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Six genes involved in cellulose synthesis in Rhizobium leguminosarum bv. trifolii were identified using Tn5 mutagenesis. Four of them displayed homology to the previously cloned and sequenced Agrobacteriurn turnefaciens cellulose genes celA, celf3, ce/C and celE These genes are organized similarly in R. legurninosarum bv. trifolii. In addition, there were strong indications that two tandemly located genes, celRl and celR2, probably organized as one operon, are involved in the regulation of cellulose synthesis. The deduced amino acid sequences of these genes displayed a high degree of similarity to the Caulobacter crescentus DivK and PleD proteins that belong to the family of two-component response regulators. This is to our knowledge the first report of genes involved in the regulation of cellulose synthesis. Results from attachment assays and electron microscopic studies indicated that cellulose synthesis in R. leguminosarum bv. trifolii is induced upon close contact with plant roots during the attachment process.

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A new Lactobacillus species from pig small intestine has been identified. In an attempt to isolate Lactobacillus reuteri strains carrying the putative colonization-factor gene (mub, for mucus binding) a mub-derived gene probe was used to screen pig intestinal material. A number of isolates were obtained and primary characterization showed that they were Gram-positive, catalasenegative, non-spore-forming, non-motile rods. Growth occurred at 45 SC but not at 15 SC and the DNA GMC content was 46 mol %. Cell wall analysis together with DNA-DNA hybridization and analysis of the 16S rRNA sequence revealed that the new isolates represent a previously undescribed Lactobacillus species closely related to L. reuteri, Lactobacillus fermentum and Lactobacillus pontis. The name Lactobacillus mucosae is proposed for this species and the type strain is S32 T . Keywords : Lactobacillus mucosae, new species, mucus binding INTRODUCTIONThe use of lactic acid bacteria (LAB) in the production and preservation of food and animal feed dates back several thousand years. Today this use is manifested by various dairy products such as cheese and yoghurt, fermented sausages, vegetables and silage for animal feed. Recently, a great deal of interest has been focused on some members of the LAB with regard to their use as probiotics (Fuller, 1989 ;Marteau & Rambaud, 1993 ;Salminen et al., 1996). The term ' probiotic ' refers to live organisms that are administered to animals or humans via feed or food products and are in some way beneficial to health (Fuller, 1989). The LAB that are currently used as probiotics are primarily species of Lactobacillus and Bifidobacterium. In this context, bifidobacteria are often included in the LAB group since they have many features in common with this group. However, in contrast to the LAB group in general, bifidobacteria belong to the high GjC group of Gram-positive bacteria. The positive effects attributed to these probiotic organisms include stabilization of the normal microflora, protection against pathogens, lowering of cholesterol levels, The GenBank/EMBL/DDBJ accession number for the 16S rDNA gene sequence of strain S32T is AF126738.immune stimulation and protection against certain forms of cancer (Elmer et al., 1996 ;Isolauri et al., 1998 ;Lichtenstein & Goldin, 1998). One of the basic assumptions regarding the important features of probiotic micro-organisms is the need for colonization ability (Huis in't Veld et al., 1994 ;Brassart & Schiffrin, 1997). In contrast to the case of many pathogenic bacteria, little is known about the mechanisms by which LAB interact with host components in the intestinal tract. However, in recent years several reports have begun to establish a knowledge base on how lactobacilli adhere to the intestinal mucosa (Adlerberth et al., 1996 ;Roos et al., 1996 ;Yamamoto et al., 1996 ;Granato et al., 1999 Wadstro$ m et al., 1987) were shown to belong to this new species. These isolates also harbour mub and possess the ability to adhere to pig mucus in vitro. An oligonucleotide pro...

To be able to function as a probiotic, bacteria have to survive the passage through the gastrointestinal tract. We have examined survival and gene expression of Lactobacillus reuteri ATCC 55730 after a sudden shift in environmental acidity to a pH close to the conditions in the human stomach. More than 80% of the L. reuteri cells survived at pH 2.7 for 1 h. A genomewide expression analysis experiment using microarrays displayed 72 differentially expressed genes at this pH. The early response to severe acid shock in L. reuteri differed from long-term acid adaptation to milder acid stress studied in other lactic acid bacteria. The genes induced included the following: clpL, genes putatively involved in alterations of the cell membrane and the cell wall; genes encoding transcriptional regulators; phage genes; and genes of unknown function. Two genes, clpL, encoding an ATPase with chaperone activity, and lr1516, encoding a putative esterase, were selected for mutation analyses. The mutants were significantly more sensitive to acid than the wild type was. Thus, these genes could contribute to the survival of L. reuteri in the gastrointestinal tract.

Wet wheat distillers' grain (WWDG), a residue from ethanol fermentation, was examined from a microbiological perspective. After storage, WWDG was characterized by a high content of lactobacilli, nondetectable levels of other bacteria, occasional occurrence of yeasts, and a pH of about 3.6 and contained a mixture of lactic acid, acetic acid, and ethanol. The composition of lactobacilli in WWDG was simple, including primarily the species Lactobacillus amylolyticus, Lactobacillus panis, and Lactobacillus pontis, as determined by 16S rRNA gene sequencing. Since the use of WWDG as pig feed has indicated a health-promoting function, some relevant characteristics of three strains of each of these species were examined together with basal physiological parameters, such as carbohydrate utilization and growth temperature. Seven of the strains were isolated from WWDG, and two strains from pig feces were included for comparison. It was clear that all three species could grow at temperatures of 45 to 50°C, with L. amylolyticus being able to grow at temperatures as high as 54°C. This finding could be the explanation for the simple microflora of WWDG, where a low pH together with a high temperature during storage would select for these organisms. Some strains of L. panis and L. pontis showed prolonged survival at pH 2.5 in synthetic stomach juice and good growth in the presence of porcine bile salt. In addition, members of all three species were able to bind to immobilized mucus material in vitro. Especially the isolates from pig feces but, interestingly, some isolates from WWDG as well possessed properties that might be of importance for colonization of the gastrointestinal tracts of pigs.

Lactobacillus reuteri, a symbiotic inhabitant of the gastrointestinal tract in humans and animals, is marketed as a probiotic. The ability to adhere to intestinal epithelial cells and mucus is an interesting property with regard to probiotic features such as colonization of the gastrointestinal tract and interaction with the host. Here, we present a study performed to elucidate the role of sortase (SrtA), four putative sortase-dependent proteins (SDPs), and one C-terminal membrane-anchored cell surface protein of Lactobacillus reuteri ATCC PTA 6475 in adhesion to Caco-2 cells and mucus in vitro. This included mutagenesis of the genes encoding these proteins and complementation of mutants. A null mutation in hmpref0536_10255 encoding srtA resulted in significantly reduced adhesion to Caco-2 cells and mucus, indicating involvement of SDPs in adhesion. Evaluation of the bacterial adhesion revealed that of the five putative surface protein mutants tested, only a null mutation in the hmpref0536_10633 gene, encoding a putative SDP with an LPxTG motif, resulted in a significant loss of adhesion to both Caco-2 cells and mucus. Complementation with the functional gene on a plasmid restored adhesion to Caco-2 cells. However, complete restoration of adhesion to mucus was not achieved. Overexpression of hmpref0536_10633 in strain ATCC PTA 6475 resulted in an increased adhesion to Caco-2 cells and mucus compared with the WT strain. We conclude from these results that, among the putative surface proteins tested, the protein encoded by hmpref0536_10633 plays a critical role in binding of Lactobacillus reuteri ATCC PTA 6475 to Caco-2 cells and mucus. Based on this, we propose that this LPxTG motif containing protein should be referred to as cell and mucus binding protein A (CmbA).

Heat shock proteins (HSPs) are a set of highly conserved proteins that can serve as intestinal gate keepers in gut homeostasis. Here, effects of a probiotic, Lactobacillus rhamnosus GG (LGG), and two novel porcine isolates, Lactobacillus johnsonii strain P47-HY and Lactobacillus reuteri strain P43-HUV, on cytoprotective HSP expression and gut barrier function, were investigated in a porcine IPEC-J2 intestinal epithelial cell line model. The IPEC-J2 cells polarized on a permeable filter exhibited villus-like cell phenotype with development of apical microvilli. Western blot analysis detected HSP expression in IPEC-J2 and revealed that L. johnsonii and L. reuteri strains were able to significantly induce HSP27, despite high basal expression in IPEC-J2, whereas LGG did not. For HSP72, only the supernatant of L. reuteri induced the expression, which was comparable to the heat shock treatment, which indicated that HSP72 expression was more stimulus specific. The protective effect of lactobacilli was further studied in IPEC-J2 under an enterotoxigenic Escherichia coli (ETEC) challenge. ETEC caused intestinal barrier destruction, as reflected by loss of cell–cell contact, reduced IPEC-J2 cell viability and transepithelial electrical resistance, and disruption of tight junction protein zonula occludens-1. In contrast, the L. reuteri treatment substantially counteracted these detrimental effects and preserved the barrier function. L. johnsonii and LGG also achieved barrier protection, partly by directly inhibiting ETEC attachment. Together, the results indicate that specific strains of Lactobacillus can enhance gut barrier function through cytoprotective HSP induction and fortify the cell protection against ETEC challenge through tight junction protein modulation and direct interaction with pathogens.