Predicting cis-acting elements of Lactobacillus plantarum by comparative genomics with different taxonomic subgroups

Cis-acting elements in Lactobacillus plantarum were predicted by comparative analysis of the upstream regions of conserved genes and predicted transcriptional units (TUs) in different bacterial genomes. TUs were predicted for two species sets, with different evolutionary distances to L.plantarum. TUs were designated ‘cluster of orthologous transcriptional units’ (COT) when >50% of the genes were orthologous in different species. Conserved DNA sequences were detected in the upstream regions of different COTs. Subsequently, conserved motifs were used to scan upstream regions of all TUs. This method revealed 18 regulatory motifs only present in lactic acid bacteria (LAB). The 18 LAB-specific candidate regulatory motifs included 13 that were not described previously. These LAB-specific different motifs were found in front of genes encoding functions varying from cold shock proteins to RNA and DNA polymerases, and many unknown functions. The best-described LAB-specific motif found was the CopR-binding site, regulating expression of copper transport ATPases. Finally, all detected motifs were used to predict co-regulated TUs (regulons) for L.plantarum, and transcriptome profiling data were analyzed to provide regulon prediction validation. It is demonstrated that phylogenetic footprinting using different species sets can identify and distinguish between general regulatory motifs and LAB-specific regulatory motifs

From transcriptional landscapes to the identification of biomarkers for robustness

The ability of microorganisms to adapt to changing environments and gain cell robustness, challenges the prediction of their history-dependent behaviour. Using our model organism Bacillus cereus, a notorious Gram-positive food spoilage and pathogenic spore-forming bacterium, a strategy will be described that allows for identification of biomarkers for robustness. First an overview will be presented of its two-component systems that generally include a transmembrane sensor histidine kinase and its cognate response regulator, allowing rapid and robust responses to fluctuations in the environment. The role of the multisensor hybrid kinase RsbK and the PP2C-type phosphatase RsbY system in activation of the general stress sigma factor σB is highlighted. An extensive comparative analysis of transcriptional landscapes derived from B. cereus exposed to mild stress conditions such as heat, acid, salt and oxidative stress, revealed that, amongst others σB regulated genes were induced in most conditions tested. The information derived from the transcriptome data was subsequently implemented in a framework for identifying and selecting cellular biomarkers at their mRNA, protein and/or activity level, for mild stressinduced microbial robustness towards lethal stresses. Exposure of unstressed and mild stress-adapted cells to subsequent lethal stress conditions (heat, acid and oxidative stress) allowed for quantification of the robustness advantage provided by mild stress pretreatment using the plate-count method. The induction levels of the selected candidate-biomarkers, σB protein, catalase activity and transcripts of certain proteases upon mild stress treatment, were significantly correlated to mild stress-induced enhanced robustness towards lethal thermal, oxidative and acid stresses, and were therefore suitable to predict these adaptive traits. Cellular biomarkers that are quantitatively correlated to adaptive behavior will facilitate our ability to predict the impact of adaptive behavior on cell robustness and will allow to control and/or exploit these adaptive traits. Extrapolation to other species and genera is discussed such as avenues towards mechanism-based design of microbial fitness and robustness

Comparative genomic analysis of the multispecies probiotic-marketed product VSL#3

Several probiotic-marketed formulations available for the consumers contain live lactic acid bacteria and/or bifidobacteria. The multispecies product commercialized as VSL#3 has been used for treating various gastro-intestinal disorders. However, like many other products, the bacterial strains present in VSL#3 have only been characterized to a limited extent and their efficacy as well as their predicted mode of action remain unclear, preventing further applications or comparative studies. In this work, the genomes of all eight bacterial strains present in VSL#3 were sequenced and characterized, to advance insights into the possible mode of action of this product and also to serve as a basis for future work and trials. Phylogenetic and genomic data analysis allowed us to identify the 7 species present in the VSL#3 product as specified by the manufacturer. The 8 strains present belong to the species Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus helveticus, Bifidobacterium breve and B. animalis subsp. lactis (two distinct strains). Comparative genomics revealed that the draft genomes of the S. thermophilus and L. helveticus strains were predicted to encode most of the defence systems such as restriction modification and CRISPR-Cas systems. Genes associated with a variety of potential probiotic functions were also identified. Thus, in the three Bifidobacterium spp., gene clusters were predicted to encode tight adherence pili, known to promote bacteria-host interaction and intestinal barrier integrity, and to impact host cell development. Various repertoires of putative signalling proteins were predicted to be encoded by the genomes of the Lactobacillus spp., i.e. surface layer proteins, LPXTG-containing proteins, or sortase-dependent pili that may interact with the intestinal mucosa and dendritic cells. Taken altogether, the individual genomic characterization of the strains present in the VSL#3 product confirmed the product specifications, determined its coding capacity as well as identified potential probiotic functions.Peer reviewe

Identification of Genetic Loci in Lactobacillus plantarum That Modulate the Immune Response of Dendritic Cells Using Comparative Genome Hybridization

Contains fulltext : 88219.pdf (publisher's version ) (Open Access)BACKGROUND: Probiotics can be used to stimulate or regulate epithelial and immune cells of the intestinal mucosa and generate beneficial mucosal immunomodulatory effects. Beneficial effects of specific strains of probiotics have been established in the treatment and prevention of various intestinal disorders, including allergic diseases and diarrhea. However, the precise molecular mechanisms and the strain-dependent factors involved are poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we aimed to identify gene loci in the model probiotic organism Lactobacillus plantarum WCFS1 that modulate the immune response of host dendritic cells. The amounts of IL-10 and IL-12 secreted by dendritic cells (DCs) after stimulation with 42 individual L. plantarum strains were measured and correlated with the strain-specific genomic composition using comparative genome hybridisation and the Random Forest algorithm. This in silico "gene-trait matching" approach led to the identification of eight candidate genes in the L. plantarum genome that might modulate the DC cytokine response to L. plantarum. Six of these genes were involved in bacteriocin production or secretion, one encoded a bile salt hydrolase and one encoded a transcription regulator of which the exact function is unknown. Subsequently, gene deletions mutants were constructed in L. plantarum WCFS1 and compared to the wild-type strain in DC stimulation assays. All three bacteriocin mutants as well as the transcription regulator (lp_2991) had the predicted effect on cytokine production confirming their immunomodulatory effect on the DC response to L. plantarum. Transcriptome analysis and qPCR data showed that transcript level of gtcA3, which is predicted to be involved in glycosylation of cell wall teichoic acids, was substantially increased in the lp_2991 deletion mutant (44 and 29 fold respectively). CONCLUSION: Comparative genome hybridization led to the identification of gene loci in L. plantarum WCFS1 that modulate the immune response of DCs

Comparative Genome Analysis of Lactococcus lactis Indicates Niche Adaptation and Resolves Genotype/Phenotype Disparity

Lactococcus lactis is one of the most important micro-organisms in the dairy industry for the fermentation of cheese and buttermilk. Besides the conversion of lactose to lactate it is responsible for product properties such as flavor and texture, which are determined by volatile metabolites, proteolytic activity and exopolysaccharide production. While the species Lactococcus lactis consists of the two subspecies lactis and cremoris their taxonomic position is confused by a group of strains that, despite of a cremoris genotype, display a lactis phenotype. Here we compared and analyzed the (draft) genomes of 43 L. lactis strains, of which 19 are of dairy and 24 are of non-dairy origin. Machine-learning algorithms facilitated the identification of orthologous groups of protein sequences (OGs) that are predictors for either the taxonomic position or the source of isolation. This allowed the unambiguous categorization of the genotype/phenotype disparity of ssp. lactis and ssp. cremoris strains. A detailed analysis of phenotypic properties including plasmid-encoded genes indicates evolutionary changes during niche adaptations. The results are consistent with the hypothesis that dairy isolates evolved from plant isolates. The analysis further suggests that genomes of cremoris phenotype strains are so eroded that they are restricted to a dairy environment. Overall the genome comparison of a diverse set of strains allowed the identification of niche and subspecies specific genes. This explains evolutionary relationships and will aid the identification and selection of industrial starter cultures

The Variable Regions of Lactobacillus rhamnosus Genomes Reveal the Dynamic Evolution of Metabolic and Host-Adaptation Repertoires

Lactobacillus rhamnosus is a diverse Gram-positive species with strains isolated from different ecological niches. Here, we report the genome sequence analysis of 40 diverse strains of L. rhamnosus and their genomic comparison, with a focus on the variable genome. Genomic comparison of 40 L. rhamnosus strains discriminated the conserved genes ( core genome) and regions of plasticity involving frequent rearrangements and horizontal transfer ( variome). The L. rhamnosus core genome encompasses 2,164 genes, out of 4,711 genes in total ( the pan-genome). The accessory genome is dominated by genes encoding carbohydrate transport and metabolism, extracellular polysaccharides ( EPS) biosynthesis, bacteriocin production, pili production, the cas system, and the associated clustered regularly interspaced short palindromic repeat ( CRISPR) loci, and more than 100 transporter functions and mobile genetic elements like phages, plasmid genes, and transposons. A clade distribution based on amino acid differences between core ( shared) proteins matched with the clade distribution obtained from the presence-absence of variable genes. The phylogenetic and variome tree overlap indicated that frequent events of gene acquisition and loss dominated the evolutionary segregation of the strains within this species, which is paralleled by evolutionary diversification of core gene functions. The CRISPR-Cas system could have contributed to this evolutionary segregation. Lactobacillus rhamnosus strains contain the genetic and metabolic machinery with strain-specific gene functions required to adapt to a large range of environments. A remarkable congruency of the evolutionary relatedness of the strains' core and variome functions, possibly favoring interspecies genetic exchanges, underlines the importance of gene-acquisition and loss within the L. rhamnosus strain diversification.Peer reviewe

Cell Surface Properties of Lactococcus lactis Reveal Milk Protein Binding Specifically Evolved in Dairy Isolates

Surface properties of bacteria are determined by the molecular composition of the cell wall and they are important for interactions of cells with their environment. Well-known examples of bacterial interactions with surfaces are biofilm formation and the fermentation of solid materials like food and feed. Lactococcus lactis is broadly used for the fermentation of cheese and buttermilk and it is primarily isolated from either plant material or the dairy environment. In this study, we characterized surface hydrophobicity, charge, emulsification properties, and the attachment to milk proteins of 55 L. lactis strains in stationary and exponential growth phases. The attachment to milk protein was assessed through a newly developed flow cytometry-based protocol. Besides finding a high degree of biodiversity, phenotype-genotype matching allowed the identification of candidate genes involved in the modification of the cell surface. Overexpression and gene deletion analysis allowed to verify the predictions for three identified proteins that altered surface hydrophobicity and attachment of milk proteins. The data also showed that lactococci isolated from a dairy environment bind higher amounts of milk proteins when compared to plant isolates. It remains to be determined whether the alteration of surface properties also has potential to alter starter culture functionalities

Lactiplantibacillus plantarum strain:Lp900 Genome sequencing and assembly

Understanding carbohydrate metabolism in L. plantaru

Lactiplantibacillus plantarum strain:Lp900 Genome sequencing and assembly

Understanding carbohydrate metabolism in L. plantaru

The dynamic transcriptome of natural competence in Streptococcus suis

Streptococcus suis is a major pig pathogen as well as an emerging zoonotic pathogen. In a previous study (Zaccaria et al. Plos One DOI: 10.1371/journal.pone.0099394) we identified the natural pheromone-induced competence system of S. suis. To identify the mechanisms enabling competence, gene expression analysis of bacteria induced or not for competence, and in presence or absence of exogenous DNA, was assessed at 5, 15 and 45 minutes after pheromone addition. The transcriptomes showed how competence was induced and eventually shut down, and which metabolic pathways were repressed or induced when competence was active or shut down