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Lactobacillus is the bacterial genus that contains the highest number of characterized probiotics. Lactobacilli in general can utilize a great variety of carbohydrates. This characteristic is an essential trait for their survival in highly competitive environments such as the gastrointestinal tract of animals. In particular, the ability of some strains to utilize complex carbohydrates such as milk oligosaccharides as well as their precursor monosaccharides, confer upon lactobacilli a competitive advantage. For this reason, many of these carbohydrates are considered as prebiotics. Genome sequencing of many lactobacilli strains has revealed a great variety of genes involved in the metabolism of carbohydrates and some of them have already been characterized. In this review, the current knowledge at biochemical and genetic levels on the catabolic pathways of complex carbohydrates utilized by lactobacilli will be summarized.

Lacto-N-biose (LNB) and galacto-N-biose (GNB) are major building blocks of free oligosaccharides and glycan moieties of glyco-complexes present in human milk and gastrointestinal mucosa. We have previously characterized the phospho-β-galactosidase GnbG from Lactobacillus casei BL23 that is involved in the metabolism of LNB and GNB. GnbG has been used here in transglycosylation reactions, and it showed the production of LNB and GNB with N-acetylglucosamine and N-acetylgalactosamine as acceptors, respectively. The reaction kinetics demonstrated that GnbG can convert 69 ± 4 and 71 ± 1 % of o-nitrophenyl-β-D-galactopyranoside into LNB and GNB, respectively. Those reactions were performed in a semi-preparative scale, and the synthesized disaccharides were purified. The maximum yield obtained for LNB was 10.7 ± 0.2 g/l and for GNB was 10.8 ± 0.3 g/l. NMR spectroscopy confirmed the molecular structures of both carbohydrates and the absence of reaction byproducts, which also supports that GnbG is specific for β1,3-glycosidic linkages. The purified sugars were subsequently tested for their potential prebiotic properties using Lactobacillus species. The results showed that LNB and GNB were fermented by the tested strains of L. casei, Lactobacillus rhamnosus (except L. rhamnosus strain ATCC 53103), Lactobacillus zeae, Lactobacillus gasseri, and Lactobacillus johnsonii. DNA hybridization experiments suggested that the metabolism of those disaccharides in 9 out of 10 L. casei strains, all L. rhamnosus strains and all L. zeae strains tested relies upon a phospho-β-galactosidase homologous to GnbG. The results presented here support the putative role of human milk oligosaccharides for selective enrichment of beneficial intestinal microbiota in breast-fed infants.

Polyphosphate (poly-P) is a polymer of phosphate residues synthesized and in some cases accumulated by microorganisms, where it plays crucial physiological roles such as the participation in the response to nutritional stringencies and environmental stresses. Poly-P metabolism has received little attention in Lactobacillus, a genus of lactic acid bacteria of relevance for food production and health of humans and animals. We show that among 34 strains of Lactobacillus, 18 of them accumulated intracellular poly-P granules, as revealed by specific staining and electron microscopy. Poly-P accumulation was generally dependent on the presence of elevated phosphate concentrations in the culture medium, and it correlated with the presence of polyphosphate kinase (ppk) genes in the genomes. The ppk gene from Lactobacillus displayed a genetic arrangement in which it was flanked by two genes encoding exopolyphosphatases of the Ppx-GppA family. The ppk functionality was corroborated by its disruption (LCABL_27820 gene) in Lactobacillus casei BL23 strain. The constructed ppk mutant showed a lack of intracellular poly-P granules and a drastic reduction in poly-P synthesis. Resistance to several stresses was tested in the ppk-disrupted strain, showing that it presented a diminished growth under high-salt or low-pH conditions and an increased sensitivity to oxidative stress. These results show that poly-P accumulation is a characteristic of some strains of lactobacilli and may thus play important roles in the physiology of these microorganisms.

Members of the genus Lactobacillus are commonly found at the gastrointestinal tract and other mucosal surfaces of humans. This genus includes various species with a great number of potentially probiotic bacteria. Other often-used probiotic species belong to Bifidobacterium , a genus almost exclusively associated with the gut. As probiotics must survive and be metabolically active at their target sites, namely host mucosal surfaces, consumption of host-produced glycans is a key factor for their survival and activity. The ability to metabolize glycans such as human milk oligosaccharides (HMOs), glycosaminoglycans and the glycan moieties of glycoproteins and glycolipids found at the mucosal surfaces grants a competitive advantage to lactobacilli and bifidobacteria. The analyses of the great number of sequenced genomes from these bacteria have revealed that many of them encode a wide assortment of genes involved in the metabolism and transport of carbohydrates, including several glycoside hydrolases required for metabolizing the carbohydrate moieties of mucins and HMOs. Here, the current knowledge on the genetic mechanisms, known catabolic pathways and biochemical properties of enzymes involved in the utilization of host-produced glycans by lactobacilli and bifidobacteria will be summarized. [ABSTRACT FROM AUTHOR]

Two-component systems (TCSs) are widespread signal transduction pathways mainly found in bacteria where they play a major role in adaptation to changing environmental conditions. TCSs generally consist of sensor histidine kinases that autophosphorylate in response to a specific stimulus and subsequently transfer the phosphate group to their cognate response regulators thus modulating their activity, usually as transcriptional regulators. In this review we present the current knowledge on the physiological role of TCSs in species of the families Lactobacillaceae and Leuconostocaceae of the group of lactic acid bacteria (LAB). LAB are microorganisms of great relevance for health and food production as the group spans from starter organisms to pathogens. Whereas the role of TCSs in pathogenic LAB (most of them belonging to the family Streptococcaceae) has focused the attention, the roles of TCSs in commensal LAB, such as most species of Lactobacillaceae and Leuconostocaceae, have been somewhat neglected. However, evidence available indicates that TCSs are key players in the regulation of the physiology of these bacteria. The first studies in food-associated LAB showed the involvement of some TCSs in quorum sensing and production of bacteriocins, but subsequent studies have shown that TCSs participate in other physiological processes, such as stress response, regulation of nitrogen metabolism, regulation of malate metabolism, and resistance to antimicrobial peptides, among others. [ABSTRACT FROM AUTHOR]

The predominant form of life for microorganisms in their natural habitats is the biofilm mode of growth. The adherence and colonization of probiotic bacteria are considered as essential factors for their immunoregulatory function in the host. Here, we show that L actobacillus casei ATCC334 adheres to and colonizes the gut of zebrafish larvae. The abundance of pro-inflammatory cytokines and the recruitment of macrophages were low when inflammation was induced in probiotic-fed animals, suggesting that these bacteria have anti-inflammatory properties. We treated human macrophage-differentiated monocytic THP-1 cells with supernatants of L . casei ATCC334 grown in either biofilm or planktonic cultures. TNF-α production was suppressed and the NF-κ B pathway was inhibited only in the presence of supernatants from biofilms. We identified GroEL as the biofilm supernatant compound responsible, at least partially, for this anti-inflammatory effect. Gradual immunodepletion of GroEL demonstrated that the abundance of GroEL and TNF-α were inversely correlated. We confirmed that biofilm development in other L actobacillus species affects the immune response. The biofilms supernatants of these species also contained large amounts of GroEL. Thus, our results demonstrate that the biofilm enhances the immunomodulatory effects of L actobacillus sp. and that secreted GroEL is involved in this beneficial effect. [ABSTRACT FROM AUTHOR]

The genomes of Lactobacillus casei/paracasei and Lactobacillus rhamnosus strains carry two genes encoding homologues of p40 and p75 from L. rhamnosus GG, two secreted proteins which display anti-apoptotic and cell protective effects on human intestinal epithelial cells. p40 and p75 carry cysteine, histidine-dependent aminohydrolase/peptidase (CHAP) and NLPC/P60 domains, respectively, which are characteristic of proteins with cell-wall hydrolase activity. In L. casei BL23 both proteins were secreted to the growth medium and were also located at the bacterial cell surface. The genes coding for both proteins were inactivated in this strain. Inactivation of LCABL_00230 (encoding p40) did not result in a significant difference in phenotype, whereas a mutation in LCABL_02770 (encoding p75) produced cells that formed very long chains. Purified glutathione-S-transferase (GST)-p40 and -p75 fusion proteins were able to hydrolyze the muropeptides from L. casei cell walls. Both fusions bound to mucin, collagen and to intestinal epithelial cells and, similar to L. rhamnosus GG p40, stimulated epidermal growth factor receptor phosphorylation in mouse intestine ex vivo. These results indicate that extracellular proteins belonging to the machinery of cell-wall metabolism in the closely related L. casei/paracasei-L. rhamnosus group are most likely involved in the probiotic effects described for these bacteria Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Sorbitol is a sugar alcohol largely used in the food industry as a low-calorie sweetener. We have previously described a sorbitol-producing Lactobacillus casei (strain BL232) in which the gutF gene, encoding a sorbitol-6-phosphate dehydrogenase, was expressed from the lactose operon. Here, a complete deletion of the ldh1 gene, encoding the main l-lactate dehydrogenase, was performed in strain BL232. In a resting cell system with glucose, the new strain, named BL251, accumulated sorbitol in the medium that was rapidly metabolized after glucose exhaustion. Reutilization of produced sorbitol was prevented by deleting the gutB gene of the phosphoenolpyruvate: sorbitol phosphotransferase system (PTSGut) in BL251. These results showed that the PTSGut did not mediate sorbitol excretion from the cells, but it was responsible for uptake and reutilization of the synthesized sorbitol. A further improvement in sorbitol production was achieved by inactivation of the mtlD gene, encoding a mannitol-1-phosphate dehydrogenase. The new strain BL300 ( lac::gutF Δ ldh1 Δ gutB mtlD) showed an increase in sorbitol production whereas no mannitol synthesis was detected, avoiding thus a polyol mixture. This strain was able to convert lactose, the main sugar from milk, into sorbitol, either using a resting cell system or in growing cells under pH control. A conversion rate of 9.4% of lactose into sorbitol was obtained using an optimized fed-batch system and whey permeate, a waste product of the dairy industry, as substrate. [ABSTRACT FROM AUTHOR]

Adhesion to intestinal epithelium is an outcome property for the selection of probiotic lactic acid bacteria strains. We have analyzed the adhesion properties of a collection of Lactobacillus casei strains from different origins, ranging from cheese isolates to commercial probiotics. Analysis of the surface characteristics of the strains by measuring adhesion to solvents (MATS test) showed that most of the strains have a basic and hydrophobic surface. The strains were able to bind ex vivo to human colon fragments at different levels and, in most cases, this adhesion correlated with the ability to in vitro binding of mucin. Attachment to this later substrate was not enhanced by growing the cells in the presence of mucin and was independent of proteinaceous factors. On the contrary, adhesion to other extracellular matrix components, such as collagen, fibronectin, or fibrinogen was partially or totally dependent on the presence of surface proteins. These results show that most of L. casei strains have in their surfaces factors that promote binding to intestinal epithelium, however, no clear correlation appears to exist between the origin of the strains and their adhesion capacities. [ABSTRACT FROM AUTHOR]

The article cites a study which identified and characterized phosphotransferase system (PTS) of Lactobacillus casei. In this study, several genes of the man operon were deleted, and the effect of these mutations on sugar uptake and carbon catabolite repression was examined. Researchers have investigated its involvement in glucose repression and regulation of sugar metabolism.

A new esterase-encoding gene was found in the draft genome sequence of Lactobacillus casei BL23 (CECT5275). It is located in an operon together with genes encoding the EIIA, EIIB, EIIC, and EIID proteins of a mannose class phosphoenolpyruvate:sugar phosphotransferase system. After overproduction in Escherichia coli and purification, the esterase could hydrolyze acetyl sugars, hence the operon was named esu for esterase-sugar uptake genes. Upstream of the genes encoding the EII components (esuABCD) and the esterase (esuE), two genes transcribed in the opposite sense were found which encode a Bacillus subtilis LevR-like transcriptional activator (esuR) and a σ54-like transcriptional factor (rpoN). As compared with the wild-type strain, elevated fructose phosphorylation was detected in L. casei mutants constitutively expressing the esu operon. However, none of the many sugars tested could induce the esu operon. The fact that EsuE exhibits esterase activity on acetyl sugars suggests that this operon could be involved in the uptake and metabolism of esterified sugars. Expression of the esu operon is similar to that of the B. subtilis lev operon: it contains a –12,–24 consensus promoter typical of σ54-regulated genes, and EsuR and RpoN are essential for its transcription which is negatively regulated by EIIBEsu. The esuABCDE transcription unit represents the first σ54-regulated operon in lactobacilli. Furthermore, replacement of His852 in the phosphoenolpyruvate:sugar phosphotransferase system regulation domain II of EsuR with Ala indicated that the transcription activator function of EsuR is inhibited by EIIBEsu-mediated phosphorylation at His852. Copyright © 2004 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The utilization of lactic acid bacteria has been proposed to mitigate the burden of heavy metal exposure through processes probably involving chelation and reduced metal bioaccessibility. We evaluated the effects of daily intake of two strains of lactobacilli (Lactobacillus intestinalis LE1 or Lactobacillus johnsonii LE2) on intestinal toxicity during methylmercury (MeHg) exposure through drinking water (5 mg/L) for two months in mice. MeHg exposure resulted in inflammation and oxidative stress at the colon, as well as an increase in intestinal permeability accompanied by decreased fecal short-chain fatty acids (SCFA). The administration of the strains resulted in a differential protective effect that, based on their chelation capacity, supported the existence of additional mechanisms of action besides chelation. Both strains reduced IL-1β levels and oxidative stress, while LE1 lowered TNF-α, diminished MeHg-induced mucus over-secretion triggered by the IL-4/IL-13/STAT6 pathway, reduced intestinal permeability, and ameliorated inflammation and oxidative stress, probably by acting on the Keap1/Nrf2/ARE pathway. Administration of LE1 partially restored SCFA contents, which could be partly responsible for the positive effects of this strain in alleviating MeHg toxicity. These results demonstrate that lactobacilli strains can be useful tools in reducing the intestinal toxicity of MeHg, the main mercurial form conveyed by food. • Specific lactobacilli strains alleviate intestinal MeHg toxicity. • LE1 and LE2 strains reduce intestinal inflammation triggered by MeHg. • LE1 reduces mucus over-secretion and recovers impaired intestinal barrier function. • LE1 strain probably acts on the IL-4/IL-13/STAT6 and Keap1/Nrf2/ARE pathways. [ABSTRACT FROM AUTHOR]

A bicameral model consisting of Caco-2 and HT29-MTX intestinal epithelial cells and THP-1-derived macrophages has been used to test the ability of two strains of Lactobacillus to protect from damage caused by mercury. Exposure to 1 mg/ml mercury [Hg(II) or methyl-Hg] for seven days in this model resulted in an inflammatory and pro-oxidant response mainly driven by macrophages. This led to an impairment in the intestinal barrier, defective tight-junctions, increased permeability and mucus hypersecretion. In addition, the wound-healing capacity of the epithelial monolayer was also diminished. However, the presence of heat-killed Lactobacillus intestinalis or Lactobacillus johnsonii cells during Hg exposure reverted these effects, and most of the parameters recovered values similar to control cells. Both lactobacilli showed the capacity to bind Hg(II) and methyl-Hg under the cell culture conditions. This points to Hg sequestration as a likely mechanism that counteracted Hg toxicity. However, differences in the Hg binding capacity and in the effects between both strains suggest that other probiotic-mediated mechanisms may play a role in the alleviation of the damage elicited by Hg. These results show the potential of the bicameral intestinal epithelial model for screening of effective strains for their use in later in vivo studies. • An in vitro model of intestinal mucosa was employed to assess the effect of probiotics on Hg toxicity. • Inflammatory/oxidant response triggered by Hg was relieved by lactobacilli. • Lactobacilli recovered impaired intestinal barrier function and epithelial repair. • Hg sequestration by lactobacilli was the main mechanism, together with additional strain-dependent factors. [ABSTRACT FROM AUTHOR]

Abstract: In lactic acid bacteria, conversion of pyruvic to lactic acid through the activity of lactate dehydrogenase (Ldh) constitutes the final step of the homofermentative pathway. Lactobacillus casei has two characterized genes encoding Ldh activities. The ldhL gene codes for an L-Ldh, which specifically catalyzes the formation of l-lactate, whereas the hicD gene codes for a d-hydroxyisocaproate dehydrogenase (HicDH), which catalyzes the conversion of pyruvate into d-lactate. In L. casei cells fermenting glucose, a mixture of l-/d-lactate with a 97:3% ratio was formed. Inactivation of hicD led to undetectable d-lactate levels after glucose fermentation, while l-lactate levels remained constant. Inactivation of ldhL did not abolish the production of l-lactate, but the lactate final concentration decreased about 25% compared to the wild type, suggesting the presence of at least a second L-Ldh. Moreover, part of the pyruvate flux was rerouted and half of the lactate produced was in the d-isomer form. ldhL inactivation in L. casei showed additional interesting effects. First, the glycolytic flux from pyruvate to lactate was redirected and other fermentation products, including acetate, acetoin, pyruvate, ethanol, diacetyl, mannitol and CO2, were produced. Second, a lack of carbon catabolite repression of lactose metabolism and N-acetyl-glucosaminidase activity was observed. This second effect could be partly avoided by growing the cells under aeration, since NADH oxidases could account for NAD+ regeneration. [Copyright &y& Elsevier]

This study evaluates the potential of strains of Lactobacillus and Saccharomyces cerevisiae to reduce inorganic arsenic [iAs, As(III)+As(V)] bioavailability. Removal of iAs from aqueous solutions by these strains was evaluated and their effect on iAs bioaccessibility was analyzed by in vitro gastrointestinal digestion. The effect of the strains on iAs absorption was also tested, using intestinal cells. The results showed that iAs removal by the yeast strains was low (<10%). In contrast, some lactobacilli substantially reduced As(V) concentration in solution (25–40%) but only when the concentration of phosphate was ≤1 mM. Both microorganisms were able to reduce cellular transport of iAs added as aqueous solution (11–58%). Moreover, a strain of Lactobacillus plantarum reduced cellular transport of iAs from rice by up to 45%. We conclude that, in vitro , some lactobacilli and yeasts could reduce intestinal absorption of inorganic As present in water and food, and consequently reduce its bioavailability. • The effect of microorganisms on the bioavailability of inorganic arsenic was tested. • As(III) and As(V) removal from solution by Saccharomyces strains was low (<10%). • As(V) removal by lactobacilli was substantial (25–40%) when inorganic phosphate concentration was ≤1 mM. • Lactobacilli and Saccharomyces strains reduced cellular transport of As(III)/As(V) in aqueous solutions. • Some strains of microorganism also reduced intestinal transport of the inorganic arsenic from rice. [ABSTRACT FROM AUTHOR]

The entire genome of Lactobacillus casei BL23, a strain with probiotic properties, has been sequenced. The genomes of BL23 and the industrially used probiotic strain Shirota YIT 9029 (Yakult) seem to be very similar. [ABSTRACT FROM AUTHOR]