Surface Properties of Wild-Type Rhizobium leguminosarum bv. trifolii Strain 24.2 and Its Derivatives with Different Extracellular Polysaccharide Content.

Jolanta Cieśla,Magdalena Kopycińska,Andrzej Bieganowski,Małgorzata Łukowska,Monika Janczarek,Francisco Martinez-Abarca

doi:10.1371/journal.pone.0165080

Abstract

Rhizobium leguminosarum bv. trifolii is a soil bacterium able to establish symbiosis with agriculturally important legumes, i.e., clover plants (Trifolium spp.). Cell surface properties of rhizobia play an essential role in their interaction with both biotic and abiotic surfaces. Physicochemical properties of bacterial cells are underpinned by the chemical composition of their envelope surrounding the cells, and depend on various environmental conditions. In this study, we performed a comprehensive characterization of cell surface properties of a wild-type R. leguminosarum bv. trifolii strain 24.2 and its derivatives producing various levels of exopolysaccharide (EPS), namely, pssA mutant Rt5819 deficient in EPS synthesis, rosR mutant Rt2472 producing diminished amounts of this polysaccharide, and two EPS-overproducing strains, Rt24.2(pBA1) and Rt24.2(pBR1), under different growth conditions (medium type, bacterial culture age, cell viability, and pH). We established that EPS plays an essential role in the electrophoretic mobility of rhizobial cells, and that higher amounts of EPS produced resulted in greater negative electrophoretic mobility and higher acidity (lower pKapp,av) of the bacterial cell surface. From the tested strains, the electrophoretic mobility was lowest in EPS-deficient pssA mutant. Moreover, EPS produced by rhizobial strains resulted not only in an increase of negative surface charge but also in increased hydrophobicity of bacterial cell surface. This was determined by measurements of water contact angle, surface free energy, and free energy of bacterial surface–water–bacterial surface interaction. Electrophoretic mobility of the studied strains was also affected by the structure of the bacterial population (i.e., live/dead cell ratio), medium composition (ionic strength and mono- and divalent cation concentrations), and pH.

Highlights

Rhizobium leguminosarum bv. trifolii is a soil gram-negative α-proteobacterium that establishes symbiosis with agriculturally important legumes, clovers (Trifolium spp.), under nitrogen-limited conditions
Growth kinetics of the wild-type R. leguminosarum bv. trifolii strain 24.2 and its derivatives were determined in two energy-rich media, TY and 79CA, over 96 h
Rt2472 and Rt5819 mutants grew more slowly than Rt24.2 and they did not achieve wild-type strain growth even after 96 h. These data indicated that the composition of 79CA medium favored the rhizobial growth more than that of TY, and rosR and pssA gene mutations negatively affected the growth of R. leguminosarum bv. trifolii

Summary

Introduction

Rhizobium leguminosarum bv. trifolii is a soil gram-negative α-proteobacterium that establishes symbiosis with agriculturally important legumes, clovers (Trifolium spp.), under nitrogen-limited conditions. Acidic exopolysaccharide (EPS) produced in large amounts by R. leguminosarum forms the outermost layer surrounding bacterial cells and plays an essential role in protecting the cells against desiccation and other stress factors [8]. This polysaccharide is crucial for the attachment and biofilm formation on both abiotic surfaces and plant roots, as well as the establishment of effective symbiosis, especially with legumes that form indeterminate-type nodules (e.g., clover, pea, vetch, alfalfa) [2,9,10,11]. EPS of S. fredii HH103 is not required for nodulation of Glycyrhiza uralensis that forms indeterminate-type nodules [16,17]

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