I.G. Young

Role of quinones in electron transport to oxygen and nitrate in Escherichia coli. Studies with a ubiA− menA− double quinone mutant

A ubiA- menA- double quinone mutant of Escherichia coli K12 was constructed together with other isogenic strains lacking either ubiquinone or menaquinone. These strains were used to study the role of quinones in electron transport to oxygen and nitrate. Each of the four oxidases examined (NADH, D-lactate, alpha-glycerophosphate and succinate) required a quinone for activity. Ubiquinone was active in each oxidase system while menaquinone gave full activity in alpha-glycerophosphate oxidase, partial activity in D-lactate oxidase but was inactive in NADH and succinate oxidation. The aerobic growth rates, growth yields and products of glucose metabolism of the quinone-deficient strains were also examined. The growth rate and growth yield of the ubi+menA- strain was the same as the wild-type strain, whereas the ubiA-men+ strain grew more slowly on glucose, had a lower growth yield (30% of wild type) and accumulated relatively large quantities of acetate and lactate. The growth of the ubiA-menA- strain was even more severely affected than that of the ubiA-men+ strain. Electron transport from formate, D-lactate, alpha-glycerophosphate and NADH to nitrate was also highly dependent on the presence of a quinone. Either ubiquinone or menaquinone was active in electron transport from formate and the activity of the quinones in electron transport from the other substrates was the same as for the oxidase systems. In contrast, quinones were not obligatory carriers in the anaerobic formate hydrogenlyase system. It is concluded that the quinones serve to link the various dehydrogenases with the terminal electron transport systems to oxygen and nitrate and that the dehydrogenases possess a degree of selectivity with respect to the quinone acceptors.

The isolation, identification and properties of isochorismic acid. An intermediate in the biosynthesis of 2,3-dihydroxybenzoic acid

Abstract A new intermediate in the biosynthesis of 2,3-dihydroxybenzoic acid in Aerobacter aerogenes has been isolated and identified as 2-hydroxy-3-(1′-carboxyvinyloxy)-2,3-dihydrobenzoic acid for which the trivial name isochorismic acid is suggested. Methods for the assay of isochorismic acid are described. Salicylic acid and 3-carboxyphenylpyruvic acid are readily produced chemically from isochorismic acid under mild conditions. It is suggested that isochorismic acid may be the metabolic precursor of these compounds in other organisms.

Regulation of the enzymes involved in the biosynthesis of 2,3-dihydroxybenzoic acid in Aerobacter aerogenes and Escherichia coli

Abstract Assays for the enzymes in Aerobacter aerogenes converting chorismate into 2,3-dihydroxybenzoate (isochorismate synthetase, 2,3-dihydro-2,3-dihydroxybenzoate synthetase and 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase) have been developed. Isochorismate synthetase is stimulated by Mg2+ and 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase requires NAD for activity. Isochorismate synthetase has been separated from the other two enzymes by column chromatography on DEAE-cellulose. The addition of iron to the culture medium of A. aerogenes represses each of the enzymes. An experiment with a multiple aromatic auxotroph of Escherichia coli indicates that iron alone, not complexed with any 2,3-dihydroxybenzoate derivative, represses the enzymes concerned with 2,3-dihydroxybenzoate synthesis.

Amplification of the respiratory nadh dehydrogenase of escherichia coli by gene cloning

A relatively simple method has been used to clone the gene coding for the respiratory NADH dehydrogenase (NADH-ubiquinone oxidoreductase) of Escherichia coli from unfractionated chromosomal DNA. The restriction endonucleases EcoRI, BamI and HindIII were used to construct three hybrid plasmid pools from total E. coli DNA and the amplifiable plasmids pSF2124 and pGM706. Three different restriction endonucleases were used to increase the chances of cloning the ndh gene intact. Mobilization by the plasmid F was used to transfer the hybrid plasmids into ndh mutants and selection was made for Apr and complementation of ndh. DNA fragments complementing ndh were isolated from both the EcoRI and HindIII hybrid plasmid pools. The strain carrying the hybrid plasmid constructed with EcoRI produced about 8--10 times the normal level of the respiratory NADH dehydrogenase in the cytoplasmic membrane. Treating the cells with chloramphenicol to increase the plasmid copy number allowed the level of NADH dehydrogenase in the membrane to be increased to 50--60 times the level in the wild type. The results indicate the potential of gene cloning for the specific amplification of particular proteins prior to their purification.

2,3-Dihydroxybenzoate as a bacterial growth factor and its route of biosynthesis.

Abstract 2,3-Dihydroxybenzoate has been shown to be a growth factor in shaken cultures of certain multiple aromatic auxotrophs of Escherichia coli and Aerobacter aerogenes . The requirement for 2,3-dihydroxybenzoate is influenced by the presence of iron and citrate in the medium. The effects of the latter compounds differ with an auxotrophic strain derived from E. coli W and auxotrophic strains derived from E. coli K12. In the case of the former strain, certain metal ions or anaerobic growth will eliminate the 2,3-duhydroxybenzoate requirement; in the latter strains citrate replaces 2,3-dihydroxybenzoate. 2,3-Dihydroxybenzoate is formed via the shikimic acid pathway from chrismate in E. coli and A. aerogenes . The enzyme system forming 2,3-dihydroxybenzoate from choristmate in A. aerogenes has been studied and shown to require NAD and Mg 2+ for activity. At least two steps appear to be involved in the overall reaction. The enzyme system converting chorismate into 2,3-dihydroxybenzoate is strongly repressed by low concentrations of iron or cobalt and is also affected by the presence of salicylate or citrate in the growth medium.

Aerobic respiration in mutants of Escherichia coli accumulating quinone analogues of ubiquinone.

The ability of three naturally occurring analogues of ubiquinone to function in aerobic respiration in Escherichia coli has been studied. The compounds, which differ from ubiquinone in terms of the substituents on the quinone ring, accumulate in the cytoplasmic membranes of ubiE-, ubiF- and ubiG- mutants. One of the analogues (2-octaprenyl-3-methyl-6-methoxy-1,4-benzoquinone, NMQ), which lacks the 5-methoxyl group of the benzoquinone ring of ubiquinone promoted the oxidation of NADH, D-lactate and alpha-glycerophosphate but not succinate. Electron transport supported by MMQ was found to be coupled to phosphorylation. In contrast, 2-octaprenyl-6-methoxy-1,4-benzoquinone, which lacks both the 3-methyl and 5-methoxyl groups of ubiquinone, and 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone, in which the 5-methoxyl group of ubiquinone is replaced by an hydroxyl group, were virtually inactive in the oxidases tested. The ability of MMQ to function in respiration in isolated membranes is consistent with the findings that the growth rate and yield of a ubiF- strain, unlike other ubi- strains, were only slightly lower than those of a ubiF+ strain. The fact that MMQ is active in some but not all oxidases provides further support for the concept that the quinones link the individual dehydrogenases to the respiratory chain and that each dehydrogenase has specific structural requirements for quinone acceptors.

The isolation, identification and properties of 2,3-dihydro-2,3-dihydroxybenzoic acid. An intermediate in the biosynthesis of 2,3-dihydroxybenzoic acid☆

Abstract An intermediate in the conversion of chorismate into 2,3-dihydroxybenzoate has been formed using cell-free extracts of Aerobacter aerogenes . The new compound has been isolated and identified as 5,6-dihydroxycyclohexa-1,3-diene-1-carboxylic acid. The trivial name 2,3-dihydro-2,3-dihydroxybenzoic acid is suggested for this compound. Nuclear magnetic resonance, infrared, ultraviolet and mass spectra, together with some other properties of the new intermediate are described. 2,3-Dihydro-2,3-dihydroxybenzoic acid is dehydrated by heating under acid or alkaline conditions to give mainly 3-hydroxybenzoic acid together with some salicylic acid.

Membrane-associated reactions in ubiquinone biosynthesis in Escherichia coli. 3-octaprenyl-4-hydroxybenzoate carboxy-lyase

A sensitive and quantitative assay for 3-octaprenyl-4-hydroxybenzoate carboxy-lyase has been developed. This enzyme, which catalyses the third reaction in ubiquinone biosynthesis in Escherichia coli, was partially purified and some of its properties determined. It was found that a considerable proportion of the carboxylyase activity could be separated from the membrane fraction in cell extracts prepared using a French press. Gel filtration showed the molecular weight of the enzyme to be about 340 000. For optimal activity the carboxy-lase was shown to require Mn2+, washed membranes or an extract of phospholipids, and an unidentified heat stable factor of molecular weight less than 10 000. The carboxy-lyase reaction was also shown to be strongly stimulated by dithiothreitol and methanol. The properties of the carboxy-lyase are compared with the three other enzymes concerned with ubiquinone biosynthesis in E. coli which have been studied in vitro. The fact that the substrate of the carboxy-lyase is membrane-bound and the enzyme is stimulated by phospholipid suggests that it normally functions in association with the cytoplasmic membrane in vivo.

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. Strain 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 mutantions 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.

[34] Isolation of enterochelin from Escherichia coli

Publisher Summary This chapter discusses that Enterochelin is a cyclic trimer of 2,3-dihydroxy-N-benzoyl-L-serine is a natural iron carder produced by all strains of enteric bacteria so far examined and forms a very stable hexadentate chelate with iron. The enterochelin system of iron transport has been extensively studied in Escherichia coli and is believed to operate when the intracellular levels of iron fall and enterochelin is synthesized and excreted into the medium where it solubilizes polymeric forms of iron. The ferricenterochelin complex is then transported into the cell and the ligand is hydrolyzed by ferric-enterochelin esterase giving less stable complexes, which allow the iron to be released and utilized for cell metabolism. It describes a convenient method for the isolation of gram quantities of enterochelin using a mutant strain of E. coli . The mutant used is unable to transport ferric-enterochelin into the cell and therefore is iron-deficient and excretes relatively large quantities of enterochelin into the growth medium. The synthesis of enaterochelin by the mutant strain is not repressed by the addition of iron to the growth medium in contrast to the normal repression of enterochelin synthesis by iron in wild-type strains. The enterochelin that is excreted is accumulated as ferric-enterochelin. The iron complex is more stable than free enterochelin and, because it is negatively charged, it can be concentrated and purified using DEAE-cellulose. It is conveniently followed during chromatography by its deep red color and is dissociated under acid conditions and the free enterochelin extracted into ethyl acetate. Other acids that have accompanied the enterochelin during purification are removed by washing with phosphate buffer, and the enterochelin is finally crystallized.