ABSTRACT Insertion sequences (ISs) are key components of most bacterial genomes and play a crucial role in bacterial mutagenesis. In this study, we observed the insertion of an IS element, ISLrh, from the Lacticaseibacillus rhamnosus M1 genome into plasmid pGK12, resulting in the generation of a new plasmid, pTRK829. This insertion enabled pTRK829 to replicate in hosts previously incompatible with pGK12, including L. rhamnosus M1, L. rhamnosus GG (LGG), Lacticaseibacillus casei ATCC 393, and Lacticaseibacillus paracasei ATCC 25598. However, the ISLrh-inserted plasmid, pTRK829, was unstable and underwent a spontaneous deletion, resulting in a smaller and more stable variant, pTRK830, which retained ISLrh. Characterization of pTRK829 and pTRK830 across several host strains showed that ISLrh insertion led to a dramatic alteration in host range and impacted plasmid stability and copy number. Sequence and functional analysis of pTRK830 revealed that the terminal inverted repeats (IRs) of the inserted ISLrh and its insertion location were essential for plasmid replication in LGG. Finally, pTRK830 was successfully used as an expression vector for heterologous β-glucuronidase expression in LGG, L. casei ATCC 393, and L. paracasei ATCC 25598. In conclusion, this study demonstrated that the insertion of the IRs from ISLrh at a specific location can directly change the host range and stability of pGK12. Furthermore, we also demonstrated the potential of pTRK830 as a new cloning and expression vector for genetically intractable lactobacilli. IMPORTANCE This study highlights the significant impact of insertion sequence (IS) elements on plasmid replication in lactobacilli. The spontaneous integration of an IS element from the Laticaseibacillus rhamnosus genome into plasmid pGK12 not only expands its host range in previously incompatible strains but also changes plasmid stability and copy number. This expansion of the plasmid's host range is crucial for developing versatile genetic tools across diverse lactobacilli species. Additionally, the stable plasmid variant of pGK12 with the IS element insertion offers a valuable tool for cloning and gene expression in lactobacilli. These findings enhance our understanding of plasmid-IS element interactions and may provide insight into a new method to expand the host range of existing plasmids.

Lactobacillus acidophilus NCFM is a probiotic strain commonly used in dairy products and dietary supplements. Postgenome in vitro studies of NCFM thus far have linked potential key genotypes to its probiotic-relevant attributes, including gut survival, prebiotic utilization, host interactions, and immunomodulatory activities. To corroborate and extend beyond previous in vivo and in vitro functional studies, we employed a dual RNA sequencing (RNA-seq) transcriptomic approach to identify genes potentially driving the gut fitness and activities of L. acidophilus NCFM in vivo, and in parallel, examine the ileal transcriptional response of its murine hosts during monocolonization. Spatial expression profiling of NCFM from the ileum through the colon revealed a set of 134 core genes that were consistently overexpressed during gut transit. These in vivo core genes are predominantly involved in the metabolism of carbohydrates, amino acids, and nucleotides, along with mucus-binding proteins and adhesion factors, confirming their functionally important roles in nutrient acquisition and gut retention. Functional characterization of the highly expressed major S-layer-encoding gene established its indispensable role as a cell shape determinant and maintenance of cell surface integrity, essential for viability and probiotic attributes. Host colonization by L. acidophilus resulted in significant downregulation of several proinflammatory cytokines and tight junction proteins. Genes related to redox signaling, mucin glycosylation, and circadian rhythm modulation were induced, suggesting impacts on intestinal development and immune functions. Metagenomic analysis of NCFM populations postcolonization demonstrated the genomic stability of L. acidophilus as a gut transient and further established its safety as a probiotic and biotherapeutic delivery platform.IMPORTANCE To date, our basis for comprehending the probiotic mechanisms of Lactobacillus acidophilus, one of the most widely consumed probiotic microbes, was largely limited to in vitro functional genomic studies. Using a germfree murine colonization model, in vivo-based transcriptional studies provided the first view of how L. acidophilus survives in the mammalian gut environment, including gene expression patterns linked to survival, efficient nutrient acquisition, stress adaptation, and host interactions. Examination of the host ileal transcriptional response, the primary effector site of L. acidophilus, has also shed light into the mechanistic roles of this probiotic microbe in promoting anti-inflammatory responses, maintaining intestinal epithelial homeostasis and modulation of the circadian-metabolic axis in its host.

Background. Our recent publication (Chey et al., Nutrients 2020) showed that a 30-day administration of pure galacto-oligosaccharides (GOS) significantly reduced symptoms and altered the fecal microbiome in patients with lactose intolerance (LI). Results. In this addendum, we performed an in-depth analysis of the fecal microbiome of the 377 LI patients randomized to one of two GOS doses (Low, 10–15 grams/day or High, 15–20 grams/day), or placebo in a multi-center, double-blinded, placebo-controlled trial. Sequencing of 16S rRNA amplicons was done on GOS or placebo groups at weeks zero (baseline), four (end of treatment), nine, 16 and 22. Taxa impacted by treatment and subsequent dairy consumption included lactose-fermenting species of Bifidobacterium, Lactobacillus, Lactococcus, and Streptococcus. Increased secondary fermentation microorganisms included Coprococcus and Ruminococcus species, Blautia producta, and Methanobrevibacterium. Finally, tertiary fermenters that use acetate to generate butyrate were also increased, including Faecalibacterium prausnitzii, Roseburia faecis, and C. eutactus. Conclusions. Results confirmed and expanded data on GOS microbiome modulation in LI individuals. Microbiome analysis at 16 and 22 weeks after treatment further suggested relatively long-term benefits when individuals continued consumption of dairy products.

Probiotic bacteria have long been believed to influence general health and well-being through their association with the gastrointestinal tract (GIT) and its normal microbiota. The microbiotas of humans, animals, and fowl vary considerably with the architecture of their GITs. Species of microorganisms are located at different locations throughout the GIT and include strains that are either harmful or beneficial to the host depending on the circumstances and specific strains involved. Probiotic microorganisms typically designed for delivery in dairy foods are most often members of the Lactobacillus or Bifidobacterium genus. This chapter discusses the effects of probiotics on GIT ecology, and deals with the appropriateness, technological suitability, competitiveness, and performance and functionality, as the criteria for selection of probiotic cultures. Prebiotics stimulate the growth and activity of beneficial bacteria in an individual's intestinal microbiota. The best-known prebiotics are fructo-oligosaccharides derived from food sources. Production of designer prebiotics can offer multiple activities in retarding undesirable microorganisms, better promoting the native desirable microbiota, or stimulating the growth or activity of synbiotic cultures. Expansion of avenues for incorporation into appropriate food vehicles and improved stimulation of beneficial microfloras are some of the aspects that are good targets for development of prebiotics.

The Klaenhammer group at North Carolina State University pioneered genomic applications in food microbiology and beneficial lactic acid bacteria used as starter cultures and probiotics. Dr. Todd Klaenhammer was honored to be the first food scientist elected to the National Academy of Sciences (2001). The program was recognized with the highest research awards presented by the American Dairy Science Association (Borden Award 1996), the Institute of Food Technologists (Nicholas Appert Medal, 2007), and the International Dairy Federation (Eli Metchnikoff Award in Biotechnology, 2010) as well as with the Outstanding Achievement Award from the University of Minnesota (2001) and the Oliver Max Gardner Award (2009) for outstanding research across the 16-campus University of North Carolina system. Dr. Klaenhammer is a fellow of the American Association for the Advancement of Science, the American Dairy Science Association, and the Institute of Food Technology. Over his career, six of his PhD graduate students were awarded the annual Kenneth Keller award for the outstanding PhD dissertation that year in the College of Agriculture and Life Sciences. He championed the use of basic microbiology and genomic approaches to set a platform for translational applications of beneficial microbes in foods and their use in food preservation and probiotics and as oral delivery vehicles for vaccines and biotherapeutics. Dr. Klaenhammer was also a founding and co-chief editor of the Annual Review of Food Science and Technology.

Lactobacillus gasseri is a human commensal which carries CRISPR-Cas, an adaptive immune system that protects the cell from invasive mobile genetic elements (MGEs). However, MGEs occasionally escape CRISPR targeting due to DNA mutations that occur in sequences involved in CRISPR interference. To better understand CRISPR escape processes, a plasmid interference assay was used to screen for mutants that escape CRISPR-Cas targeting. Plasmids containing a target sequence and a protospacer adjacent motif (PAM) were transformed for targeting by the native CRISPR-Cas system. Although the primary outcome of the assay was efficient interference, a small proportion of the transformed population overcame targeting. Mutants containing plasmids that had escaped were recovered to investigate the genetic routes of escape and their relative frequencies. Deletion of the targeting spacer in the native CRISPR array was the dominant pattern of escape, accounting for 52-70 % of the mutants from two L. gasseri strains. We repeatedly observed internal deletions in the chromosomal CRISPR array, characterized by polarized excisions from the leader end that spanned 1-15 spacers, and systematically included the leader-proximal targeting spacer. This study shows that deletions of spacers within CRISPR arrays constitute a key escape mechanism to evade CRISPR targeting, while preserving the functionality of the CRISPR-Cas system. This mechanism enables cells to maintain an active immune system, but allows the uptake of potentially beneficial plasmids. Our study revealed the co-occurrence of other genomic mutations associated with various phenotypes, showing how this selection process uncovers population diversification.

Safe and efficacious orally-delivered mucosal vaccine platforms are desperately needed to combat the plethora of mucosally transmitted pathogens. Lactobacillus spp. have emerged as attractive candidates to meet this need and are known to activate the host innate immune response in a species- and strain-specific manner. For selected bacterial isolates and mutants, we investigated the role of key innate immune pathways required for induction of innate and subsequent adaptive immune responses. Co-culture of murine macrophages with L. gasseri (strain NCK1785), L. acidophilus (strain NCFM), or NCFM-derived mutants—NCK2025 and NCK2031—elicited an M2b-like phenotype associated with TH2 skewing and immune regulatory function. For NCFM, this M2b phenotype was dependent on expression of lipoteichoic acid and S layer proteins. Through the use of macrophage genetic knockouts, we identified Toll-like receptor 2 (TLR2), the cytosolic nucleotide-binding oligomerization domain containing 2 (NOD2) receptor, and the inflammasome-associated caspase-1 as contributors to macrophage activation, with NOD2 cooperating with caspase-1 to induce inflammasome derived interleukin (IL)-1β in a pyroptosis-independent fashion. Finally, utilizing an NCFM-based mucosal vaccine platform with surface expression of human immunodeficiency virus type 1 (HIV-1) Gag or membrane proximal external region (MPER), we demonstrated that NOD2 signaling is required for antigen-specific mucosal and systemic humoral responses. We show that lactobacilli differentially utilize innate immune pathways and highlight NOD2 as a key mediator of macrophage function and antigen-specific humoral responses to a Lactobacillus acidophilus mucosal vaccine platform.

Introduction: Lactose Intolerance (LI) causes gastrointestinal (GI) symptoms in affected individuals. A novel galacto-oligosaccharide (RP-G28) shifts the human gut microbiota by altering the relative abundance of specific bacteria. These changes remove undigested lactose from the gut, thus reducing the gas production and water secretion that produce LI symptoms. Methods: 377 LI patients in a double-blind, multi-centered Phase 2b clinical trial received either low (n=127) or high (n=123) doses of RP-G28 or placebo (n=127). Changes in gut microbiome were assessed by 16S rRNA amplicon sequencing at baseline (day 0) and 30-days post treatment (day 31). Supervised learning analysis (Random Forest) was used to build a model of taxa for investigation. High-throughput quantitative polymerase chain reaction (qPCR) identified the relative abundances of Bifidobacterium and Lactobacillus sp. Results: Treatment with RP-G28 resulted in adaptations of the microbiota and abundance of 28 bacterial species. Specifically, an increase of lactose-fermenting Bifidobacterium was observed. qPCR data showed a significant increase in the relative abundance of the phylum Actinobacteria, family Bifidobacteriaceae, genus Bifidobacterium and family Lactobacillaceae after 30 days of treatment, in both low- and high-dose RP-G28 patients, but not in the placebo group. Specifically, abundances of B. angulatum, B. gallicum, B. longum, B. bifidum, B. breve, and B. catenulatum increased by RP-G28. Actinobacteria of the family Bifidobacteriaceae increased in a higher proportion of treated patients. Bifidobacteria increased in 77.7% (77/99) of patients in the RP-G28 groups compared to 52.1% (49/94) in the placebo group (p=<.001), correlating with a prior 85-subject Phase 2a trial of RP-G28. Pediococcus, species of the Lactobacillales order, Enterococcaceae, and Delftia were increased in responders in both low- and high-dose groups. Conclusion: The results confirm that RP-G28 promoted a beneficial adaptation of the gut microbiome. RP-G28 promoted a significant increase in lactose-metabolizing Bifidobacterium providing a plausible explanation for the clinical benefits observed with this therapeutic in LI patients. Whether the changes in gut microbiome induced by RP-G28 offers other health benefits for the GI tract or immune system deserves further study.

Lactobacillus buchneri is a Gram-positive, obligate heterofermentative, facultative anaerobe commonly affiliated with spoilage of food products. Notably, L. buchneri is able to metabolize lactic acid into acetic acid and 1,2-propanediol. Although beneficial to the silage industry, this metabolic capability is detrimental to preservation of cucumbers by fermentation. The objective of this study was to characterize isolates of L. buchneri purified from both industrial and experimental fermented cucumber after the onset of secondary fermentation. Genotypic and phenotypic characterization included 16S rRNA sequencing, DiversiLab((R)) rep-PCR, colony morphology, API 50 CH carbohydrate analysis, and ability to degrade lactic acid in modified MRS and fermented cucumber media. Distinct groups of isolates were identified with differing colony morphologies that varied in color (translucent white to opaque yellow), diameter (1 mm-11 mm), and shape (umbonate, flat, circular or irregular). Growth rates in MRS revealed strain differences, and a wide spectrum of carbon source utilization was observed. Some strains were able to ferment as many as 21 of 49 tested carbon sources, including inulin, fucose, gentiobiose, lactose, mannitol, potassium ketogluconate, saccharose, raffinose, galactose, and xylose, while others metabolized as few as eight carbohydrates as the sole source of carbon. All isolates degraded lactic acid in both fermented cucumber medium and modified MRS, but exhibited differences in the rate and extent of lactate degradation. I Isolates clustered into eight distinct groups based on rep-PCR fingerprints with 20 of 36 of the isolates exhibiting > 97% similarity. Although isolated from similar environmental niches, significant phenotypic and genotypic diversity was found among the L. buchneri cultures. A collection of unique L. buchneri strains was identified and characterized, providing the basis for further analysis of metabolic and genomic capabilities of this species to enable control of lactic acid degradation in fermented plant materials.

Comprehension of underlying mechanisms of probiotic action will support rationale selection of probiotic strains and targeted clinical study design with a higher likelihood of success. This will consequently contribute to better substantiation of health claims. Here, we aim to provide a perspective from a microbiology point of view that such comprehensive understanding is not straightforward. We show examples of well-documented probiotic effector molecules in Lactobacillus and Bifidobacterium strains, including surface-located molecules such as specific pili, S-layer proteins, exopolysaccharides, muropeptides, as well as more widely produced metabolites such as tryptophan-related and histamine-related metabolites, CpG-rich DNA, and various enzymes such as lactase and bile salt hydrolases. We also present recent advances in genetic tool development, microbiome analyses and model systems, as well as perspectives on how the field could further progress. This opinion is based on a discussion group organized at the annual meeting of the International Scientific Association on Probiotics and Prebiotics (ISAPP) in June 2017.

Directed modulation of the colonic bacteria to metabolize lactose effectively is a potentially useful approach to improve lactose digestion and tolerance. A randomized, double-blind, multisite placebo-controlled trial conducted in human subjects demonstrated that administration of a highly purified (>95%) short-chain galactooligosaccharide (GOS), designated "RP-G28," significantly improved clinical outcomes for lactose digestion and tolerance. In these individuals, stool samples were collected pretreatment (day 0), after GOS treatment (day 36), and 30 d after GOS feeding stopped and consumption of dairy products was encouraged (day 66). In this study, changes in the fecal microbiome were investigated using 16S rRNA amplicon pyrosequencing and high-throughput quantitative PCR. At day 36, bifidobacterial populations were increased in 27 of 30 of GOS subjects (90%), demonstrating a bifidogenic response in vivo. Relative abundance of lactose-fermenting Bifidobacterium, Faecalibacterium, and Lactobacillus were significantly increased in response to GOS. When dairy was introduced into the diet, lactose-fermenting Roseburia species increased from day 36 to day 66. The results indicated a definitive change in the fecal microbiome of lactose-intolerant individuals, increasing the abundance of lactose-metabolizing bacteria that were responsive to dietary adaptation to GOS. This change correlated with clinical outcomes of improved lactose tolerance.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins form adaptive immune systems that occur in many bacteria and most archaea. In addition to protecting bacteria from phages and other invasive mobile genetic elements, CRISPR-Cas molecular machines can be repurposed as tool kits for applications relevant to the food industry. A primary concern of the food industry has long been the proper management of food-related bacteria, with a focus on both enhancing the outcomes of beneficial microorganisms such as starter cultures and probiotics and limiting the presence of detrimental organisms such as pathogens and spoilage microorganisms. This review introduces CRISPR-Cas as a novel set of technologies to manage food bacteria and offers insights into CRISPR-Cas biology. It primarily focuses on the applications of CRISPR-Cas systems and tools in starter cultures and probiotics, encompassing strain-typing, phage resistance, plasmid vaccination, genome editing, and antimicrobial activity.

Lactobacillus acidophilus NCFM is a well-characterized probiotic microorganism, supported by a decade of genomic and functional phenotypic investigations. L. acidophilus deficient in lipoteichoic acid (LTA), a major immunostimulant in Gram-positive bacteria, has been shown to shift immune system responses in animal disease models. However, the pleiotropic effects of removing LTA from the cell surface in lactobacilli are unknown. In this study, we surveyed the global transcriptional and extracellular protein profiles of two strains of L. acidophilus deficient in LTA. Twenty-four differentially expressed genes specific to the LTA-deficient strains were identified, including a predicted heavy metal resistance operon and several putative peptidoglycan hydrolases. Cell morphology and manganese sensitivity phenotypes were assessed in relation to the putative functions of differentially expressed genes. LTA-deficient L. acidophilus exhibited elongated cellular morphology and their growth was severely inhibited by elevated manganese concentrations. Exoproteomic surveys revealed distinct changes in the composition and relative abundances of several extracellular proteins and showed a bias of intracellular proteins in LTA-deficient strains of L. acidophilus. Taken together, these results elucidate the impact of ltaS deletion on the transcriptome and extracellular proteins of L. acidophilus, suggesting roles of LTA in cell morphology and ion homeostasis as a structural component of the Gram positive cell wall.

ABSTRACT Therapeutically active glycosylated phytochemicals are ubiquitous in the human diet. The human gut microbiota (HGM) modulates the bioactivities of these compounds, which consequently affect host physiology and microbiota composition. Despite a significant impact on human health, the key players and the underpinning mechanisms of this interplay remain uncharacterized. Here, we demonstrate the growth of Lactobacillus acidophilus on mono- and diglucosyl dietary plant glycosides (PGs) possessing small aromatic aglycones. Transcriptional analysis revealed the upregulation of host interaction genes and identified two loci that encode phosphotransferase system (PTS) transporters and phospho-β-glucosidases, which mediate the uptake and deglucosylation of these compounds, respectively. Inactivating these transport and hydrolysis genes abolished or severely reduced growth on PG, establishing the specificity of the loci to distinct groups of PGs. Following intracellular deglucosylation, the aglycones of PGs are externalized, rendering them available for absorption by the host or for further modification by other microbiota taxa. The PG utilization loci are conserved in L. acidophilus and closely related lactobacilli, in correlation with versatile growth on these compounds. Growth on the tested PG appeared more common among human gut lactobacilli than among counterparts from other ecologic niches. The PGs that supported the growth of L. acidophilus were utilized poorly or not at all by other common HGM strains, underscoring the metabolic specialization of L. acidophilus. These findings highlight the role of human gut L. acidophilus and select lactobacilli in the bioconversion of glycoconjugated phytochemicals, which is likely to have an important impact on the HGM and human host. IMPORTANCE Thousands of therapeutically active plant-derived compounds are widely present in berries, fruits, nuts, and beverages like tea and wine. The bioactivity and bioavailability of these compounds, which are typically glycosylated, are altered by microbial bioconversions in the human gut. Remarkably, little is known about the bioconversion of PGs by the gut microbial community, despite the significance of this metabolic facet to human health. Our work provides the first molecular insights into the metabolic routes of diet relevant and therapeutically active PGs by Lactobacillus acidophilus and related human gut lactobacilli. This taxonomic group is adept at metabolizing the glucoside moieties of select PG and externalizes their aglycones. The study highlights an important role of lactobacilli in the bioconversion of dietary PG and presents a framework from which to derive molecular insights into their metabolism by members of the human gut microbiota.

Health-promoting aspects attributed to probiotic microorganisms, including adhesion to intestinal epithelia and modulation of the host mucosal immune system, are mediated by proteins found on the bacterial cell surface. Notably, certain probiotic and commensal bacteria contain a surface (S-) layer as the outermost stratum of the cell wall. S-layers are non-covalently bound semi-porous, crystalline arrays of self-assembling, proteinaceous subunits called S-layer proteins (SLPs). Recent evidence has shown that multiple proteins are non-covalently co-localized within the S-layer, designated S-layer associated proteins (SLAPs). In Lactobacillus acidophilus NCFM, SLP and SLAPs have been implicated in both mucosal immunomodulation and adhesion to the host intestinal epithelium. In this study, a S-layer associated serine protease homolog, PrtX (prtX, lba1578), was deleted from the chromosome of L. acidophilus NCFM. Compared to the parent strain, the PrtX-deficient strain (ΔprtX) demonstrated increased autoaggregation, an altered cellular morphology, and pleiotropic increases in adhesion to mucin and fibronectin, in vitro. Furthermore, ΔprtX demonstrated increased in vitro immune stimulation of IL-6, IL-12, and IL-10 compared to wild-type, when exposed to mouse dendritic cells. Finally, in vivo colonization of germ-free mice with ΔprtX led to an increase in epithelial barrier integrity. The absence of PrtX within the exoproteome of a ΔprtX strain caused morphological changes, resulting in a pleiotropic increase of the organisms’ immunomodulatory properties and interactions with some intestinal epithelial cell components.

ABSTRACT Of the few predicted extracellular glycan-active enzymes, glycoside hydrolase family 13 subfamily 14 (GH13_14) pullulanases are the most common in human gut lactobacilli. These enzymes share a unique modular organization, not observed in other bacteria, featuring a catalytic module, two starch binding modules, a domain of unknown function, and a C-terminal surface layer association protein (SLAP) domain. Here, we explore the specificity of a representative of this group of pullulanases, Lactobacillus acidophilus Pul13_14 ( La Pul13_14), and its role in branched α-glucan metabolism in the well-characterized Lactobacillus acidophilus NCFM, which is widely used as a probiotic. Growth experiments with L. acidophilus NCFM on starch-derived branched substrates revealed a preference for α-glucans with short branches of about two to three glucosyl moieties over amylopectin with longer branches. Cell-attached debranching activity was measurable in the presence of α-glucans but was repressed by glucose. The debranching activity is conferred exclusively by La Pul13_14 and is abolished in a mutant strain lacking a functional La Pul13_14 gene. Hydrolysis kinetics of recombinant La Pul13_14 confirmed the preference for short-branched α-glucan oligomers consistent with the growth data. Curiously, this enzyme displayed the highest catalytic efficiency and the lowest K m reported for a pullulanase. Inhibition kinetics revealed mixed inhibition by β-cyclodextrin, suggesting the presence of additional glucan binding sites besides the active site of the enzyme, which may contribute to the unprecedented substrate affinity. The enzyme also displays high thermostability and higher activity in the acidic pH range, reflecting adaptation to the physiologically challenging conditions in the human gut. IMPORTANCE Starch is one of the most abundant glycans in the human diet. Branched α-1,6-glucans in dietary starch and glycogen are nondegradable by human enzymes and constitute a metabolic resource for the gut microbiota. The role of health-beneficial lactobacilli prevalent in the human small intestine in starch metabolism remains unexplored in contrast to colonic bacterial residents. This study highlights the pivotal role of debranching enzymes in the breakdown of starchy branched α-glucan oligomers (α-limit dextrins) by human gut lactobacilli exemplified by Lactobacillus acidophilus NCFM, which is one of the best-characterized strains used as probiotics. Our data bring novel insight into the metabolic preference of L. acidophilus for α-glucans with short α-1,6-branches. The unprecedented affinity of the debranching enzyme that confers growth on these substrates reflects its adaptation to the nutrient-competitive gut ecological niche and constitutes a potential advantage in cross-feeding from human and bacterial dietary starch metabolism.

ABSTRACT Autolysins, also known as peptidoglycan hydrolases, are enzymes that hydrolyze specific bonds within bacterial cell wall peptidoglycan during cell division and daughter cell separation. Within the genome of Lactobacillus acidophilus NCFM, there are 11 genes encoding proteins with peptidoglycan hydrolase catalytic domains, 9 of which are predicted to be functional. Notably, 5 of the 9 putative autolysins in L. acidophilus NCFM are S-layer-associated proteins (SLAPs) noncovalently colocalized along with the surface (S)-layer at the cell surface. One of these SLAPs, AcmB, a β- N -acetylglucosaminidase encoded by the gene lba0176 ( acmB ), was selected for functional analysis. In silico analysis revealed that acmB orthologs are found exclusively in S-layer- forming species of Lactobacillus . Chromosomal deletion of acmB resulted in aberrant cell division, autolysis, and autoaggregation. Complementation of acmB in the Δ acmB mutant restored the wild-type phenotype, confirming the role of this SLAP in cell division. The absence of AcmB within the exoproteome had a pleiotropic effect on the extracellular proteins covalently and noncovalently bound to the peptidoglycan, which likely led to the observed decrease in the binding capacity of the Δ acmB strain for mucin and extracellular matrices fibronectin, laminin, and collagen in vitro . These data suggest a functional association between the S-layer and the multiple autolysins noncovalently colocalized at the cell surface of L. acidophilus NCFM and other S-layer-producing Lactobacillus species. IMPORTANCE Lactobacillus acidophilus is one of the most widely used probiotic microbes incorporated in many dairy foods and dietary supplements. This organism produces a surface (S)-layer, which is a self-assembling crystalline array found as the outermost layer of the cell wall. The S-layer, along with colocalized associated proteins, is an important mediator of probiotic activity through intestinal adhesion and modulation of the mucosal immune system. However, there is still a dearth of information regarding the basic cellular and evolutionary function of S-layers. Here, we demonstrate that multiple autolysins, responsible for breaking down the cell wall during cell division, are associated with the S-layer. Deletion of the gene encoding one of these S-layer-associated autolysins confirmed its autolytic role and resulted in reduced binding capacity to mucin and intestinal extracellular matrices. These data suggest a functional association between the S-layer and autolytic activity through the extracellular presentation of autolysins.

Probiotic research has progressed greatly in recent years, to the extent that full genomic sequencing is becoming commonplace for strains used in intervention trials. Unfortunately, examples of ina...

Whole cell and surface proteomes were analyzed together with adhesive properties of the probiotic bacterium Lactobacillus acidophilus NCFM (NCFM) grown on the emerging prebiotic raffinose, exemplifying a synbiotic. Adhesion of NCFM to mucin and intestinal HT‐29 cells increased three‐fold after culture with raffinose versus glucose, as also visualized by scanning electron microscopy. Comparative proteomics using 2D‐DIGE showed 43 unique proteins to change in relative abundance in whole cell lysates from NCFM grown on raffinose compared to glucose. Furthermore, 14 unique proteins in 18 spots of the surface subproteome underwent changes identified by differential 2DE, including elongation factor G, thermostable pullulanase, and phosphate starvation inducible stress‐related protein increasing in a range of +2.1 − +4.7 fold. By contrast five known moonlighting proteins decreased in relative abundance by up to −2.4 fold. Enzymes involved in raffinose catabolism were elevated in the whole cell proteome; α‐galactosidase (+13.9 fold); sucrose phosphorylase (+5.4 fold) together with metabolic enzymes from the Leloir pathway for galactose utilization and the glycolysis; β‐galactosidase (+5.7 fold); galactose (+2.9/+3.1 fold) and fructose (+2.8 fold) kinases. The insights at the molecular and cellular levels contributed to the understanding of the interplay of a synbiotic composed of NCFM and raffinose with the host.