Lactic acid-producing bacteria are associated with various plant and animal niches and play a key role in the production of fermented foods and beverages. We report nine genome sequences representing the phylogenetic and functional diversity of these bacteria. The small genomes of lactic acid bacteria encode a broad repertoire of transporters for efficient carbon and nitrogen acquisition from the nutritionally rich environments they inhabit and reflect a limited range of biosynthetic capabilities that indicate both prototrophic and auxotrophic strains. Phylogenetic analyses, comparison of gene content across the group, and reconstruction of ancestral gene sets indicate a combination of extensive gene loss and key gene acquisitions via horizontal gene transfer during the coevolution of lactic acid bacteria with their habitats. evolutionary genomics ͉ fermentation L actic acid bacteria (LAB) are historically defined as a group of microaerophilic, Gram-positive organisms that ferment hexose sugars to produce primarily lactic acid. This functional classification includes a variety of industrially important genera, including Lactococcus, Enterococcus, Oenococcus, Pediococcus, Streptococcus, Leuconostoc, and Lactobacillus species. The seemingly simplistic metabolism of LAB has been exploited throughout history for the preservation of foods and beverages in nearly all societies dating back to the origins of agriculture (1). Domestication of LAB strains passed down through various culinary traditions and continuous passage on food stuffs has resulted in modern-day cultures able to carry out these fermentations. Today, LAB play a prominent role in the world food supply, performing the main bioconversions in fermented dairy products, meats, and vegetables. LAB also are critical for the production of wine, coffee, silage, cocoa, sourdough, and numerous indigenous food fermentations (2).LAB species are indigenous to food-related habitats, including plant (fruits, vegetables, and cereal grains) and milk environments. In addition, LAB are naturally associated with the mucosal surfaces of animals, e.g., small intestine, colon, and vagina. Isolates of the same species often are obtained from plant, dairy, and animal habitats, implying wide distribution and specialized adaptation to these diverse environments. LAB species employ two pathways to metabolize hexose: a homofermentative pathway in which lactic acid is the primary product and a heterofermentative pathway in which lactic acid, CO 2 , acetic acid, and͞or ethanol are produced (3).Complete genome sequences have been published for eight fermentative and commensal LAB species: Lactococcus lactis, Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus acidophilus, Lactobacillus sakei, Lactobacillus bulgaricus, Lactobacillus salivarius, and Streptococcus thermophilus (4-11). This study examines nine other LAB genomes representing the phylogenetic and functional diversity of lactic acid-producing microorganisms. The LAB have small genomes encoding a range of biosynthe...

The prebiotic concept was introduced twenty years ago, and despite several revisions to the original definition, the scientific community has continued to debate what it means to be a prebiotic. How prebiotics are defined is important not only for the scientific community, but also for regulatory agencies, the food industry, consumers and healthcare professionals. Recent developments in community-wide sequencing and glycomics have revealed that more complex interactions occur between putative prebiotic substrates and the gut microbiota than previously considered. A consensus among scientists on the most appropriate definition of a prebiotic is necessary to enable continued use of the term.

In silico genome analysis of Lactobacillus acidophilus NCFM coupled with gene expression studies have identified putative genes and regulatory networks that are potentially important to this organism's survival, persistence, and activities in the gastrointestinal tract. Correlation of key genotypes to phenotypes requires an efficient gene replacement system. In this study, use of the upp-encoded uracil phosphoribosyltransferase (UPRTase) of L. acidophilus NCFM was explored as a counterselection marker to positively select for recombinants that have resolved from chromosomal integration of pORI-based plasmids. An isogenic mutant carrying a upp gene deletion was constructed and was resistant to 5-fluorouracil (5-FU), a toxic uracil analog that is also a substrate for UPRTase. A 3.0-kb pORI-based counterselectable integration vector bearing a upp expression cassette, pTRK935, was constructed and introduced into the ⌬upp host harboring the pTRK669 helper plasmid. Extrachromosomal replication of pTRK935 complemented the mutated chromosomal upp allele and restored sensitivity to 5-FU. This host background provides a platform for a two-step plasmid integration and excision strategy that can select for plasmid-free recombinants with either the wild-type or mutated allele of the targeted gene in the presence of 5-FU. The efficacy of the system was demonstrated by in-frame deletion of the slpX gene (LBA0512) encoding a novel 51-kDa secreted protein associated with the S-layer complex of L. acidophilus. The resulting ⌬slpX mutant exhibited lower growth rates, increased sensitivity to sodium dodecyl sulfate, and greater resistance to bile. Overall, this improved gene replacement system represents a valuable tool for investigating the mechanisms underlying the probiotic functionality of L. acidophilus.Lactobacillus acidophilus NCFM is a commercially established probiotic bacterium that is widely used in dietary supplements and in milk and fermented dairy products (47). Originally a human intestinal isolate from the 1970s (8), this strain has since been examined extensively for various desirable traits. Due to the importance of understanding the molecular mechanisms involved in probiotic functions, the complete genome sequence of L. acidophilus NCFM was determined (2). The NCFM genome sequence serves as a blueprint for in silico identification of candidate gene loci and gene regulatory networks that may play essential roles in the survival and host interactions of this microorganism in the gastrointestinal (GI) tract, including genes involved in acid tolerance (5), bile tolerance (38, 42), adherence factors (20), environmental sensing and response (7), prebiotic sugar utilization (9), polysaccharide biosynthesis, oxalate degradation (6), and bacteriocin production (22). In addition, ongoing microarray gene expression studies have revealed specific gene sets of interest that are being investigated further. The fundamental approach for establishing functional roles for important gene features involves inactivation of the genes ...

This study presents the complete genome sequence of Lactobacillus gasseri ATCC 33323, a neotype strain of human origin and a native species found commonly in the gastrointestinal tracts of neonates and adults. The plasmid-free genome was 1,894,360 bp in size and predicted to encode 1,810 genes. The GC content was 35.3%, similar to the GC content of its closest relatives, L. johnsonii NCC 533 (34%) and L. acidophilus NCFM (34%). Two identical copies of the prophage LgaI (40,086 bp), of the Sfi11-like Siphoviridae phage family, were integrated tandomly in the chromosome. A number of unique features were identified in the genome of L. gasseri that were likely acquired by horizontal gene transfer and may contribute to the survival of this bacterium in its ecological niche. L. gasseri encodes two restriction and modification systems, which may limit bacteriophage infection. L. gasseri also encodes an operon for production of heteropolysaccharides of high complexity. A unique alternative sigma factor was present similar to that of B. caccae ATCC 43185, a bacterial species isolated from human feces. In addition, L. gasseri encoded the highest number of putative mucusbinding proteins (14) among lactobacilli sequenced to date. Selected phenotypic characteristics that were compared between ATCC 33323 and other human L. gasseri strains included carbohydrate fermentation patterns, growth and survival in bile, oxalate degradation, and adhesion to intestinal epithelial cells, in vitro. The results from this study indicated high intraspecies variability from a genome encoding traits important for survival and retention in the gastrointestinal tract.

Bacterial surface (S-) layers are crystalline arrays of self-assembling, proteinaceous subunits called S-layer proteins (Slps), with molecular masses ranging from 40 to 200 kDa. The S-layer-forming bacterium Lactobacillus acidophilus NCFM expresses three major Slps: SlpA (46 kDa), SlpB (47 kDa) and SlpX (51 kDa). SlpA has a demonstrated role in adhesion to Caco-2 intestinal epithelial cells in vitro, and has been shown to modulate dendritic cell (DC) and T-cell functionalities with murine DCs. In this study, a modification of a standard lithium chloride S-layer extraction revealed 37 proteins were solubilized from the S-layer wash fraction. Of these, 30 have predicted cleavage sites for secretion, 24 are predicted to be extracellular, six are lipid-anchored, three have N-terminal hydrophobic membrane spanning regions and four are intracellular, potentially moonlighting proteins. Some of these proteins, designated S-layer associated proteins (SLAPs), may be loosely associated with or embedded within the bacterial S-layer complex. Lba-1029, a putative SLAP gene, was deleted from the chromosome of L. acidophilus. Phenotypic characterization of the deletion mutant demonstrated that the SLAP LBA1029 contributes to a pro-inflammatory TNF-α response from murine DCs. This study identified extracellular proteins and putative SLAPs of L. acidophilus NCFM using LC-MS/MS. SLAPs appear to impart important surface display features and immunological properties to microbes that are coated by S-layers.

BackgroundStreptococcus thermophilus represents the only species among the streptococci that has “Generally Regarded As Safe” status and that plays an economically important role in the fermentation of yogurt and cheeses. We conducted comparative genome analysis of S. thermophilus LMD-9 to identify unique gene features as well as features that contribute to its adaptation to the dairy environment. In addition, we investigated the transcriptome response of LMD-9 during growth in milk in the presence of Lactobacillus delbrueckii ssp. bulgaricus, a companion culture in yogurt fermentation, and during lytic bacteriophage infection.ResultsThe S. thermophilus LMD-9 genome is comprised of a 1.8 Mbp circular chromosome (39.1% GC; 1,834 predicted open reading frames) and two small cryptic plasmids. Genome comparison with the previously sequenced LMG 18311 and CNRZ1066 strains revealed 114 kb of LMD-9 specific chromosomal region, including genes that encode for histidine biosynthetic pathway, a cell surface proteinase, various host defense mechanisms and a phage remnant. Interestingly, also unique to LMD-9 are genes encoding for a putative mucus-binding protein, a peptide transporter, and exopolysaccharide biosynthetic proteins that have close orthologs in human intestinal microorganisms. LMD-9 harbors a large number of pseudogenes (13% of ORFeome), indicating that like LMG 18311 and CNRZ1066, LMD-9 has also undergone major reductive evolution, with the loss of carbohydrate metabolic genes and virulence genes found in their streptococcal counterparts. Functional genome distribution analysis of ORFeomes among streptococci showed that all three S. thermophilus strains formed a distinct functional cluster, further establishing their specialized adaptation to the nutrient-rich milk niche. An upregulation of CRISPR1 expression in LMD-9 during lytic bacteriophage DT1 infection suggests its protective role against phage invasion. When co-cultured with L. bulgaricus, LMD-9 overexpressed genes involved in amino acid transport and metabolism as well as DNA replication.ConclusionsThe genome of S. thermophilus LMD-9 is shaped by its domestication in the dairy environment, with gene features that conferred rapid growth in milk, stress response mechanisms and host defense systems that are relevant to its industrial applications. The presence of a unique exopolysaccharide gene cluster and cell surface protein orthologs commonly associated with probiotic functionality revealed potential probiotic applications of LMD-9.

Recent insights into the relationship between the human gut and its resident microbiota have revolutionized our appreciation of this symbiosis and its impact on health and disease development. Accumulating evidence on probiotic and prebiotic interventions has demonstrated promising effects on promoting gastrointestinal health by modulating the microbiota toward the enrichment of beneficial microorganisms. However, the precise mechanisms of how prebiotic nondigestible oligosaccharides are metabolized by these beneficial microbes in vivo remain largely unknown. Genome sequencing of probiotic lactobacilli and bifidobacteria has revealed versatile carbohydrate metabolic gene repertoires dedicated to the catabolism of various oligosaccharides. In this review, we highlight recent findings on the genetic mechanisms involved in the utilization of prebiotic fructooligosaccharides, β-galactooligosaccharides, human milk oligosaccharides, and other prebiotic candidates by these probiotic microbes.

Intestinal immune regulatory signals govern gut homeostasis. Breakdown of such regulatory mechanisms may result in inflammatory bowel disease (IBD). Lactobacillus acidophilus contains unique surface layer proteins (Slps), including SlpA, SlpB, SlpX, and lipoteichoic acid (LTA), which interact with pattern recognition receptors to mobilize immune responses. Here, to elucidate the role of SlpA in protective immune regulation, the NCK2187 strain, which solely expresses SlpA, was generated. NCK2187 and its purified SlpA bind to the C-type lectin SIGNR3 to exert regulatory signals that result in mitigation of colitis, maintenance of healthy gastrointestinal microbiota, and protected gut mucosal barrier function. However, such protection was not observed in Signr3 À/À mice, suggesting that the SlpA/SIGNR3 interaction plays a key regulatory role in colitis. Our work presents critical insights into SlpA/SIGNR3-induced responses that are integral to the potential development of novel biological therapies for autoinflammatory diseases, including IBD.