RESUMEN
The acidic exopolysaccharide (EPS) secreted in large amounts by the symbiotic nitrogen-fixing bacterium Rhizobium leguminosarum bv. trifolii is required for the establishment of an effective symbiosis with the host plant Trifolium spp. EPS biosynthesis in rhizobia is a very complex process regulated at both transcriptional and post-transcriptional levels and influenced by various nutritional and environmental conditions. The R. leguminosarum bv. trifolii rosR gene encodes a transcriptional regulator with a C(2)H(2) type zinc-finger motif involved in positive regulation of EPS synthesis. In silico sequence analysis of the 450-bp long rosR upstream region revealed the presence of several inverted repeats (IR1 to IR6) and motifs with significant identity to consensus sequences recognized by PhoB and LysR-type proteins associated with phosphate- and flavonoid-dependent gene regulation in R. leguminosarum. Using a set of sequentially truncated rosR-lacZ transcriptional fusions, the role of the individual motifs and the effect of phosphate and clover root exudates on rosR expression were established. In addition, the significance of IR4 inverted repeats in the repression, and P2-10 hexamer in the activation of rosR transcription, respectively, was found. The expression of rosR increased in the presence of phosphate (0.1-20 mM) and clover root exudates (10 µM). PHO boxes and the LysR motif located upstream of the rosR translation start site were engaged in the regulation of rosR transcription. The synthesis of EPS and biofilm formation decreased at high phosphate concentrations, but increased in the presence of clover root exudates, indicating a complex regulation of these processes.
Asunto(s)
Proteínas Bacterianas/metabolismo , Medicago/química , Fosfatos/farmacología , Extractos Vegetales/farmacología , Polisacáridos Bacterianos/metabolismo , Rhizobium leguminosarum/fisiología , Transcripción Genética/efectos de los fármacos , Regiones no Traducidas 5' , Proteínas Bacterianas/genética , Secuencia de Bases , Biopelículas/efectos de los fármacos , Exudados y Transudados/química , Exudados y Transudados/metabolismo , Flavonoides/química , Flavonoides/farmacología , Medicago/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Fosfatos/química , Extractos Vegetales/química , Raíces de Plantas/química , Raíces de Plantas/metabolismo , Regiones Promotoras Genéticas , ARN/química , ARN/metabolismo , Rhizobium leguminosarum/genética , Simbiosis/efectos de los fármacosRESUMEN
Exopolysaccharide production by Rhizobium leguminosarum bv. trifolii is required for successful establishment of nitrogen-fixing symbiosis with clover (Trifolium pratense L.). Using plasmid-borne transcriptional fusions of promoters of pss genes with promoterless lacZ the effect of root exudate, phosphate, and ammonia on expression of pssT, pssN, pssO, and pssP genes in wild-type strain RtTA1 background was determined. A stimulating effect of these environmental factors on pssO and pssP gene expression was observed. The putative pssO gene promoter was determined to be a strong promoter within which the divergent nod-box element was identified. The pssO promoter was slightly inducible in a flavonoid-dependent manner in wild-type R. leguminosarum bv. trifolii strains RtTA1 and ANU843 and very weakly active in a mutant of strain ANU843 that lacks the regulatory nodD gene. The expression of pssO and pssP genes in planta was investigated using plasmid-borne pssO-gusA and pssP-gusA fusions under different phosphate availability to clover. The level of pssO-gusA fusion expression was shown to be dependent on phosphate concentration in the plant growth medium.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Polisacáridos Bacterianos/biosíntesis , Rhizobium leguminosarum/genética , Fusión Artificial Génica , Secuencia de Bases , Flavonoides/metabolismo , Orden Génico , Genes Bacterianos , Genes Reporteros , Glucuronidasa/genética , Glucuronidasa/metabolismo , Medicago/microbiología , Datos de Secuencia Molecular , Fosfatos/metabolismo , Extractos Vegetales/metabolismo , Regiones Promotoras Genéticas , Compuestos de Amonio Cuaternario/metabolismo , Simbiosis , Transcripción Genética , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
The specific interaction between rhizobia and their hosts requires many genes that influence both early and late steps in symbiosis. Three new genes, designated prsD, prsE (protein secretion) and orf3, were identified adjacent to the exo133 mutation in a cosmid carrying the genomic DNA of Rhizobium leguminosarum bv. trifolii TA1. The prsDE genes share significant homology to the genes encoding ABC transporter proteins PrtDE from Erwinia chrysanthemi and AprDE from Pseudomonas aeruginosa which export the proteases in these bacteria. PrsD shows at least five potential transmembrane hydrophobic regions and a large hydrophilic domain containing an ATP/GTP binding cassette. PrsE has only one potential transmembrane hydrophobic domain in the N-terminal part and is proposed to function as an accessory factor in the transport system. ORF3, like PrtF and AprF, has a typical N-terminal signal sequence but has no homology to these proteins. The insertion of a kanamycin resistance cassette into the prsD gene of the R. leguminosarum bv. trifolii TA1 wild-type strain created a mutant which produced a normal amount of exopolysaccharide but was not effective in the nodulation of clover plants.