Exploring the Regulatory Network and Physiological Significance of the Dipeptide Transport System During Anaerobic Adaptation in Escherichia Coli

This dissertation, "Exploring the Regulatory Network and Physiological Significance of the Dipeptide Transport System During Anaerobic Adaptation in Escherichia Coli" by Xiang, Gao, 高翔, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled "EXPLORING THE REGULATORY NETWORK AND PHYSIOLOGICAL SIGNIFICANCE OF THE DIPEPTIDE TRANSPORT SYSTEM DURING ANAEROBIC ADAPTATION IN ESCHERICHIA COLI" Submitted by Gao Xiang for the Degree of Doctor of Philosophy at The University of Hong Kong in August 2014 Escherichia coli (E. coli) and other facultative anaerobes are genetically programmed to adapt to both aerobic and anaerobic growth conditions. Among the various genes which expression is altered during the transition from aerobic to anaerobic growth, those involved in the nutrients uptake and waste product extrusion is of particular essential to the physiology of E. coli. Genome-wide studies have suggested that the dipeptide transporter system Dpp which can also transport heme and its precursor ALA (δ-aminolevulinic acid) is one of the anaerobically repressed transporters during anaerobiosis. To understand the mechanisms of this regulation, the roles of the anaerobic global regulators FNR, the two-component system ArcAB, and the sRNA GcvB on the expression of the dpp operon were investigated. Results of translational fusion of Pdpp-lacZ, RT-qPCR, and Western blot analysis showed that FNR repressed the expression of dpp since Δfnr caused increased expression of Dpp under anaerobic conditions in LB medium. However, mutation of the FNR putative binding site on Pdpp did not cause de-repression and no direct binding of FNR to Pdpp was observed in EMSA, suggesting that FNR may not directly regulate dpp expression. Interestingly, deletion of arcA or gcvB caused increased expression of Dpp and ΔgcvB ΔarcA double deletion led to a further increase of Dpp protein level, indicating the simultaneous repression of Dpp by both ArcA and GcvB. EMSA demonstrated direct binding of the phosphorylated ArcA (ArcA P) to Pdpp and PgcvB, and ΔarcA caused decreased level of GcvB under anaerobic conditions, suggesting that ArcA can both directly repress dpp expression and through its activation of GcvB. These results together indicated a "coherent feed-forward regulatory loop" on the expression of Dpp in which ArcA and GcvB co-repress dpp, and ArcA also activates GcvB. To investigate the physiological significance of the repression of the Dpp expression under anaerobic condition, the Dpp system was overexpressed and examined on the growth and viability of E. coli under various anaerobic growth conditions. It was found that overexpression of the system in ΔtolC E. coli background strain caused accumulation of red fluorescent substances and photosensitivity to near-UV irradiation (366 nm) in the presence of 3 μg/ml ALA. Deletion of fnr, arcA, gcvB, and both gcvB and arcA caused similarly decreased viability to photo-irradiation. However, under aerobic conditions, except for the ΔgcvB ΔtolC strain, none of these strains displayed increased photosensitivity even in the presence of 10 μg/ml ALA, suggesting that E. coli cells were more tolerant to near-UV irradiation under anaerobic conditions and the importance of repressing the dpp expression under this condition. Consistent with the viability assay, the concentration of intracellular coproporphyrin was highest in E. coli ΔtolC background containing ΔgcvB ΔarcA (24.9 μM), followed by ΔgcvB, ΔarcA, and Δfnr (17, 16.7, and 14.8 μM) respectively, suggesting that the increas