Mutations affecting the reduced nicotinamide adenine dinucleotide dehydrogenase complex of Escherichia coli

A strain carrying a point mutation affecting the NADH dehydrogenase complex of Escherichia coli has been isolated and its properties examined. The gene carrying the mutation (designated ndh) was located on the E. coli chromosome at about minute 23 and was shown to be cotransducible with the pyrC gene. Strains carrying the ndh^? allele were found to be unable to grow on mannitol and to grow very poorly on glucose unless the medium was supplemented with succinate, acetate or casamino acids.The following properties of strains carrying the ndh^? allele were established which suggest that the mutation affects the NADH dehydrogenase complex but apparently not the primary dehydrogenase. Membrane preparations possess normal to elevated levels of d-lactate oxidase and succinate oxidase activities but NADH oxidase is absent. NADH is unable to reduce ubiquinone in the aerobic steady state and reduces cytochrome b very slowly when the membranes become anaerobic. NADH dehydrogenase, measured as NADH-dichlorophenolindophenol reductase is reduced but not absent. NADH oxidase is stimulated by menadione although not by Q-3 or MK-1 and in the presence of menadione, cytochrome b is reduced normally by NADH.Further mutants affected in NADH oxidase were isolated using a screening procedure based on the growth characteristics of the original ndh^? strain. The mutations carried by these strains were all cotransducible with the pyrC gene and the biochemical properties of the additional mutants were similar to those of the original mutant.The properties of the group of ndh^? mutants established so far suggest that they are affected in the transfer of reducing equivalents from the NADH dehydrogenase complex to ubiquinone.     

Reversible interconversion of the functional state of the gene regulator FNR from Escherichia coli in vivo by O2 and iron availability

FNR, the gene regulator of anaerobic respiratory genes of Escherichia coli is converted in vivo by O₂ and by chelating agents to an inactive state. The interconversion process was studied in vivo in a strain with temperature controlled synthesis of FNR by measuring the expression of the frd (fumarate reductase) operon and the reactivity of FNR with the alkylating agent iodoacetic acid. FNR from aerobic bacteria is, after arresting FNR synthesis and shifting to anaerobic conditions, able to activate frd expression and behaves in the alkylation assay like anaerobic FNR. After shift from anaerobic to aerobic conditions, FNR no longer activates the expression of frd and reacts similar to aerobic FNR in the alkylation assay. The conversion of aerobic (inactive) to anaerobic (active) FNR occurs in the presence of chloramphenicol, an inhibitor of protein synthesis. Anaerobic FNR can also be converted post-translationally to inactive, metal-depleted FNR by growing the bacteria in the presence of chelating agents. The reverse is also possible by incubating metal-depleted bacteria with Fe²⁺. From the experiments it is concluded that the aerobic and the metal-depleted form of FNR can be transferred post-translationally and reversibly to the anaerobic (active) form. The response of FNR to changes in O₂ supply therefore occurs at the FNR protein level in a reversible mode.Abbreviation BV_red = reduced benzyl viologen     

Engineering a native homoethanol pathway in Escherichia coli B for ethanol production

A native homoethanol pathway (pyruvate-to-acetyl-CoA-to-acetaldehyde-to-ethanol) was engineered in Escherichia coli B. The competing fermentation pathways were eliminated by chromosomal deletions of the genes encoding for fumarate reductase (frdABCD), lactate dehydrogenase (ldhA), acetate kinase (ackA), and pyruvate formate lyase (pflB). For redox balance and anaerobic cell growth, the pyruvate dehydrogenase complex (aceEF-lpd, a typical aerobically-expressed operon) was highly expressed anaerobically using a native anaerobic inducible promoter. The resulting strain SZ420 (ΔfrdBC ΔldhA ΔackA ΔfocA-pflB ΔpdhR::pflBp6-pflBrbs-aceEF-lpd) contains no foreign genes and/or promoters and efficiently ferments glucose and xylose into ethanol with a yield of 90% under anaerobic conditions.     

Anaerobic Expression of Escherichia coli Succinate Dehydrogenase: Functional Replacement of Fumarate Reductase in the Respiratory Chain during Anaerobic Growth

Succinate-ubiquinone oxidoreductase (SQR) from Escherichia coli is expressed maximally during aerobic growth, when it catalyzes the oxidation of succinate to fumarate in the tricarboxylic acid cycle and reduces ubiquinone in the membrane. The enzyme is similar in structure and function to fumarate reductase (menaquinol-fumarate oxidoreductase [QFR]), which participates in anaerobic respiration by E. coli. Fumarate reductase, which is proficient in succinate oxidation, is able to functionally replace SQR in aerobic respiration when conditions are used to allow the expression of the frdABCD operon aerobically. SQR has not previously been shown to be capable of supporting anaerobic growth of E. coli because expression of the enzyme complex is largely repressed by anaerobic conditions. In order to obtain expression of SQR anaerobically, plasmids which utilize the P_FRD promoter of the frdABCD operon fused to the sdhCDAB genes to drive expression were constructed. It was found that, under anaerobic growth conditions where fumarate is utilized as the terminal electron acceptor, SQR would function to support anaerobic growth of E. coli. The levels of amplification of SQR and QFR were similar under anaerobic growth conditions. The catalytic properties of SQR isolated from anaerobically grown cells were measured and found to be identical to those of enzyme produced aerobically. The anaerobic expression of SQR gave a greater yield of enzyme complex than was found in the membrane from aerobically grown cells under the conditions tested. In addition, it was found that anaerobic expression of SQR could saturate the capacity of the membrane for incorporation of enzyme complex. As has been seen with the amplified QFR complex, E. coli accommodates the excess SQR produced by increasing the amount of membrane. The excess membrane was found in tubular structures that could be seen in thin-section electron micrographs.