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The Rhizobium leguminosarum bv. trifolii RosR: Transcriptional Regulator Involved in Exopolysaccharide Production

Department of General Microbiology, University of M. Curie-Skłodowska, Akademicka 19, 20-033 Lublin, Poland.
Molecular Plant-Microbe Interactions (Impact Factor: 4.46). 08/2007; 20(7):867-81. DOI: 10.1094/MPMI-20-7-0867
Source: PubMed

ABSTRACT The acidic exopolysaccharide is required for the establishment of symbiosis between the nitrogen-fixing bacterium Rhizobium leguminosarum bv. trifolii and clover. Here, we describe RosR protein from R. leguminosarum bv. trifolii 24.2, a homolog of transcriptional regulators belonging to the family of Ros/MucR proteins. R. leguminosarum bv. trifolii RosR possesses a characteristic Cys2His2 type zinc-finger motif in its C-terminal domain. Recombinant (His)6RosR binds to an RosR-box sequence located up-stream of rosR. Deletion analysis of the rosR upstream region resulted in identification of two -35 to -10 promoter sequences, two conserved inverted palindromic pentamers that resemble the cAMP-CRP binding site of Escherichia coli, inverted repeats identified as a RosR binding site, and other regulatory sequence motifs. When assayed in E. coli, a transcriptional fusion of the cAMP-CRP binding site containing the rosR upstream region and lacZ gene was moderately responsive to glucose. The sensitivity of the rosR promoter to glucose was not observed in E. coli deltacyaA. A rosR frame-shift mutant of R. leguminosarum bv. trifolii formed dry, wrinkled colonies and induced nodules on clover, but did not fix nitrogen. In the rosR mutant, transcription of pssA-lacZ fusion was decreased, indicating positive regulation of the pssA gene by RosR. Multiple copies of rosR in R. leguminosarum bv. trifolii 24.2 increased exopolysaccharide production.

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    • "Besides the RosR-box, several regulatory sites have been identified in the rosR upstream region, including two P1 and P2 promoters and three motifs resembling the E. coli cAMP-CRP binding site, indicating a complex regulation of rosR expression [23,29]. RosR binding to the RosR-box negatively regulates transcription of its own gene [23]. In the presence of glucose, the transcriptional activity of the rosR is significantly reduced, showing that the expression of this gene is regulated by catabolic repression. "
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    ABSTRACT: Rhizobium leguminosarum bv. trifolii is a symbiotic nitrogen-fixing bacterium that elicits nodules on roots of host plants Trifolium spp. Bacterial surface polysaccharides are crucial for establishment of a successful symbiosis with legumes that form indeterminate-type nodules, such as Trifolium, Pisum, Vicia, and Medicago spp. and aid the bacterium in withstanding osmotic and other environmental stresses. Recently, the R. leguminosarum bv. trifolii RosR regulatory protein which controls exopolysaccharide production has been identified and characterized. In this work, we extend our earlier studies to the characterization of rosR mutants which exhibit pleiotropic phenotypes. The mutants produce three times less exopolysaccharide than the wild type, and the low-molecular-weight fraction in that polymer is greatly reduced. Mutation in rosR also results in quantitative alterations in the polysaccharide constituent of lipopolysaccharide. The rosR mutants are more sensitive to surface-active detergents, antibiotics of the beta-lactam group and some osmolytes, indicating changes in the bacterial membranes. In addition, the rosR mutants exhibit significant decrease in motility and form a biofilm on plastic surfaces, which differs significantly in depth, architecture, and bacterial viability from that of the wild type. The most striking effect of rosR mutation is the considerably decreased attachment and colonization of root hairs, indicating that the mutation affects the first stage of the invasion process. Infection threads initiate at a drastically reduced rate and frequently abort before they reach the base of root hairs. Although these mutants form nodules on clover, they are unable to fix nitrogen and are outcompeted by the wild type in mixed inoculations, demonstrating that functional rosR is important for competitive nodulation. This report demonstrates the significant role RosR regulatory protein plays in bacterial stress adaptation and in the symbiotic relationship between clover and R. leguminosarum bv. trifolii 24.2.
    BMC Microbiology 11/2010; 10(1):284. DOI:10.1186/1471-2180-10-284 · 2.98 Impact Factor
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    • "trifolii and viciae is rosR, encoding a regulatory protein which positively regulates EPS production. rosR mutant produces three times less EPS than wild type strain and induces nodules incapable of nitrogen-fixation (Janczarek and Skorupska 2007). rosR mutant of R. etli formed colonies with altered morphology but retained the ability to induce nitrogen-fixing nodules on common bean (Phaseolus vulgaris), which forms a determinate type of nodules (Bittinger et al. 1997). "
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    ABSTRACT: Rhizobium leguminosarum bv. trifolii exopolysaccharide (EPS) plays an important role in determining symbiotic competence. The pssA gene encoding the first glucosyl-IP-transferase and rosR encoding a positive transcriptional regulator are key genes involved in the biosynthesis and regulation of EPS production. Mutation in pssA resulted in deficiency in EPS production and rosR mutation substantially decreased the amount of EPS. Both mutants induced nodules but the bacteria were unable to fix nitrogen. Defective functions of pssA and rosR mutants were fully restored by wild type copies of the respective genes. Introduction of multiple rosR and pssA gene copies on the plasmid vector pBBR1MCS-2 into five R. leguminosarum bv. trifolii nodule isolates resulted in significantly increased growth rates, EPS production and the number of nodules on clover roots. Increase in fresh and dry shoot mass of clovers and nodule occupation was also statistically significant. Interestingly, additional copies of pssA but particularly rosR gene, increased strains' competitiveness in relation to the wild type parental strains nearly twofold. Overall, experimental evidence is provided that increased amount of EPS beneficially affects R. leguminosarum bv. trifolii competitiveness and symbiosis with clover.
    Antonie van Leeuwenhoek 08/2009; 96(4):471-86. DOI:10.1007/s10482-009-9362-3 · 2.14 Impact Factor
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    • "These data confirmed that the sequence located immediately upstream of the À 35 hexamer plays an important role in rosR transcription from P1 and is successfully recognized by RNAP in both genetic backgrounds. Apart from the rosR regulatory elements described above, two cAMP–CRP binding sites have been identified previously (Janczarek & Skorupska, 2007). cAMP–CRP binding site I located upstream of the P1 promoter 95 bp from TS1 contained the first perfect and the second imperfect pentamers (AAGC-TGTGA-N 7 -TTGCT-GGA), as compared with E. coli consensus AA-TGTGA-N 6 -TCACA-T (Fig. 1). "
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    ABSTRACT: Rhizobium leguminosarum bv. trifolii rosR gene encodes a transcriptional regulator involved in the positive regulation of exopolysaccharide synthesis. Transcription of rosR is directed by two promoters, distal P1 and proximal P2, of different strengths. We demonstrated that rosR P1 functions as the main promoter and, besides the -35 and -10 sequences, it contains two other important regulatory elements, an extended -10 motif and an upstream promoter element, that play a significant role in the initiation of transcription. Two cAMP-CRP binding sites (I and II) have been identified upstream of P1, both necessary for optimal rosR expression. cAMP-CRP binding site III is located within the P2 promoter and also influences rosR transcription. rosR transcription levels are dependent both on the presence of the cAMP-CRP complex and on the carbon source, indicating regulation of transcription and exopolysaccharide production by catabolite repression.
    FEMS Microbiology Letters 02/2009; 291(1):112-9. DOI:10.1111/j.1574-6968.2008.01443.x · 2.72 Impact Factor
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