Genomic and transcriptomic analysis of Ligilactobacillus salivarius IBB3154-in search of new promoters for vaccine construction.

IMPORTANCE: The genome of the strain Ligilactobacillus salivarius IBB3154 was sequenced, and transcriptome analysis was carried out at two different temperatures, allowing the determination of gene expression levels in response to environmental changes (temperature). Genes with higher expression at 42°C were identified. The use of a reporter gene (ß- glucuronidase) did not confirm the transcriptomic results; it was found that the promoters of the genes sasA1 and sasA2 were active in the presence of bile salts. This opens up new opportunities for the overexpression of genes of other bacterial species in Ligilactobacillus cells in the intestinal environment.

Health-Promoting Nature of Lactococcus lactis IBB109 and Lactococcus lactis IBB417 Strains Exhibiting Proliferation Inhibition and Stimulation of Interleukin-18 Expression in Colorectal Cancer Cells.

Lactic acid bacteria (LAB) are Gram-positive bacteria which are considered for use as adjuvant therapeutics in management of various disease ailments, including obesity, irritable bowel syndrome, lactose intolerance and cancer. To investigate the possible use of Lactococcus lactis strains from our collection in treatment of gastrointestinal cancer, we tested them for the ability to arrest proliferation of human colorectal adenocarcinoma cells (Caco-2). Results of the BrdU assay showed that the anti-proliferative activity of L. lactis cells is strain-specific. We found that particularly, two strains, L. lactis IBB109 and L. lactis IBB417, exhibited the most potent inhibitory effect. Moreover, both strains triggered interleukin 18 gene expression, normally inhibited in Caco-2 (cancer) cells. To examine the probiotic potential of the two strains, we tested them for bile salts and acid tolerance, as well as adhesion properties. Both isolates exhibited probiotic potential-they survived in the presence of 0.3% bile salts and tolerated exposure to low pH and osmotic stress. Notably, we found that L. lactis IBB417 displayed better adherence to mucus and Caco-2 cells than L. lactis IBB109. Additionally, by microdilution tests we confirmed that both strains are sensitive to all nine antibiotics of human and veterinary importance listed by the European Food Safety Authority. Finally, by in silico investigations of whole genome sequencing data, we revealed the genetic features of L. lactis IBB109 and L. lactis IBB417 that can be associated with functional (e.g., adhesion and carbohydrate metabolic genes) and safety (e.g., virulence and antibiotic resistance) aspects of the strains, confirming their health-promoting potential.

An Adenosine Triphosphate- Dependent 5'-3' DNA Helicase From sk1-Like Lactococcus lactis F13 Phage.

Here, we describe functional characterization of an early gene (gp46) product of a virulent Lactococcus lactis sk1-like phage, vB_Llc_bIBBF13 (abbr. F13). The GP46 F13 protein carries a catalytically active RecA-like domain belonging to the P-loop NTPase superfamily. It also retains features characteristic for ATPases forming oligomers. In order to elucidate its detailed molecular function, we cloned and overexpressed the gp46 gene in Escherichia coli. Purified GP46 F13 protein binds to DNA and exhibits DNA unwinding activity on branched substrates in the presence of adenosine triphosphate (ATP). Size exclusion chromatography with multi-angle light scattering (SEC-MALS) experiments demonstrate that GP46 F13 forms oligomers, and further pull-down assays show that GP46 F13 interacts with host proteins involved in replication (i.e., DnaK, DnaJ, topoisomerase I, and single-strand binding protein). Taking together the localization of the gene and the obtained results, GP46 F13 is the first protein encoded in the early-expressed gene region with helicase activity that has been identified among lytic L. lactis phages up to date.

LactococcusCeduovirus Phages Isolated from Industrial Dairy Plants-from Physiological to Genomic Analyses.

LactococcusCeduovirus (formerly c2virus) bacteriophages are among the three most prevalent phage types reported in dairy environments. Phages from this group conduct a strictly lytic lifestyle and cause substantial losses during milk fermentation processes, by infecting lactococcal host starter strains. Despite their deleterious activity, there are limited research data concerning Ceduovirus phages. To advance our knowledge on this specific phage group, we sequenced and performed a comparative analysis of 10 new LactococcuslactisCeduovirus phages isolated from distinct dairy environments. Host range studies allowed us to distinguish the differential patterns of infection of L. lactis cells for each phage, and revealed a broad host spectrum for most of them. We showed that 40% of the studied Ceduovirus phages can infect both cremoris and lactis strains. A preference to lyse strains with the C-type cell wall polysaccharide genotype was observed. Phage whole-genome sequencing revealed an average nucleotide identity above 80%, with distinct regions of divergence mapped to several locations. The comparative approach for analyzing genomic data and the phage lytic spectrum suggested that the amino acid sequence of the orf8-encoded putative tape measure protein correlates with host range. Phylogenetic studies revealed separation of the sequenced phages into two subgroups. Finally, we identified three types of phage origin of replication regions, and showed they are able to support plasmid replication without additional phage proteins.

Bacterial community dynamics in spontaneous sourdoughs made from wheat, spelt, and rye wholemeal flour.

Sourdough fermentation is a traditional process that is used to improve bread quality. A spontaneous sourdough ecosystem consists of a mixture of flour and water that is fermented by endogenous lactic acid bacteria (LAB) and yeasts. The aim of this study was to identify bacterial diversity during backslopping of spontaneous sourdoughs prepared from wheat, spelt, or rye wholemeal flour. Culture-dependent analyses showed that the number of LAB (109  CFU/ml) was higher by three orders of magnitude than the number of yeasts (106  CFU/ml), irrespective of the flour type. These results were complemented by next-generation sequencing of the 16S rDNA V3 and V4 variable regions. The dominant phylum in all sourdough samples was Firmicutes, which was represented exclusively by the Lactobacillales order. The two remaining and less abundant phyla were Proteobacteria and Bacteroidetes. The culture-independent approach allowed us to detect changes in microbial ecology during the 72-hr fermentation period. Weissella sp. was the most abundant genus after 24 hr of fermentation of the rye sourdough, but as the process progressed, its abundance decreased in favor of the Lactobacillus genus similarly as in wheat and spelt sourdoughs. The Lactobacillus genus was dominant in all sourdoughs after 72 hr, which was consistent with our results obtained using culture-dependent analyses. This work was carried out to determine the microbial biodiversity of sourdoughs that are made from wheat, spelt, and rye wholemeal flour and can be used as a source of strains for specific starter cultures to produce functional bread.

Potential of Lactobacillus plantarum IBB3036 and Lactobacillus salivarius IBB3154 to persistence in chicken after in ovo delivery.

The aim of this study was to characterize and compare selected Lactobacillus strains originating from different environments (cow milk and hen feces) with respect to their applicative potential to colonize gastrointestinal track of chickens before hatching from an egg. In vitro phenotypic characterization of lactobacilli strains included the investigation of the important prerequisites for persistence in gastrointestinal tract, such as a capability to survive in the presence of bile salts and at low pH, enzymatic and sugar metabolic profiles, adhesion abilities, and resistance to osmolytes, temperature, and antibiotics. Regarding the resistance of lactobacilli to most of the various stress factors tested, the milk isolate Lactobacillus plantarum IBB3036 showed better abilities than the chicken feces isolate Lactobacillus salivarius IBB3154. However, regarding the acidification tolerance and adherence ability, L. salivarius IBB3154 revealed better characteristics. Use of these two selected lactobacilli isolates together with proper prebiotics resulted in the preparation of two S1 and S2 bioformulations, which were injected in ovo into hen Cobb500 FF fertilized eggs. Furthermore, in vivo tests assessing the persistence of L. plantarum IBB3036 and L. salivarius IBB3154 in the chicken gastrointestinal tract was monitored by PCR-based classical and quantitative techniques and revealed the presence of both strains in fecal samples collected 3 days after hatching. Subsequently, the number of L. salivarius IBB3154 increased significantly in the chicken intestine, whereas the presence of L. plantarum IBB3036 was gradually decreased.

Toxicity of Food-Grade TiO2 to Commensal Intestinal and Transient Food-Borne Bacteria: New Insights Using Nano-SIMS and Synchrotron UV Fluorescence Imaging.

Titanium dioxide (TiO2) is commonly used as a food additive (E171 in the EU) for its whitening and opacifying properties. However, a risk of intestinal barrier disruption, including dysbiosis of the gut microbiota, is increasingly suspected because of the presence of a nano-sized fraction in this additive. We hypothesized that food-grade E171 and Aeroxyde P25 (identical to the NM-105 OECD reference nanomaterial in the European Union Joint Research Centre) interact with both commensal intestinal bacteria and transient food-borne bacteria under non-UV-irradiated conditions. Based on differences in their physicochemical properties, we expect a difference in their respective effects. To test these hypotheses, we chose a panel of eight Gram-positive/Gram-negative bacterial strains, isolated from different biotopes and belonging to the species Escherichia coli, Lactobacillus rhamnosus, Lactococcus lactis (subsp. lactis and cremoris), Streptococcus thermophilus, and Lactobacillus sakei. Bacterial cells were exposed to food-grade E171 vs. P25 in vitro and the interactions were explored with innovative (nano)imaging methods. The ability of bacteria to trap TiO2 was demonstrated using synchrotron UV fluorescence imaging with single cell resolution. Subsequent alterations in the growth profiles were shown, notably for the transient food-borne L. lactis and the commensal intestinal E. coli in contact with food-grade TiO2. However, for both species, the reduction in cell cultivability remained moderate, and the morphological and ultrastructural damages, observed with electron microscopy, were restricted to a small number of cells. E. coli exposed to food-grade TiO2 showed some internalization of TiO2 (7% of cells), observed with high-resolution nano-secondary ion mass spectrometry (Nano-SIMS) chemical imaging. Taken together, these data show that E171 may be trapped by commensal and transient food-borne bacteria within the gut. In return, it may induce some physiological alterations in the most sensitive species, with a putative impact on gut microbiota composition and functioning, especially after chronic exposure.

In vitro characteristics of Lactobacillus spp. strains isolated from the chicken digestive tract and their role in the inhibition of Campylobacter colonization.

Campylobacter jejuni/coli infections are the leading cause of bacterial diarrheal illnesses in humans. Many epidemiological studies indicate that improperly prepared meat from chickens that carry a high load of Campylobacter in their intestinal tracts is the key source of human infections. LAB, mainly members of the Lactococcus and Lactobacillus genera, increasingly have been tested as vehicles for the delivery of heterologous bacterial or viral antigens to animal mucosal immune systems. Thus, the objective of this study was to isolate, identify, and characterize Lactobacillus spp. strains isolated from chickens bred in Poland. Their ability to decrease the level of bird gut colonization by C. jejuni strain was also analyzed. First, the influence of the different chicken rearing systems was evaluated, especially the effect of diets on the Lactobacillus species that colonize the gut of chickens. Next, selected strains were analyzed in terms of their anti-Campylobacter activity in vitro; potential probiotic traits such as adhesion properties, bile and low pH tolerance; and their ability to grow on a defined carbon source. Given that improperly prepared chicken meat is the main source of human infection by Campylobacter, the selected strains were also assessed for their ability to inhibit Campylobacter colonization in the bird's intestine. These experiments revealed enormous physiological diversity among the Lactobacillus genus strains. Altogether, our results showed that L. plantarum strains isolated from the digestive tracts of chickens bred in Poland displayed some probiotic attributes in vitro and were able to decrease the level of bird gut colonization by C. jejuni strain. This suggests that they can be employed as vectors to deliver Campylobacter immunodominant proteins to the bird's immune system to strengthen the efficacy of in ovo vaccination.

Termination factor Rho: From the control of pervasive transcription to cell fate determination in Bacillus subtilis.

In eukaryotes, RNA species originating from pervasive transcription are regulators of various cellular processes, from the expression of individual genes to the control of cellular development and oncogenesis. In prokaryotes, the function of pervasive transcription and its output on cell physiology is still unknown. Most bacteria possess termination factor Rho, which represses pervasive, mostly antisense, transcription. Here, we investigate the biological significance of Rho-controlled transcription in the Gram-positive model bacterium Bacillus subtilis. Rho inactivation strongly affected gene expression in B. subtilis, as assessed by transcriptome and proteome analysis of a rho-null mutant during exponential growth in rich medium. Subsequent physiological analyses demonstrated that a considerable part of Rho-controlled transcription is connected to balanced regulation of three mutually exclusive differentiation programs: cell motility, biofilm formation, and sporulation. In the absence of Rho, several up-regulated sense and antisense transcripts affect key structural and regulatory elements of these differentiation programs, thereby suppressing motility and biofilm formation and stimulating sporulation. We dissected how Rho is involved in the activity of the cell fate decision-making network, centered on the master regulator Spo0A. We also revealed a novel regulatory mechanism of Spo0A activation through Rho-dependent intragenic transcription termination of the protein kinase kinB gene. Altogether, our findings indicate that distinct Rho-controlled transcripts are functional and constitute a previously unknown built-in module for the control of cell differentiation in B. subtilis. In a broader context, our results highlight the recruitment of the termination factor Rho, for which the conserved biological role is probably to repress pervasive transcription, in highly integrated, bacterium-specific, regulatory networks.

Contribution of plasmid-encoded peptidase S8 (PrtP) to adhesion and transit in the gut of Lactococcus lactis IBB477 strain.

The ability of Lactococcus lactis to adhere to the intestinal mucosa can potentially prolong the contact with the host, and therefore favour its persistence in the gut. In the present study, the contribution of plasmid-encoded factors to the adhesive and transit properties of the L. lactis subsp. cremoris IBB477 strain was investigated. Plasmid-cured derivatives as well as deletion mutants were obtained and analysed. Adhesion tests were performed using non-coated polystyrene plates, plates coated with mucin or fibronectin and mucus-secreting HT29-MTX intestinal epithelial cells. The results indicate that two plasmids, pIBB477a and b, are involved in adhesion of the IBB477 strain. One of the genes localised on plasmid pIBB477b (AJ89_14230), which encodes cell wall-associated peptidase S8 (PrtP), mediates adhesion of the IBB477 strain to bare, mucin- and fibronectin-coated polystyrene, as well as to HT29-MTX cells. Interactions between bacteria and mucus secreted by HT29-MTX cells were further investigated by fluorescent staining and confocal microscopy. Confocal images showed that IBB477 forms dense clusters embedded in secreted mucus. Finally, the ability of IBB477 strain and its ΔprtP deletion mutant to colonise the gastrointestinal tract of conventional C57Bl/6 mice was determined. Both strains were present in the gut for up to 72 h. In summary, adhesion and persistence of IBB477 were analysed by in vitro and in vivo approaches, respectively. Our studies revealed that plasmidic genes encoding cell surface proteins are more involved in the adhesion of IBB477 strain than in the ability to confer a selective advantage in the gut.

Recombinant Lactococcus lactis Expressing Haemagglutinin from a Polish Avian H5N1 Isolate and Its Immunological Effect in Preliminary Animal Trials.

Lactic acid bacteria (LAB) are Gram-positive, nonpathogenic microorganisms that are gaining much interest as antigen producers for development of live vaccine vectors. Heterologous proteins of different origin have been successfully expressed in various LAB species, including Lactococcus lactis. Recombinant L. lactis strains have been shown to induce specific local and systemic immune responses against various antigens. Our study aimed at constructing a L. lactis strain expressing haemagglutinin of a Polish avian H5H1 influenza isolate and examining its effect on animals. Expression of the cloned H5 gene was achieved using the nisin-controlled gene expression system. Detection of the intracellular H5 antigen produced in L. lactis was performed by Western blot analysis and confirmed using mass spectrometry. The potential of L. lactis recombinant cells to induce an immune response was examined by setting up preliminary immunization trials on chickens and mice. Obtained sera were tested for specific antibodies by ELISA assays. The results of these studies are a promising step toward developing a vaccine against the bird flu using Lactococcus lactis cells as bioreactors for efficient antigen production and delivery to the mucosal surface.

Adhesion of the genome-sequenced Lactococcus lactis subsp. cremoris IBB477 strain is mediated by specific molecular determinants.

Understanding the nature of mucus-microbe interactions will provide important information that can help to elucidate the mechanisms underlying probiotic adhesion. This study focused on the adhesive properties of the Lactococcus lactis subsp. cremoris IBB477 strain, previously shown to persist in the gastrointestinal tract of germ-free rats. The shear flow-induced detachment of L. lactis cells was investigated under laminar flow conditions. Such a dynamic approach demonstrated increased adhesion to bare and mucin-coated polystyrene for IBB477, compared to that observed for the MG1820 control strain. To identify potential genetic determinants giving adhesive properties to IBB477, the improved high-quality draft genome sequence comprising chromosome and five plasmids was obtained and analysed. The number of putative adhesion proteins was determined on the basis of surface/extracellular localisation and/or the presence of adhesion domains. To identify proteins essential for the IBB477 specific adhesion property, nine deletion mutants in chromosomal genes have been constructed and analysed using adhesion tests on bare polystyrene as well as mucin-, fibronectin- or collagen IV-coated polystyrene plates in comparison to the wild-type strain. These experiments demonstrated that gene AJ89_07570 encoding a protein containing DUF285, MucBP and four Big_3 domains is involved in adhesion to bare and mucin-coated polystyrene. To summarise, in the present work, we characterised the adhesion of IBB477 under laminar flow conditions; identified the putative adherence factors present in IBB477, which is the first L. lactis strain exhibiting adhesive and mucoadhesive properties to be sequenced and demonstrated that one of the proteins containing adhesion domains contributes to adhesion.

Transcription termination factor Rho: a hub linking diverse physiological processes in bacteria.

Factor-dependent termination of transcription in bacteria relies on the activity of a specific RNA helicase, the termination factor Rho. Rho is nearly ubiquitous in bacteria, but the extent to which its physiological functions are conserved throughout the different phyla remains unknown. Most of our current knowledge concerning the mechanism of Rho's activity and its physiological roles comes from the model micro-organism Escherichia coli, where Rho is essential and involved in the control of several important biological processes. However, the rather comprehensive knowledge about the general mechanisms of action and activities of Rho based on the E. coli paradigm cannot be directly extrapolated to other bacteria. Recent studies performed in different species favour the view that Rho-dependent termination plays a significant role even in bacteria where Rho is not essential. Here, we summarize the current state of the ever-increasing knowledge about the various aspects of the physiological functions of Rho, such as limitation of deleterious foreign DNA expression, control of gene expression, suppression of pervasive transcription, prevention of R-loops and maintenance of chromosome integrity, focusing on similarities and differences of the activities of Rho in various bacterial species.

Genomic and Functional Characterization of the Unusual pLOCK 0919 Plasmid Harboring the spaCBA Pili Cluster in Lactobacillus casei LOCK 0919.

Here, we report the extensive bioinformatic and functional analyses of the unusual pLOCK 0919, a plasmid originating from the probiotic Lactobacillus casei LOCK 0919 strain. This plasmid is atypical because it harbors the spaCBA-srtC gene cluster encoding SpaCBA pili. We show that all other spaCBA-srtC sequences of the Lactobacillus genus that have been previously described and deposited in GenBank are present in the chromosomal DNA. Another important observation for pLOCK 0919 is that the spaCBA-srtC gene cluster and its surrounding genes are highly similar to the respective DNA region that is present in the most well-known and active SpaCBA pili producer, the probiotic Lactobacillus rhamnosus GG strain. Our results demonstrate that the spaCBA-srtC clusters of pLOCK 0919 and L. rhamnosus GG are genealogically similar, located in DNA regions that are rich in transposase genes and are poorly conserved among the publicly available sequences of Lactobacillus sp. In contrast to chromosomally localized pilus gene clusters from L. casei and Lactobacillus paracasei, the plasmidic spaC of L. casei LOCK 0919 is expressed and undergoes a slight glucose-induced repression. Moreover, results of series of in vitro tests demonstrate that L. casei LOCK 0919 has an adhesion potential, which is largely determined by the presence of the pLOCK 0919 plasmid. In particular, the plasmid occurrence positively influenced the hydrophobicity and aggregation abilities of L. casei LOCK 0919. Moreover, in vivo studies indicate that among the three Lactobacillus strains used to colonize the gastrointestinal tract of germ-free mice, already after 2 days of colonization, L. casei LOCK 0919 became the dominant strain and persisted there for at least 48 days.

Identification and characterization of tetracycline resistance in Lactococcus lactis isolated from Polish raw milk and fermented artisanal products.

To assess the occurrence of antibiotic-resistant Lactic Acid Bacteria (LAB) in Polish raw milk and fermented artisanal products, a collection comprising 500 isolates from these products was screened. Among these isolates, six strains (IBB28, IBB160, IBB161, IBB224, IBB477 and IBB487) resistant to tetracycline were identified. The strains showing atypical tetracycline resistance were classified as Lactococcus lactis: three of them were identified as L. lactis subsp. cremoris (IBB224, IBB477 and IBB487) and the other three (IBB28, IBB160, IBB161) were identified as L. lactis subsp. lactis. The mechanism involving Ribosomal Protection Proteins (RPP) was identified as responsible for tetracycline resistance. Three of the tested strains (IBB28, IBB160 and IBB224) had genes encoding the TetS protein, whereas the remaining three (IBB161, IBB477 and IBB487) expressed TetM. The results also demonstrated that the genes encoding these proteins were located on genetic mobile elements. The tet(S) gene was found to be located on plasmids, whereas tet(M) was found within the Tn916 transposon.

Oral Administration of Lactococcus lactis Expressing Synthetic Genes of Myelin Antigens in Decreasing Experimental Autoimmune Encephalomyelitis in Rats.

BACKGROUND: Multiple sclerosis is a human autoimmunological disease that causes neurodegeneration. One of the potential ways to stop its development is induction of oral tolerance, whose effect lies in decreasing immune response to the fed antigen. It was shown in animal models that administration of specific epitopes of the three main myelin proteins - myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), and proteolipid protein (PLP) - results in induction of oral tolerance and suppression of disease symptoms. Use of bacterial cells to produce and deliver antigens to gut mucosa seems to be an attractive method for oral tolerance induction in treatment of diseases with autoimmune background. MATERIAL AND METHODS: Synthetic genes of MOG35-55, MBP85-97, and PLP139-151 myelin epitopes were generated and cloned in Lactococcus lactis under a CcpA-regulated promoter. The tolerogenic effect of bacterial preparations was tested on experimental autoimmune encephalomyelitis, which is the animal model of MS. EAE was induced in rats by intradermal injection of guinea pig spinal cord homogenate into hind paws. RESULTS: Rats were administered preparations containing whole-cell lysates of L. lactis producing myelin antigens using different feeding schemes. Our study demonstrates that 20-fold, but not 4-fold, intragastric administration of autoantigen-expressing L. lactis cells under specific conditions reduces the clinical symptoms of EAE in rats. CONCLUSIONS: The present study evaluated the use of myelin antigens produced in L. lactis in inhibiting the onset of experimental autoimmune encephalomyelitis in rats. Obtained results indicate that application of such recombinant cells can be an attractive method of oral tolerance induction.

Lactic acid bacteria--20 years exploring their potential as live vectors for mucosal vaccination.

Lactic acid bacteria (LAB) are a diverse group of Gram-positive, nonsporulating, low G + C content bacteria. Many of them have been given generally regarded as safe status. Over the past two decades, intensive genetic and molecular research carried out on LAB, mainly Lactococcus lactis and some species of the Lactobacillus genus, has revealed new, potential biomedical LAB applications, including the use of LAB as adjuvants, immunostimulators, or therapeutic drug delivery systems, or as factories to produce therapeutic molecules. LAB enable immunization via the mucosal route, which increases effectiveness against pathogens that use the mucosa as the major route of entry into the human body. In this review, we concentrate on the encouraging application of Lactococcus and Lactobacillus genera for the development of live mucosal vaccines. First, we present the progress that has recently been made in the field of developing tools for LAB genetic manipulations, which has resulted in the successful expression of many bacterial, parasitic, and viral antigens in LAB strains. Next, we discuss the factors influencing the efficacy of the constructed vaccine prototypes that have been tested in various animal models. Apart from the research focused on an application of live LABs as carriers of foreign antigens, a lot of work has been recently done on the potential usage of nonliving, nonrecombinant L. lactis designated as Gram-positive enhancer matrix (GEM), as a delivery system for mucosal vaccination. The advantages and disadvantages of both strategies are also presented.

Lactic acid bacteria as a surface display platform for Campylobacter jejuni antigens.

BACKGROUND: Food poisoning and diarrheal diseases continue to pose serious health care and socioeconomic problems worldwide. Campylobacter spp. is a very widespread cause of gastroenteritis. Over the past decade there has been increasing interest in the use of lactic acid bacteria (LAB) as mucosal delivery vehicles. They represent an attractive opportunity for vaccination in addition to vaccination with attenuated bacterial pathogens. METHODS: We examined the binding ability of hybrid proteins to nontreated or trichloroacetic acid (TCA)-pretreated LAB cells by immunofluorescence and Western blot analysis. RESULTS: In this study we evaluated the possibility of using GEM (Gram-positive enhancer matrix) particles of Lactobacillus salivarius as a binding platform for 2 conserved, immunodominant, extracytoplasmic Campylobacter jejuni proteins: CjaA and CjaD. We analyzed the binding ability of recombinant proteins that contain C. jejuni antigens (CjaA or CjaD) fused with the protein anchor (PA) of the L. lactis peptidoglycan hydrolase AcmA, which comprises 3 LysM motifs and determines noncovalent binding to the cell wall peptidoglycan. Both fused proteins, i.e. 6HisxCjaAx3LysM and 6HisxCjaDx3LysM, were able to bind to nontreated or TCA-pretreated L. salivarius cells. CONCLUSION: Our results documented that the LysM-mediated binding system allows us to construct GEM particles that present 2 C. jejuni antigens.

ClaR--a novel key regulator of cellobiose and lactose metabolism in Lactococcus lactis IL1403.

In a number of previous studies, our group has discovered an alternative pathway for lactose utilization in Lactococcus lactis that, in addition to a sugar-hydrolyzing enzyme with both P-ß-glucosidase and P-ß-galactosidase activity (BglS), engages chromosomally encoded components of cellobiose-specific PTS (PTS(Cel-Lac)), including PtcA, PtcB, and CelB. In this report, we show that this system undergoes regulation via ClaR, a novel activator protein from the RpiR family of transcriptional regulators. Although RpiR proteins are widely distributed among lactic acid bacteria, their roles have yet to be confirmed by functional assays. Here, we show that ClaR activity depends on intracellular cellobiose-6-phosphate availability, while other sugars such as glucose or galactose have no influence on it. We also show that ClaR is crucial for activation of the bglS and celB expression in the presence of cellobiose, with some limited effects on ptcA and ptcB activation. Among 190 of carbon sources tested, the deletion of claR reduces L. lactis growth only in lactose- and/or cellobiose-containing media, suggesting a narrow specificity of this regulator within the context of sugar metabolism.