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Probiotic bacteria are being explored for the in situ delivery of various therapeutic agents. In this study, we aimed to express two HIV-inhibiting lectins, actinohivin (AH) and griffithsin (GRFT), in the probiotic strains Lactobacillus rhamnosus GG and L. rhamnosus GR-1 for gastrointestinal and vaginal mucosal delivery, respectively. Constructs were generated for the intracellular and extracellular production of AH and GRFT under the control of the promoter of their Major Secreted Protein Msp1. Also, intracellular expression of GRFT was investigated under the control of the nisA promoter from the inducible nisin-controlled expression (NICE) system. For the extracellular localization, the signal leader peptide of Msp1/p75 from L. rhamnosus GG was translationally fused with the genes encoding AH and GRFT. Construction of recombinant strains expressing the AH monomer and dimer was unsuccessful, probably due to the intracellular toxicity of AH for the lactobacilli. On the other hand, recombinant strains for intra- and extracellular production of GRFT by L. rhamnosus GG and GR-1 were successfully constructed. The highest expression levels of recombinant GRFT were observed for the constructs under the control of the inducible nisA promoter and we demonstrated anti-HIV activity against an M-tropic and a T-tropic HIV-1 strain. We can conclude that recombinant Lactobacillus expressing anti-HIV lectins could contribute to the development of enhanced probiotic strains that are able to inhibit HIV transmission and subsequent replication, although further research and development are required. ispartof: INTERNATIONAL JOURNAL OF ANTIMICROBIAL AGENTS vol:52 issue:5 pages:599-607 ispartof: location:Netherlands status: published

The use of plant growth-promoting rhizobacteria as a sustainable alternative for chemical nitrogen fertilizers has been explored for many economically important crops. For one such strain isolated from rice rhizosphere and endosphere, nitrogen-fixing Pseudomonas stutzeri A15, unequivocal evidence of the plant growth-promoting effect and the potential contribution of biological nitrogen fixation (BNF) is still lacking. In this study, we investigated the effect of P. stutzeri A15 inoculation on the growth of rice seedlings in greenhouse conditions. P. stutzeri A15 induced significant growth promotion compared to uninoculated rice seedlings. Furthermore, inoculation with strain A15 performed significantly better than chemical nitrogen fertilization, clearly pointing to the potential of this bacterium as biofertilizer. To assess the contribution of BNF to the plant growth-promoting effect, rice seedlings were also inoculated with a nitrogen fixation-deficient mutant. Our results suggest that BNF (at best) only partially contributes to the stimulation of plant growth.

There is an urgent need for new antibacterial drugs that are robust against the development of resistance. Drug repurposing is a cost-effective strategy to fast-track the drug development process. Here we examine why the nucleoside and nucleobase analogue drugs in particular present an attractive class for repurposing. Some of these drugs have already been evaluated for their potential as antibacterial agents. In addition to inhibiting bacterial growth and survival, some also act synergistically with antibiotics, and as such can enhance the therapeutic spectrum of currently available antibiotics. Furthermore, nucleoside and nucleobase analogue drugs can inhibit bacterial virulence and biofilm formation. Biofilms are known to impart antibiotic tolerance and are associated with chronic infections. Targeting biofilm formation thus renders pathogens more susceptible to antibiotic treatment and host immune defences. Moreover, specific analogues have properties that make them less susceptible to the development of resistance. Thus, nucleoside and nucleobase analogue drugs ought to be considered as new weapons in our fight against pathogenic bacteria.

Probiotic Lactobacillus strains are widely used to benefit human and animal health, although the exact mechanisms behind their interactions with the host and the microbiota are largely unknown. Fluorescent tagging of live probiotic cells is an important tool to unravel their modes of action. In this study, the implementation of different heterologously expressed fluorescent proteins for the labelling of the model probiotic strains Lactobacillus rhamnosusGG (gastrointestinal) and Lactobacillus rhamnosusGR-1 (vaginal) was explored. Heterologous expression of mTagBFP2 and mCherry resulted in long-lasting fluorescence of L. rhamnosusGG and GR-1 cells, using the nisin-controlled expression (NICE) system. These novel fluorescent strains were then used to study in vitro aspects of their microbe-microbe and microbe-host interactions. Lactobacillus rhamnosusGG and L. rhamnosusGR-1 expressing mTagBFP2 and mCherry could be visualized in mixed-species biofilms, where they inhibited biofilm formation by Salmonella Typhimurium-gfpmut3 expressing the green fluorescent protein. Likewise, fluorescent L. rhamnosusGG and L. rhamnosusGR-1 were implemented for the visualization of their adhesion patterns to intestinal epithelial cell cultures. The fluorescent L. rhamnosus strains developed in this study can therefore serve as novel tools for the study of probiotic interactions with their environment. ispartof: Microbial Biotechnology vol:11 issue:2 pages:317-331 ispartof: location:United States status: published

Plant growth-promoting rhizobacteria (PGPR) have been isolated from legume nodules, displaying a potential to enhance nodulation, growth and yield of legume plants when co-inoculated with Rhizobium. This study genetically characterizes bacteria isolated from bean root nodules in Cuba and investigates the effect of Rhizobium–Pseudomonas co-inoculation on common bean (Phaseolus vulgaris L.) genotypes under two different Cuban soil conditions. Bacteria were identified by partial sequencing of the 16S rDNA gene. Growth-promoting effects of Rhizobium pisi–Pseudomonas monteilii co-inoculation were evaluated under field conditions in consecutive years. Two contrasting bean genotypes (BAT-477 and DOR-364) were co-inoculated and grown in two different soil types (Cambisol and Oxisol) in Cuban farm areas. Several growth parameters were evaluated at three time points during the crop cycle (21, 42 and 87 days after planting). In total, 20 strains were isolated belonging to six different genera, i.e. Arthrobacter, Chryseobacterium, Enterobacter, Stenotrophomonas, Pseudomonas and Rhizobium. It was observed that native Rhizobium–Pseudomonas co-inoculation as compared to single Rhizobium inoculation increased the nodulation, growth parameters and yield of the different genotypes. The response of BAT-477 was more pronounced than that of DOR-364. These findings contribute to the understanding of the interplay between Rhizobium, PGPR and the plant host under different soil conditions. Importantly, co-inoculation with R. pisi and P. monteilii could be an effective biofertilization strategy for common bean production.

Biofilm-associated infections, particularly those caused by Staphylococcus aureus, are a major cause of implant failure. Covalent coupling of broad-spectrum antimicrobials to implants is a promising approach to reduce the risk of infections. In this study, we developed titanium substrates on which the recently discovered antibacterial agent SPI031, a N-alkylated 3, 6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol, was covalently linked (SPI031-Ti). We found that SPI031-Ti substrates prevent biofilm formation of S. aureus and Pseudomonas aeruginosa in vitro, as quantified by plate counting and fluorescence microscopy. To test the effectiveness of SPI031-Ti substrates in vivo, we used an adapted in vivo biomaterial-associated infection model in mice in which SPI031-Ti substrates were implanted subcutaneously and subsequently inoculated with S. aureus. Using this model, we found a significant reduction in biofilm formation (up to 98%) on SPI031-Ti substrates compared to control substrates. Finally, we demonstrated that the functionalization of the titanium surfaces with SPI031 did not influence the adhesion and proliferation of human cells important for osseointegration and bone repair. In conclusion, these data demonstrate the clinical potential of SPI031 to be used as an antibacterial coating for implants, thereby reducing the incidence of implant-associated infections. ispartof: Journal of Orthopaedic Research vol:34 issue:12 pages:2191-2198 ispartof: location:United States status: published

The auxin-producing bacterium Azospirillum brasilense Sp245 can promote the growth of several plant species. The model plant Arabidopsis thaliana was chosen as host plant to gain an insight into the molecular mechanisms that govern this interaction. The determination of differential gene expression in Arabidopsis roots after inoculation with either A. brasilense wild-type or an auxin biosynthesis mutant was achieved by microarray analysis. Arabidopsis thaliana inoculation with A. brasilense wild-type increases the number of lateral roots and root hairs, and elevates the internal auxin concentration in the plant. The A. thaliana root transcriptome undergoes extensive changes on A. brasilense inoculation, and the effects are more pronounced at later time points. The wild-type bacterial strain induces changes in hormone- and defense-related genes, as well as in plant cell wall-related genes. The A. brasilense mutant, however, does not elicit these transcriptional changes to the same extent. There are qualitative and quantitative differences between A. thaliana responses to the wild-type A. brasilense strain and the auxin biosynthesis mutant strain, based on both phenotypic and transcriptomic data. This illustrates the major role played by auxin in the Azospirillum-Arabidopsis interaction, and possibly also in other bacterium-plant interactions.

Recent developments demonstrate that the combination of microbiology with micro- and nanoelectronics is a successful approach to develop new miniaturized sensing devices and other technologies. In the last decade, there has been a shift from the optimization of the abiotic components, for example, the chip, to the improvement of the processing capabilities of cells through genetic engineering. The synthetic biology approach will not only give rise to systems with new functionalities, but will also improve the robustness and speed of their response towards applied signals. To this end, the development of new genetic circuits has to be guided by computational design methods that enable to tune and optimize the circuit response. As the successful design of genetic circuits is highly dependent on the quality and reliability of its composing elements, intense characterization of standard biological parts will be crucial for an efficient rational design process in the development of new genetic circuits. Microengineered devices can thereby offer a new analytical approach for the study of complex biological parts and systems. By summarizing the recent techniques in creating new synthetic circuits and in integrating biology with microdevices, this review aims at emphasizing the power of combining synthetic biology with microfluidics and microelectronics.

The urogenital tract appears to be the only niche of the human body that shows clear differences in microbiota between men and women. The female reproductive tract has special features in terms of immunological organization, an epithelial barrier, microbiota, and influence by sex hormones such as estrogen. While the upper genital tract is regarded as free of microorganisms, the vagina is colonized by bacteria dominated by Lactobacillus species, although their numbers vary considerably during life. Bacterial vaginosis is a common pathology characterized by dysbiosis, which increases the susceptibility for HIV infection and transmission. On the other hand, HIV infections are often characterized by a disturbed vaginal microbiota. The endogenous vaginal microbiota may protect against HIV by direct production of antiviral compounds, through blocking of adhesion and transmission by ligands such as lectins, and/or by stimulation of immune responses. The potential role of probiotics in the prevention of HIV infections and associated symptoms, by introducing them to the vaginal and gastrointestinal tract (GIT), is also discussed. Of note, the GIT is a site of considerable HIV replication and CD4(+) T-cell destruction, resulting in both local and systemic inflammation. Finally, genetically engineered lactobacilli show promise as new microbicidal agents against HIV.

International audience; Interactions between microbial cells and milk proteins are important for cell location into dairy matrices. In this study, interactions between two probiotic strains, Lactobacillus rhamnosus GG and Lactobacillus rhamnosus GR-1, and milk proteins (micellar casein, native and denatured whey proteins) were studied. The bacterial surface characterization was realized with X-ray photoelectron spectroscopy (XPS) to evaluate surface composition (in terms of proteins, polysaccharides and lipid-like compounds) and electrophoretic mobility that provide information on surface charge of both bacteria and proteins along the 3-7 pH range. In addition, atomic force microscopy (AFM) enabled the identification of specific interactions between bacteria and whey proteins, in contrast to the observed nonspecific interactions with micellar casein. These specific events appeared to be more important for the GG strain than for the GR-1 strain, showing that matrix interaction is strain-specific. Furthermore, our study highlighted that in addition to the nature of the strains, many other factors influence the bacterial interaction with dairy matrix including the nature of the proteins and the pH of the media.

Knowledge of the mechanisms by which bacterial pili adhere to host cells and withstand external forces is critical to our understanding of their functional roles and offers exciting avenues in biomedicine for controlling the adhesion of bacterial pathogens and probiotics. While much progress has been made in the nanoscale characterization of pili from Gram-negative bacteria, the adhesive and mechanical properties of Gram-positive bacterial pili remain largely unknown. Here, we use single-molecule atomic force microscopy to unravel the binding mechanism of pili from the probiotic Gram-positive bacterium Lactobacillus rhamnosus GG (LGG). First, we show that SpaC, the key adhesion protein of the LGG pilus, is a multifunctional adhesin with broad specificity. SpaC forms homophilic trans-interactions engaged in bacterial aggregation and specifically binds mucin and collagen, two major extracellular components of host epithelial layers. Homophilic and heterophilic interactions display similar binding strengths and dissociation rates. Next, pulling experiments on living bacteria demonstrate that LGG pili exhibit two unique mechanical responses, that is, zipper-like adhesion involving multiple SpaC molecules distributed along the pilus length and nanospring properties enabling pili to resist high force. These mechanical properties may represent a generic mechanism among Gram-positive bacterial pili for strengthening adhesion and withstanding shear stresses in the natural environment. The single-molecule experiments presented here may help us to design molecules capable of promoting or inhibiting bacterial-host interactions. © 2013 American Chemical Society.

Probiotic bacteria have a strong potential in biomedicine owing to their ability to induce various beneficial health effects. Bacterial cell surface constituents play a key role in establishing tight interactions between probiotics and their host. Yet, little is known about the spatial organization and biophysical properties of the individual molecules. In this paper, we discuss how we have been using atomic force microscopy (AFM) imaging and force spectroscopy to probe the nanoscale surface properties of Gram-positive lactic acid bacteria, with an emphasis on probiotic strains. Topographic imaging has enabled us to visualize bacterial cell surface structures (peptidoglycan, teichoic acids, pili, polysaccharides) under physiological conditions and with unprecedented resolution. In parallel, single molecule force spectroscopy has been used to localize and force probe single cell surface constituents, providing novel insights into their spatial distribution and molecular elasticity. ispartof: Micron vol:43 issue:12 pages:1323-1330 ispartof: location:England status: published

To characterize the interaction potential of the human vaginal isolate Lactobacillus plantarum CMPG5300, its genome was mined for genes encoding lectin-like proteins. cmpg5300.05_29 was identified as the gene encoding a putative mannose-binding lectin. Phenotypic analysis of a gene knock-out mutant of cmpg5300.05_29 showed that expression of this gene is important for auto-aggregation, adhesion to the vaginal epithelial cells, biofilm formation and binding to mannosylated glycans. Purification of the predicted lectin domain of Cmpg5300.05_29 and characterization of its sugar binding capacity confirmed the specificity of the lectin for high- mannose glycans. Therefore, we renamed Cmpg5300.05_29 as a mannose-specific lectin (Msl). The purified lectin domain of Msl could efficiently bind to HIV-1 glycoprotein gp120 and Candida albicans, and showed an inhibitory activity against biofilm formation of uropathogenic Escherichia coli, Staphylococcus aureus and Salmonella Typhimurium. Thus, using a combination of molecular lectin characterization and functional assays, we could show that lectin-sugar interactions play a key role in host and pathogen interactions of a prototype isolate of the vaginal Lactobacillus microbiota.

Background: Biofilm formation is an important survival strategy of Salmonella in all environments. By mutant screening, we showed a knock-out mutant of fabR, encoding a repressor of unsaturated fatty acid biosynthesis (UFA), to have impaired biofilm formation. In order to unravel how this regulator impinges on Salmonella biofilm formation, we aimed at elucidating the S. Typhimurium FabR regulon. Hereto, we applied a combinatorial high-throughput approach, combining ChIP-chip with transcriptomics.Results: All the previously identified E. coli FabR transcriptional target genes (fabA, fabB and yqfA) were shown to be direct S. Typhimurium FabR targets as well. As we found a fabB overexpressing strain to partly mimic the biofilm defect of the fabR mutant, the effect of FabR on biofilms can be attributed at least partly to FabB, which plays a key role in UFA biosynthesis. Additionally, ChIP-chip identified a number of novel direct FabR targets (the intergenic regions between hpaR/hpaG and ddg/ydfZ) and yet putative direct targets (i.a. genes involved in tRNA metabolism, ribosome synthesis and translation). Next to UFA biosynthesis, a number of these direct targets and other indirect targets identified by transcriptomics (e.g. ribosomal genes, ompA, ompC, ompX, osmB, osmC, sseI), could possibly contribute to the effect of FabR on biofilm formation.Conclusion: Overall, our results point at the importance of FabR and UFA biosynthesis in Salmonella biofilm formation and their role as potential targets for biofilm inhibitory strategies.

Glycoproteins form an interesting class of macromolecules involved in bacterial-host interactions, but they are not yet widely explored in Gram-positive and beneficial species. Here, an integrated and widely applicable approach was followed to identify putative bacterial glycoproteins, combining proteome fractionation with 2D protein and glycostained gels and lectin blots. This approach was validated for the microbiota isolate Lactobacillus rhamnosus GG. The approach resulted in a list of putative glycosylated proteins receiving a 'glycosylation score'. Ultimately, we could identify 41 unique glycosylated proteins in L. rhamnosus GG (6 top-confidence, 10 high-confidence and 25 putative hits; classification based on glycosylation score). Most glycoproteins are associated with the cell wall and membrane. Identified glycoproteins include proteins involved in transport, translation, and sugar metabolism processes. A robust screening resulted in a comprehensive mapping of glycoproteins in L. rhamnosus GG. Our results reflect the glycosylation of sugar metabolism enzymes, transporters, and other proteins crucial for cell physiology. We hypothesize that protein glycosylation can confer an extra level of regulation, for example by affecting enzyme functions. This is the first systematic study of the glycoproteome of a probiotic and beneficial gut isolate.

During the last decade it has been shown that among cell variation in gene expression plays an important role within clonal populations. Here, we provide an overview of the different mechanisms contributing to gene expression variability in clonal populations. These are ranging from inherent variations in the biochemical process of gene expression itself, such as intrinsic noise, extrinsic noise and bistability to individual responses to variations in the local micro-environment, a phenomenon called phenotypic plasticity. Also genotypic variations caused by clonal evolution and phase variation can contribute to gene expression variability. Consequently, gene expression studies need to take these fluctuations in expression into account. However, frequently used techniques for expression quantification, such as microarrays, RNA sequencing, quantitative PCR and gene reporter fusions classically determine the population average of gene expression. Here, we discuss how these techniques can be adapted towards single cell analysis by integration with single cell isolation, RNA amplification and microscopy. Alternatively more qualitative selection-based techniques, such as mutant screenings, in vivo expression technology (IVET) and recombination-based IVET (RIVET) can be applied for detection of genes expressed only within a subpopulation. Finally, differential fluorescence induction (DFI), a protocol specially designed for single cell expression is discussed.

Mapping of the microbial molecules underlying microbiota-host interactions is key to understand how microbiota preserve mucosal homeostasis. A pivotal family of such bacterial molecules are pili. Pili are proteinaceous cell wall appendages with a well-documented role in adhesion, whilst their role in immune interaction with the host is less established. Gram-positive pili are often posttranslationally modified by sortase-specific cleavage reactions and the formation of intramolecular peptide bonds. Here we report glycosylation as a new level of posttranslational modification of sortase-dependent pili of a beneficial microbiota species and its role in immune modulation. We focused on the SpaCBA pili of the model probiotic and beneficial human gut microbiota isolate Lactobacillus rhamnosus GG. A unique combination of molecular techniques, nanoscale mechanical and immunological approaches led to the identification of mannose and fucose residues on the SpaCBA pili. These glycans on the pili are recognized by human dendritic cells via the C-type lectin receptor DC-SIGN, a key carbohydrate-dependent immune tailoring pattern recognition receptor. This specific lectin-sugar interaction is moreover of functional importance and modulated the cytokine response of dendritic cells. This provides insight into the direct role bacterial glycoproteins can play in the immunomodulation of the host. Modification of the complex heterotrimeric pili of a model probiotic and microbiota isolate with mannose and fucose is of importance for the functional interaction with the host immune lectin receptor DC-SIGN on human dendritic cells. Our findings shed light on the yet underappreciated role of glycoconjugates in bacteria-host interactions.

The ability of Salmonella to form complex surface-associated communities, called biofilms, contributes to its resistance and persistence in both host and non-host environments and is especially important in food processing environments. In this review, the different types of abiotic (plastic, glass, cement, rubber, and stainless steel) and biotic surfaces (plant surfaces, epithelial cells, and gallstones) on which Salmonella biofilms have been described are discussed, as well as a number of commonly used laboratory setups to study Salmonella biofilm formation (rdar morphotype, pellicle formation, and biofilms on polystyrene pegs). Furthermore, the structural components important during Salmonella biofilm formation are described (curli and other fimbriae, BapA, flagella, cellulose, colanic acid, anionic O-antigen capsule and fatty acids), with special attention to the structural variations of biofilms grown on different surfaces and under different conditions. Indeed, biofilm formation is strongly influenced by different environmental signals, via a complex regulatory network. An extensive overview is given on the current understanding of this genetic network and the interactions between its different components (CsgD, RpoS, Crl, OmpR, IHF, H-NS, CpxR, MlrA, c-di-GMP, BarA/SirA, Csr, PhoPQ, RstA, Rcs, metabolic processes and quorum sensing). To further illustrate that biofilm formation is a mechanism of Salmonella to adapt to different environments, the resistance of Salmonella biofilms against different stress factors including desiccation stress, disinfectants (e.g. hypochlorite, glutaraldehyde, cationic tensides and triclosan) and antibiotics (e.g. ciprofloxacin) is described. Finally, a number of Salmonella biofilm inhibitors, identified through bottom-up- and top-down-approaches, are discussed, such as surfactin, glucose, halogenated furanones, 4(5)-aryl 2-aminoimidazoles, furocoumarins and salicylates. Also the potential of combination therapy (e.g. combinations of triclosan and quaternary ammonium salts or halogenated furanones and antibiotics/disinfectants) and nano- and micro-emulsions to inhibit Salmonella biofilm formation is discussed. Insight into the pathogen's complex biofilm process will eventually lead to further unraveling of its intricacies and more efficient strategies to combat Salmonella biofilms. ispartof: FOOD RESEARCH INTERNATIONAL vol:45 issue:2 pages:502-531 status: published

Sorafenib is a relatively new cytostatic drug approved for the treatment of renal cell and hepatocellular carcinoma. In this report we describe the synthesis of sorafenib derivatives 4a-e which differ from sorafenib in their amide part. A 4-step synthetic pathway includes preparation of 4-chloropyridine-2-carbonyl chloride hydrochloride (1), 4-chloro-pyridine-2-carboxamides 2a-e, 4-(4-aminophenoxy)-pyridine-2-carboxamides 3a-e and the target compounds 4-[4-[[4-chloro-3-(trifluoromethyl)phenyl]carbamoylamino]-phenoxy]-pyridine-2-carboxamides 4a-e. All compounds were fully chemically characterized and evaluated for their cytostatic activity against a panel of carcinoma, lymphoma and leukemia tumour cell lines. In addition, their antimetabolic potential was investigated as well. The most prominent antiproliferative activity was obtained for compounds 4a-e (IC(50) = 1-4.3 μmol·L-1). Their potency was comparable to the potency of sorafenib, or even better. The compounds inhibited DNA, RNA and protein synthesis to a similar extent and did not discriminate between tumour cell lines and primary fibroblasts in terms of their anti-proliferative activity. ispartof: Molecules vol:17 issue:1 pages:1124-1137 ispartof: location:Switzerland status: published

In inflammatory bowel diseases (IBD), it is known that besides genetic and environmental factors (e.g. diet, drugs, stress), the microbiota play an important role in the pathogenesis. Patients with IBD have an altered microbiota (dysbiosis) and therefore, probiotics, defined as 'live micro-organisms that when administered in adequate amounts can confer a health benefit on the host', have been suggested as nutritional supplements to restore these imbalances. The best response on probiotics among the different types of IBD appears to be in the case of ulcerative colitis. Although probiotics show promise in IBD in both clinical and animal studies, further mechanistic studies are necessary to optimize the use of probiotics as supporting therapy in IBD. Murine models of experimental colitis have been used for decades to study this pathology, and these models have been proven useful to search for new therapeutic approaches. The purpose of this review is to summarize probiotic-host interaction studies in murine models of experimental colitis and to evaluate how these models can further help in understanding these complex interactions. Unraveling the molecular mechanisms behind the beneficial effects will assist in better and possibly more efficient probiotic formulations. ispartof: Molecular Nutrition & Food Research vol:55 issue:10 pages:1441-1453 ispartof: location:Germany status: published

Bacterial biofilm formation is an important cause of environmental persistence of food-borne pathogens, such as Salmonella Typhimurium. As the ensemble of bacterial cells within a biofilm represents different physiological states, even for monospecies biofilms, gene expression patterns in these multicellular assemblages show a high degree of heterogeneity. This heterogeneity might mask differential gene expression that occurs only in subpopulations of the entire biofilm population when using methods that average expression output. In an attempt to address this problem and to refine expression analysis in biofilm studies, we used the Differential Fluorescence Induction (DFI) technique to gain more insight in S. Typhimurium biofilm gene expression. Using this single cell approach, we were able to identify 26 genetic loci showing biofilm specific increased expression. For a selected number of identified genes, we confirmed the DFI results by the construction of defined promoter fusions, measurement of relative gene expression levels and construction of mutants. Overall, we have shown for the first time that the DFI technique can be used in biofilm research. The fact that this analysis revealed genes that have not been linked with Salmonella biofilm formation in previous studies using different approaches illustrates that no single technique, in casu biofilm formation, is able to identify all genes related to a given phenotype.

Azospirillum influences growth and development of several crops by producing phytohormones such as auxins which have a major impact on root development. An improved root system leads to better water and nutrient uptake that in turn may influence yield positively. In this study, two agronomically contrasting sugarcane cvs R 570 and M 1176/77 adapted to different agroclimatic zones were inoculated with Azospirillum sp., with and without drought stress, to gauge how far they could benefit from this bacterial association. As early as 103 days after planting, cv M 1176/77 responded positively to inoculation with 15% improved growth (shoot height) and 75% more root dry mass when subjected to drought stress, whereas cv R 570 responded negatively particularly in the absence of drought stress. The significant interaction of cultivar x water regime x Azospirillum inoculation suggests a complex interplay of these factors, possibly involving the indigenous plant auxin pool. Therefore, plant genotype needs to be taken into account when recommending bacterial inoculation for direct plant growth promotion. Furthermore, enhanced growth under sub-optimal water conditions shows clearly the benefits that could be obtained in semi-arid conditions where water deficits frequently occur.

Summary Probiotic bacteria are administered as live microorganisms to provide a health benefit to the host. Insight into the adaptation factors that promote the survival and persistence of probiotics in the gastrointestinal tract (GIT) is important to understand their performance. In this study, the role of the long galactose-rich exopolysaccharides (EPS) of the prototypical probiotic strain Lactobacillus rhamnosus GG (LGG) was investigated. In a competition experiment with wild type, the isogenic EPS mutant CMPG5351 exhibited a reduced persistence in the murine GIT, especially in the lower parts of the intestine. This was surprising as our previous in vitro studies had shown an increased adhesion capacity for this EPS mutant. Follow-up assays indicated that this mutant is more sensitive towards host innate defence molecules, such as the LL-37 antimicrobial peptide and complement factors. This suggests that EPS forms a protective shield for LGG against these molecules in the GIT. Moreover, culturing LGG wild-type in subinhibitory concentrations of host defence factors such as LL-37 resulted in increased production of EPS, indicating that bacterial EPS production is modulated in the host to fine-tune the balance between adhesion and immune evasion. These observations are of interest in understanding the dynamics of adaptation of probiotics to the host environments.

The probiotic Lactobacillus rhamnosus GG, first isolated from healthy human gut microbiota, has been reported to adhere very well to components of the intestinal mucosa, thereby enabling transient colonization of the gastrointestinal tract (GIT). In a search for the genes responsible for the good adherence capacity of this strain, a genomic region encoding a protein with homology to putative adhesion proteins (LGG_01865) and its putative regulator (LGG_01866) was identified. The sequence of the L. rhamnosus GG LGG_01865 encodes a polypeptide of 2419 amino acid residues containing 26 repetitive DUF1542 domains and a C-terminal LPxTG cell wall-anchoring motif. Phenotypic analyses of a dedicated LGG_01865 knockout mutant revealed a reduced biofilm formation capacity on abiotic surfaces and decreased adhesion to intestinal epithelial cells and tissues of the murine GIT. This suggests a modulating role for LGG_01865 in L. rhamnosus GG-host interactions. Therefore, we propose a new name for LGG_01865, i.e. MabA, modulator of adhesion and biofilm. Expression analysis indicated that LGG_01866 plays a conditional role in the regulation of LGG_01865 expression, i.e. when cells are grown under conditions of sugar starvation. At the time of the experiments, M.P.V. held a PhD grant from the Interfaculty Council for Development Cooperation of the K.U. Leuven (IRO-16302). I.C. holds a PhD grant from the Institute for Science and Technology (IWT, Belgium). Additionally, this work was partially supported by the FWO-Vlaanderen through project G.0236.07 and by the Federal Office for Scientific, Technical and Cultural Affairs (Interuniversity Poles of Attraction Programme). We gratefully acknowledge K. Zhou, D. Verstraeten, K. Schrijvers and E. Dillissen for technical assistance. We thank D. De Coster for his skilled assistance with the qRT-PCR experiments. Dr I. Nagy and Prof. D. Bullens are acknowledged for useful suggestions. Prof. J. Ceuppens and Dr C. Shen are thanked for their guidance with the mouse experiments. We also thank M. Danielsen, P. Augustijns and R. Mols for kindly providing the plasmids and CaCo-2 cells used in this study.

The second-order nonlinear optical properties of two monomeric red fluorescent proteins, mStrawberry and mCherry, have been experimentally determined by frequency-resolved femtosecond hyper-Rayleigh scattering. These proteins were found to exhibit a stronger nonlinear response than the previously described eGFP, eYFP and DsRed,1 confirming the trend that fluorophores with a more extended conjugated system, or a lower-energy band gap between ground and excited state, exhibit a larger static hyperpolarizability (β0). Furthermore, these experimental data were complemented with quantum chemical calculations. A discrepancy was observed between experimental and theoretical results, but this could be explained by the chromophore model extracted from the available X-ray diffraction data. While eGFP showed a larger dynamic experimental response (βHRS) due to the highest resonance enhancement, we measured an even higher signal for mCherry. Furthermore, mCherry also shows a better separation of the second harmonic signal and two-photon excited fluorescent signal, making this the preferred fluorescent protein for second harmonic imaging at 800 nm so far.

The interaction between both beneficial and pathogenic microbes and their host has been the subject of many studies. Although the field of systems biology is rapidly evolving, the use of a systems biology approach by means of high-throughput techniques to study host–microbe interactions is just beginning to be explored. In this review, we discuss some of the most recently developed high-throughput ‘omics’ techniques and their use in the context of host–microbe interaction. Moreover, we highlight studies combining several techniques that are pioneering the integration of ‘omics’ data related to host–microbe interactions. Finally, we list the major challenges ahead for successful systems biology research on host–microbe interactions.

To unravel the biological function of the widely used probiotic bacterium Lactobacillus rhamnosus GG, we compared its 3.0-Mbp genome sequence with the similarly sized genome of L. rhamnosus LC705, an adjunct starter culture exhibiting reduced binding to mucus. Both genomes demonstrated high sequence identity and synteny. However, for both strains, genomic islands, 5 in GG and 4 in LC705, punctuated the colinearity. A significant number of strain-specific genes were predicted in these islands (80 in GG and 72 in LC705). The GG-specific islands included genes coding for bacteriophage components, sugar metabolism and transport, and exopolysaccharide biosynthesis. One island only found in L. rhamnosus GG contained genes for 3 secreted LPXTG-like pilins ( spaCBA ) and a pilin-dedicated sortase. Using anti-SpaC antibodies, the physical presence of cell wall-bound pili was confirmed by immunoblotting. Immunogold electron microscopy showed that the SpaC pilin is located at the pilus tip but also sporadically throughout the structure. Moreover, the adherence of strain GG to human intestinal mucus was blocked by SpaC antiserum and abolished in a mutant carrying an inactivated spaC gene. Similarly, binding to mucus was demonstrated for the purified SpaC protein. We conclude that the presence of SpaC is essential for the mucus interaction of L. rhamnosus GG and likely explains its ability to persist in the human intestinal tract longer than LC705 during an intervention trial. The presence of mucus-binding pili on the surface of a nonpathogenic Gram-positive bacterial strain reveals a previously undescribed mechanism for the interaction of selected probiotic lactobacilli with host tissues.

The knowledge of molecular mechanisms underlying the adhesive and mechanical properties of cell surface-associated molecules is a key to understanding their functions. In this context, single-molecule force spectroscopy (SMFS) has recently offered new opportunities for probing the adhesion and mechanics of polysaccharides and proteins on live cells. Here we present a protocol that we have used to analyze polysaccharide chains of different nature on the bacterium Lactobacillus rhamnosus GG. We describe procedures (i) for functionalizing atomic force microscopy (AFM) tips with Pseudomonas aeruginosa-I or concanavalin A lectins, (ii) for stretching specific polysaccharide molecules on live bacteria using SMFS with lectin tips and (iii) for mapping the localization, adhesion and extension of individual polysaccharide chains. We also discuss data treatment, emphasizing how to gain insight into the elasticity of the stretched macromolecules using the extended freely jointed chain model. Even though the presented protocol is for L. rhamnosus, it can be easily modified for other cell types. For users having expertise in the field, the entire protocol can be completed in about 5 d.

In the rhizosphere and their interaction with plants rhizobia encounter many different plant compounds, including phytohormones like auxins. Moreover, some rhizobial strains are capable of producing the auxin, indole-3-acetic acid (IAA). However, the role of IAA for the bacterial partner in the legume– Rhizobium symbiosis is not known. To identify the effect of IAA on rhizobial gene expression, a transposon (mTn 5gusA - oriV ) mutant library of Rhizobium etli , enriched for mutants that show differential gene expression under microaerobiosis and/or addition of nodule extracts as compared with control conditions, was screened for altered gene expression upon IAA addition. Four genes were found to be regulated by IAA. These genes appear to be involved in plant signal processing, motility or attachment to plant roots, clearly demonstrating a distinct role for IAA in legume– Rhizobium interactions.

Quorum sensing (QS) systems are bacterial cell-to-cell communication systems that use small molecules as signals. Since QS is involved in the regulation of virulence and biofilm formation in several pathogenic bacteria, it has been suggested as a new target for the development of novel antibacterial therapies. As such, interference with the signal receptors by using chemical compounds has been proposed as an alternative strategy for treatment of bacterial infections and has already shown promising results in combination with traditional antibiotic treatments. In Gram-negative bacteria, the best studied QS systems use N-acyl homoserine lactones (AHLs) as signal molecules. This review provides an overview of all new chemical structure types that inhibit AHL-mediated QS systems as reported during the last three years in scientific journals and in the patent literature. The compounds were classified into three main groups depending on their structure: AHL analogues, 2(5H)-furanones, and compounds that are not structurally related to AHLs. We discuss the biological assays used and the different strategies applied to discover these molecules, including new approaches such as molecular docking for in silico identification of lead structures and random high-throughput screening of large libraries of chemicals. Finally, we elaborate on structure-activity relationships and on the new insights in the mechanisms of action of the identified inhibitors, highlighting the potential of these small molecules in medicine.

Despite the increasing number of scientific reports describing adhesion of Lactobacillus to components of the human intestinal mucosa, information on the surface molecules mediating this adhesion and their corresponding receptors is fragmentary. This MiniReview compiles present knowledge of the genetically and functionally characterized Lactobacillus factors responsible for mediating adhesion to different components of the human gastrointestinal tract. In addition, for the proteins among these factors, the domain structure is discussed, and where appropriate the results of in silico analyses are reported.

Lactobacillus rhamnosus GG (ATCC 53103) is one of the clinically best-studied probiotic organisms. Moreover, L. rhamnosus GG displays very good in vitro adherence to epithelial cells and mucus. Here, we report that L. rhamnosus GG is able to form biofilms on abiotic surfaces, in contrast to other strains of the Lactobacillus casei group tested under the same conditions. Microtiter plate biofilm assays indicated that in vitro biofilm formation by L. rhamnosus GG is strongly modulated by culture medium factors and conditions related to the gastrointestinal environment, including low pH; high osmolarity; and the presence of bile, mucins, and nondigestible polysaccharides. Additionally, phenotypic analysis of mutants affected in exopolysaccharides ( wzb ), lipoteichoic acid ( dltD ), and central metabolism ( luxS ) showed their relative importance in biofilm formation by L. rhamnosus GG.

Aims: Considering the significant rise in the probiotic market in Columbia, and given the lack of reports concerning the microbial population and strain performance in products from different producers, this study aims at determining the number of viable starter bacteria and probiotics in bio-yoghurts available at the Columbian market, identifying the species and analysing the performance of the isolated strains in bile acid resistance, antagonistic activity against pathogens, and adherence capacity to human intestinal epithelial cells. Methods and Results: Seven bio-yoghurts were analysed for the bacterial species present. Species identification was carried out using 16S rRNA gene targeted PCR. The cultured bacteria were tested for bile acid resistance, adherence to a human intestinal epithelial cell line, and antagonism against the pathogen Salmonella enterica serovar Typhimurium. A total of 17 different strains were identified. Based on plate counting, all bio-yoghurts have at least total viable cells of ∼107 CFU ml−1. Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus were the most frequently isolated bacteria. Viable Bifidobacterium was only recovered from one product. However, after PCR analysis, DNA of this genus was confirmed in five out of seven products. Major differences were found for S. typhimurium antagonism. The adherence capacity to Caco-2 cells was observed in 10 of the isolated strains. In general, low survival to simulated gastric juice was observed. Conclusions: Some of the isolated strains have probiotic potential, although not all of them were present in the advised amount to exert beneficial health effects. However, the full correct scientific name of the isolated bacteria and their viable counts were not included on the product label. Significance and Impact of the Study: This is the first report describing the identification and functionality of starter bacteria and probiotics present in dairy products on the Columbian market.

Lipoteichoic acid (LTA) is a macroamphiphile molecule which performs several functions in gram-positive bacteria, such as maintenance of cell wall homeostasis. d -Alanylation of LTA requires the proteins encoded by the dlt operon, and this process is directly related to the charge properties of this polymer strongly contributing to its function. The insertional inactivation of dltD of the probiotic strain Lactobacillus rhamnosus GG (ATCC 53103) resulted in the complete absence of d -alanyl esters in the LTA as confirmed by nuclear magnetic resonance analysis. This was reflected in modifications of the bacterial cell surface properties. The dltD strain showed 2.4-fold-increased cell length, a low survival capacity in response to gastric juice challenge, an increased sensitivity to human beta-defensin-2, an increased rate of autolysis, an increased capacity to initiate growth in the presence of an anionic detergent, and a decreased capacity to initiate growth in the presence of cationic peptides compared to wild-type results. However, in vitro experiments revealed no major differences for adhesion to human intestinal epithelial cells, biofilm formation, and immunomodulation. These properties are considered to be important for probiotics. The role of the dlt operon in lactobacilli is discussed in view of these results.

Swarming motility is considered to be a social phenomenon that enables groups of bacteria to move coordinately atop solid surfaces. The differentiated swarmer cell population is embedded in an extracellular slime layer, and the phenomenon has previously been linked with biofilm formation and virulence. The gram-negative nitrogen-fixing soil bacterium Rhizobium etli CNPAF512 was previously shown to display swarming behavior on soft agar plates. In a search for novel genetic determinants of swarming, a detailed analysis of the swarming behavior of 700 miniTn5 mutants of R. etli was performed. Twenty-four mutants defective in swarming or displaying abnormal swarming patterns were identified and could be divided into three groups based on their swarming pattern. Fourteen mutants were completely swarming deficient, five mutants showed an atypical swarming pattern with no completely smooth edge and local extrusions, and five mutants displayed an intermediate swarming phenotype. Sequence analysis of the targeted genes indicated that the mutants were likely affected in quorum-sensing, polysaccharide composition or export, motility, and amino acid and polyamines metabolism. Several of the identified mutants displayed a reduced symbiotic nitrogen fixation activity.

Sulfate modification on Rhizobium Nod factor signaling molecules is not a prerequisite for successful symbiosis with the common bean (Phaseolus vulgaris L.). However, many bean-nodulating rhizobia, including the broad host strain Sinorhizobium sp. BR816, produce sulfated Nod factors. Here, we show that the nodH gene, encoding a sulfotransferase, is responsible for the transfer of sulfate to the Nod factor backbone in Sinorhizobium sp. BR816, as was shown for other rhizobia. Interestingly, inactivation of nodH enables inoculated bean plants to fix significantly more nitrogen under different experimental setups. Our studies show that nodH in the wild-type strain is still expressed during the later stages of symbiosis. This is the first report on enhanced nitrogen fixation by blocking Nod factor sulfation.

The last decades, the research on cell-cell communication or quorum sensing has been quite intense. Bacterial communication allows bacteria to coordinate their behavior and to act as one entity. Quorum sensing controls microbiological functions of medical, agricultural and industrial importance and a better understanding of the underlying mechanisms and the conditions under which the signaling occurs, offers possibilities for new applications. In this paper the production of the AI-2 signaling molecule by Salmonella Typhimurium is investigated in a controlled environment (bioreactor). The experimental results are applied to build a macroscopic model that describes the evolution of the AI- 2 production and uptake by Salmonella Typhimurium in relation to the growth kinetics and biomass concentration. Copyright c 2007 IFAC