A variety of strains of the genus Lactobacillus was investigated with respect to the structure, softness, and interactions of their outer surface layers in order to construct structure-property relations of the Gram-positive bacterial cell wall. The role of the conformational properties of the constituents of the outer cell-wall layers and their spatial distribution on the cell wall is emphasized. Atomic force microscopy was used to resolve the surface structure, interactions, and softness of the bacterial cell wall at nanometer-length scales and upwards. The pH-dependence of the electrophoretic mobility and a novel interfacial adhesion assay were used to analyze the average physicochemical properties of the bacterial strains. The bacterial surface is smooth when a compact layer of globular proteins constitutes the outer surface, e.g., the S-layer of L. crispatus DSM20584. In contrast, for two other S-layer containing strains (L. helveticus ATCC12046 and L. helveticus ATCC15009), the S-layer is covered by polymeric surface constituents which adopt a much more extended conformation and which confer a certain roughness to the surface. Consequently, the S-layer is important for the overall surface properties of L. crispatus, but not for the surface properties of L. helveticus. Both surface proteins (L. crispatus DSM20584) and (lipo)teichoic acids (L. johnsonii ATCC332) confer hydrophobic properties to the bacterial surface whereas polysaccharides (L. johnsonii DSM20533 and L. johnsonii ATCC 33200) render the bacterial surface hydrophilic. Using the interfacial adhesion assay, it was demonstrated that hydrophobic groups within the cell wall adsorb limited quantities of hydrophobic compounds. The present work demonstrates that the impressive variation in surface properties displayed by even a limited number of genetically-related bacterial strains can be understood in terms of established colloidal concepts, provided that sufficiently detailed structural, chemical, and conformational information on the surface constituents is available.
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Bacterial surfaces are soft systems, which display an impressive variation in physicochemical properties. Apart from the chemical nature of the surface constituents and the organization of these constituents within the cell wall, these properties are determined by the conformational degrees of freedom of the polymeric surface constituents. We have probed the surface properties of a number of strains of lactic acid bacteria using a variety of microscopic and physicochemical techniques with emphasis on the elucidation of the global physicochemical nature of the outer layer of the cell wall, the conformation of the surface macromolecules, and the susceptibility of the surface toward external perturbations (interfacial behavior, micromechanical forces). These three factors essentially determine the propensity of a bacterium to adhere to surfaces and to bind polymeric constituents of the growth medium; and they are also implied in bacterial auto- and co-aggregation and clustering. Cell-wall heterogeneities can strongly influence the colloidal properties of the bacteria. Such heterogeneities are difficult to detect using classical physicochemical techniques, but AFM is particularly suitable to analyze their nature. The relevant aspects of bacterial surface roughness show up at a length scale which is typically much smaller than the characteristic size of a bacterium, but which is, at the same time, much larger than the typical dimensions of a surface protein or a single surface polymer. In the case where the outer surface is made up of a regular lattice of globular proteins, like an S-layer, the surface is smooth on length scales larger than the typical size of the surface protein (a few nm). When the outer surface is made up of single polymers of fairly equal contour length, the surface is also smooth at these length scales, but may appear fuzzy because of thermal fluctuations of the surface polymers. Spatially varying distributions of surface polymers, which are also possibly crosslinked, result in heterogeneous and rough surfaces. This is the case if the outer surface contains polysaccharides and (lipo)teichoic acids. The presence of a dominant surface constituent can be inferred by combining the various physicochemical and microscopic analyses. The presence of surface proteins in lactobacilli can be deducted from the elevated isoelectric point and the high hydrophobicity of the surface. (Lipo)teichoic acids render the surface strongly negatively charged and hydrophobic at the same time. Surfaces rich in polysaccharides are generally weakly charged and are hydrophilic. Hydrophobic compounds like hexadecane can adsorb on sites on or within the cell wall. If the absorbing moieties are at the outer surface, this will render the bacterial surface very hydrophobic. In summary, we have found that the diversity in surface properties of lactobacilli strains can be fruitfully analyzed using a combination of classical physicochemical techniques and advanced microscopic techniques. In particular, AFM is a tool, which is highly suitable to study bacterial surface properties because spatial heterogeneities in surface structure, softness, and interaction forces can be detected at the same time. We expect that our findings will be helpful in increasing the understanding of the structure-property relations of the bacterial cell wall—in particular, with respect to bacterial interactions. For the AFM experiments, the support of Alcon Laboratories (Fort Worth, Texas), is acknowledged. In particular, we thank Gerald Cagle for enabling us to work at Alcon, and Andrew Griggs for carrying out a number of AFM experiments. For microbiological support, we are indebted to Marco Ventura and Nicola D’Amico. We are grateful for experimental support received from Marie-Lise Dillmann (TEM analysis) and Christophe Schmitt (electrophoresis). The force-volume data were analyzed using a MatLab macro originally developed by Urs Ziegler (University of Zürich) and adapted with help from Christoph Schär (ETH Zürich). We thank Conrad Woldringh (University of Amsterdam) for discussions on the bacterial cell cycle and Yves Dufrêne (Université Catholique de Louvain) for discussions on the analysis of biological specimens by AFM. Elisabeth Prior is thanked for a critical reading of the manuscript. The management of Nestec Ltd. is acknowledged for the permission to publish the work. Appendix