Food phenolics and Lactiplantibacillus plantarum.

Phenolic compounds are important constituents of plant food products. These compounds play a key role in food characteristics such as flavor, astringency and color. Lactic acid bacteria are naturally found in raw vegetables, being Lactiplantibacillus plantarum the most commonly used commercial starter for the fermentation of plant foods. Hence, the metabolism of phenolic compounds of L. plantarum has been a subject of study in recent decades. Such studies confirm that L. plantarum, in addition to presenting catalytic capacity to transform aromatic alcohols and phenolic glycosides, exhibits two main differentiated metabolic routes that allow the biotransformation of dietary hydroxybenzoic and hydroxycinnamic acid-derived compounds. These metabolic pathways lead to the production of new compounds with new biological and organoleptic properties. The described metabolic pathways involve the action of specialized esterases, decarboxylases and reductases that have been identified through genetic analysis and biochemically characterized. The purpose of this review is to provide a comprehensive and up-to-date summary of the current knowledge of the metabolism of food phenolics in L. plantarum.

Intracellular glycogen accumulation by human gut commensals as a niche adaptation trait.

The human gut microbiota is a key contributor to host metabolism and physiology, thereby impacting in various ways on host health. This complex microbial community has developed many metabolic strategies to colonize, persist and survive in the gastrointestinal environment. In this regard, intracellular glycogen accumulation has been associated with important physiological functions in several bacterial species, including gut commensals. However, the role of glycogen storage in shaping the composition and functionality of the gut microbiota offers a novel perspective in gut microbiome research. Here, we review what is known about the enzymatic machinery and regulation of glycogen metabolism in selected enteric bacteria, while we also discuss its potential impact on colonization and adaptation to the gastrointestinal tract. Furthermore, we survey the presence of such glycogen biosynthesis pathways in gut metagenomic data to highlight the relevance of this metabolic trait in enhancing survival in the highly competitive and dynamic gut ecosystem.

Maternal-infant antibiotic resistance genes transference: what do we know?

Resistance to antibiotics is becoming a worldwide threat as infections caused by multidrug-resistant pathogenic microorganisms can overcome antibiotic treatments and spread quickly in the population. In the context of early life, newborns are at increased risk as their immune system is still under development, so infections and acquisition of resistance during childhood have short- and long-term consequences for the health. The moment of birth is the first exposure of infants to possible antibiotic-resistant microorganisms that may colonize their gut and other body sites. Different factors including mode of delivery, previous antibiotic exposure of the mother, gestational age and consumption of antibiotics in early-life have been described to modulate the neonate's microbiota, and thus, the resistome. Other factors, such as lactation, also impact the establishment and development of gut microbiota, but little is known about the role of breastmilk in transferring Antibiotic Resistant Genes (ARG). A deeper understanding of vertical transmission of antibiotic resistance from mothers to their offspring is necessary to determine the most effective strategies for reducing antibiotic resistance in the early life. In this review, we aim to present the current perspective on antibiotic resistances in mother-infant dyads, as well as a new insight on the study of the human gut and breastmilk resistome, and current strategies to overcome this public health problem, toward highlighting the gaps of knowledge that still need to be closed.

Human milk microbiota: what did we learn in the last 20 years?

Human milk (HM) is the gold standard for infant nutrition during the first months of life. Beyond its nutritional components, its complex bioactive composition includes microorganisms, their metabolites, and oligosaccharides, which also contribute to gut colonization and immune system maturation. There is growing evidence of the beneficial effects of bacteria present in HM. However, current research presents limited data on the presence and functions of other organisms. The potential biological impacts on maternal and infant health outcomes, the factors contributing to milk microbes' variations, and the potential functions in the infant's gut remain unclear. This review provides a global overview of milk microbiota, what the actual knowledge is, and what the gaps and challenges are for the next years.

Bifidobacterium breve Exopolysaccharide Blocks Dendritic Cell Maturation and Activation of CD4+ T Cells.

Exopolysaccharide (EPS) is a bacterial extracellular carbohydrate moiety which has been associated with immunomodulatory activity and host protective effects of several gut commensal bacteria. Bifidobacterium breve are early colonizers of the human gastrointestinal tract (GIT) but the role of EPS in mediating their effects on the host has not been investigated for many strains. Here, we characterized EPS production by a panel of human B. breve isolates and investigated the effect of EPS status on host immune responses using human and murine cell culture-based assay systems. We report that B. breve EPS production is heterogenous across strains and that immune responses in human THP-1 monocytes are strain-specific, but not EPS status-specific. Using wild type and isogenic EPS deficient mutants of B. breve strains UCC2003 and JCM7017 we show that EPS had strain-specific divergent effects on cytokine responses from murine bone marrow derived macrophages (BMDMs) and dendritic cells (BMDCs). The B. breve UCC2003 EPS negative (EPS-) strain increased expression of cytokine genes (Tnfa, Il6, Il12a, and Il23a) relative to untreated BMDCs and BMDCs treated with wild type strain. B. breve UCC2003 and JCM7017 EPS- strains increased expression of dendritic cell (DC) activation and maturation marker genes (Cd80, Cd83, and Cd86) relative to untreated BMDCs. Consistent with this, BMDCs co-cultured with B. breve UCC2003 and JCM7017 EPS- strains engineered to express OVA antigen activated OVA-specific OT-II CD4+ T-cells in a co-culture antigen-presentation assay while EPS proficient strains did not. Collectively, these data indicate that B. breve EPS proficient strains use EPS to prevent maturation of DCs and activation of antigen specific CD4+ T cells responses to B. breve. This study identifies a new immunomodulatory role for B. breve EPS and suggests it may be important for immune evasion of adaptive immunity by B. breve and contribute to host-microbe mutualism.

Isolation of Chromosomal and Plasmid DNA from Bifidobacteria.

Rapid and efficient protocols aimed at the isolation and purification of DNA for the purpose of downstream applications, such as cloning, PCR, Southern blotting, or sequencing, are essential for genetic, biochemical, and molecular biological analyses of a given bacterium. The protocols herein presented provide a robust and efficient method for the isolation of chromosomal and plasmid DNA from Bifidobacterium strains by organic extraction. The methods are simple, and the yield, purity, and quality of the DNA are adequate to perform various downstream applications including next-generation sequencing.

A Resource for Cloning and Expression Vectors Designed for Bifidobacteria: Overview of Available Tools and Biotechnological Applications.

Bifidobacteria represent an important group of (mostly) commensal microorganisms, which have enjoyed increasing scientific and industrial attention due to their purported health-promoting attributes. For the latter reason, several species have been granted "generally recognized as safe" (GRAS) and "qualified presumption of safety" (QPS) status by the Food and Drugs Administration (FDA) and European Food Safety Authority (EFSA) organizations. Increasing scientific evidence supports their potential as oral delivery vectors to produce bioactive and therapeutic molecules at intestinal level. In order to achieve an efficient utilization of bifidobacterial strains as health-promoting (food) ingredients, it is necessary to provide evidence on the molecular mechanisms behind their purported beneficial and probiotic traits, and precise mechanisms of interaction with their human (or other mammalian) host. In this context, developing appropriate molecular tools to generate and investigate recombinant strains is necessary. While bifidobacteria have long remained recalcitrant to genetic manipulation, a wide array of Bifidobacterium-specific replicating vectors and genetic modification procedures have been described in literature. The current chapter intends to provide an updated overview on the vectors used to genetically modify and manipulate bifidobacteria, including their general characteristics, reviewing examples of their use to successfully generate recombinant bifidobacterial strains for specific purposes, and providing a general workflow and cautions to design and conduct heterologous expression in bifidobacteria. Knowledge gaps and fields of research that may help to widen the molecular toolbox to improve the functional and technological potential of bifidobacteria are also discussed.

Metabolism of biosynthetic oligosaccharides by human-derived Bifidobacterium breve UCC2003 and Bifidobacterium longum NCIMB 8809.

This work aimed to investigate the ability of two human-derived bifidobacterial strains, i.e. Bifidobacterium breve UCC2003 and Bifidobacterium longum NCIMB 8809, to utilize various oligosaccharides (i.e., 4-galactosyl-kojibiose, lactulosucrose, lactosyl-oligofructosides, raffinosyl-oligofructosides and lactulose-derived galacto-oligosaccharides) synthesized by means of microbial glycoside hydrolases. With the exception of raffinosyl-oligofructosides, these biosynthetic oligosaccharides were shown to support growth acting as a sole carbon and energy source of at least one of the two studied strains. Production of short-chain fatty acids (SCFAs) as detected by HPLC analysis corroborated the suitability of most of the studied novel oligosaccharides as fermentable growth substrates for the two bifidobacterial strains, showing that acetic acid is the main metabolic end product followed by lactic and formic acids. Transcriptomic and functional genomic approaches carried out for B. breve UCC2003 allowed the identification of key genes encoding glycoside hydrolases and carbohydrate transport systems involved in the metabolism of 4-galactosyl-kojibiose and lactulosucrose. In particular, the role of ß-galactosidases in the hydrolysis of these particular trisaccharides was demonstrated, highlighting their importance in oligosaccharide metabolism by human bifidobacterial strains.

A Diverse Range of Human Gut Bacteria Have the Potential To Metabolize the Dietary Component Gallic Acid.

The human gut microbiota contains a broad variety of bacteria that possess functional genes, with resultant metabolites that affect human physiology and therefore health. Dietary gallates are phenolic components that are present in many foods and beverages and are regarded as having health-promoting attributes. However, the potential for metabolism of these phenolic compounds by the human microbiota remains largely unknown. The emergence of high-throughput sequencing (HTS) technologies allows this issue to be addressed. In this study, HTS was used to assess the incidence of gallate-decarboxylating bacteria within the gut microbiota of healthy individuals for whom bacterial diversity was previously determined to be high. This process was facilitated by the design and application of degenerate PCR primers to amplify a region encoding the catalytic C subunit of gallate decarboxylase (LpdC) from total metagenomic DNA extracted from human fecal samples. HTS resulted in the generation of a total of 3,261,967 sequence reads and revealed that the primary gallate-decarboxylating microbial phyla in the intestinal microbiota were Firmicutes (74.6%), Proteobacteria (17.6%), and Actinobacteria (7.8%). These reads corresponded to 53 genera, i.e., 47% of the bacterial genera detected previously in these samples. Among these genera, Anaerostipes and Klebsiella accounted for the majority of reads (40%). The usefulness of the HTS-lpdC method was demonstrated by the production of pyrogallol from gallic acid, as expected for functional gallate decarboxylases, among representative strains belonging to species identified in the human gut microbiota by this method.IMPORTANCE Despite the increasing wealth of sequencing data, the health contributions of many bacteria found in the human gut microbiota have yet to be elucidated. This study applies a novel experimental approach to predict the ability of gut microbes to carry out a specific metabolic activity, i.e., gallate metabolism. The study showed that, while gallate-decarboxylating bacteria represented 47% of the bacterial genera detected previously in the same human fecal samples, no gallate decarboxylase homologs were identified from representatives of Bacteroidetes The presence of functional gallate decarboxylases was demonstrated in representative Proteobacteria and Firmicutes strains from the human microbiota, an observation that could be of considerable relevance to the in vivo production of pyrogallol, a physiologically important bioactive compound.

Comparative genomics and genotype-phenotype associations in Bifidobacterium breve.

Bifidobacteria are common members of the gastro-intestinal microbiota of a broad range of animal hosts. Their successful adaptation to this particular niche is linked to their saccharolytic metabolism, which is supported by a wide range of glycosyl hydrolases. In the current study a large-scale gene-trait matching (GTM) effort was performed to explore glycan degradation capabilities in B. breve. By correlating the presence/absence of genes and associated genomic clusters with growth/no-growth patterns across a dataset of 20 Bifidobacterium breve strains and nearly 80 different potential growth substrates, we not only validated the approach for a number of previously characterized carbohydrate utilization clusters, but we were also able to discover novel genetic clusters linked to the metabolism of salicin and sucrose. Using GTM, genetic associations were also established for antibiotic resistance and exopolysaccharide production, thereby identifying (novel) bifidobacterial antibiotic resistance markers and showing that the GTM approach is applicable to a variety of phenotypes. Overall, the GTM findings clearly expand our knowledge on members of the B. breve species, in particular how their variable genetic features can be linked to specific phenotypes.

Transcriptional Reprogramming at Genome-Scale of Lactobacillus plantarum WCFS1 in Response to Olive Oil Challenge.

Dietary fats may exert selective pressures on Lactobacillus species, however, knowledge on the mechanisms of adaptation to fat stress in these organisms is still fragmentary. This study was undertaken to gain insight into the mechanisms of adaptation of Lactobacillus plantarum WCFS1 to olive oil challenge by whole genome transcriptional profiling using DNA microarrays. A set of 230 genes were differentially expressed by L. plantarum WCFS1 to respond to this vegetable oil. This response involved elements typical of the stringent response, as indicated by the induction of genes involved in stress-related pathways and downregulation of genes related to processes associated with rapid growth. A set of genes involved in the transport and metabolism of compatible solutes were downregulated, indicating that this organism does not require osmoprotective mechanisms in presence of olive oil. The fatty acid biosynthetic pathway was thoroughly downregulated at the transcriptional level, which coincided with a diminished expression of genes controlled by this pathway in other organisms and that are required for the respiratory function, pyruvate dehydrogenase activity, RNA processing and cell size setting. Finally, a set of genes involved in host-cell signaling by L. plantarum were differentially regulated indicating that olive oil can influence the expression of metabolic traits involved in the crosstalk between this bacterium and the host.

The Lp_3561 and Lp_3562 Enzymes Support a Functional Divergence Process in the Lipase/Esterase Toolkit from Lactobacillus plantarum.

Lactobacillus plantarum species is a good source of esterases since both lipolytic and esterase activities have been described for strains of this species. No fundamental biochemical difference exists among esterases and lipases since both share a common catalytic mechanism. L. plantarum WCFS1 possesses a protein, Lp_3561, which is 44% identical to a previously described lipase, Lp_3562. In contrast to Lp_3562, Lp_3561 was unable to degrade esters possessing a chain length higher than C4 and the triglyceride tributyrin. As in other L. plantarum esterases, the electrostatic potential surface around the active site in Lp_3561 is predicted to be basic, whereas it is essentially neutral in the Lp_3562 lipase. The fact that the genes encoding both proteins were located contiguously in the L. plantarum WCFS1 genome, suggests that they originated by tandem duplication, and therefore are paralogs as new functions have arisen during evolution. The presence of the contiguous lp_3561 and lp_3562 genes was studied among L. plantarum strains. They are located in a 8,903 bp DNA fragment that encodes proteins involved in the catabolism of sialic acid and are predicted to increase bacterial adaptability under certain growth conditions.

Enantioselective oxidation of galactitol 1-phosphate by galactitol-1-phosphate 5-dehydrogenase from Escherichia coli.

Galactitol-1-phosphate 5-dehydrogenase (GPDH) is a polyol dehydrogenase that belongs to the medium-chain dehydrogenase/reductase (MDR) superfamily. It catalyses the Zn(2+)- and NAD(+)-dependent stereoselective dehydrogenation of L-galactitol 1-phosphate to D-tagatose 6-phosphate. Here, three crystal structures of GPDH from Escherichia coli are reported: that of the open state of GPDH with Zn(2+) in the catalytic site and those of the closed state in complex with the polyols Tris and glycerol, respectively. The closed state of GPDH reveals no bound cofactor, which is at variance with the conformational transition of the prototypical mammalian liver alcohol dehydrogenase. The main intersubunit-contacting interface within the GPDH homodimer presents a large internal cavity that probably facilitates the relative movement between the subunits. The substrate analogue glycerol bound within the active site partially mimics the catalytically relevant backbone of galactitol 1-phosphate. The glycerol binding mode reveals, for the first time in the polyol dehydrogenases, a pentacoordinated zinc ion in complex with a polyol and also a strong hydrogen bond between the primary hydroxyl group and the conserved Glu144, an interaction originally proposed more than thirty years ago that supports a catalytic role for this acidic residue.

A Lactobacillus plantarum esterase active on a broad range of phenolic esters.

Lactobacillus plantarum is the lactic acid bacterial species most frequently found in the fermentation of food products of plant origin on which phenolic compounds are abundant. L. plantarum strains showed great flexibility in their ability to adapt to different environments and growth substrates. Of 28 L. plantarum strains analyzed, only cultures from 7 strains were able to hydrolyze hydroxycinnamic esters, such as methyl ferulate or methyl caffeate. As revealed by PCR, only these seven strains possessed the est_1092 gene. When the est_1092 gene was introduced into L. plantarum WCFS1 or L. lactis MG1363, their cultures acquired the ability to degrade hydroxycinnamic esters. These results support the suggestion that Est_1092 is the enzyme responsible for the degradation of hydroxycinnamic esters on the L. plantarum strains analyzed. The Est_1092 protein was recombinantly produced and biochemically characterized. Surprisingly, Est_1092 was able to hydrolyze not only hydroxycinnamic esters, since all the phenolic esters assayed were hydrolyzed. Quantitative PCR experiments revealed that the expression of est_1092 was induced in the presence of methyl ferulate, an hydroxycinnamic ester, but was inhibited on methyl gallate, an hydroxybenzoic ester. As Est_1092 is an enzyme active on a broad range of phenolic esters, simultaneously possessing feruloyl esterase and tannase activities, its presence on some L. plantarum strains provides them with additional advantages to survive and grow on plant environments.

Genetic and biochemical approaches towards unravelling the degradation of gallotannins by Streptococcus gallolyticus.

BACKGROUND: Herbivores have developed mechanisms to overcome adverse effects of dietary tannins through the presence of tannin-resistant bacteria. Tannin degradation is an unusual characteristic among bacteria. Streptococcus gallolyticus is a common tannin-degrader inhabitant of the gut of herbivores where plant tannins are abundant. The biochemical pathway for tannin degradation followed by S. gallolyticus implies the action of tannase and gallate decarboxylase enzymes to produce pyrogallol, as final product. From these proteins, only a tannase (TanBSg) has been characterized so far, remaining still unknown relevant proteins involved in the degradation of tannins. RESULTS: In addition to TanBSg, genome analysis of S. gallolyticus subsp. gallolyticus strains revealed the presence of an additional protein similar to tannases, TanASg (GALLO_0933). Interestingly, this analysis also indicated that only S. gallolyticus strains belonging to the subspecies "gallolyticus" possessed tannase copies. This observation was confirmed by PCR on representative strains from different subspecies. In S. gallolyticus subsp. gallolyticus the genes encoding gallate decarboxylase are clustered together and close to TanBSg, however, TanASg is not located in the vicinity of other genes involved in tannin metabolism. The expression of the genes enconding gallate decarboxylase and the two tannases was induced upon methyl gallate exposure. As TanBSg has been previously characterized, in this work the tannase activity of TanASg was demonstrated in presence of phenolic acid esters. TanASg showed optimum activity at pH 6.0 and 37°C. As compared to the tannin-degrader Lactobacillus plantarum strains, S. gallolyticus presented several advantages for tannin degradation. Most of the L. plantarum strains possessed only one tannase enzyme (TanBLp), whereas all the S. gallolytcius subsp. gallolyticus strains analyzed possesses both TanASg and TanBSg proteins. More interestingly, upon methyl gallate induction, only the tanB Lp gene was induced from the L. plantarum tannases; in contrast, both tannase genes were highly induced in S. gallolyticus. Finally, both S. gallolyticus tannase proteins presented higher activity than their L. plantarum counterparts. CONCLUSIONS: The specific features showed by S. gallolyticus subsp. gallolyticus in relation to tannin degradation indicated that strains from this subspecies could be considered so far the best bacterial cellular factories for tannin degradation.

In vitro bactericidal and bacteriolytic activity of ceragenin CSA-13 against planktonic cultures and biofilms of Streptococcus pneumoniae and other pathogenic streptococci.

Ceragenin CSA-13, a cationic steroid, is here reported to show a concentration-dependent bactericidal/bacteriolytic activity against pathogenic streptococci, including multidrug-resistant Streptococcus pneumoniae. The autolysis promoted by CSA-13 in pneumococcal cultures appears to be due to the triggering of the major S. pneumoniae autolysin LytA, an N-acetylmuramoyl-L-alanine amidase. CSA-13 also disintegrated pneumococcal biofilms in a very efficient manner, although at concentrations slightly higher than those required for bactericidal activity on planktonic bacteria. CSA-13 has little hemolytic activity which should allow testing its antibacterial efficacy in animal models.

Characterization of a cold-active esterase from Lactobacillus plantarum suitable for food fermentations.

Lactobacillus plantarum is a lactic acid bacteria that can be found in numerous fermented foods. Esterases from L. plantarum exert a fundamental role in food aroma. In the present study, the gene lp_2631 encoding a putative esterase was cloned and expressed in Escherichia coli BL21 (DE3) and the overproduced Lp_2631 protein has been biochemically characterized. Lp_2631 exhibited optimal esterase activity at 20 °C and more than 90% of maximal activity at 5 °C, being the first cold-active esterase described in a lactic acid bacteria. Lp_2631 exhibited 40% of its maximal activity after 2 h of incubation at 65 °C. Lp_2631 also showed marked activity in the presence of compounds commonly found in food fermentations, such as NaCl, ethanol, or lactic acid. The results suggest that Lp_2631 might be a useful esterase to be used in food fermentations.

Characterization of a versatile arylesterase from Lactobacillus plantarum active on wine esters.

The gene lp_1002 from Lactobacillus plantarum WCFS1 encoding a putative lipase/esterase was cloned and overexpressed in Escherichia coli BL21(DE3). The purified Lp_1002 protein was biochemically characterized. Lp_1002 is an arylesterase which showed high hydrolytic activity on phenyl acetate. Although to a lesser extent, Lp_1002 also hydrolyzed most of the esters assayed including relevant wine aroma compounds. Importantly, Lp_1002 exhibited hydrolytic activity at winemaking conditions, although optimal catalytic activity is observed at 40 °C and pH 5-7. The effect of wine compounds on Lp_1002 activity was assayed. From the compounds assayed (ethanol, sodium metabisulfite, and malic, tartaric, lactic and citric acids), only malic acid slightly inhibited Lp_1002 activity. Lp_1002 is the first arylesterase described in a wine lactic acid bacteria and possessed suitable biochemical properties to be used during winemaking.

Esterase LpEst1 from Lactobacillus plantarum: a novel and atypical member of the αß hydrolase superfamily of enzymes.

The genome of the lactic acid bacterium Lactobacillus plantarum WCFS1 reveals the presence of a rich repertoire of esterases and lipases highlighting their important role in cellular metabolism. Among them is the carboxylesterase LpEst1 a bacterial enzyme related to the mammalian hormone-sensitive lipase, which is known to play a central role in energy homeostasis. In this study, the crystal structure of LpEst1 has been determined at 2.05 Å resolution; it exhibits an αß-hydrolase fold, consisting of a central ß-sheet surrounded by α-helices, endowed with novel topological features. The structure reveals a dimeric assembly not comparable with any other enzyme from the bacterial hormone-sensitive lipase family, probably echoing the specific structural features of the participating subunits. Biophysical studies including analytical gel filtration and ultracentrifugation support the dimeric nature of LpEst1. Structural and mutational analyses of the substrate-binding pocket and active site together with biochemical studies provided insights for understanding the substrate profile of LpEst1 and suggested for the first time the conserved Asp173, which is adjacent to the nucleophile, as a key element in the stabilization of the loop where the oxyanion hole resides.