Christophe J. P. Boonaert

Surface of Lactic Acid Bacteria: Relationships between Chemical Composition and Physicochemical Properties

ABSTRACT The surface chemical composition and physicochemical properties (hydrophobicity and zeta potential) of two lactic acid bacteria,Lactococcus lactis subsp. lactis bv. diacetilactis and Lactobacillus helveticus, have been investigated using cells harvested in exponential or stationary growth phase. The surface composition determined by X-ray photoelectron spectroscopy (XPS) was converted into a molecular composition in terms of proteins, polysaccharides, and hydrocarbonlike compounds. The concentration of the last was always below 15% (wt/wt), which is related to the hydrophilic character revealed by water contact angles of less than 30°. The surfaces of L. lactis cells had a polysaccharide concentration about twice that of proteins. The S-layer of L. helveticus was either interrupted or crossed by polysaccharide-rich compounds; the concentration of the latter was higher in the stationary growth phase than in the exponential growth phase. Further progress was made in the interpretation of XPS data in terms of chemical functions by showing that the oxygen component at 531.2 eV contains a contribution of phosphate in addition to the main contribution of the peptide link. The isoelectric points were around 2 and 3, and the electrophoretic mobilities above pH 5 (ionic strength, 1 mM) were about −3.0 × 10−8 and −0.6 × 10−8 m2 s−1 V−1 forL. lactis and L. helveticus, respectively. The electrokinetic properties of the latter reveal the influence of carboxyl groups, while the difference between the two strains is related to a difference between N/P surface concentration ratios, reflecting the relative exposure of proteins and phosphate groups at the surface.

Direct probing by atomic force microscopy of the cell surface softness of a fibrillated and nonfibrillated oral streptococcal strain

In this paper, direct measurement by atomic force microscopy (AFM) of the cell surface softness of a fibrillated oral streptococcal strain Streptococcus salivarius HB and of a nonfibrillated strain S. salivarius HBC12 is presented, and the data interpretation is validated by comparison with results from independent techniques. Upon approach of the fibrillated strain in water, the AFM tip experienced a long-range repulsion force, starting at approximately 100 nm, attributed to the compression of the soft layer of fibrils present at the cell surface. In 0.1 M KCl, repulsion was only experienced when the tip was closer than approximately 10 nm, reflecting a stiffer cell surface due to collapse of the fibrillar mass. Force-distance curves indicated that the nonfibrillated strain, probed both in water and in 0.1 M KCl, was much stiffer than the fibrillated strain in water, and a repulsion force was experienced by the tip at close approach only (20 nm in water and 10 nm in 0.1 M KCl). Differences in cell surface softness were further supported by differences in cell surface morphology, the fibrillated strain imaged in water being the only specimen that showed characteristic topographical features attributable to fibrils. These results are in excellent agreement with previous indirect measurements of cell surface softness by dynamic light scattering and particulate microelectrophoresis and demonstrate the potential of AFM to directly probe the softness of microbial cell surfaces.

Adhesion of Lactococcus lactis to model substrata: direct study of the interface

The adhesion of Lactococcus lactis harvested in the exponential and stationary growth phases was examined on glass and on polystyrene with cells resuspended in water. A low adhesion density was observed with exponential cells whatever the substratum. Stationary cells adhered in large amount on polystyrene and not on glass. This adhesion behavior cannot be explained simply in terms of physicochemical properties of the surfaces. The attention is drawn on particular aspects which are not taken into consideration in classical approaches of cell adhesion. Atomic force microscopy showed that, after a forced contact, the bacterial surface sticks to the retracting AFM probe, indicating that the force applied when the cell comes in contact with the substratum may influence its adhesion. This may affect the relevance of adhesion tests in laminar conditions with respect to real situations. The X-ray photoelectron spectroscopy analysis of the substratum surface after detachment of adhering cells, on the one hand, and of evaporated cell supernatants, on the other hand, revealed that extracellular substances were released by L. lactis in the aqueous phase and at cell-substratum interface. They may have a critical effect on cell adhesion by increasing the ionic strength of the solution confined between the cells and the substrata and by bringing macromolecules at the interface

Adhesion of Azospirillum brasilense: role of proteins at the cell-support interface

The adhesion of the soil bacterium Azospirillum brasilense to polystyrene has been investigated using a parallel-plate chamber in conditions allowing cell transport to the support by sedimentation and rinsing under controlled hydrodynamic conditions. The adhesion pattern, which was heterogeneous, and the density of adhering cells were determined by cell aggregation at the support surface and detachment of aggregates upon rinsing. Determination of the support surface composition by X-ray photoelectron spectroscopy after detachment of adhering cells and analysis of the supernatant of cell suspensions revealed that, during the course of adhesion, extracellular proteins are released progressively into the aqueous phase and adsorb at the support surface. Support preconditioning by contact with a cell suspension promoted adhesion after a short contact time (2 h) due to protein adsorption at the support surface. Cell ageing prior to the adhesion test also enhanced adhesion after 2 h due to protein accumulation at the cell surface. Moreover, when supports were preconditioned and when cells were aged prior to the test, adhesion was still dependent on cell-support contact time, which pointed to the influence of in situ secretion of proteins by the adhering cells. It was therefore concluded that the role played by proteins at the cell-support interface is twofold. In the first stage, proteins accumulate at the cell surface, are liberated into the solution and adsorb at the support surface; the increase of the protein concentration at the interface promotes initial adhesion. In the next stage, in situ secretion of proteins during the prolonged contact between the cells and the support strengthens adhesion.

Surface properties of microbial cells probed at the nanometre scale with atomic force microscopy

Atomic force microscopy (AFM) was used to characterize, under water, the surface morphology and molecular interactions of two types of microbial cells: fungal spores (Phanerochaete chrysosporium) and bacteria (Lactococcus lactis), High-resolution deflection images showed that the spore surface was uniformly covered with patterns of rodlets that were several hundred nanometres in length and had a periodicity of similar to 10 nm, Such surface organization was not detected on the bacterial surface, which showed a sponge-like structure. Force-distance curves revealed very different molecular interactions for the two microorganisms: upon approach, no significant curvature was seen in the contact region for spores, contrary to bacteria, pointing to a difference in cell softness; and upon retraction, no adhesion forces were detected on spores but multiple unbinding events and elongation forces attributed to macromolecular bridging were observed on bacteria. These results show that AFM is a powerful tool for probing the surface properties of native microbial cells on the nanometre scale. Copyright (C) 2000 John Wiley & Sons, Ltd.

Surface analysis by X-ray photoelectron spectroscopy in study of bioadhesion and biofilms

Publisher Summary This chapter presents the various applications offered by X-ray photoelectron spectroscopy (XPS) in bioadhesion and biofilm studies. The chapter includes a presentation of the XPS technique (basic principles, spectroscopic aspects, and data interpretation) and a description of a case study concerning the adhesion of the bacterium Azospirillum brasilense to model substrata. The chapter also provides an overview of the various applications of XPS and relevant methodologies in the study of bioadhesion and biofilms, based on experience developed in the laboratory. XPS can be applied to analyze the chemical composition of substratum surfaces, microbial cell surfaces, and surface-active molecules in the liquid phase in terms of elemental, functional, and, to a certain extent, molecular composition. Correlations among XPS data, surface properties and interfacial behaviors provide a better understanding of bioadhesion and biofilm formation.

Deformation of Lactococcus lactis surface in atomic force microscopy study

The surface of the bacterium Lactococcus lactis was investigated under water by contact mode atomic force microscopy (AFM) while varying the imaging parameters (imaging force, scanning velocity, scanning direction). Height images were three-dimensionally (3-D) reconstructed using GOCAD(C) software; this revealed grooves oriented along the scanning direction. Although the grooves were created by the scanning probe, they were not due to a single passage of the probe; the periodicity of the grooves was indeed always three to four times larger than the scanning line periodicity (ratio between the image size and the number of scanning lines). Upon repeated imaging at low force, the grooves were re-created each time with the same morphology; groove depth was increased at higher imaging force. The groove formation is tentatively explained by a deformation of the surface, due to compression or to adhesion, and its slow relaxation. While the grooves reveal a perturbation of the cell surface by the AFM probe, they provide pertinent information about the nanomechanical properties of the cell surface

Role of Proteins in the Adhesion of Azospirillum Brasilense to Model Substrata

In the natural environment, microbial cells adhere to a large variety of solid surfaces, from inanimate materials to living tissues (Savage and Fletcher, 1985; Characklis and Marshall, 1990; Marshall, 1991). In aquatic habitats (e.g. streams, lakes, oceans), microorganisms accumulate on living organisms, suspended particles, rocks and sediments. In many instances, these surfaces are rapidly colonized by bacteria and biofilms are formed as a result of cell multiplication and production of extracellular substances. Soil is undoubtedly the most complex microbial habitat (Stotzky, 1985) because of the high variability of the composition and size of solid constituents, the amount of water, nutrients and gases, and the physicochemical characteristics (e.g. pH, Eh, ionic strength). Soil particles that are colonized by bacteria are often coated by clay minerals, hydrous metal oxides, and organic matter. Clay minerals, and surfaces in general, may affect microbial activity in different ways, e.g., by modifying the physicochemical characteristics of the microbial habitat (Filip and Hattori, 1984; Rouxhet and Mozes, 1990; Mozes and Rouxhet, 1991, 1992). In the environment of plant roots (rhizosphere), bacteria benefit from root exudates as carbon and energy sources and, in turn, may promote plant growth by nitrogen fixation and production of various substances (Pueppke and Kluepfel, 1985).