying the recAo('c) mutation were used to study the role of amplified levels of recA protein in the expression of certain SOS functions. The recAo('c) mutation did not suppress the UV inhibitory effect of the lexA3 mutation on the expression of UV induced cellular mutagenesis, and the reactivation and mutagenesis of UV irradiated phage (lamda). The expression of these functions in lexA('+) strains was not enhanced by the recAo('c) mutation. Constitutive recA synthesis did not result in lethal filamentous growth. These results are consistent with those reported elsewhere that the expression of SOS function is not dependent on high levels of recA protein and that the various "SOS genes" are repressed by the lexA protein as is the recA gene. Thus, recA protein is required in SOS expression for the inactivation of lexA protein and recA amplification is a consequence, not a cause of SOS expression. The DNA sequence of the recA operator region from a (lamda)precA transducing phage thought to carry the recAo('c) mutation isolated here, was determined. No difference was detected between the supposed mutant DNA and wild type controls. The significance of these results and the possibility that the recAo('c) mutation was not transferred to the phage are discussed.

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.

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.