Structure and biosynthesis of two exopolysaccharides produced by Lactobacillus johnsonii FI9785.

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)-α-Glcp-(1-3)-ß-Glcp-(1-5)-ß-Galf-(1-6)-α-Glcp-(1-4)-ß-Galp-(1-4)-ß-Glcp-(1-. Sites on 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 (ΔepsE) 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 ΔepsE 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.

CsiA is a bacterial cell wall synthesis inhibitor contributing to DNA translocation through the cell envelope.

Conjugation is a widely spread mechanism allowing bacteria to adapt and evolve by acquiring foreign DNA. The chromosome of Lactococcus lactis MG 1363 contains a 60 kb conjugative element called the sex factor capable of high-frequency DNA transfer. Yet, little is known about the proteins involved in this process. Comparative genomics revealed a close relationship between the sex factor and elements found in Gram-positive pathogenic cocci. Among the conserved gene products, CsiA is a large protein that contains a highly conserved domain (HCD) and a C-terminal cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) domain in its C-terminal moiety. Here, we show that CsiA is required for DNA transfer. Surprisingly, increased expression of CsiA affects cell viability and the cells become susceptible to lysis. Point mutagenesis of HCD reveals that this domain is responsible for the observed phenotypes. Growth studies and electron microscope observations suggest that CsiA is acting as a cell wall synthesis inhibitor. In vitro experiments reveal the capacity of CsiA to bind d-Ala-d-Ala analogues and to prevent the action of penicillin binding proteins. Our results strongly suggest that CsiA sequesters the peptidoglycan precursor and prevents the final stage of cell wall biosynthesis to enable the localized assembly of the DNA transfer machinery through the cell wall.

Strain-specific diversity of mucus-binding proteins in the adhesion and aggregation properties of Lactobacillus reuteri.

Mucus-binding proteins (MUBs) have been revealed as one of the effector molecules involved in mechanisms of the adherence of lactobacilli to the host; mub, or mub-like, genes are found in all of the six genomes of Lactobacillus reuteri that are available. We recently reported the crystal structure of a Mub repeat from L. reuteri ATCC 53608 (also designated strain 1063), revealing an unexpected recognition of immunoglobulins. In the current study, we explored the diversity of the ATCC 53608 mub gene, and MUB expression levels in a large collection of L. reuteri strains isolated from a range of vertebrate hosts. This analysis revealed that the MUB was only detectable on the cell surface of two highly related isolates when using antibodies that were raised against the protein. There was considerable variation in quantitative mucus adhesion in vitro among L. reuteri strains, and mucus binding showed excellent correlation with the presence of cell-surface ATCC 53608 MUB. ATCC 53608 MUB presence was further highly associated with the autoaggregation of L. reuteri strains in washed cell suspensions, suggesting a novel role of this surface protein in cell aggregation. We also characterized MUB expression in representative L. reuteri strains. This analysis revealed that one derivative of strain 1063 was a spontaneous mutant that expressed a C-terminally truncated version of MUB. This frameshift mutation was caused by the insertion of a duplicated 13 nt sequence at position 4867 nt in the mub gene, producing a truncated MUB also lacking the C-terminal LPxTG region, and thus unable to anchor to the cell wall. This mutant, designated 1063N (mub-4867(i)), displayed low mucus-binding and aggregation capacities, further providing evidence for the contribution of cell-wall-anchored MUB to such phenotypes. In conclusion, this study provided novel information on the functional attributes of MUB in L. reuteri, and further demonstrated that MUB and MUB-like proteins, although present in many L. reuteri isolates, show a high genetic heterogeneity among strains.

Controlled release of protein from viable Lactococcus lactis cells.

Overexpression of the lactococcal CsiA protein affects the cell wall integrity of growing cells and leads to leakage of intracellular material. This property was optimized and exploited for the targeted release of biologically active compounds into the extracellular environment, thereby providing a new delivery system for bacterial proteins and peptides. The effects of different levels of CsiA expression on the leakage of endogenous lactate dehydrogenase and nucleic acids were measured and related to the impact of CsiA expression on Lactococcus lactis cell viability and growth. A leakage phenotype was obtained from cells expressing both recombinant and nonrecombinant forms of CsiA. As proof of principle, we demonstrated that CsiA promotes the efficient release of the heterologous Listeria bacteriophage endolysin LM4 in its active form. Under optimized conditions, native and heterologous active-molecule release is possible without affecting cell viability. The ability of CsiA to release intracellular material by controlled lysis without the requirement for an external lytic agent provides a technology for the control of both the extent of lysis and its timing. Taken together, these results demonstrate the potential of this novel approach for applications including product recovery in industrial fermentations, food processing, and medical therapy.

Recognition of galactan components of pectin by galectin-3.

It has been reported that modified forms of pectin possess anticancer activity. To account for this bioactivity, it has been proposed that fragments of pectin molecules can act by binding to and inhibiting the various roles of the mammalian protein galectin 3 (Gal3) in cancer progression and metastasis. Despite this clear molecular hypothesis and evidence for the bioactivity of modified pectin, the structural origins of the "bioactive fragments" of pectin molecules are currently ill defined. By using a combination of fluorescence microscopy, flow cytometry, and force spectroscopy, it has been possible to demonstrate, for the first time, specific binding of a pectin galactan to the recombinant form of human Gal3. Present studies suggest that bioactivity resides in the neutral sugar side chains of pectin polysaccharides and that these components could be isolated and modified to optimize bioactivity.

The transcriptional programme of Salmonella enterica serovar Typhimurium reveals a key role for tryptophan metabolism in biofilms.

BACKGROUND: Biofilm formation enhances the capacity of pathogenic Salmonella bacteria to survive stresses that are commonly encountered within food processing and during host infection. The persistence of Salmonella within the food chain has become a major health concern, as biofilms can serve as a reservoir for the contamination of food products. While the molecular mechanisms required for the survival of bacteria on surfaces are not fully understood, transcriptional studies of other bacteria have demonstrated that biofilm growth triggers the expression of specific sets of genes, compared with planktonic cells. Until now, most gene expression studies of Salmonella have focused on the effect of infection-relevant stressors on virulence or the comparison of mutant and wild-type bacteria. However little is known about the physiological responses taking place inside a Salmonella biofilm. RESULTS: We have determined the transcriptomic and proteomic profiles of biofilms of Salmonella enterica serovar Typhimurium. We discovered that 124 detectable proteins were differentially expressed in the biofilm compared with planktonic cells, and that 10% of the S. Typhimurium genome (433 genes) showed a 2-fold or more change in the biofilm compared with planktonic cells. The genes that were significantly up-regulated implicated certain cellular processes in biofilm development including amino acid metabolism, cell motility, global regulation and tolerance to stress. We found that the most highly down-regulated genes in the biofilm were located on Salmonella Pathogenicity Island 2 (SPI2), and that a functional SPI2 secretion system regulator (ssrA) was required for S. Typhimurium biofilm formation. We identified STM0341 as a gene of unknown function that was needed for biofilm growth. Genes involved in tryptophan (trp) biosynthesis and transport were up-regulated in the biofilm. Deletion of trpE led to decreased bacterial attachment and this biofilm defect was restored by exogenous tryptophan or indole. CONCLUSIONS: Biofilm growth of S. Typhimurium causes distinct changes in gene and protein expression. Our results show that aromatic amino acids make an important contribution to biofilm formation and reveal a link between SPI2 expression and surface-associated growth in S. Typhimurium.

Characterization of planktonic and biofilm communities of day-of-hatch chicks cecal microflora and their resistance to Salmonella colonization.

Recent concerns about the use of antimicrobials in food animals have increased interest in the microbial ecology and biofilms within their gastrointestinal tract. This work used a continuous-flow chemostat system to model the microbial community within the ceca from day-of-hatch chicks and its ability to resist colonization by Salmonella enterica serovar Typhimurium. We characterized the biofilm and planktonic communities from five cultures by using automated ribotyping. Eight species from six different genera were identified. Overall, the planktonic communities were more diverse, with 40% of the cultures containing four or more bacterial species. Eighty percent of the biofilm communities contained only one or two species of bacteria. Enterococcus faecalis was the only species isolated from all communities. None of the resulting microbial communities was able to resist colonization by S. enterica serovar Typhimurium. This is the first study to provide a molecular-based characterization of the biofilm and planktonic communities found in day-of-hatch chicken cecal microflora cultures.

Planktonic and biofilm community characterization and Salmonella resistance of 14-day-old chicken cecal Microflora-derived continuous-flow cultures.

This study evaluated the composition of gastrointestinal bacterial communities in birds during an age in which their susceptibility to Salmonella is highly diminished. One of the challenges to developing probiotics is to develop an efficacious culture of minimal diversity that includes bacteria that are vital contributors to protection from pathogens, but excludes unnecessary species. This study used in vitro continuous-flow culture techniques to test the ability of mixed bacterial cultures acquired from in vivo sources, to resist colonization by a marker Salmonella enterica serovar Typhimurium, and then characterized the constituents of both biofilm and planktonic communities by biochemical, phenotypic, and molecular methods. These cultures, initiated from 14-day-old chicks, were all able to restrict colonization by Salmonella in an average of 10 days. Eighteen species of bacteria from 10 different genera were characterized. However, each culture contained a mixture of only 11 species, which included lactic acid bacteria. Biofilms contained less than 50% of the species found in the planktonic communities. Although not adults, the diversity of microbes within the cecal cultures from 14-day-old birds represents a community complex enough to oppose colonization by a nonindigenous bacteria in vitro. These results describe bacterial mixtures containing less diversity than in previously described avian protective cultures.

Nisin-controlled extracellular production of interleukin-2 in Lactococcus lactis strains, without the requirement for a signal peptide sequence.

Secretion of the cytokine interleukin-2 (IL-2) was investigated in Lactococcus lactis using the secretory machinery of the bacteriocin lactococcin A. Surprisingly, the lcnCD transport genes were not essential for mouse IL-2 secretion. Furthermore, expression of a mature mouse IL-2 gene resulted in interleukin secretion without the requirement for a leader sequence.

Conservation of σ28-Dependent Non-Coding RNA Paralogs and Predicted σ54-Dependent Targets in Thermophilic Campylobacter Species.

Assembly of flagella requires strict hierarchical and temporal control via flagellar sigma and anti-sigma factors, regulatory proteins and the assembly complex itself, but to date non-coding RNAs (ncRNAs) have not been described to regulate genes directly involved in flagellar assembly. In this study we have investigated the possible role of two ncRNA paralogs (CjNC1, CjNC4) in flagellar assembly and gene regulation of the diarrhoeal pathogen Campylobacter jejuni. CjNC1 and CjNC4 are 37/44 nt identical and predicted to target the 5' untranslated region (5' UTR) of genes transcribed from the flagellar sigma factor σ54. Orthologs of the σ54-dependent 5' UTRs and ncRNAs are present in the genomes of other thermophilic Campylobacter species, and transcription of CjNC1 and CNC4 is dependent on the flagellar sigma factor σ28. Surprisingly, inactivation and overexpression of CjNC1 and CjNC4 did not affect growth, motility or flagella-associated phenotypes such as autoagglutination. However, CjNC1 and CjNC4 were able to mediate sequence-dependent, but Hfq-independent, partial repression of fluorescence of predicted target 5' UTRs in an Escherichia coli-based GFP reporter gene system. This hints towards a subtle role for the CjNC1 and CjNC4 ncRNAs in post-transcriptional gene regulation in thermophilic Campylobacter species, and suggests that the currently used phenotypic methodologies are insufficiently sensitive to detect such subtle phenotypes. The lack of a role of Hfq in the E. coli GFP-based system indicates that the CjNC1 and CjNC4 ncRNAs may mediate post-transcriptional gene regulation in ways that do not conform to the paradigms obtained from the Enterobacteriaceae.

Green fluorescent protein as a marker for conditional gene expression in bacterial cells.

To date, the majority of studies of bacterial gene expression have been carried out on large communities, as techniques for analysis of expression in individual cells have not been available. Recent developments now allow us to use reporter genes to monitor gene expression in individual bacterial cells. Conventional reporters are not suitable for studies of living single cells. However, variants of GFP have proved to be ideal for the study of development, cell biology, and pathogenesis and are now the reporters of choice for microbial studies. In combination with techniques such as DFI and IVET and the use of flow cytometry and advanced fluorescence microscopy, the latest generation of GFP reporters allows the investigation of gene expression in individual bacterial cells within particular environments. These studies promise to bring a new level of understanding to the fields of bacterial pathogenesis and environmental microbiology.

A bacterial homolog of a eukaryotic inositol phosphate signaling enzyme mediates cross-kingdom dialog in the mammalian gut.

Dietary InsP6 can modulate eukaryotic cell proliferation and has complex nutritive consequences, but its metabolism in the mammalian gastrointestinal tract is poorly understood. Therefore, we performed phylogenetic analyses of the gastrointestinal microbiome in order to search for candidate InsP6 phosphatases. We determined that prominent gut bacteria express homologs of the mammalian InsP6 phosphatase (MINPP) and characterized the enzyme from Bacteroides thetaiotaomicron (BtMinpp). We show that BtMinpp has exceptionally high catalytic activity, which we rationalize on the basis of mutagenesis studies and by determining its crystal structure at 1.9 Å resolution. We demonstrate that BtMinpp is packaged inside outer membrane vesicles (OMVs) protecting the enzyme from degradation by gastrointestinal proteases. Moreover, we uncover an example of cross-kingdom cell-to-cell signaling, showing that the BtMinpp-OMVs interact with intestinal epithelial cells to promote intracellular Ca(2+) signaling. Our characterization of BtMinpp offers several directions for understanding how the microbiome serves human gastrointestinal physiology.

Effect of acidic pH on flow cytometric detection of bacteria stained with SYBR Green I and their distinction from background.

Unspecific background caused by biotic or abiotic particles, cellular debris, or autofluorescence is a well-known interfering parameter when applying flow cytometry to the detection of microorganisms in combination with fluorescent dyes. We present here an attempt to suppress the background signal intensity and thus to improve the detection of microorganisms using the nucleic acid stain SYBR® Green I. It has been observed that the fluorescent signals from SYBR Green I are greatly reduced at acidic pH. When lowering the pH of pre-stained samples directly prior to flow cytometric analysis, we hypothesized that the signals from particles and cells with membrane damage might therefore be reduced. Signals from intact cells, temporarily maintaining a neutral cytosolic pH, should not be affected. We show here that this principle holds true for lowering background interference, whereas the signals of membrane-compromised dead cells are only affected weakly. Signals from intact live cells at low pH were mostly comparable to signals without acidification. Although this study was solely performed with SYBR® Green I, the principle of low pH flow cytometry (low pH-FCM) might hold promise when analyzing complex matrices with an abundance of non-cellular matter, especially when expanded to non-DNA binding dyes with a stronger pH dependence of fluorescence than SYBR Green I and a higher pKa value.

Anticancer activity of olive oil hydroxytyrosyl acetate in human adenocarcinoma Caco-2 cells.

The anticancer activity of hydroxytyrosyl acetate (HTy-Ac) has been studied in human colon adenocarcinoma cells. Gene expression of proteins involved in cell cycle (p21, p53, cyclin B1, and cyclin G2) and programmed cell death (BNIP3, BNIP3L, PDCD4, and ATF3), as well as phase I and phase II detoxifying enzymes CYPA1 and UGT1A10, were evaluated by reverse transcription polymerase chain reaction after 24 h of exposure of Caco-2/TC7 cells to 5, 10, and 50 µM of HTy-Ac. The results show that HTy-Ac inhibited cell proliferation and arrested cell cycle by enhancing p21 and CCNG2 and lowering CCNB1 protein expression. HTy-Ac also affected the transcription of genes involved in apoptosis up-regulating of BNIP3, BNIP3L, PDCD4, and ATF3 and activating caspase-3. In addition, HTy-Ac also up-regulated xenobiotic metabolizing enzymes CYP1A1 and UGT1A10, thus enhancing carcinogen detoxification. In conclusion, these results highlight that HTy-Ac has the potential to modulate biomarkers involved in colon cancer.

Spontaneous mutation reveals influence of exopolysaccharide on Lactobacillus johnsonii surface characteristics.

As a competitive exclusion agent, Lactobacillus johnsonii FI9785 has been shown to prevent the colonization of selected pathogenic bacteria from the chicken gastrointestinal tract. During growth of the bacterium a rare but consistent emergence of an altered phenotype was noted, generating smooth colonies in contrast to the wild type rough form. A smooth colony variant was isolated and two-dimensional gel analysis of both strains revealed a protein spot with different migration properties in the two phenotypes. The spot in both gels was identified as a putative tyrosine kinase (EpsC), associated with a predicted exopolysaccharide gene cluster. Sequencing of the epsC gene from the smooth mutant revealed a single substitution (G to A) in the coding strand, resulting in the amino acid change D88N in the corresponding gene product. A native plasmid of L. johnsonii was engineered to produce a novel vector for constitutive expression and this was used to demonstrate that expression of the wild type epsC gene in the smooth mutant produced a reversion to the rough colony phenotype. Both the mutant and epsC complemented strains had increased levels of exopolysaccharides compared to the wild type strain, indicating that the rough phenotype is not solely associated with the quantity of exopolysaccharide. Another gene in the cluster, epsE, that encoded a putative undecaprenyl-phosphate galactosephosphotransferase, was deleted in order to investigate its role in exopolysaccharide biosynthesis. The ΔepsE strain exhibited a large increase in cell aggregation and a reduction in exopolysaccharide content, while plasmid complementation of epsE restored the wild type phenotype. Flow cytometry showed that the wild type and derivative strains exhibited clear differences in their adhesive ability to HT29 monolayers in tissue culture, demonstrating an impact of EPS on surface properties and bacteria-host interactions.

Intestinal intraepithelial lymphocyte-enterocyte crosstalk regulates production of bactericidal angiogenin 4 by Paneth cells upon microbial challenge.

Antimicrobial proteins influence intestinal microbial ecology and limit proliferation of pathogens, yet the regulation of their expression has only been partially elucidated. Here, we have identified a putative pathway involving epithelial cells and intestinal intraepithelial lymphocytes (iIELs) that leads to antimicrobial protein (AMP) production by Paneth cells. Mice lacking γδ iIELs (TCRδ(-/-)) express significantly reduced levels of the AMP angiogenin 4 (Ang4). These mice were also unable to up-regulate Ang4 production following oral challenge by Salmonella, leading to higher levels of mucosal invasion compared to their wild type counterparts during the first 2 hours post-challenge. The transfer of γδ iIELs from wild type (WT) mice to TCRδ(-/-) mice restored Ang4 production and Salmonella invasion levels were reduced to those obtained in WT mice. The ability to restore Ang4 production in TCRδ(-/-) mice was shown to be restricted to γδ iIELs expressing Vγ7-encoded TCRs. Using a novel intestinal crypt co-culture system we identified a putative pathway of Ang4 production initiated by exposure to Salmonella, intestinal commensals or microbial antigens that induced intestinal epithelial cells to produce cytokines including IL­23 in a TLR-mediated manner. Exposure of TCR-Vγ7(+) γδ iIELs to IL-23 promoted IL­22 production, which triggered Paneth cells to secrete Ang4. These findings identify a novel role for γδ iIELs in mucosal defence through sensing immediate epithelial cell cytokine responses and influencing AMP production. This in turn can contribute to the maintenance of intestinal microbial homeostasis and epithelial barrier function, and limit pathogen invasion.

Mucin-lectin interactions assessed by flow cytometry.

The O-glycosylated domains of mucins and mucin-type glycoproteins contain 50-80% of carbohydrate and possess expanded conformations. Herein, we describe a flow cytometry (FCM) method for determining the carbohydrate-binding specificities of lectins to mucin. Biotinylated mucin was immobilized on streptavidin-coated beads, and the binding specificities of the major mucin sugar chains, as determined by GC-MS and MALDI-ToF, were monitored using fluorescein-labeled lectins. The specificities of lectins toward specific biotinylated glycans were determined as controls. The advantage of flexibility, multiparametric data acquisition, speed, sensitivity, and high-throughput capability makes flow cytometry a valuable tool to study diverse interactions between glycans and proteins.

Bacterial Concentration and Diversity within Repetitive Aliquots Collected from Replicate Continuous-Flow Bioreactor Cultures.

The aim of this study was to determine the reproducibility of small volume repeat sampling from replicate bioreactors with stabilized continuous-flow chicken cecal bacterial communities. Bacterial concentration and diversity were analyzed by phenotypic, biochemical and ribotype analysis. Significant differences in concentrations and variations in diversity were found in replicate bioreactors.