Exopolysaccharide Carbohydrate Structure and Biofilm Formation by Rhizobium leguminosarum bv. trifolii Strains Inhabiting Nodules of Trifoliumrepens Growing on an Old Zn-Pb-Cd-Polluted Waste Heap Area.

Ewa Oleńska,Sofie Thijs,Jaco Vangronsveld,Izabela Swiecicka,Urszula Kotowska,Jerzy Wydrych,Weronika Polińska,Wanda Małek

doi:10.3390/ijms22062808

Abstract

Heavy metals polluting the 100-year-old waste heap in Bolesław (Poland) are acting as a natural selection factor and may contribute to adaptations of organisms living in this area, including Trifolium repens and its root nodule microsymbionts—rhizobia. Exopolysaccharides (EPS), exuded extracellularly and associated with bacterial cell walls, possess variable structures depending on environmental conditions; they can bind metals and are involved in biofilm formation. In order to examine the effects of long-term exposure to metal pollution on EPS structure and biofilm formation of rhizobia, Rhizobium leguminosarum bv. trifolii strains originating from the waste heap area and a non-polluted reference site were investigated for the characteristics of the sugar fraction of their EPS using gas chromatography mass-spectrometry and also for biofilm formation and structural characteristics using confocal laser scanning microscopy under control conditions as well as when exposed to toxic concentrations of zinc, lead, and cadmium. Significant differences in EPS structure, biofilm thickness, and ratio of living/dead bacteria in the biofilm were found between strains originating from the waste heap and from the reference site, both without exposure to metals and under metal exposure. Received results indicate that studied rhizobia can be assumed as potentially useful in remediation processes.

Highlights

Exopolysaccharides (EPS) represent a group of polysaccharides synthesized and secreted to the external environment or synthesized extracellularly by cell wall-associated enzymes of many Gram-positive and Gram-negative bacteria [1,2,3,4]
They were produced by R. leguminosarum bv. trifolii strains originating from root nodules of T. repens from the polluted Bolesław waste heap and the non-polluted reference area in Bolestraszyce
Our results indicate significant differences in EPS carbohydrate profiles and strongly confirm the presence of morphological adaptations in EPS layers resulting in biofilm characteristics, which have a role in coping with the toxic metals concentrations present in the waste heap area of Bolesław

Summary

Introduction

Exopolysaccharides (EPS) represent a group of polysaccharides synthesized and secreted to the external environment or synthesized extracellularly by cell wall-associated enzymes of many Gram-positive and Gram-negative bacteria [1,2,3,4]. Many bacteria usually do not occur as single-celled planktonic organisms but exist in highly organized multi-cellular associations This is mainly due to the unique properties of the EPS, involving improvement of inter-cellular as well as cell-surface adhesion [11,12,13], where they communicate via quorum sensing (QS) pathways [14]. Such a multi-organismal composition offers to its inhabitants a significantly higher resistance to multiple and changing environmental factors than individual planktonic forms represent [15,16]. It was revealed that bacteria in a biofilm form tolerate a wide range of challenging fluctuations in temperature, humidity, salinity, and pH, as well as the presence of bacteriocins, antibiotics, or toxic metal ions in the environment [17,18]

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Conclusion