RESUMO
The nifL gene product of Klebsiella pneumoniae inhibits the activity of the positive activator protein NifA in response to increased levels either of fixed nitrogen or of oxygen in the medium. In order to demonstrate that the responses to these two effectors are discrete we have subjected nifL to hydroxylamine mutagenesis and isolated nifL mutants that are impaired in their ability to respond to oxygen but not to fixed nitrogen. Two such mutations were sequenced and shown to be single base pair changes located in different parts of nifL. The amino acid sequence of NifL shows limited homology to the histidine protein kinases which comprise the sensing component of bacterial two-component regulatory systems. In the light of the location of one of the oxygen-insensitive mutations (Leu294Phe) we have reassessed this homology and we suggest that the Gln273-Leu317 region of NifL may facilitate interactions between NifL and NifA.
Assuntos
Genes Bacterianos , Klebsiella pneumoniae/genética , Fixação de Nitrogênio/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Análise Mutacional de DNA , DNA Bacteriano/genética , Histidina Quinase , Klebsiella pneumoniae/metabolismo , Dados de Sequência Molecular , Mutação , Oxigênio/metabolismo , Fenótipo , Proteínas Quinases/genética , Homologia de Sequência de AminoácidosRESUMO
Cassette mutagenesis has been used to study the role of a helix-turn-helix (HTH) motif in the novel RNA polymerase sigma factor sigma 54 of Klebsiella pneumoniae. Of the four residues which are predicted to be solvent-exposed in the second helix, the first (Glu-378) tolerated all substitutions, and some mutations of this residue increased expression from sigma 54-dependent promoters. Certain substitutions in the third exposed residue (Ser-382) produced a promoter-specific phenotype and all substitutions in the fourth residue (Arg-383) inactivated the protein, identifying this residue as being likely to be involved in base-specific interactions with the promoter. In vivo footprinting indicated that the inactive HTH mutants of sigma 54 were defective in interaction with both the -24 and -12 regions of the glnAp2 promoter.
Assuntos
Klebsiella pneumoniae/genética , Regiões Promotoras Genéticas/fisiologia , Fator sigma/química , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Sítios de Ligação/fisiologia , Dados de Sequência Molecular , Mutagênese Insercional , Conformação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Fator sigma/genética , Fator sigma/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Two open reading frames (ORFs), designated ORF95 and ORF162, downstream of the Klebsiella pneumoniae sigma 54 structural gene (rpoN) have been sequenced and shown to encode polypeptides of 12 kD and 16 kD, respectively. ORFs homologous to ORF95 are present downstream of four out of five rpoN genes sequenced to date from a range of Gram-negative bacteria, and ORF162 is also conserved, at least in Pseudomonas putida. Chromosomal mutations have been created in each gene using a kan cassette and both have the same phenotype, i.e. they cause an increase in the level of expression from sigma 54-dependent promoters. We propose that the products of both genes function to modulate the activity of E sigma 54, although a physiological role for these proteins has not yet been identified.
