Selection and characterization of a SpaCBA pilus-secreting food-grade derivative of Lacticaseibacillus rhamnosus GG.

Many studies have established the functional properties of Lacticaseibacillus rhamnosus GG, previously known as Lactobacillus rhamnosus GG, marketed worldwide as a probiotic. The extraordinary capacity of L. rhamnosus GG to bind to human mucus and influence the immune system especially stand out. Earlier, we have shown the key role of its SpaCBA sortase-dependent pili encoded by the spaCBA-srtC1 gene cluster herein. These heterotrimeric pili consist of a shaft pilin SpaA, a basal pilin SpaB, and tip pilin SpaC that contains a mucus-binding domain. Here, we set out to characterize a food-grade non-GMO mutant of L. rhamnosus GG, strain PA11, which secretes its pilins, rather than coupling them to the cell surface, due to a defect in the housekeeping sortase A. The sortase-negative strain PA11 was extensively characterized using functional genomics and biochemical approaches and found to secrete the SpaCBA pili into the supernatant. Given the functional importance and uniqueness of the mucus-binding pili of L. rhamnosus GG, strain PA11 offers novel opportunities towards the characterization and further therapeutic application of SpaCBA pili and their low-cost, large-scale production. KEY POINTS: •Creation of pilus-secreting mutant (PA11) of the key probiotic LGG. •Strain PA11 is defective in a functional housekeeping sortase SrtA. •Strain PA11 opens novel biotherapeutic application avenues. Graphical abstract.

Lactobacillus rhamnosus GG Outcompetes Enterococcus faecium via Mucus-Binding Pili: Evidence for a Novel and Heterospecific Probiotic Mechanism.

UNLABELLED: Vancomycin-resistant enterococci (VRE) have become a major nosocomial threat. Enterococcus faecium is of special concern, as it can easily acquire new antibiotic resistances and is an excellent colonizer of the human intestinal tract. Several clinical studies have explored the potential use of beneficial bacteria to weed out opportunistic pathogens. Specifically, the widely studied Lactobacillus rhamnosus strain GG has been applied successfully in the context of VRE infections. Here, we provide new insight into the molecular mechanism underlying the effects of this model probiotic on VRE decolonization. Both clinical VRE isolates and L. rhamnosus GG express pili on their cell walls, which are the key modulators of their highly efficient colonization of the intestinal mucosa. We found that one of the VRE pilus clusters shares considerable sequence similarity with the SpaCBA-SrtC1 pilus cluster of L. rhamnosus GG. Remarkable immunological and functional similarities were discovered between the mucus-binding pili of L. rhamnosus GG and those of the clinical E. faecium strain E1165, which was characterized at the genome level. Moreover, E. faecium strain E1165 bound efficiently to mucus, which may be prevented by the presence of the mucus-binding SpaC protein or antibodies against L. rhamnosus GG or SpaC. These results present experimental support for a novel probiotic mechanism, in which the mucus-binding pili of L. rhamnosus GG prevent the binding of a potential pathogen to the host. Hence, we provide a molecular basis for the further exploitation of L. rhamnosus GG and its pilins for prophylaxis and treatment of VRE infections. IMPORTANCE: Concern about vancomycin-resistant Enterococcus faecium causing nosocomial infections is rising globally. The arsenal of antibiotic strategies to treat these infections is nearly exhausted, and hence, new treatment strategies are urgently needed. Here, we provide molecular evidence to underpin reports of the successful clinical application of Lactobacillus rhamnosus GG in VRE decolonization strategies. Our results provide support for a new molecular mechanism, in which probiotics can perform competitive exclusion and possibly immune interaction. Moreover, we spur further exploration of the potential of intact L. rhamnosus GG and purified SpaC pilin as prophylactic and curative agents of the VRE carrier state.

Polymorphisms, Chromosomal Rearrangements, and Mutator Phenotype Development during Experimental Evolution of Lactobacillus rhamnosus GG.

UNLABELLED: Lactobacillus rhamnosus GG is a lactic acid bacterium widely marketed by the food industry. Its genomic analysis led to the identification of a gene cluster encoding mucus-binding SpaCBA pili, which is located in a genomic island enriched in insertion sequence (IS) elements. In the present study, we analyzed by genome-wide resequencing the genomic integrity of L. rhamnosus GG in four distinct evolutionary experiments conducted for approximately 1,000 generations under conditions of no stress or salt, bile, and repetitive-shearing stress. Under both stress-free and salt-induced stress conditions, the GG population (excluding the mutator lineage in the stress-free series [see below]) accumulated only a few single nucleotide polymorphisms (SNPs) and no frequent chromosomal rearrangements. In contrast, in the presence of bile salts or repetitive shearing stress, some IS elements were found to be activated, resulting in the deletion of large chromosomal segments that include the spaCBA-srtC1 pilus gene cluster. Remarkably, a high number of SNPs were found in three strains obtained after 900 generations of stress-free growth. Detailed analysis showed that these three strains derived from a founder mutant with an altered DNA polymerase subunit that resulted in a mutator phenotype. The present work confirms the stability of the pilus production phenotype in L. rhamnosus GG under stress-free conditions, highlights the possible evolutionary scenarios that may occur when this probiotic strain is extensively cultured, and identifies external factors that affect the chromosomal integrity of GG. The results provide mechanistic insights into the stability of GG in regard to its extensive use in probiotic and other functional food products. IMPORTANCE: Lactobacillus rhamnosus GG is a widely marketed probiotic strain that has been used in numerous clinical studies to assess its health-promoting properties. Hence, the stability of the probiotic functions of L. rhamnosus GG is of importance, and here we studied the impact of external stresses on the genomic integrity of L. rhamnosus GG. We studied three different stresses that are relevant for understanding its robustness and integrity under both ex vivo conditions, i.e., industrial manufacturing conditions, and in vivo conditions, i.e., intestinal tract-associated stress. Overall, our findings contribute to predicting the genomic stability of L. rhamnosus GG and its ecological performance.

Isolation and whole genome sequencing of a Ruminococcus-like bacterium, associated with irritable bowel syndrome.

In our previous studies on the intestinal microbiota in irritable bowel syndrome (IBS), we identified a bacterial phylotype with higher abundance in patients suffering from diarrhea than in healthy controls. In the present work, we have isolated in pure culture strain RT94, belonging to this phylotype, determined its whole genome sequence and performed an extensive genomic analysis and phenotypical testing. This revealed strain RT94 to be a strict anaerobe apparently belonging to a novel species with only 94% similarity in the 16S rRNA gene sequence to the closest relatives Ruminococcus torques and Ruminococcus lactaris. The G + C content of strain RT94 is 45.2 mol% and the major long-chain cellular fatty acids are C16:0, C18:0 and C14:0. The isolate is metabolically versatile but not a mucus or cellulose utilizer. It produces acetate, ethanol, succinate, lactate and formate, but very little butyrate, as end products of glucose metabolism. The mechanisms underlying the association of strain RT94 with diarrhea-type IBS are discussed.

Functional identification of conserved residues involved in Lactobacillus rhamnosus strain GG sortase specificity and pilus biogenesis.

In Gram-positive bacteria, sortase-dependent pili mediate the adhesion of bacteria to host epithelial cells and play a pivotal role in colonization, host signaling, and biofilm formation. Lactobacillus rhamnosus strain GG, a well known probiotic bacterium, also displays on its cell surface mucus-binding pilus structures, along with other LPXTG surface proteins, which are processed by sortases upon specific recognition of a highly conserved LPXTG motif. Bioinformatic analysis of all predicted LPXTG proteins encoded by the L. rhamnosus GG genome revealed a remarkable conservation of glycine residues juxtaposed to the canonical LPXTG motif. Here, we investigated and defined the role of this so-called triple glycine (TG) motif in determining sortase specificity during the pilus assembly and anchoring. Mutagenesis of the TG motif resulted in a lack or an alteration of the L. rhamnosus GG pilus structures, indicating that the TG motif is critical in pilus assembly and that they govern the pilin-specific and housekeeping sortase specificity. This allowed us to propose a regulatory model of the L. rhamnosus GG pilus biogenesis. Remarkably, the TG motif was identified in multiple pilus gene clusters of other Gram-positive bacteria, suggesting that similar signaling mechanisms occur in other, mainly pathogenic, species.

Genomic characterization of non-mucus-adherent derivatives of Lactobacillus rhamnosus GG reveals genes affecting pilus biogenesis.

Lactobacillus rhamnosus GG is one of the best-characterized lactic acid bacteria and can be considered a probiotic paradigm. Comparative and functional genome analysis showed that L. rhamnosus GG harbors a genomic island including the spaCBA-srtC1 gene cluster, encoding the cell surface-decorating host-interacting pili. Here, induced mutagenesis was used to study pilus biogenesis in L. rhamnosus GG. A combination of two powerful approaches, mutation selection and next-generation sequencing, was applied to L. rhamnosus GG for the selection of pilus-deficient mutants from an enriched population. The isolated mutants were first screened by immuno-dot blot analysis using antiserum against pilin proteins. Relevant mutants were selected, and the lack of pili was confirmed by immunoelectron microscopy. The pilosotype of 10 mutant strains was further characterized by analyzing pilin expression using Western blot, dot blot, and immunofluorescence methods. A mucus binding assay showed that the mutants did not adhere to porcine intestinal mucus. Comparative genome sequence analysis using the Illumina MiSeq platform allowed us to determine the nature of the mutations in the obtained pilus-deficient derivatives. Three major classes of mutants with unique genotypes were observed: class I, with mutations in the srtC1 gene; class II, with a deletion containing the spaCBA-srtC1 gene cluster; and class III, with mutations in the spaA gene. Only a limited number of collateral mutations were observed, and one of the pilus-deficient derivatives with a deficient srtC1 gene contained 24 other mutations. This strain, PB12, can be considered a candidate for human trials addressing the impact of the absence of pili.

Bifidobacterium animalis subsp. lactis Bi-07 supports lactose digestion in vitro and in randomized, placebo- and lactase-controlled clinical trials.

BACKGROUND: Probiotics may alleviate lactose maldigestion. OBJECTIVES: The objective was to select a probiotic with high lactase activity and compare it with lactase and placebo in clinical trials. METHODS: Bacterial cultures were screened for lactase activity in a model of the upper gastrointestinal (GI) tract. Bifidobacterium animalis subsp. lactis Bi-07 (Bi-07) counts were adjusted in subsequent experiments to correspond to 4500 Food Chemicals Codex (FCC) units of lactase, the amount in the European Food Safety Authority (EFSA)-approved health claim. Two crossover clinical trials, Booster Alpha and Booster Omega, were performed in participants with lactose intolerance, where 2 × 1012 CFUs Bi-07, 4662 FCC lactase, or placebo was consumed simultaneously with a lactose challenge, with 1-wk washouts between challenges. The trial designs were identical except for the source of lactose. Breath hydrogen concentration (BHC) was measured to assess the effect of the investigational products on lactose digestion, for which incremental area under the curve (iAUC) was the primary outcome. Peak BHC, cumulative BHC, and GI symptoms were secondary outcomes. RESULTS: Bi-07 was superior to placebo in reducing BHC [iAUC, parts per million (ppm) ∙ h] in both trials (Booster Alpha: geometric least square mean ratio: 0.462; 95% CI: 0.249, 0.859; P = 0.016; Booster Omega: 0.227; 95% CI: 0.095, 0.543; P = 0.001). Lactase was superior to placebo in Booster Alpha (0.190; 95% CI: 0.102, 0.365; P < 0.001) but not Booster Omega (0.493; 95% CI: 0.210, 1.156; P = 0.102). Noninferiority of Bi-07 compared with lactase was observed in Booster Omega (0.460; 95% CI: 0.193, 1.096; P = 0.079; CI upper limit < 1.25 noninferiority margin). Odds of abdominal pain (compared with placebo: 0.32, P = 0.036) and flatulence (compared with placebo: 0.25, P = 0.007) were lower with lactase in Booster Alpha. Increased odds of nausea were seen with Bi-07 (compared with placebo: 4.0, P = 0.005) in Booster Omega. CONCLUSIONS: Bi-07 has high lactase activity, and in 2 clinical trials, it supported lactose digestion in individuals with lactose intolerance.These trials were registered at clinicaltrials.gov as NCT03659747 (Booster Alpha) and NCT03814668 (Booster Omega).

Identification and Characterization of a Novel Species of Genus Akkermansia with Metabolic Health Effects in a Diet-Induced Obesity Mouse Model.

Akkermansia muciniphila is a well-known bacterium with the ability to degrade mucin. This metabolic capability is believed to play an important role in the colonization of this bacterium in the gut. In this study, we report the identification and characterization of a novel Akkermansia sp. DSM 33459 isolated from human feces of a healthy donor. Phylogenetic analysis based on the genome-wide average nucleotide identity indicated that the Akkermansia sp. DSM 33459 has only 87.5% similarity with the type strain A. muciniphila ATCC BAA-835. Akkermansia sp. DSM 33459 showed significant differences in its fatty acid profile and carbon utilization as compared to the type strain. The Akkermansia sp. DSM 33459 strain was tested in a preclinical obesity model to determine its effect on metabolic markers. Akkermansia sp. DSM 33459 showed significant improvement in body weight, total fat weight, and resistin and insulin levels. Interestingly, these effects were more pronounced with the live form as compared to a pasteurized form of the strain. The strain showed production of agmatine, suggesting a potential novel mechanism for supporting metabolic and cognitive health. Based on its phenotypic features and phylogenetic position, it is proposed that this isolate represents a novel species in the genus Akkermansia and a promising therapeutic candidate for the management of metabolic diseases.

Characterization of Highly Mucus-Adherent Non-GMO Derivatives of Lacticaseibacillus rhamnosus GG.

Lacticaseibacillus rhamnosus GG is one of the best studied lactic acid bacteria in the context of probiotic effects. L. rhamnosus GG has been shown to prevent diarrhea in children and adults and has been implicated to have mitigating or preventive effects in several disorders connected to microbiota dysbiosis. The probiotic effects are largely attributed to its adhesive heterotrimeric sortase-dependent pili, encoded by the spaCBA-srtC1 gene cluster. Indeed, the strain-specific SpaCBA pili have been shown to contribute to adherence, biofilm formation and host signaling. In this work we set out to generate non-GMO derivatives of L. rhamnosus GG that adhere stronger to mucus compared to the wild-type strain using chemical mutagenesis. We selected 13 derivatives that showed an increased mucus-adherent phenotype. Deep shotgun resequencing of the strains enabled division of the strains into three classes, two of which revealed SNPs (single nucleotide polymorphisms) in the spaA and spaC genes encoding the shaft and tip adhesive pilins, respectively. Strikingly, the other class derivatives demonstrated less clear genotype - phenotype relationships, illustrating that pili biogenesis and structure is also affected by other processes. Further characterization of the different classes of derivatives was performed by PacBio SMRT sequencing and RNAseq analysis, which resulted in the identification of molecular candidates driving pilin biosynthesis and functionality. In conclusion, we report on the generation and characterization of three classes of strongly adherent L. rhamnosus GG derivatives that show an increase in adhesion to mucus. These are of special interest as they provide a window on processes and genes driving piliation and its control in L. rhamnosus GG and offer a variety of non-GMO derivatives of this key probiotic strain that are applicable in food products.

Bifidobacterium animalis subsp. lactis 420 for Metabolic Health: Review of the Research.

The growing worldwide epidemic of obesity and associated metabolic health comorbidities has resulted in an urgent need for safe and efficient nutritional solutions. The research linking obesity with gut microbiota dysbiosis has led to a hypothesis that certain bacterial strains could serve as probiotics helping in weight management and metabolic health. In the search for such strains, the effect of Bifidobacterium animalis subsp. lactis 420 (B420) on gut microbiota and metabolic health, and the mechanisms of actions, has been investigated in a variety of in vitro, pre-clinical, and clinical studies. In this review, we aim to highlight the research on B420 related to obesity, metabolic health, and the microbiota. Current research supports the hypothesis that gut dysbiosis leads to an imbalance in the inflammatory processes and loss of epithelial integrity. Bacterial components, like endotoxins, that leak out of the gut can invoke low-grade, chronic, and systemic inflammation. This imbalanced state is often referred to as metabolic endotoxemia. Scientific evidence indicates that B420 can slow down many of these detrimental processes via multiple signaling pathways, as supported by mechanistic in vitro and in vivo studies. We discuss the connection of these mechanisms to clinical evidence on the effect of B420 in controlling weight gain in overweight and obese subjects. The research further indicates that B420 may improve the epithelial integrity by rebalancing a dysbiotic state induced by an obesogenic diet, for example by increasing the prevalence of lean phenotype microbes such as Akkermansia muciniphila. We further discuss, in the context of delivering the health benefits of B420: the safety and technological aspects of the strain including genomic characterization, antibiotic resistance profiling, stability in the product, and survival of the live probiotic in the intestine. In summary, we conclude that the clinical and preclinical studies on metabolic health suggest that B420 may be a potential candidate in combating obesity; however, further clinical studies are needed.

Effects of Soluble Corn Fiber Alone or in Synbiotic Combination with Lactobacillus rhamnosus GG and the Pilus-Deficient Derivative GG-PB12 on Fecal Microbiota, Metabolism, and Markers of Immune Function: A Randomized, Double-Blind, Placebo-Controlled, Crossover Study in Healthy Elderly (Saimes Study).

BACKGROUND: The aging process leads to a potential decline in immune function and adversely affects the gut microbiota. To date, many in vitro and in vivo studies focused on the application of synbiotics (prebiotics combined with probiotics) as a promising dietary approach to affect gut microbiota composition and improved functioning of the immune system. However, studies using synbiotic preparations often have the limitation that it remains unclear whether any effect observed is a result of the prebiotic or probiotic or a synergistic effect of the combined supplement. OBJECTIVES: We investigated the effects of a probiotic Lactobacillus rhamnosus GG and pilus-deficient L. rhamnosus GG-PB12 combined with Promitor™ Soluble Corn Fiber (SCF, a candidate prebiotic) on fecal microbiota, metabolism, immunity, and blood lipids in healthy elderly persons. A prospective, double-blind, placebo controlled, randomized, single-centered, crossover study in 40 healthy elderly subjects (aged 60-80 years) was carried out. Volunteers were randomized to consume either probiotic and prebiotic as synbiotic, prebiotic or placebo (maltodextrin) during 3 weeks. Three-week washout periods separated all the treatments. We assessed effects upon blood lipids, glucose, cytokines, natural killer (NK) cell activity, phenotype, and intestinal microbiota composition. SCF decreased IL-6, which was not observed with the synbiotics. RESULTS: Consumption of L. rhamnosus GG combined with SCF increased NK cell activity compared to baseline in females and the older group. In the fecal microbiota analyses, the strongest community shifts were due to L. rhamnosus GG combined with SCF and SCF treatments. L. rhamnosus GG combined with SCF and L. rhamnosus GG-PB12 combined with SCF significantly increased the genus Parabacteroides. L. rhamnosus GG combined with SCF and SCF increased concentrations of Ruminococcaceae Incertae Sedis. Oscillospira and Desulfovibrio slightly decreased in the L. rhamnosus GG combined with SCF group, whereas Desulfovibrio decreased also in the L. rhamnosus GG-PB12 combined with SCF group. L. rhamnosus GG combined with SCF reduced total cholesterol and LDL-cholesterol in volunteers with initially elevated concentrations. C-reactive protein significantly decreased during L. rhamnosus GG-PB12 combined with SCF intervention compared to baseline. CONCLUSION: In conclusion, the synbiotic combination of L. rhamnosus GG with SCF showed a tendency to promote innate immunity by increasing NK cell activity in elderly women and in 70 to 80-year-old volunteers and decreased TC and LDL-c in hypercholesterolemic patients. In addition, L. rhamnosus GG-PB12 combined with SCF demonstrated an increase in NK cell activity compared to SCF alone in older volunteers. We also found significant positive effects on the immune response, evidenced by a decrease of the pro-inflammatory cytokine IL-6. Therefore, dietary intervention with L. rhamnosus GG combined with SCF could be of importance in elderly as an attractive option for enhancement of both the microbial and immune systems.

Genotypic and phenotypic diversity of Lactobacillus rhamnosus clinical isolates, their comparison with strain GG and their recognition by complement system.

Lactobacillus rhamnosus strains are ubiquitous in fermented foods, and in the human body where they are commensals naturally present in the normal microbiota composition of gut, vagina and skin. However, in some cases, Lactobacillus spp. have been implicated in bacteremia. The aim of the study was to examine the genomic and immunological properties of 16 clinical blood isolates of L. rhamnosus and to compare them to the well-studied L. rhamnosus probiotic strain GG. Blood cultures from bacteremic patients were collected at the Helsinki University Hospital laboratory in 2005-2011 and L. rhamnosus strains were isolated and characterized by genomic sequencing. The capacity of the L. rhamnosus strains to activate serum complement was studied using immunological assays for complement factor C3a and the terminal pathway complement complex (TCC). Binding of complement regulators factor H and C4bp was also determined using radioligand assays. Furthermore, the isolated strains were evaluated for their ability to aggregate platelets and to form biofilms in vitro. Genomic comparison between the clinical L. rhamnosus strains showed them to be clearly different from L. rhamnosus GG and to cluster in two distinct lineages. All L. rhamnosus strains activated complement in serum and none of them bound complement regulators. Four out of 16 clinical blood isolates induced platelet aggregation and/or formed more biofilms than L. rhamnosus GG, which did not display platelet aggregation activity nor showed strong biofilm formation. These findings suggest that clinical L. rhamnosus isolates show considerable heterogeneity but are clearly different from L. rhamnosus GG at the genomic level. All L. rhamnosus strains are still normally recognized by the human complement system.

Correction: Genotypic and phenotypic diversity of Lactobacillus rhamnosus clinical isolates, their comparison with strain GG and their recognition by complement system.

[This corrects the article DOI: 10.1371/journal.pone.0176739.].

The N-Terminal GYPSY Motif Is Required for Pilin-Specific Sortase SrtC1 Functionality in Lactobacillus rhamnosus Strain GG.

Predominantly identified in pathogenic Gram-positive bacteria, sortase-dependent pili are also found in commensal species, such as the probiotic-marketed strain Lactobacillus rhamnosus strain GG. Pili are typically associated with host colonization, immune signalling and biofilm formation. Comparative analysis of the N-terminal domains of pilin-specific sortases from various piliated Gram-positive bacteria identified a conserved motif, called GYPSY, within the signal sequence. We investigated the function and role of the GYPSY residues by directed mutagenesis in homologous (rod-shaped) and heterologous (coccoid-shaped) expression systems for pilus formation. Substitutions of some of the GYPSY residues, and more specifically the proline residue, were found to have a direct impact on the degree of piliation of Lb. rhamnosus GG. The present findings uncover a new signalling element involved in the functionality of pilin-specific sortases controlling the pilus biogenesis of Lb. rhamnosus GG and related piliated Gram-positive species.

Probiotic Gut Microbiota Isolate Interacts with Dendritic Cells via Glycosylated Heterotrimeric Pili.

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.

Genome Sequence of the Butyrate-Producing Anaerobic Bacterium Anaerostipes hadrus PEL 85.

Anaerostipes hadrus PEL 85, which was isolated from human feces, is a Gram-positive rod-shaped bacterium. The species may play an important role in gut health, as it was previously reported to produce butyric acid. Here, we present the genome assembly of PEL 85, a novel strain of A. hadrus.

Expanding the biotechnology potential of lactobacilli through comparative genomics of 213 strains and associated genera.

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.