Lactobacilli are a diverse group of species that occupy diverse nutrient-rich niches associated with humans, animals, plants and food. They are used widely in biotechnology and food preservation, and are being explored as therapeutics. Exploiting lactobacilli has been complicated by metabolic diversity, unclear species identity and uncertain relationships between them and other commercially important lactic acid bacteria. The capacity for biotransformations catalysed by lactobacilli is an untapped biotechnology resource. Here we report the genome sequences of 213 Lactobacillus strains and associated genera, and their encoded genetic catalogue for modifying carbohydrates and proteins. In addition, we describe broad and diverse presence of novel CRISPR-Cas immune systems in lactobacilli that may be exploited for genome editing. We rationalize the phylogenomic distribution of host interaction factors and bacteriocins that affect their natural and industrial environments, and mechanisms to withstand stress during technological processes. We present a robust phylogenomic framework of existing species and for classifying new species.

Lactobacillus rhamnosus is a lactic acid bacterium that is found in a large variety of ecological habitats, including artisanal and industrial dairy products, the oral cavity, intestinal tract or vagina. To gain insights into the genetic complexity and ecological versatility of the species L. rhamnosus, we examined the genomes and phenotypes of 100 L. rhamnosus strains isolated from diverse sources. The genomes of 100 L. rhamnosus strains were mapped onto the L. rhamnosus GG reference genome. These strains were phenotypically characterized for a wide range of metabolic, antagonistic, signalling and functional properties. Phylogenomic analysis showed multiple groupings of the species that could partly be associated with their ecological niches. We identified 17 highly variable regions that encode functions related to lifestyle, i.e. carbohydrate transport and metabolism, production of mucus-binding pili, bile salt resistance, prophages and CRISPR adaptive immunity. Integration of the phenotypic and genomic data revealed that some L. rhamnosus strains possibly resided in multiple niches, illustrating the dynamics of bacterial habitats. The present study showed two distinctive geno-phenotypes in the L. rhamnosus species. The geno-phenotype A suggests an adaptation to stable nutrient-rich niches, i.e. milk-derivative products, reflected by the alteration or loss of biological functions associated with antimicrobial activity spectrum, stress resistance, adaptability and fitness to a distinctive range of habitats. In contrast, the geno-phenotype B displays adequate traits to a variable environment, such as the intestinal tract, in terms of nutrient resources, bacterial population density and host effects.

f Four Lactobacillus strains were isolated from marketed probiotic products, including L. rhamnosus strains from Vifit (Friesland Campina) and Idoform (Ferrosan) and L. casei strains from Actimel (Danone) and Yakult (Yakult Honsa Co.). Their genomes and phenotypes were characterized and compared in detail with L. casei strain BL23 and L. rhamnosus strain GG. Phenotypic analysis of the new isolates indicated differences in carbohydrate utilization between L. casei and L. rhamnosus strains, which could be linked to their genotypes. The two isolated L. rhamnosus strains had genomes that were virtually identical to that of L. rhamnosus GG, testifying to their genomic stability and integrity in food products. The L. casei strains showed much greater genomic heterogeneity. Remarkably, all strains contained an intact spaCBA pilus gene cluster. However, only the L. rhamnosus strains produced mucus-binding SpaCBA pili under the conditions tested. Transcription initiation mapping demonstrated that the insertion of an iso-IS30 element upstream of the pilus gene cluster in L. rhamnosus strains but absent in L. casei strains had constituted a functional promoter driving pilus gene expression. All L. rhamnosus strains triggered an NF-B response via Tolllike receptor 2 (TLR2) in a reporter cell line, whereas the L. casei strains did not or did so to a much lesser extent. This study demonstrates that the two L. rhamnosus strains isolated from probiotic products are virtually identical to L. rhamnosus GG and further highlights the differences between these and L. casei strains widely marketed as probiotics, in terms of genome content, mucus-binding and metabolic capacities, and host signaling capabilities.

Genome analysis using next generation sequencing technologies has revolutionized the characterization of lactic acid bacteria and complete genomes of all major groups are now available. Comparative genomics has provided new insights into the natural and laboratory evolution of lactic acid bacteria and their environmental interactions. Moreover, functional genomics approaches have been used to understand the response of lactic acid bacteria to their environment. The results have been instrumental in understanding the adaptation of lactic acid bacteria in artisanal and industrial food fermentations as well as their interactions with the human host. Collectively, this has led to a detailed analysis of genes involved in colonization, persistence, interaction and signaling towards to the human host and its health. Finally, massive parallel genome re-sequencing has provided new opportunities in applied genomics, specifically in the characterization of novel non-GMO strains that have potential to be used in the food industry. Here, we provide an overview of the state of the art of these functional genomics approaches and their impact in understanding, applying and designing lactic acid bacteria for food and health.

Bifidobacteria are natural inhabitants of the human gastrointestinal tract and well known for their health-promoting effects. Tolerance to bile stress is crucial for bifidobacteria to survive in the colon and to exert their beneficial actions. In this work, RNA-Seq transcriptomic analysis complemented with proteomic analysis was used to investigate the cellular response to bile in Bifidobacterium longum BBMN68. The transcript levels of 236 genes were significantly changed (> threefold, p < 0.001) and 44 proteins were differentially abundant (>1.6-fold, p < 0.01) in B. longum BBMN68 when exposed to 0.75 g l ؊1 ox-bile. The hemolysin-like protein and bile efflux systems were significantly over produced, which might prevent bile adsorption and exclude bile, respectively. The cell membrane composition was modified probably by an increase of cyclopropane fatty acid and a decrease of transmembrane proteins, resulting in a cell membrane more impermeable to bile salts. Our hypothesis was later confirmed by surface hydrophobicity assay. The transcription of genes related to xylose utilization and bifid shunt were up-regulated, which increased the production of ATP and reducing equivalents to cope with bile-induced damages in a xylan-rich colon environment. Bile salts signal the B. longum BBMN68 to gut entrance and enhance the expression of esterase and sortase associated with adhesion and colonization in intestinal tract, which was supported by a fivefold increased adhesion ability to HT-29 cells by BBMN68 upon bile exposure. Notably, bacterial one-hybrid and EMSA assay revealed that the two-component system senX3-regX3 controlled the expression of pstS in bifidobacteria and the role of this target gene in bile resistance was further verified by heterologous expression in Lactococcus lactis. Taken altogether, this study established a model for global response mechanisms in B. longum to bile. Molecular & Cellular