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.

Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus gasseri and Lactobacillus johnsonii. The objective of this work was to develop a rapid and de®nitive DNA sequencebased identi®cation system for unknown isolates of the Lact. acidophilus complex. A I500 bp region of the 16S rRNA gene, which contained the V1 and V2 variable regions, was ampli®ed from the isolates by the polymerase chain reaction. The sequence of this region of the 16S rRNA gene from the type strains of the Lact. acidophilus complex was suf®ciently variable to allow for clear differentiation amongst each of the strains. As an initial step in the characterization of potentially probiotic strains, this technique was successfully used to identify a variety of unknown human intestinal isolates. The approach described here represents a rapid and de®nitive method for the identi®cation of Lact. acidophilus complex members.

CRISPR-Cas systems are now widely used for genome editing and transcriptional regulation in diverse organisms. The compact and portable nature of class 2 single effector nucleases, such as Cas9 or Cas12, has facilitated directed genome modifications in plants, animals, and microbes. However, most CRISPR-Cas systems belong to the more prevalent class 1 category, which hinges on multiprotein effector complexes. In the present study, we detail how the native type I-E CRISPR-Cas system, with a 5′-AAA-3′ protospacer adjacent motif (PAM) and a 61-nucleotide guide CRISPR RNA (crRNA) can be repurposed for efficient chromosomal targeting and genome editing in Lactobacillus crispatus, an important commensal and beneficial microbe in the vaginal and intestinal tracts. Specifically, we generated diverse mutations encompassing a 643-base pair (bp) deletion (100% efficiency), a stop codon insertion (36%), and a single nucleotide substitution (19%) in the exopolysaccharide priming-glycosyl transferase (p-gtf). Additional genetic targets included a 308-bp deletion (20%) in the prophage DNA packaging Nu1 and a 730-bp insertion of the green fluorescent protein gene downstream of enolase (23%). This approach enables flexible alteration of the formerly genetically recalcitrant species L. crispatus, with potential for probiotic enhancement, biotherapeutic engineering, and mucosal vaccine delivery. These results also provide a framework for repurposing endogenous CRISPR-Cas systems for flexible genome targeting and editing, while expanding the toolbox to include one of the most abundant and diverse systems found in nature.

The Lactobacillus acidophilus homology group comprises Gram-positive species that include L. acidophilus, L. helveticus, L. crispatus, L. amylovorus, L. gallinarum, L. delbrueckii subsp. bulgaricus, L. gasseri, and L. johnsonii. While these bacteria are closely related, they have varied ecological lifestyles as dairy and food fermenters, allochthonous probiotics, or autochthonous commensals of the host gastrointestinal tract. Bacterial cell surface components play a critical role in the molecular dialogue between bacteria and interaction signaling with the intestinal mucosa. Notably, the L. acidophilus complex is distinguished in two clades by the presence or absence of S-layers, which are semiporous crystalline arrays of self-assembling proteinaceous subunits found as the outermost layer of the bacterial cell wall. In this study, S-layer-associated proteins (SLAPs) in the exoproteomes of various S-layer-forming Lactobacillus species were proteomically identified, genomically compared, and transcriptionally analyzed. Four gene regions encoding six putative SLAPs were conserved in the S-layer-forming Lactobacillus species but not identified in the extracts of the closely related progenitor, L. delbrueckii subsp. bulgaricus, which does not produce an S-layer. Therefore, the presence or absence of an S-layer has a clear impact on the exoproteomic composition of Lactobacillus species. This proteomic complexity and differences in the cell surface properties between S-layer- and non-S-layer-forming lactobacilli reveal the potential for SLAPs to mediate intimate probiotic interactions and signaling with the host intestinal mucosa.

Lactobacillus crispatus and Lactobacillus gasseri are two of the main Lactobacillus species found in the healthy vaginal microbiome and have also previously been identified and isolated from the human gastrointestinal (GI) tract. These two ecological niches are fundamentally different, notably with regards to the epithelial cell type, nutrient availability, environmental conditions, pH, and microbiome composition. Given the dramatic differences between these two environments, we characterized strains within the same Lactobacillus species isolated from either the vaginal or intestinal tract to assess whether they are phenotypically and genetically different. We compared the genomes of the Lactobacillus strains selected in this study for genetic features of interest, and performed a series of comparative phenotypic assays including small intestinal juice and acid resistance, carbohydrate fermentation profiles, lactic acid production, and host interaction with intestinal Caco-2 and vaginal VK2 cell lines. We also developed a simulated vaginal fluid (SVF) to study bacterial growth in a proxy vaginal environment and conducted differential transcriptomic analysis between SVF and standard laboratory MRS medium. Overall, our results show that although strain-specific variation is observed, some phenotypic differences seem associated with the isolation source. We encourage future probiotic formulation to include isolation source and take into consideration genetic and phenotypic features for use at various body sites.