Genetic characterization of the Pss region and the role of PssS in exopolysaccharide production and symbiosis of Rhizobium leguminosarum bv. trifolii with clover

Monika Janczarek,Kamila Rachwał,Joanna Kopcińska

doi:10.1007/s11104-015-2567-5

Monika Janczarek, Kamila Rachwał + Show 1 more

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https://doi.org/10.1007/s11104-015-2567-5

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Abstract

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). 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. 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. EPS secreted by Rt24.2 is required for both adaptation to soil conditions and the establishment of effective symbiosis with clover plants.

Highlights

Extracellular polysaccharides (EPS) are produced by a wide range of microorganisms
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
Some data concerning genetic characteristics of a few genes involved in EPS synthesis in R. leguminosarum have been published previously, no comprehensive studies on the transcriptional activity of genes grouped in the EPS synthesis region have been performed so far

Summary

Introduction

Extracellular polysaccharides (EPS) are produced by a wide range of microorganisms. Various functions are ascribed to these polymers, among them protection against stress factors and plant antimicrobial compounds, nutrient gathering, attachment to both abiotic and biotic surfaces, and biofilm formation, which ensures adaptation of bacterial cells to changing environmental conditions (Fujishige et al 2006; Flemming and Wingender 2010; Jaszek et al 2014; Janczarek et al 2015a). The initiation of EPS biosynthesis is conducted by a glucosyl-IP-transferase, which transfers glucose-1-phosphate from UDP-glucose to a C55-isoprenylphosphate (IP) carrier anchored at the cytoplasmic site of the inner membrane (Pollock et al 1998) This enzyme is encoded by the pssA gene, which is located in the chromosome at a long distance from other EPS synthesis genes and transcribed as a monocistronic mRNA (Borthakur et al 1988; Ivashina et al 1994; van Workum et al 1997). Glucuronosyl-(β1-4)-glucosyl transferase PssDE and glucuronosyl-(β1-4)-glucuronosyl transferase PssC are involved in the second and the third step of the unit synthesis, respectively (Pollock et al 1998) These proteins are encoded by the pssC, pssD, and pssE genes located in a cluster named Pss-I (Król et al 2007).

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