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  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Archives of microbiology 142 (1985), S. 289-294 
    ISSN: 1432-072X
    Keywords: Proteus mirabilis ; Nitrogen fixation ; nif genes ; nif plasmids ; Klebsiella pneumoniae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Self-transmissible plasmids carryinghis andnif genes fromKlebsiella pneumoniae have been introduced into threehis mutants ofProteus mirabilis: strains 5006-1, WR19 and WR20. Expression ofhis 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 allownif expression and no nitrogenase MoFe-protein peptide was detected immunologically after exposure to conditions in which diazotrophic enterobacteria, normal or genetically constructed, derepressnif. One strain,P. mirabilis WR19, carrying thehis nif Kmr plasmid pMF250 was examined in detail. Thenif activator genenifA 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 ofnif 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 ofK. 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 (anif-related phenotype of this plasmid) in aerobic glucose minimal medium. Expression ofnif inP. mirabilis WR19 (pCK1, pMF250) was NH 4 + -repressible despite the constitutivenifA character of pCK1 and introduction of thentrA + plasmid pMM17 did not alter this phenotype. However, pCK1 did not give rise to NH 4 + -constitutive diazotrophy in the wild-typeK. pneumoniae M5al. A construct of WR19 carrying pMF250 and constitutiventrC plasmid (pMD45) remained Nif- even after pre-growth on glucose-minimal media. We conclude (a) thatP. mirabilis forms a gene product functionally equivalent to that ofntrA inK. pneumoniae, (b) that it forms no functional equivalent of thentrC product in our growth conditions. The need for pre-conditioning on aerobic glucose media remains perplexing.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 4 (1990), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Using directed mutagenesis, amino acid substitutions have been made in the α- and β-subunits of the Klebsiella pneumoniae nitrogenase component 1 at positions normally occupied by conserves cysteine or tyrosine residues. Nif', Nif and intermediate pheno-types have been obtained. To extend our earlier biochemical characterization (Kent et al, 1989) the electrophoretic mobiliy of component 1 of the mutant and wild-type nitrogenases has been analysed by non-denaturing gel electrophoresis. The major and minor forms of component 1 separated by this methodology have been probed for by using both polyclonal and monoclonal antibodies. All Nif mutants exhibited a distribution of electrophoretic forms of component 1 comparable to the wild type, and the abundance of the major from found in purified nitrogennase correlated approximately with the specific activity of the extract. In contrast, after electrophoresis, component 1 from Nif mutants exhibited either a major low-mobility from or a fast-moving from. Analysis of co-factor (FeMoco) allowed us to conclude that changing cysteine 275 to alanine in the α-submit produces component 1 defective in its interaction with FeMoco. Substitution of other con-served cysteine residues by alanine appears to prevent early steps in nitrogebnase assembly or to promote degradation. Two single mutations (cysteine89 to alanine in the β-subunit) which are tightly Nif can be combined to produce a weakly active nitrogenase, indicating regions involved in the interaction between subunits.
    Type of Medium: Electronic Resource
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