The effect of the simple and complex basic components of a fermentation medium on the surface properties of Lactobacillus acidophilus NCC2628 is studied by physicochemical methods, such as electrophoresis, interfacial adhesion, and X-ray photonelectron spectroscopy, and by transmission electron microscopy. Starting from an optimized complete medium, the effect of carbohydrates, peptones, and yeast extracts on the physicochemical properties of the cell wall is systematically investigated by consecutively omitting one of the principal components from the fermentation medium at the time. The physicochemical properties and structure of the bacterial cell wall remain largely unchanged if the carbohydrate content of the fermentation medium is strongly reduced, although the concentration of surface proteins increases slightly. Both peptone and yeast extract have a considerable influence on the bacterial cell wall, as witnessed by changes in surface charge, hydrophobicity, and the nitrogen-to-carbon ratio. Both zeta potential and the cell wall hydrophobicity show a positive correlation with the nitrogen-to-carbon ratio of the bacterial surfaces, indicative of the important role of surface proteins in the overall surface physical chemistry. The hydrophobicity of the cell wall, which is low for the cultures grown in the complete medium and in the absence of carbohydrates, becomes fairly high for the cultures grown in the medium without peptones and the medium without yeast extract. UV spectrophotometry and sodium dodecyl sulfate-polyacrylamide gel electrophoresis combined with liquid chromatographytandem mass spectrometry are used to analyze the effect of medium composition on LiCl-extractable cell wall proteins, confirming the major change in protein composition of the cell wall for the culture fermented in the medium without peptones. In particular, it is found that expression of the S-layer protein is dependent on the protein source of the fermentation medium.

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The genes responsible for exopolysaccharide (EPS) synthesis in Streptococcus thermophilus Sfi39 were identified on a 20-kb genomic fragment. The two genes, epsE and epsG, were shown to be involved in EPS synthesis as their disruption lead to the loss of the ropy phenotype. Several naturally selected nonropy mutants were isolated, one acquired an insertion sequence (IS)-element (IS905) in the middle of the eps gene cluster. The eps gene cluster was cloned and transferred into a nonEPS-producing heterologous host, Lactococcus lactis MG1363. The EPS produced was shown by chemical analysis and NMR spectroscopy to be identical to the EPS produced by S. thermophilus Sfi39.This demonstrated first that all genes needed for EPS production and export were present in the S. thermophilus Sfi39 eps gene cluster, and second that the heterologous production of an EPS was possible by transfer of the complete eps gene cluster alone, provided that the heterologous host possessed all necessary genetic information for precursor synthesis.Keywords: eps gene cluster; exopolysaccharide; lactic acid bacteria; nuclear magnetic resonance; Streptococcus thermophilus.The food industry's interest in microbial polysaccharides was initially triggered by the texturizing properties that these molecules confer to fermented food products combined with their 'generally recognized as safe' (GRAS) status [1,2]. Detailed structural analysis of these polysaccharides from species including lactobacilli [3 -5] and streptococci [6][7][8][9][10][11] over the last 10 years has revealed an immense variety of structures in terms of monosaccharide components, stereo-and regio-specific linkages. This high diversity is reminiscent of the capsule variation present in pathogenic streptococci such as Streptococcus pneumoniae and initiated interest in analysis of the biochemistry and the genetic basis of polysaccharide biosynthesis. The aims are to harness this wealth of biological information to develop applications in carbohydrate engineering that could range from improving technological aspects of polysaccharides, such as texturing, stabilizing, gellification and designing novel bioactive properties such as immune-stimulation and infection-blocking compounds.The gene cluster responsible for exopolysaccharide (EPS) biosynthesis has been characterized in a few lactic acid bacteria strains, and includes the initial work on S. thermophilus Sfi39 is a strain with ropy phenotype associated with fast acidification and an uncommon resistance to pervasive industrial phages. The repeating unit of the EPS produced has been shown to be a tetrasaccharide composed of only glucose and galactose in equimolar ratio with the structureIn this work, we present the identification and characterization of the gene cluster involved in EPS biosynthesis in S. thermophilus Sfi39, as well as the first successful heterologous EPS expression by transfer of the complete eps gene cluster into a nonropy strain of L. lactis MG1363. Finally, the structural analysis of the heterologuous EPS confi...

A mozzarella cheese factory using an undefined, milk-derived Streptococcus thermophilus starter system was monitored longitudinally for 2 years to determine whether the diversity of the resident bacteriophage population arose from environmental sources or from genetic changes in the resident phage in the factory. The two hypotheses led to different predictions about the genetic diversity of the phages. With respect to host range, 12 distinct phage types were observed. With two exceptions, phages belonging to different lytic groups showed clearly distinct restriction patterns and multiple isolates of phages showing the same host range exhibited identical or highly related restriction patterns. Sequencing studies in a conserved region of the phage genome revealed no point mutations in multiple isolates of the same phage type, while up to 12% nucleotide sequence diversity was observed between the different phage types. This diversity is as large as that between the most different sequences from phages in our collection. These observations make unlikely a model that postulates a single phage invasion event and diversification of the phage during its residence in the factory. In the second stage of our factory study, a defined starter system was introduced that could not propagate the resident factory phage population. Within a week, three new phage types were observed in the factory while the resident phage population was decreased but not eliminated. Raw milk was the most likely source of these new phages, as phages with identical host ranges and restriction patterns were isolated from raw milk delivered to the factory during the intervention trial. Apparently, all of the genetic diversity observed in the S. thermophilus phages isolated during our survey was already created in their natural environment. A better understanding of the raw-milk ecology of S. thermophilus phages is thus essential for successful practical phage control.