Jolanta Cieśla

Determination of the electrokinetic potential of Rhizobium leguminosarum bv trifolii Rt24.2 using Laser Doppler Velocimetry — A methodological study

Electrokinetic potential (ζ, zeta potential) is one of the parameters which characterize the physicochemical properties of the bacterial cell envelope. The term is often used in the context of adhesiveness of bacteria and biofilm formation. This work presents the methodological aspects of zeta potential determination in strain Rt24.2 of Rhizobium leguminosarum using Laser Doppler Velocimetry combined with Phase Analysis Light Scattering and changed electric field techniques. The influence of media (0.9% NaCl, 0.2% NaCl, TY, GYM, 79CA, 20E and M1), temperature of measurement, number of measurement repetitions, phase of culture, concentration of bacteria, and storage at low temperature on the value of electrokinetic potential was investigated and a comparison was drawn between live and dead bacteria. All of those factors modified the zeta potential, showing that these parameters should be precisely specified in studies of bacterial electrokinetic potential, which is not always done. The obtained results also indicated that the zeta potential of Rhizobium leguminosarum should be determined directly in samples without storage at a defined bacterial density. The measurement should be done only once in a sample inserted into the cell of a measuring device to eliminate changes occurring in the sample (increase of electrolytic conductivity) under the electric field used.

Production of exopolysaccharide by Rhizobium leguminosarum bv. trifolii and its role in bacterial attachment and surface properties

Background and aimsThe acidic exopolysaccharide (EPS) produced by Rhizobium leguminosarum bv. trifolii is required for the establishment of effective symbiosis with compatible host plants (Trifolium spp.). In the rhizobium-legume interaction, early stages of root infection and nodule development have been well studied from a genetic standpoint. However, factors important for colonization of several surfaces by rhizobia, including soil particles and roots, have not yet been thoroughly investigated. The aim of this study was establishing of environmental factors affecting production of EPS by R. leguminosarum bv. trifolii strain 24.2 and the role of this polysaccharide in bacterial surface properties and attachment ability.MethodsBesides the wild-type strain, its derivatives differing in the level of EPS produced were used to these analyses. The ability of attachment to abiotic and biotic surfaces of these strains were established using CFU counting experiments. Three-dimensional structure and other parameters of biofilms formed were characterized in confocal laser scanning microscopy. Electrokinetic (zeta) potential of rhizobial cells were determined using Laser Doppler Velocimetry.ResultsIt was evidenced that the ability of R. leguminosarum bv. trifolii to produce EPS significantly affected bacterial attachment and biofilm formation on both abiotic and biotic surfaces. In addition, the presence of this polysaccharide influenced the zeta potential of rhizobial cells. Mutant strains having a mutation in genes involved in EPS synthesis were significantly impaired in attachment, whereas strains overproducing this polysaccharide showed higher adhesion efficiency to all of the tested materials. EPS facilitated attachment of bacterial cells to the tested surfaces most probably due to hydrophobic interactions and heterogeneity of the envelope surface.ConclusionsEPS produced by R. leguminosarum bv. trifolii plays a significant role in attachment and biofilm formation to both abiotic and biotic surfaces as well as bacterial surface properties.

Use of a Dynamic Light Scattering Technique for SDS/Water/Pentanol Studies

The interpretation of micelle/aggregate size obtained by use of the DLS technique for SDS/water/pentanol systems was discussed by comparison of the results of measurement with theoretical data. For most of the studied systems, the apparent radii (R h,app ) did not satisfactorily characterize the size of the aggregates (R h,app < 1 nm). The use of a correction factor (f = 0.26) confirmed that the discrepancies were associated with the electrostatic intermicellar interactions. However, the fuzzy optical interface between dispersed and dispersing phases can also be the reason of such results. An increase of pentanol content caused a decrease of the droplet radius in w/o systems but in o/w systems the changes were negligible.

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

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.

An Interaction of Rhamnolipids with Cu2+ Ions

This study was focused on the description of interaction between Cu2+ ions and the 1:1 mono- and dirhamnolipid mixtures in the premicellar and aggregated state in water and 20 mM KCl solution at pH 5.5 and 6.0. The critical micelle concentration of biosurfactants was determined conductometrically and by the pH measurements. Hydrodynamic diameter and electrophoretic mobility were determined in micellar solutions using dynamic light scattering and laser Doppler electrophoresis, respectively. The copper immobilization by rhamnolipids, methylglycinediacetic acid (MGDA), and ethylenediaminetetraacetic acid (EDTA) was estimated potentiometrically for the Cu2+ to chelating agent molar ratio from 16:100 to 200:100. The degree of ion binding and the complex stability constant were calculated at a 1:1 metal to chelant molar ratio. The aggregates of rhamnolipids (diameter of 43–89 nm) were negatively charged. Biosurfactants revealed the best chelating activities in premicellar solutions. For all chelants studied the degree of metal binding decreased with the increasing concentration of the systems. The presence of K+ lowered Cu2+ binding by rhamnolipids, but did not modify the complex stability significantly. Immobilization of Cu2+ by biosurfactants did not cause such an increase of acidification as that observed in MGDA and EDTA solutions. Rhamnolipids, even in the aggregated form, can be an alternative for the classic chelating agents.

Effectiveness of Parachlorella kessleri cell disruption evaluated with the use of laser light scattering methods

The main objective of this study is to demonstrate the possibilities of using laser light scattering methods, dynamic light scattering and laser Doppler electrophoresis, as suitable methods in investigations of algal production biosystems and biotechnology. This paper highlights the innovative use of the dynamic light scattering (DLS) methods for monitoring the destruction of Parachlorella kessleri cells. Additionally, these results indicate electrophoretic mobility as a new parameter to investigate the effectiveness of cell disruption prior to extraction conducted to optimise the biotechnological processes of recovery of microalgal intracellular metabolites. The efficacy of P. kessleri cell disintegration by ultrasound was determined by measurements of the number of cells with the algal cell reduction (CRns), relative mean hydrodynamic diameter (Rdt) and electrophoretic mobility after applying different lengths of ultrasound exposure to a cell suspension. It was found that stationary-phase cells were the most resistant to the ultrasound treatment, especially at low values of the optical density. Both the relative hydrodynamic diameter and the electrophoretic mobility of cells were correlated statistically significantly with the time of sonication (t) and the algal cell reduction. The relationships allowed estimation of the sonication time needed for total cell disruption.

Extracellular polysaccharide protects Rhizobium leguminosarum cells against zinc stress in vitro and during symbiosis with clover: EPS protects rhizobial cells against zinc stress

Rhizobium leguminosarum bv. trifolii is a soil bacterium that establishes symbiosis with clover (Trifolium spp.) under nitrogen-limited conditions. This microorganism produces exopolysaccharide (EPS), which plays an important role in symbiotic interactions with the host plant. The aim of the current study was to establish the role of EPS in the response of R. leguminosarum bv. trifolii cells, free-living and during symbiosis, to zinc stress. We show that EPS-deficient mutants were more sensitive to Zn2+ exposure than EPS-producing strains, and that EPS overexpression conferred some protection onto the strains beyond that observed in the wild type. Exposure of the bacteria to Zn2+ ions stimulated EPS and biofilm production, and increased cell hydrophobicity. However, zinc stress negatively affected the motility and attachment of bacteria to clover roots, as well as the symbiosis with the host plant. In the presence of Zn2+ ions, cell viability, root attachment, biofilm formation and symbiotic efficiency of EPS-overproducing strains were significantly higher than those of the EPS-deficient mutants. We conclude that EPS plays an important role in the adaptation of rhizobia to zinc stress, in both the free-living stage and during symbiosis.