Article

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: 3.94). 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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Available from: Monika Janczarek
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    • "These data indicated that the RosR protein was engaged in regulation of transcription of the pssO, pssN, pssP, and pssF genes. In previous studies, it was confirmed that RosR recognized a sequence motif, called the RosR-box, located in the upstream regions of the rosR and pssA genes and affected their transcription (Janczarek and Skorupska 2007;Janczarek and Urbanik-Sypniewska 2013). The RosR-box is a 22-bp-long sequence containing 9-bp inverted repeats separated by a 4-bp spacer (Fig. 2). "
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    ABSTRACT: Background and aims In the symbiotic bacterium Rhizobium leguminosarum bv. trifolii, a majority of proteins involved in exopolysaccharide (EPS) synthesis are encoded by genes located in a large polysaccharide synthesis cluster (Pss). The aim of this study was genetic characterization of the Pss region in the Rt24.2 strain in the context of EPS production and symbiosis with red clover (Trifolium pratense). Methods The expression of genes located in the Pss cluster was determined using constructed pss-lacZ transcriptional fusions. The role of transcriptional regulator RosR in pss transcription was confirmed using a rosR mutant and the Rt24.2(pBR1) strain carrying multiple rosR copies. An EPS-deficient mutant, Rt770 was obtained using a random mutagenesis and mTn5SSgusA40 transposon. Symbiotic properties of the Rt770 strain in interaction with clover were characterized in inoculation experiments. Infection of host roots and nodule occupancy by this mutant were investigated using both light and electron microscopy. Results Transcriptional levels of particular pss genes differed significantly; the genes encoding glycosyltransferases and enzymes modifying EPS have promoters of weak activities, whereas those encoding proteins involved in EPS polymerization and export possess stronger promoters. Furthermore, RosR affected expression of some pss genes. A mutation in Rt24.2 pssS encoding glucosyltransferase totally abolished EPS synthesis, decreased motility, and increased sensitivity to some stressors. The pssS mutant Rt770 induced formation of nodules on clover roots, which were ineffective in nitrogen fixation. Conclusion EPS secreted by Rt24.2 is required for both adaptation to soil conditions and the establishment of effective symbiosis with clover plants.
    Preview · Article · Jul 2015 · Plant and Soil
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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.
    Full-text · Article · Nov 2010 · BMC Microbiology
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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.
    Full-text · Article · Aug 2009 · Antonie van Leeuwenhoek
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