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Lactobacillus johnsonii FI9785 has an eps gene cluster which is required for the biosynthesis of homopolymeric exopolysaccharides (EPS)-1 and heteropolymeric EPS-2 as a capsular layer. The first gene of the cluster, epsA, is the putative transcriptional regulator. In this study we showed the crucial role of epsA in EPS biosynthesis by demonstrating that deletion of epsA resulted in complete loss of both EPS-1 and EPS-2 on the cell surface. Plasmid complementation of the epsA gene fully restored EPS production, as confirmed by transmission electron microscopy and nuclear magnetic resonance (NMR) analysis. Furthermore, this complementation resulted in a twofold increase in the expression levels of this gene, which almost doubled amounts of EPS production in comparison with the wild-type strain. Analysis of EPS by NMR showed an increased ratio of the heteropolysaccharide to homopolysaccharide in the complemented strain and allowed identification of the acetylated residue in EPS-2 as the (1,4)-linked Glcp unit, with the acetyl group located at O-6. These findings indicate that epsA is a positive regulator of EPS production and that EPS production can be manipulated by altering its expression.

Exopolysaccharides were isolated and purified from Lactobacillus johnsonii FI9785, which has previously been shown to act as a competitive exclusion agent to control Clostridium perfringens in poultry. Structural analysis by NMR spectroscopy revealed that L. johnsonii FI9785 can produce two types of exopolysaccharide: EPS-1 is a branched dextran with the unusual feature that every backbone residue is substituted with a 2-linked glucose unit, and EPS-2 was shown to have a repeating unit with the following structure: -6)-alpha-Glcp-(1-3)-beta-Glcp-(1-5)-beta-Galf-(1-6)-alpha-Glcp-(1-4)-beta-Galp-(1-4)-beta-Glcp-(1-. Siteson both polysaccharides were partially occupied by substituent groups: 1-phosphoglycerol and O-acetyl groups in EPS-1 and a single O-acetyl group in EPS-2. Analysis of a deletion mutant (Delta epsE) lacking the putative priming glycosyltransferase gene located within a predicted eps gene cluster revealed that the mutant could produce EPS-1 but not EPS-2, indicating that epsE is essential for the biosynthesis of EPS-2. Atomic force microscopy confirmed the localization of galactose residues on the exterior of wild type cells and their absence in the Delta epsE mutant. EPS2 was found to adopt a random coil structural conformation. Deletion of the entire 14-kb eps cluster resulted in an acapsular mutant phenotype that was not able to produce either EPS-2 or EPS-1. Alterations in the cell surface properties of the EPS-specific mutants were demonstrated by differences in binding of an anti-wild type L. johnsonii antibody. These findings provide insights into the biosynthesis and structures of novel exopolysaccharides produced by L. johnsonii FI9785, which are likely to play an important role in biofilm formation, protection against harsh environment of the gut, and colonization of the host.

The traditionally perceived function of nutrition includes supplying the consumer with the appropriate quantity and quality of substrates. As nutritional substrates, proteins are prone to spontaneously occurring non-enzymatic glycosylation (glycation) which can alter their molecular structure, making them highly bioactive. Glycated food proteins are able to modify the bacterial intestinal ecosystem, which is of great importance for the optimal usage of nutrients and maintenance of both intestinal homeostasis and balanced health status of the consumer. This study aimed to determine the impact of glycated pea proteins on the intestinal bacteria from a healthy human. The analyses were conducted with the use of experimental batch-type simulator models imitating human intestinal conditions. The glycated pea proteins affected the growth of gut commensal bacteria, particularly lactobacilli and bifidobacteria, whose levels increased significantly. There was a corresponding shift in the bacterial metabolites with increased levels of the short chain fatty acids (SCFAs); acetate, propionate lactate and butyrate. Intestinal bacteria were able to utilize these pea proteins thus indicating that the energy encrypted in glycated pea proteins, partially inaccessible for gastric enzymes, may be salvaged by gut microbiota. Such changes in microbial composition may beneficially impact the intestinal environment and exert a health-promoting effect in humans.

The bacteriophage ΦCD27 is capable of lysing Clostridium difficile, a pathogenic bacterium that is a major cause for nosocomial infection. A recombinant CD27L endolysin lyses C. difficile in vitro, and represents a promising alternative as a bactericide. To better understand the lysis mechanism, we have determined the crystal structure of an autoproteolytic fragment of the CD27L endolysin. The structure covers the C-terminal domain of the endolysin, and represents a novel fold that is identified in a number of lysins that target Clostridia bacteria. The structure indicates endolysin cleavage occurs at the stem of the linker connecting the catalytic domain with the C-terminal domain. We also solved the crystal structure of the C-terminal domain of a slow cleaving mutant of the CTP1L endolysin that targets C. tyrobutyricum. Two distinct dimerization modes are observed in the crystal structures for both endolysins, despite a sequence identity of only 22% between the domains. The dimers are validated to be present for the full length protein in solution by right angle light scattering, small angle X-ray scattering and cross-linking experiments using the cross-linking amino acid p-benzoyl-L-phenylalanine (pBpa). Mutagenesis on residues contributing to the dimer interfaces indicates that there is a link between the dimerization modes and the autocleavage mechanism. We show that for the CTP1L endolysin, there is a reduction in lysis efficiency that is proportional to the cleavage efficiency. We propose a model for endolysin triggering, where the extended dimer presents the inactive state, and a switch to the side-by-side dimer triggers the cleavage of the C-terminal domain. This leads to the release of the catalytic portion of the endolysin, enabling the efficient digestion of the bacterial cell wall., Author Summary Clostridium difficile infection is a common cause of hospital-acquired diarrhea, following broad-spectrum antibiotic treatment particularly in elderly patients. Bacteriophage therapy could provide an alternative treatment, but a better understanding of the viral components that lyse the bacterial cell is necessary. Here, we report on the activation of two endolysins from bacteriophages that lyse Clostridia. The structures of autoproteolytic fragments of two endolysins were determined by X-ray crystallography. Based on the structures, we introduced mutations that affect the autolytic cleavage of the enzymatic portion of the endolysins, and we show that two oligomeric states have an effect on the cleavage mechanism. Moreover, the lysis activity is affected when autocleavage is inhibited for one endolysin. We propose that the cleavage and oligomerization are linked, and they provide the endolysin with a trigger and release mechanism that leads to activation. The identification of a trigger and release factor may not only be relevant to Clostridia endolysins, but could be an important factor in the triggering of many bacteriophage endolysins. A fuller understanding of this activation mechanism will help in the design of recombinant endolysins or bacteriophages with a more efficient therapeutic potential.