RESUMEN
Rhizobium tropici strain CIAT899 induces nitrogen-fixing nodules on the roots of a wide range of tropical legumes, including Phaseolus vulgaris and Leucaena leucocephala. Previously, a DNA region of the CIAT899 pSym plasmid containing the common nodulation genes nodABC and one of the nodD alleles was characterized (P. van Rhijn, B. Feys, C. Verreth, and J. Vanderleyden, J. Bacteriol. 175: 438-447, 1993). As reported here, the region immediately downstream of nodC contains the nodSU genes. The nucleotide sequence of these genes is presented. CIAT899 nodS and nodU mutants were constructed. The nodS mutant was completely deficient in nodulation on the host plants P. vulgaris and L. leucocephala. The nodU mutation caused a decrease in nodulation on Leucaena but resorted no effect on Phaseolus. Introduction of the CIAT899 nodABCSU region in R. etli CE-3, a strain that only nodulates P. vulgaris, caused an extension of the host range of strain CE-3 to L. leucocephala.
Asunto(s)
Transferasas de Carboxilo y Carbamoilo , Fabaceae/microbiología , Genes Bacterianos , Plantas Medicinales , Rhizobium/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Secuencia de Bases , Mapeo Cromosómico , ADN Bacteriano/genética , Prueba de Complementación Genética , Lipopolisacáridos/metabolismo , Metiltransferasas/genética , Metiltransferasas/fisiología , Datos de Secuencia Molecular , Mutación , N-Acetilglucosaminiltransferasas/genética , Rhizobium/fisiología , Homología de Secuencia de AminoácidoRESUMEN
Transcription of the Rhizobium meliloti fixK gene is induced in symbiotic and microaerobic growth conditions by the FixL/FixJ modulator/effector pair. Transcription of fixK is also negatively autoregulated. By 5' deletion analysis, the involvement in negative regulation of a DNA region between -514 and -450 with respect to the transcription start was demonstrated. Site-directed mutagenesis allowed us to show that a sequence homologous to the binding site of the Escherichia coli Fnr protein, centred at position -487, participates in this effect. However, deletion or mutagenesis of this Fnr-like sequence does not completely eliminate FixK-dependent repression, which suggests that either an additional DNA region is involved in negative regulation or that it is mediated at the level of fixLJ transcription. Deletion analysis also allowed the definition of a DNA region involved in FixJ-mediated activation of the fixK promoter, between -79 and -42. Different point mutations in the -60, -45 and -35 regions were shown to affect promoter activity. In some cases, the activity of mutant promoters could be partly or fully restored by increasing the expression of the fixLJ regulatory genes, in an E. coli strain harbouring a plasmid with fixLJ under the control of an inducible (p-tac) promoter.
Asunto(s)
Regulación Bacteriana de la Expresión Génica/fisiología , Genes Bacterianos , Regiones Promotoras Genéticas/genética , Sinorhizobium meliloti/genética , Transcripción Genética/genética , Proteínas Bacterianas/genética , Secuencia de Bases , Regulación Bacteriana de la Expresión Génica/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fijación del Nitrógeno/genética , Regiones Promotoras Genéticas/fisiología , Transcripción Genética/fisiologíaRESUMEN
The fixLJ genes of Rhizobium leguminosarum biovar phaseoli CNPAF512 were identified by DNA hybridization of a genomic library with an internal fragment of the Rhizobium meliloti fixJ gene. The nucleotide sequence was determined and the corresponding amino acid sequence was aligned with the amino acid sequences of the FixL proteins of R. meliloti, Bradyrhizobium japonicum and Azorhizobium caulinodans. While the FixJ protein and the carboxy-terminal part of the FixL protein are highly homologous to the other FixL and FixJ proteins, the homology in the central heme-binding, oxygen-sensing domain and in the amino-terminal domain of FixL is very low. The R. leguminosarum bv. phaseoli FixL protein does not contain the heme-binding motif defined for the previously described FixL proteins. R. leguminosarum bv. phaseoli fixLJ and fixJ mutants were constructed. These mutants can still fix nitrogen, albeit at a reduced level. Expression analysis of nifA-gusA and nifH-gusA fusions in the constructed mutants revealed that the R. leguminosarum bv. phaseoli fixLJ genes are involved in microaerobic nifH expression but not in nifA expression.