Hans Jonsson

A high-molecular-mass cell-surface protein from Lactobacillus reuteri 1063 adheres to mucus components

A gene from Lactobacillus reuteri 1063 encoding a cell-surface protein, designated Mub, that adheres to mucus components in vitro has been cloned and sequenced. The deduced amino acid sequence of Mub (358 kDa) shows the presence of 14 approximately 200 aa repeats and features typical for other cell-surface proteins of Gram-positive bacteria. Fusion proteins consisting of different repeats of Mub and the maltose-binding protein (MBP) were produced. These proteins adhered to pig mucus components, with molecular masses ranging from <0.1 to >2 MDa, to pig gastric mucin and to hen intestinal mucus. The binding of Mub to mucus components occurred in the pH range 3-7.4, with maximum binding at pH 4-5 and could be partly inhibited by the glycoprotein fetuin. Affinity-purified antibodies against recombinant Mub were used in immunofluorescence microscopy to demonstrate the presence of Mub on the cell surface of strain 1063. By using the antibodies in a Western blot analysis, Mub could also be detected in the growth medium. The results implicate Mub as a cell-surface protein that is involved in Lactobacillus interactions with mucin and in colonization of the digestive tract.

Lactobacillus mucosae sp. nov., a new species with in vitro mucus-binding activity isolated from pig intestine.

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, catalase-negative, non-spore-forming, non-motile rods. Growth occurred at 45 degrees C but not at 15 degrees C and the DNA G+C 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 S32T.

Structural and putative regulatory genes involved in cellulose synthesis in Rhizobium leguminosarum bv. trifolii.

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 Agrobacterium tumefaciens cellulose genes celA, celB, celC and celE. These genes are organized similarly in R. leguminosarum bv. trifolii. In addition, there were strong indications that two tandemly located genes, celR1 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.

The Early Response to Acid Shock in Lactobacillus reuteri Involves the ClpL Chaperone and a Putative Cell Wall-Altering Esterase

ABSTRACT 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.

Autoaggregation of Lactobacillus reuteri is mediated by a putative DEAD-box helicase

We have cloned and sequenced a gene from Lactobacillus reuteri that encodes a 56 kDa protein, which mediates autoaggregation of the bacteria. Using an antiserum raised against extracellular proteins from the pig intestinal isolate L. reuteri 1063, we screened a genomic λ library derived from the same strain. Affinity purification of recombinant protein from the isolated λ clones showed that one type of clone expressed a protein that efficiently aggregated the parental strain when added to the bacteria. Subcloning and introduction of the corresponding gene, here denoted aggHinto the L. reuteri type strain markedly enhanced aggregation. Furthermore, insertional inactivation of aggH in strain 1063 resulted in an autoaggregation‐deficient phenotype. Finally, an affinity‐purified and cleaved fusion of AggH protein and the maltose‐binding protein, MBP, strongly promoted aggregation of L. reuteri 1063, whereas the uncleaved fusion protein was inactive. Sequencing of aggH revealed that the corresponding protein has extensive sequence homology to the large family of ATP‐dependent DEAD‐box helicases. These results are intriguing in view of earlier data on the promotion of genetic exchange in Lactobacillus by aggregation.

Global transcriptional response of Lactobacillus reuteri to the sourdough environment

Lactobacillus reuteri is a lactic acid bacterium that is highly adapted to the sourdough environment. It is a dominant member of industrial type II sourdoughs, and is also able to colonize the intestinal tract of mammals, including humans, and birds. In this study, the transcriptional response of L. reuteri ATCC 55730 was investigated during sourdough fermentation by using whole-genome microarrays. Significant changes of mRNA levels were found for 101 genes involved in diverse cellular processes, such as carbohydrate and energy metabolism, cell envelope biosynthesis, exopolysaccharide production, stress responses, signal transduction and cobalamin biosynthesis. The results showed extensive changes of the organisms gene expression during growth in sourdough as compared with growth in chemically defined medium, and, thus, revealed pathways involved in the adaptation of L. reuteri to the ecological niche of sourdough. The utilization of starch and non-starch carbohydrates, the remodelling of the cell wall, characterized by reduced D-alanylation, and increased amounts of cell wall-associated polysaccharides, as well as the regulatory function of two component systems for cell wall biogenesis and metabolism were suggested by the gene expression data as being important for growth in sourdough. The impact of several L. reuteri genes for effective growth in sourdough was shown by implementation of mutant strains in sourdough fermentation. This study contributes to the understanding of the molecular fundamentals of L. reuteris ecological competitiveness, and provides a basis for further exploration of genetic traits involved in adaptation to the food environment.

Weissella soli sp. nov., a lactic acid bacterium isolated from soil.

Phylogenetic analysis of the 16S rRNA gene of bacterial isolates from garden soil showed relatedness to Weissella kandleri and Weissella confusa. However, the sequences had notable differences, and DNA-DNA hybridizations confirmed that the isolates are separate from these two species. The isolates could be further distinguished from all previously described Weissella species by electrophoretic analysis of whole-cell proteins, as well as by the results from different biochemical tests. The name Weissella soli is proposed for the new species, the type strain being Mi268T (= LMG 20113T = DSM 14420T).

MAG, a novel plasma protein receptor from Streptococcus dysgalactiae

The gene encoding a plasma protein receptor from Streptococcus dysgalactiae has been cloned and sequenced. The gene product, with a predicted molecular mass of approx. 44 kDa, binds alpha 2-macroglobulin (alpha 2 M), serum albumin and immunoglobulin G (IgG). By subcloning and expressing various parts of the gene as fusion proteins, we found that the three binding activities reside in discrete domains of the protein. The single IgG-binding domain, localized in the C-terminal part of the molecule, shows high homology to streptococcal type-III Fc receptors. In the middle of the molecule, there is a stretch of 50 amino acids (aa) mediating albumin binding. This region has partial homology with the albumin-binding domains of streptococcal protein G. The alpha 2 M-binding domain is located in the N terminus of the molecule and is composed of a unique aa sequence. We call this trifunctional plasma protein receptor, MAG (binds alpha 2 M, albumin and IgG).

Effects of Lactobacillus johnsonii and Lactobacillus reuteri on gut barrier function and heat shock proteins in intestinal porcine epithelial cells

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.

Role of Lactobacillus reuteri cell and mucus-binding protein A (CmbA) in adhesion to intestinal epithelial cells and mucus in vitro

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).