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J R Postgate

Publications and source records attributed to J R Postgate.

At least 19 recordsLinked to original sources
PubMed1987

A rapid and efficient method for plasmid transformation of Klebsiella pneumoniae and Escherichia coli.

A rapid and efficient method for plasmid transformation of Klebsiella pneumoniae M5a1 and Escherichia coli K12 has been developed. The method, which uses a freeze-thaw cycle in the presence of CaCl2 to facilitate DNA uptake, is substantially more efficient for K. pneumoniae M5a1 than the conventional transformation procedure for E. coli. The simplicity and speed of the method makes it very attractive for routine transformation of K. pneumoniae M5a1 and E. coli K12.

Escherichia coli↗
PubMed1985

Expression of Klebsiella pneumoniae nif genes in Proteus mirabilis.

Self-transmissible plasmids carrying his and nif genes from Klebsiella pneumoniae have been introduced into three his mutants of Proteus mirabilis: strains 5006-1, WR19 and WR20. Expression of his by the transconjugants was unequivocal, if slightly temperature-sensitive, but none was Nif+ when tested for acetylene reduction in anaerobic glucose medium using inocula from rich or glucose-minimal aerobic agar cultures. Succinate or pyruvate in place of glucose, low glucose, lower temperature or elevated Na2MoO4 did not allow nif expression and no nitrogenase MoFe-protein peptide was detected immunologically after exposure to conditions in which diazotrophic enterobacteria, normal or genetically constructed, derepress nif. One strain, P. mirabilis WR19, carrying the his nif Kmr plasmid pMF250 was examined in detail. The nif activator gene nifA was introduced on the plasmid pCK1. Such derivatives remained Nif- when tested, after aerobic growth on rich agar media, with normal or low glucose, with succinate or with elevated Mo. However, pre-conditioning by aerobic growth on glucose-minimal agar led to subsequent anaerobic expression of nif in glucose medium from pMF250 in WR19 carrying pCK1. NH+4 or proline could serve as N-source in the glucose-minimal agar. Maximum activity was about 5% of that of K. pneumoniae in our assay conditions. Material cross-reacting with anti-serum to the nitrogenase MoFe protein was formed. Nitrogenase activity was not 'switched off' by NH+4. P. mirabilis WR19 (pCK1) showed NH+4-constitutive temperature-sensitive kanamycin resistance (a nif-related phenotype of this plasmid) in aerobic glucose minimal medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonia↗
PubMed1978

Nitrogenase synthesis in Klebsiella pneumoniae: comparison of ammonium and oxygen regulation.

Rates of nitrogenase synthesis by Klebsiella pneumoniae were measured by pulse-labelling organisms with a mixture of 14C-labelled amino acids followed by sodium dodecyl sulphate gel electrophoresis and autoradiography. Populations from an NH4+-repressed, SO42--limited chemostat (0.46 mg dry wt ml-1), when released from NH4+ repression, simultaneously synthesized detectable quantities of the three nitrogenase polypeptides 45 min before acetylene-reducing activity was observed. Exposure of populations synthesizing nitrogenase to air or NH4+ (200 microgram N ml-1) repressed synthesis of both component proteins simultaneously, the rate initially decreasing by half in 11 to 12 min; in the presence of NH4+ a second slower phase with an approximate half-life of 30 min was observed. With 5% O2 in N2 the half-lives for the decreases in the rates of synthesis were 30 min for the Fe protein and 33 min for the Mo-Fe protein. Oxygen also repressed nitrogenase in a glutamine synthetase constitutive derivative of K. pneumoniae (strain SK24) which escapes NH4+ repression. Regulation of nitrogenase by O2 may therefore be independent of glutamine synthetase.

Ammonia↗
PubMed1977

Expression of Klebsiella nif and his genes in Salmonella typhimurium.

Derivatives of Salmonella typhimurium carrying F prime or P prime plasmids with Klebsiella nif and his genes had specific nitrogenase activities similar to Klebsiella in selective conditions, even to showing "hyperinduction" under argon. No evidence was obtained for catabolite repression of normal nif expression but dibutyl cyclic AMP often augmented "hyperinduction". In non-selective conditions the Klebsiella his nif determinants were rapidly lost from the plasmids; the low levels of nif expression and temperature-sensitive his expression previously reported were probably due to ready loss of his nif in the test conditions used.

Cyclic AMP↗
PubMed1977

Expression of Klebsiella pneumoniae nitrogen fixation genes in nitrate reductase mutants of Escherichia coli.

Nitrate reductase (nar) A, B and E mutants of Escherichia coli with plasmids carrying Klebsiella pneumoniae nitrogen fixation (nif) genes reduced acetylene independently of added molybdate, but nar D mutants showed pleiotropic dependence on the concentration of added molybdate for expression of both nar and nif. No complementation of nar mutations by nif occurred; nitrite but not nitrate repressed nif in nar hosts. Derepression of nif occurred in molybdenum-deficient nar D (nif) strains since nitrogenase peptides were present. nifB mutants, thought to have a lesion in the pathway of molybdenum to nitrogenase, as well as nif deletion mutants, had normal nitrate reductase activity.

Enzyme Repression↗
PubMed1976

Derivation and properties of F-prime factors in Escherichia coli carrying nitrogen fixation genes from Klebsiella pneumoniae.

A His+ Nif+ Escherichia coli K12, Hfr strain (UNF43) was constructed by an intergeneric mating between a Klebsiella pneumoniae donor strain (HF3) and a his-HFR E. coli strain (SBI824) which transfers his as an early marker. An F-prime nif plasmid, FN39, carrying genes which correspond to the E. coli chromosomal region, metG gnd his shiA, but excluding purF and aroD, was isolated from UNF43. Translocation of carbenicillin resistance genes from a P-type R-factor, R68, to FN39 increased the stability of his and nif on the derivative F-prime, FN68. Sedimentation analysis of both F-primes in sucrose gradients revealed our covalently closed circular(CCC) DNA species of molecular weights 279 +/- 9, 136 +/- 3, 90 +/- 1 and 44 +/- 1 megadaltons. It is suggested that the two smallest CCC-DNA species are component replicons of the composite F-primes of molecular weight 136 +/- 3 megadaltons, and that the molecules of 279 +/- 9 megadaltons are CCC-dimers. FN68 was transferable in intergeneric matings to Klebsiella aerogenes, K. pneumoniae and Salmonella typhimurium but not to Proteus mirabilis; only carbenicillin resistance and sex factor activity were transferred to Erwinia herbicola. nif genes on FN68 were expressed in a Nif- mutant of K. pneumoniae and also in S. typhimurium, which in conventional tests is naturally non-nitrogen-fixing; expression of the his determinant of FN68 became temperature-sensitive in S. typhimurium.

Carbenicillin↗
PubMed1974

Nitrogenase.

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Adenosine Triphosphate↗
PubMed1973

A four-iron ferredoxin from Desulfovibrio desulfuricans.

Ferredoxin from Desulfovibrio desulfuricans was isolated, purified and crystallized. It contains four iron atoms and four sulphido or ;acid-labile' sulphur atoms for a molecule of 6000 daltons. The absorption spectrum in the u.v.-visible region and the electron-paramagnetic-resonance signals of the reduced protein are similar to those observed for other four-iron ferredoxins. The amino acid composition is different from that of Desulfovibrio gigas ferredoxin. The redox potential of -0.33V at pH7.0 was determined by dye techniques.

Amino Acids↗