RESUMEN
A novel technique was developed which may be generally well suited to the site-specific construction of mutations in Enterobacter agglomerans. The method is based on the observation that E. agglomerans can be cured of a plasmid of the incompatibility group IncQ by cultivation on citrate-containing medium. To test the applicability of this technique, we inserted a kanamycin cassette into the cloned nifB gene, transferred it into E. agglomerans, and selected for recombinants in which the wild-type nifB was replaced by the mutated gene by growing transformants on citrate medium with kanamycin. The nifB- mutants with the kanamycin cassette inserted in either orientation showed a nif- phenotype. Further, we determined the nucleotide sequence of nifB. A typical sigma 54-dependent promoter and a consensus NifA binding site were found upstream of nifB. Activation of this promoter by both heterologous and homologous NifA proteins was observed in vivo. The predicted amino acid sequence of the NifB protein showed strong similarity to the NifB sequences of other diazotrophic bacteria. The typical clustering of cysteine residues at the N-terminal end indicates its involvement in Fe-Mo cofactor biosynthesis.
Asunto(s)
Proteínas Bacterianas/genética , Enterobacter/genética , Genes Bacterianos , Fijación del Nitrógeno/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Secuencia de Bases , ADN Bacteriano , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Homología de Secuencia de AminoácidoRESUMEN
The nucleotide sequence of a plasmid-borne 3.9 kb XhoI-SmaI fragment comprising the 3'-region of the nifM gene, the nifL and nifA genes and the 5'-region of nifB gene of Enterobacter agglomerans was determined. The genes were identified by their homology to the corresponding nif genes of Klebsiella pneumoniae. A typical sigma 54-dependent promoter and a consensus NtrC-binding motif were identified upstream of nifL. The predicted amino acid sequence of NifL showed close similarities to NifL of K. pneumoniae and Azotobacter vinelandii. However, no histidine residue was found to correspond to histidine-304 of A. vinelandii NifL, which had been proposed to be required for the repressor activity of NifL. The NifA sequence with a putative DNA binding motif (Q(x3) A(x3) G(x5)I) and an ATP binding site in the C-terminal and central domains, respectively, resembles that of other known NifA proteins. The function of the nifL and nifA genes was demonstrated in vivo using a binary plasmid system by their ability to activate a nifH promoter-lacZ fusion at different temperatures and concentrations of NH4+. Maximal promoter activity occurred at 25 degrees C, and it appears that the sensitivity of NifA to elevated temperatures is independent of NifL. The expression of nifL inhibited promoter activity in the presence of NifA when the initial NH4+ concentration in the medium exceeded 4 mM.
Asunto(s)
Proteínas Bacterianas/genética , Enterobacter/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Fijación del Nitrógeno/genética , Operón/genética , Oxidorreductasas , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Enterobacter/efectos de los fármacos , Datos de Secuencia Molecular , Nitrogenasa/genética , Regiones Promotoras Genéticas , Unión Proteica , Compuestos de Amonio Cuaternario/farmacología , Proteínas Recombinantes de Fusión/biosíntesis , Mapeo Restrictivo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Temperatura , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
A cosmid library was generated to the 200-kb self-transmissible nif plasmid pEA9 isolated from Enterobacter agglomerans 339. The cosmid clone identified to contain the complete nif cluster was used to determine the nif gene organization and the physical map. The restriction pattern and nif gene organization of this nif cluster showed remarkable similarities to the nif cluster identified on the 110-kb plasmid pEA3 of Enterobacter agglomerans 333. Nucleotide sequence of several randomly selected regions of the nif cluster of pEA9 showed 96% similarity when compared to the known sequences of the nif cluster of pEA3. However, the homology ended abruptly at the flanking regions of the nif clusters and no similarity could be detected with the rest of the DNA of these plasmids. This reveals the existence of similar nif clusters on dissimilar plasmids, implying the horizontal transfer of the entire nif gene cluster.
Asunto(s)
Proteínas Bacterianas/genética , Enterobacter/genética , Plásmidos/genética , Proteínas de Unión al ARN , Factores de Transcripción/genética , Secuencia de Bases , Clonación Molecular , ADN Bacteriano/genética , Datos de Secuencia Molecular , Familia de Multigenes , Homología de Secuencia de Ácido NucleicoRESUMEN
With a length of 1221 bp and 44-bp inverted repeats with ten mismatches, IS1222 was identified as an endogenous insertion sequence in Enterobacter agglomerans 339. In this host strain, four copies were located, three on the nif plasmid pEA9 and one at the chromosome. Sequence analysis showed two consecutive open reading frames, orfA and orfB, encoding putative polypeptides of 87 and 276 amino acids. In-between both reading frames, a potential frameshift window of the homonucleotide type was postulated, followed by a pseudoknot structure and a ribosome-binding site. Based on significant homology at the sequence level and similarity of the features discussed, IS1222 was placed among the group of IS3 elements with IS407, IS476 and ISR1 being the most closely related IS. Hybridization experiments suggest that the distribution of IS1222 is limited to a group of related bacterial strains among Enterobacteriaceae.