Roles and regulation of the iron-sulphur proteins, HCP, NapG and NapH, induced during anaerobic growth of E. coli

The periplasmic nitrate reductase (Nap) has been shown to support anaerobic growth of Escherichia coli K-12 under nitrate-limiting conditions. Two of the Nap proteins, NapG and NapH, are predicted to contain four and two [4Fe-4S] clusters, respectively. In this thesis it is reported that, during fermentative growth. Nap plays a role in redox balancing. This role is most pronounced in a strain that lacks menaquinol and tiierefore cannot use the menaquinol-dependent ftunarate reductase to fulfil a redox balancing role during glucose fermentation. Nitrate stimulated the growth of both a AmenBC AnapGH and an isogenic AmenBC nap^ strain to the same extent, even although the Nap activity was extremely low. This showed that the residual 1% electron flow in the strain deleted for NapG and NapH was sufficient to fulfil this redox balancing function. Using artificial quinones, NapG and NapH were shown to be linked to oxidation of quinones with high midpoint redox potentials. NapF^ and NapF' strains were grown anaerobically after either aerobic or anaerobic growth and NapF was shown to be involved in adaptation from aerobic to anaerobic growth. The hybrid cluster protein (HCP) contains two Fe-S clusters, one of which is a hybrid [4Fe-2S-20] cluster. Despite intensive study, its physiological function is unclear. E. coli HCP is detected after anaerobic growth with nitrate or nitrite, so a possible role for it in some stage of the nitrogen cycle has been proposed. To study the regulation of HCP, an hcprlacZ fusion was constructed and transformed into_^r, arcA and norR mutant strains of E. coli. Transcription from the hep promoter was induced during anaerobic growth. Only the jhr mutant was defective in hep expression, suggesting that transcription from the promoter in response to anaerobiosis is dependent on FNR. Nitrate and nitrite fiirther induced transcription from the hep promoter. The parental strain and the narL, narP and narLmrP mutants were grown anaerobically in medium supplemented with nitrite or nitrate. The nitrite and nitrate response of the hep promoter was mediated by both of the response regulator proteins, NarL and NarP. It is argued that NarL plays a dual role at the hep promoter acting as an activator during growth in the presence of a low concentration of nitrite or nitrate and as both repressor and activator in the presence of high nitrite or nitrate concentrations. Gel retardation assays were used to show that FNR and NarL form a complex with the hep promoter, thus confirming that their effect on transcription is direct. A technique involving the rapid amplification of cDNA ends (RACE) was used to demonstrate that transcription of the hcp~hcr operon initiates at a thymine nucleotide located 31 bp upstream of the translation-initiation codon. A A/icp strain was constructed by homologous recombination. When grown in medium supplemented with nitrate, the growth rate and yield of the parental strain and the hhep mutant were the same, suggesting that HCP is not involved in nitrate-dependent growth. Both HCP^ and HCP' strains were equally sensitive to nitric oxide and hydroxylamine. It was concluded therefore that HCP is unable to protect bacteria against nitric oxide or hydroxylamine toxicity in vivo. HCP was overexpressed from a recombinant plasmid and subsequently purified on a nickel column for biochemical studies. A qualitative method using reduced methyl viologen as an electron donor was developed for use in attempts to identify a possible substrate of HCP in vitro. Nitrite, nitrate and hydroxylamine were tested, but no evidence was presented that any of them can be used as an electron acceptor.