Whole Genome Analysis of Tibetan Kefir-Derived Lactiplantibacillus Plantarum 12-3 Elucidates Its Genomic Architecture, Antimicrobial and Drug Resistance, Potential Probiotic Functionality and Safety.

BACKGROUND: Lactiplantibacillus plantarum 12-3 holds great promise as a probiotic bacterial strain, yet its full potential remains untapped. This study aimed to better understand this potential therapeutic strain by exploring its genomic landscape, genetic diversity, CRISPR-Cas mechanism, genotype, and mechanistic perspectives for probiotic functionality and safety applications. METHODS: L. plantarum 12-3 was isolated from Tibetan kefir grains and, subsequently, Illumina and Single Molecule Real-Time (SMRT) technologies were used to extract and sequence genomic DNA from this organism. After performing pan-genomic and phylogenetic analysis, Average Nucleotide Identity (ANI) was used to confirm the taxonomic identity of the strain. Antibiotic resistance gene analysis was conducted using the Comprehensive Antibiotic Resistance Database (CARD). Antimicrobial susceptibility testing, and virulence gene identification were also included in our genomic analysis to evaluate food safety. Prophage, genomic islands, insertion sequences, and CRISPR-Cas sequence analyses were also carried out to gain insight into genetic components and defensive mechanisms within the bacterial genome. RESULTS: The 3.4 Mb genome of L. plantarum 12-3, was assembled with 99.1% completeness and low contamination. A total of 3234 genes with normal length and intergenic spacing were found using gene prediction tools. Pan-genomic studies demonstrated gene diversity and provided functional annotation, whereas phylogenetic analysis verified taxonomic identity. Our food safety study revealed a profile of antibiotic resistance that is favorable for use as a probiotic. Analysis of insertional sequences, genomic islands, and prophage within the genome provided information regarding genetic components and their possible effects on evolution. CONCLUSIONS: Pivotal genetic elements uncovered in this study play a crucial role in bacterial defense mechanisms and offer intriguing prospects for future genome engineering efforts. Moreover, our findings suggest further in vitro and in vivo studies are warranted to validate the functional attributes and probiotic potential of L. plantarum 12-3. Expanding the scope of the research to encompass a broader range of L. plantarum 12-3 strains and comparative analyses with other probiotic species would enhance our understanding of this organism's genetic diversity and functional properties.

pH-dependent regulation of an acidophilic O-acetylhomoserine sulfhydrylase from Lactobacillus plantarum.

Bacteria have two routes for the l-methionine biosynthesis. In one route called the direct sulfuration pathway, acetylated l-homoserine is directly converted into l-homocysteine. The reaction using H2S as the second substrate is catalyzed by a pyridoxal 5'-phosphate-dependent enzyme, O-acetylhomoserine sulfhydrylase (OAHS). In the present study, we determined the enzymatic functions and the structures of OAHS from Lactobacillus plantarum (LpOAHS). The LpOAHS enzyme exhibited the highest catalytic activity under the weak acidic pH condition. In addition, crystallographic analysis revealed that the enzyme takes two distinct structures, open and closed forms. In the closed form, two acidic residues are sterically clustered. The proximity may cause the electrostatic repulsion, inhibiting the formation of the closed form under the neutral to the basic pH conditions. We concluded that the pH-dependent regulation mechanism using the two acidic residues contributes to the acidophilic feature of the enzyme. IMPORTANCE: In the present study, we can elucidate the pH-dependent regulation mechanism of the acidophilic OAHS. The acidophilic feature of the enzyme is caused by the introduction of an acidic residue to the neighborhood of the key acidic residue acting as a switch for the structural interconversion. The strategy may be useful in the field of protein engineering to change the optimal pH of the enzymes. In addition, this study may be useful for the development of antibacterial drugs because the l-methionine synthesis essential for bacteria is inhibited by the OAHS inhibitors. The compounds that can inhibit the interconversion between the open and closed forms of OAHS may become antibacterial drugs.

Heterologous expression and enzymatic characteristics of sulfatase from Lactobacillus plantarum dy-1.

Barley, rich in bioactive components including dietary fiber, polyphenolic compounds and functional proteins, exhibits health benefits such as regulating glucose and lipid metabolism. Previous studies have found that the content and composition of free phenolic acids in barley may be significantly changed by fermentation with the laboratory patented strain Lactobacillus plantarum dy-1 (L. p dy-1), but the mechanism of enzymatic release of phenolic acid remains to be elucidated. Based on this, this study aimed to identify the key enzyme in L. p dy-1 responsible for releasing the bound phenolic acid and to further analyze its enzymatic properties. The Carbohydrate-Active enZYmes database revealed that L. p dy-1 encodes 7 types of auxiliary enzymes, among which we have identified a membrane sulfatase. The enzyme gene LPMS05445 was heterologous to that expressed in E. coli, and a recombinant strain was induced to produce the target protein and purified. The molecular weight of the purified enzyme was about 59.9 kDa, with 578.21 U mg-1 enzyme activity. The optimal temperature and pH for LPMS05445 expression were 40 °C and 7.0, respectively. Furthermore, enzymatic hydrolysis by LPMS05445 can obviously change the surface microstructure of dietary fiber from barley bran and enhance the release of bound phenolic acid, thereby increasing the free phenolic acid content and improving its physiological function. In conclusion, sulfatase produced by Lactobacillus plantarum dy-1 plays a key role in releasing bound phenolic acids during the fermentation of barley.

Revealing the dynamics and mechanisms of bacterial interactions in cheese production with metabolic modelling.

Cheese taste and flavour properties result from complex metabolic processes occurring in microbial communities. A deeper understanding of such mechanisms makes it possible to improve both industrial production processes and end-product quality through the design of microbial consortia. In this work, we caracterise the metabolism of a three-species community consisting of Lactococcus lactis, Lactobacillus plantarum and Propionibacterium freudenreichii during a seven-week cheese production process. Using genome-scale metabolic models and omics data integration, we modeled and calibrated individual dynamics using monoculture experiments, and coupled these models to capture the metabolism of the community. This model accurately predicts the dynamics of the community, enlightening the contribution of each microbial species to organoleptic compound production. Further metabolic exploration revealed additional possible interactions between the bacterial species. This work provides a methodological framework for the prediction of community-wide metabolism and highlights the added value of dynamic metabolic modeling for the comprehension of fermented food processes.

Laboratory domestication of Lactiplantibacillus plantarum alters some phenotypic traits but causes non-novel genomic impact.

AIMS: Laboratory domestication has been negligibly examined in lactic acid bacteria (LAB). Lactiplantibacillus plantarum is a highly studied and industrially relevant LAB. Here, we passaged L. plantarum JGR2 in a complex medium to study the effects of domestication on the phenotypic properties and the acquisition of mutations. METHODS AND RESULTS: Lactiplantibacillus plantarum JGR2 was passaged in mMRS medium (deMan Rogossa Sharpe supplemented with 0.05% w/v L-cysteine) in three parallel populations for 70 days. One pure culture from each population was studied for various phenotypic properties and genomic alterations. Auto-aggregation of the evolved strains was significantly reduced, and lactic acid production and ethanol tolerance were increased. Other probiotic properties and antibiotic sensitivity were not altered. Conserved synonymous and non-synonymous mutations were observed in mobile element proteins (transposases), ß-galactosidase, and phosphoketolases in all three isolates. The evolved strains lost all the repeat regions and some of the functions associated with them. Most of the conserved mutations were found in the genomes of other wild-type strains available in a public database, indicating the non-novel genomic impact of laboratory passaging. CONCLUSIONS: Laboratory domestication can affect the phenotypic and genotypic traits of L. plantarum and similar studies are necessary for other important species of LAB.

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.

Lycium barbarum polysaccharide promotes the immunoprotective effects of a recombinant Lactobacillus plantarum vaccine expressing the Trichinella spiralis cathepsin F-like protease 1 gene.

Trichinellosis caused by Trichinella spiralis (T. spiralis) is a zoonotic disease that poses a substantial risk to human health. At present, vaccines used to prevent trichinellosis are effective, but the production of antibody levels and immunogenicity are low. Adjuvants can increase antibody levels and vaccine immunogenicity. As a result, it is critical to develop an effective adjuvant for the T. spiralis vaccine. Recent research has shown that traditional Chinese medicine polysaccharides with low-toxicity and biodegradability can act as adjuvants in vaccines. In this study, BALB/c mice were orally inoculated with a recombinant Lactobacillus plantarum (L. plantarum) vaccine expressing the T. spiralis cathepsin F-like protease 1 gene (rTs-CPF1), which was given three times at 10-day intervals. Lycium barbarum polysaccharide (LBP) was administered orally for 37 days. At 37 days after the first immunization, mice were infected with 350 T. spiralis muscle larvae (ML). Specific IgG and sIgA antibody levels against the T. spiralis CPF1 protein were increased in mice immunized with rTs-CPF1+LBP compared to those immunized with rTs-CPF1 alone. Furthermore, LBP increased IFN-γ and IL-4 expression levels, and the number of intestinal and intramuscular worms was significantly reduced in the rTs-CPF1+LBP group compared to that in the rTs-CPF1 group. In the rTs-CPF1+LBP group, the reduction rates of adult worms and muscle larvae were 47.31 % and 68.88 %, respectively. To summarize, LBP promotes the immunoprotective effects of the T. spiralis vaccine and may be considered as a novel adjuvant in parasitic vaccines.

Detection and absolute quantification of Lactiplantibacillus plantarum ATCC 202195 by quantitative real-time PCR.

IMPORTANCE: When administered for seven consecutive days shortly after birth, the probiotic bacterium Lactiplantibacillus plantarum ATCC 202195 plus fructooligosaccharide (FOS) was reported to reduce sepsis and lower respiratory tract infection events during early infancy in a randomized trial in India. Since probiotic effects are often strain specific, strain-level detection and quantification by routine molecular methods enables the monitoring of safety outcomes, such as probiotic-associated bacteremia, and allows for the quality of probiotic interventions to be assessed and monitored (i.e., verify strain identity and enumerate). Despite the potential clinical applications of L. plantarum ATCC 202195, an assay to detect and quantify this strain has not previously been described. Herein, we report the design of primer and probe sequences to detect L. plantarum ATCC 202195 and the development and optimization of a real-time PCR assay to detect and quantify the strain with high specificity and high sensitivity.

Supplementation with high-GABA-producing Lactobacillus plantarum L5 ameliorates essential tremor triggered by decreased gut bacteria-derived GABA.

BACKGROUND: The γ-aminobutyric acid (GABA) hypothesis posits a role of GABA deficiency in the central nervous system in the pathogenesis and progression of essential tremor (ET). However, the specific causative factor for GABA deficiency is not clear. The gut microbiota in mammals has recently been considered as a significant source of GABA. Furthermore, the GABA-based signals originating from the intestine can be transmitted to the brain through the "enteric nervous system-vagus nerve-brain" axis. However, the plausible contribution of gut microbiota to ET seems inspiring but remains obscure. METHODS: Fecal samples from patients with ET and healthy controls were examined by metagenomic sequencing to compare the composition of gut microbiota and the expression of genes involved in GABA biosynthesis. The impact of gut microbiota on ET was explored through transplantation of fecal microbiota from patients with ET into the murine ET model. Lactic acid bacteria producing high amounts of GABA were identified through whole-genome sequencing and ultra-performance liquid chromatography-tandem mass spectrometry. Subsequently, mice were treated with the high-GABA-producing strain Lactobacillus plantarum L5. Tremor severity, behavioral tests, pro-inflammatory cytokines, GABA concentration, and gut microbiota composition were examined in these mice. RESULTS: The gut microbiota of patients with ET demonstrated an impaired GABA-producing capacity and a reduced fecal GABA concentration. Transplantation of the gut microbiota from patients with ET induced an extension of tremor duration and impaired mobility in the murine model of ET. L5 exhibited an augmented GABA-producing capacity, with the De Man-Rogosa-Sharpe culture broth containing 262 mg/l of GABA. In addition, administration of L5 significantly decreased the tremor severity and enhanced the movement capability and grasping ability of ET mice. In vivo mechanistic experiments indicated that L5 reshaped the gut microbial composition, supplemented the mucosa-associated microbiota with GABA-producing capacity, increased the GABA concentrations in the cerebellum, and diminished inflammation in the central nervous system. CONCLUSIONS: These findings highlight that deficiency of GABA-producing gut microbes plays an essential role in the pathogenesis of ET and that L5 is a promising candidate for treating ET.

Engineered probiotic Lactobacillus plantarum WCSF I for monitoring and treatment of Staphylococcus aureus infection.

IMPORTANCE: Bacterial infection and the emergence of drug-resistant strains are major problems in clinical treatment. Staphylococcus aureus, which typically infects the skin and blood of animals, is also a potential intestinal pathogen that needs to be addressed by the emergence of a new treatment approach. Probiotic therapy is the most likely alternative to antibiotic therapy to solve the problem of bacterial drug resistance in clinical practice. In this study, the engineered Lactobacillus plantarum can not only sense the signal AIP to detect S. aureus but also kill S. aureus by secreting the lysostaphin enzyme. Our strategy employed an Agr quorum-sensing genetic circuit to simultaneously detect and treat pathogenic bacteria, which provided a theoretical possibility for solving practical clinical bacterial infection cases in the future.

Physiological and genomic characterization of Lactiplantibacillus plantarum isolated from Indri indri in Madagascar.

AIMS: Indri indri is a lemur of Madagascar which is critically endangered. The analysis of the microbial ecology of the intestine offers tools to improve conservation efforts. This study aimed to achieve a functional genomic analysis of three Lactiplantibacillus plantarum isolates from indris. METHODS AND RESULTS: Samples were obtained from 18 indri; 3 isolates of Lp. plantarum were obtained from two individuals. The three isolates were closely related to each other, with <10 single nucleotide polymorphisms, suggesting that the two individuals shared diet-associated microbes. The genomes of the three isolates were compared to 96 reference strains of Lp. plantarum. The three isolates of Lp. plantarum were not phenotypically resistant to antibiotics but shared all 17 genes related to antimicrobial resistance that are part of the core genome of Lp. plantarum. The genomes of the three indri isolates of Lp. plantarum also encoded for the 6 core genome genes coding for enzymes related to metabolism of hydroxybenzoic and hydroxycinnamic acids. The phenotype for metabolism of hydroxycinnamic acids by indri isolates of Lp. plantarum matched the genotype. CONCLUSIONS: Multiple antimicrobial resistance genes and gene coding for metabolism of phenolic compounds were identified in the genomes of the indri isolates, suggesting that Lp. plantarum maintains antimicrobial resistance in defense of antimicrobial plant secondary pathogens and that their metabolism by intestinal bacteria aids digestion of plant material by primate hosts.

Production of Lactobacillus plantarum ghosts by conditional expression of a prophage-encoded holin.

Bacterial ghosts (BGs) are nonviable empty bacterial cell envelopes with intact cellular morphology and native surface structure. BGs made from pathogenic bacteria are used for biomedical and pharmaceutical applications. However, incomplete pathogenic cell inactivation during BG preparation raises safety concerns that could limit the intended use. Therefore, safer bacterial cell types are needed for BG production. Here, we produced BGs from the food-grade Gram-positive bacterium Lactobacillus plantarum TBRC 2-4 by conditional expression of a prophage-encoded holin (LpHo). LpHo expression was regulated using the pheromone-inducible pSIP system and LpHo was localized to the cell membrane. Upon LpHo induction, a significant growth retardation and a drastic decrease in cell viability were observed. LpHo-induced cells also showed membrane pores by scanning electron microscopy, membrane depolarization by flow cytometry, and release of nucleic acid contents in the cell culture supernatant, consistent with the role of LpHo as a pore-forming protein and L. plantarum ghost formation. The holin-induced L. plantarum BG platform could be developed as a safer alternative vehicle for the delivery of biomolecules.

Probiotic and Safety Evaluation of Twelve Lactic Acid Bacteria as Future Probiotics.

The human gut flora is highly diverse. Most lactic acid bacteria (LAB) are widely used as probiotics in human and animal husbandry and have a variety of physiological benefits. This article mainly studied the bacteriostatic ability of LAB against four pathogenic bacteria, gastrointestinal environment tolerance, and adhesion ability to Caco-2 cells. The genome of Lactiplantibacillus plantarum L461 was sequenced and analyzed. The results showed that strains F512, L461, and D469 had the most significant inhibitory effects on Escherichia coli, Salmonella enterica B, Staphylococcus aureus, and Listeria monocytogenes. In addition, strains L461, C502, and P231 showed good tolerance after exposure to simulated gastric fluid for 0-4 h. Strains C502, H781, and L461 showed good tolerance in simulated intestinal fluid. Strains L461 and H781 showed good adhesion to Caco-2 cells. The number of viable bacteria was more than 60. Therefore, we screened L. plantarum L461 from 12 LAB strains through three aspects of evaluation, and conducted whole genome sequencing and analysis. Sequencing results showed that L. plantarum L461 had more defense mechanisms and phage annotation genes than L. plantarum WCFS1. Virulence factor studies showed that L. plantarum L461 has iron absorption system and adhesion-related gene annotation, indicating that L. plantarum L461 has survival advantage in intestinal tract. The predicted results showed that there were eight phages with phage resistance in L. plantarum L461. L. plantarum L461 is sensitive to several antibiotics, notably penicillin and oxacillin. In summary, the results of this study prove that L. plantarum L461 has good prebiotic function and is safe. Therefore, L. plantarum L461 can be safely used as a potential functional probiotic.

Dual 6Pß-Galactosidase/6Pß-Glucosidase GH1 Family for Lactose Metabolism in the Probiotic Bacterium Lactiplantibacillus plantarum WCFS1.

Intestinal lactic acid bacteria can help alleviate lactose maldigestion by promoting lactose hydrolysis in the small intestine. This study shows that protein extracts from probiotic bacterium Lactiplantibacillus plantarum WCFS1 possess two metabolic pathways for lactose metabolism, involving ß-galactosidase (ß-gal) and 6Pß-galactosidase (6Pß-gal) activities. As L. plantarum WCFS1 genome lacks a putative 6Pß-gal gene, the 11 GH1 family proteins, in which their 6Pß-glucosidase (6Pß-glc) activity was experimentally demonstrated,, were assayed for 6Pß-gal activity. Among them, only Lp_3525 (Pbg9) also exhibited a high 6Pß-gal activity. The sequence comparison of this dual 6Pß-gal/6Pß-glc GH1 protein to previously described dual GH1 proteins revealed that L. plantarum WCFS1 Lp_3525 belonged to a new group of dual 6Pß-gal/6Pß-glc GH1 proteins, as it possessed conserved residues and structural motifs mainly present in 6Pß-glc GH1 proteins. Finally, Lp_3525 exhibited, under intestinal conditions, an adequate 6Pß-gal activity with possible relevance for lactose maldigestion management.

[Efficient biosynthesis of γ-aminobutyric acid by rationally engineering the catalytic pH range of a glutamate decarboxylase from Lactobacillus plantarum].

γ-aminobutyric acid can be produced by a one-step enzymatic reaction catalyzed by glutamic acid decarboxylase. The reaction system is simple and environmentally friendly. However, the majority of GAD enzymes catalyze the reaction under acidic pH at a relatively narrow range. Thus, inorganic salts are usually needed to maintain the optimal catalytic environment, which adds additional components to the reaction system. In addition, the pH of solution will gradually rise along with the production of γ-aminobutyric acid, which is not conducive for GAD to function continuously. In this study, we cloned the glutamate decarboxylase LpGAD from a Lactobacillus plantarum capable of efficiently producing γ-aminobutyric acid, and rationally engineered the catalytic pH range of LpGAD based on surface charge. A triple point mutant LpGADS24R/D88R/Y309K was obtained from different combinations of 9 point mutations. The enzyme activity at pH 6.0 was 1.68 times of that of the wild type, suggesting the catalytic pH range of the mutant was widened, and the possible mechanism underpinning this increase was discussed through kinetic simulation. Furthermore, we overexpressed the Lpgad and LpgadS24R/D88R/Y309K genes in Corynebacterium glutamicum E01 and optimized the transformation conditions. An optimized whole cell transformation process was conducted under 40 ℃, cell mass (OD600) 20, 100 g/L l-glutamic acid substrate and 100 µmol/L pyridoxal 5-phosphate. The γ-aminobutyric acid titer of the recombinant strain reached 402.8 g/L in a fed-batch reaction carried out in a 5 L fermenter without adjusting pH, which was 1.63 times higher than that of the control. This study expanded the catalytic pH range of and increased the enzyme activity of LpGAD. The improved production efficiency of γ-aminobutyric acid may facilitate its large-scale production.

Structural and biosynthetic diversity of plantaricins from Lactiplantibacillus.

Lactiplantibacillus plantarum (L. plantarum) produces an antimicrobial peptide known as plantaricin. Plantaricin-producing L. plantarum is of interest for its gut-friendly nature, wide range of sugar utilization, palatability, and probiotic attributes, making it a better candidate for the food industry. Numerous strains of plantaricin-producing L. plantarum have been isolated from different ecological niches and found to follow different mechanisms for plantaricin production. The mechanism of plantaricin production is sensitive to environmental factors; therefore, any alteration in the optimum conditions can inhibit/halt bacteriocin production. To regain the lost or hidden plantaricin-producing character of the L. plantarum strains under ideal laboratory conditions, it is essential to understand the mechanism of plantaricin production. Previously, discrete information on various mechanisms of plantaricin production has been elaborated. However, based on the literature analysis, we observed that a systematic classification of plantaricins produced by L. plantarum is not explored. Hence, we aim to collect information about rapidly emerging plantaricins and distribute them among the different classes of bacteriocin, followed by classifying them based on different mechanisms of plantaricin production. This may help scaleup the bacteriocin production at industrial levels, which is otherwise challenging to achieve. This will also help the reader understand plantaricins and their mechanism of plantaricin production to a deeper extent and to characterize/reproduce the peptide where plantaricin production is a hidden character. KEY POINTS: • L. plantarum produces the antimicrobial compound plantaricin. • L. plantarum has different regulatory operons which control plantaricin production. • Based on the regulatory operon, the mechanism of plantaricin production is different.

bsh1 Gene of Lactobacillus plantarum AR113 Plays an Important Role in Ameliorating Western Diet-Aggravated Colitis.

Western diet is thought to increase susceptibility to inflammatory bowel disease (IBD), and probiotics are a potential therapeutic agent for IBD. This study revealed the effects of Lactobacillus plantarum AR113 and L. plantarum AR113Δbsh1 on a dextran sulfate sodium (DSS)-induced colitis mouse model under the Western diet (WD). After four weeks of WD and low-sugar and low-fat diet (LD) intervention, induction with 3% DSS, and intragastric administration of probiotics, we found that L. plantarum AR113 could regulate blood glucose and lipid levels and have a certain protective effect on hepatocytes. Our results suggested that the L. plantarum AR113 alleviated DSS-induced colitis under the Western diet by improving dyslipidemia, repairing intestinal barrier dysfunction, and inhibiting the TLR4/Myd88/TRAF-6/NF-κB inflammatory pathway. However, these changes were not demonstrated in the L. plantarum AR113Δbsh1, and therefore, we reasoned that the presence of bsh1 may play a crucial role in the L. plantarum AR113 exerting its anti-inflammatory function. The relationship between bile salt hydrolase (BSH) and colitis was worthy of further exploration.

Nisin controlled homologous Over-expression of an exopolysaccharide biosynthetic glycosyltransferase gene for enhanced EPS production in Lactobacillus plantarum BR2.

Glycosyltransferases synthesize a variety of exopolysaccharides (EPS) with different properties by altering the type of glycosidic linkage, degree of branching, length, mass, and conformation of the polymers. The genome analysis of an EPS-producing, Lactobacillus plantarum BR2 (Accession No: MN176402) showed twelve glycosyltransferase genes, and the gene BR2gtf (1116 bp), annotated as an EPS biosynthetic glycosyltransferase was cloned into the pNZ8148 vector. The recombinant pNZ8148 vector along with pNZ9530, a regulatory plasmid, were electroporated to L. plantarum BR2 for the over-expression of gtf gene under a nisin-controlled expression system and the glycosyltransferase activity of the recombinant and the wild-type strains were analysed. The recombinant strain showed 54.4% increase in EPS production with the maximum EPS production of 23.2 ± 0.5 g/L in a 5 L bioreactor study after 72 h of fermentation. This study shows an effective molecular strategy possibly to be adopted in lactic acid bacteria to enhance exopolysaccharide production.

Acid-tolerant Lactiplantibacillus plantarum ZDY2013 shows a colonization niche preference and interacts with enterotoxigenic Bacillus cereus in specific-pathogen-free mice.

Probiotics have long been utilized as functional food and modulate gut microbial homeostasis, but their colonization niche is mostly unclear and transient, which restrains the development of microbiome-targeted strategies. Lactiplantibacillus (L.) plantarum ZDY2013 is an allochthonous species of the human gastrointestinal tract with acid-tolerant properties. It serves as an antagonistic agent against the food-borne pathogen Bacillus (B.) cereus and a potent regulator of the gut microbiota. However, there is a knowledge gap regarding the colonization dynamics of L. plantarum ZDY2013 in the host intestine and the colonization niche of its interaction with pathogens. Here, we designed a pair of specific primers targeting L. plantarum ZDY2013 based on its whole genome sequence. We evaluated their accuracy and sensitivity with other host-derived strains and confirmed their availability with artificially spiked fecal samples from different mouse models. Additionally, the content of L. plantarum ZDY2013 was quantified by qPCR in fecal samples from BALB/c mice, followed by the analysis of its colonization niche preference. Moreover, the interactions between L. plantarum ZDY2013 and enterotoxigenic B. cereus HN001 were also elucidated. The results revealed that the newly designed primers could identify L. plantarum ZDY2013 with high specificity and were resistant to the influence of the complex fecal matrix and gut microbes from different hosts. Interestingly, the content of mixed L. plantarum ZDY2013 and B. cereus HN001 when orally administered remained higher when compared with the single strain group in BALB/c mice upon discontinuation of intragastric administration. In addition, L. plantarum ZDY2013 was mainly enriched in the large intestine during the ingestion period and maintained the highest content in the stomach after discontinuing supplementation on day 7. Moreover, L. plantarum ZDY2013 colonization neither damaged the intestine nor ameliorated the damage triggered by B. cereus in BALB/c mice. Overall, our study constructed two efficient specific primers targeting L. plantarum ZDY2013 and provided the potential to explore the underlying mechanism of competition between L. plantarum ZDY2013 and pathogens in host species.

Effect of LuxS/AI-2-mediated quorum sensing system on bacteriocin production of Lactobacillus plantarum NMD-17.

Lactobacillus plantarum NMD-17 separated from koumiss could produce a bacteriocin named plantaricin MX against Gram-positive bacteria and Gram-negative bacteria. The bacteriocin synthesis of L. plantarum NMD-17 was remarkably induced in co-cultivation with Lactobacillus reuteri NMD-86 as the increase of cell numbers and AI-2 activity, and the expressions of luxS encoding signal AI-2 synthetase, plnB encoding histidine protein kinase, plnD encoding response regulator, and plnE and plnF encoding structural genes of bacteriocin were significantly upregulated in co-cultivation, showing that the bacteriocin synthesis of L. plantarum NMD-17 in co-cultivation may be regulated by LuxS/AI-2-mediated quorum sensing system. In order to further demonstrate the role of LuxS/AI-2-mediated quorum sensing system in the bacteriocin synthesis of L. plantarum NMD-17, plasmids pUC18 and pMD18-T simple were used as the skeleton to construct the suicide plasmids pUC18-UF-tet-DF and pMD18-T simple-plnB-tet-plnD for luxS and plnB-plnD gene deletion, respectively. luxS and plnB-plnD gene knockout mutants were successfully obtained by homologous recombination. luxS gene knockout mutant lost its AI-2 synthesis ability, suggesting that LuxS protein encoded by luxS gene is key enzyme for AI-2 synthesis. plnB-plnD gene knockout mutant lost the ability to synthesize bacteriocin against Salmonella typhimurium ATCC14028, indicating that plnB-plnD gene was a necessary gene for bacteriocin synthesis of L. plantarum NMD-17. Bacteriocin synthesis, cell numbers, and AI-2 activity of luxS or plnB-plnD gene knockout mutants in co-cultivation with L. reuteri NMD-86 were obviously lower than those of wild-type strain in co-cultivation at 6-9 h (P < 0.01). The results showed that LuxS/AI-2-mediated quorum sensing system played an important role in the bacteriocin synthesis of L. plantarum NMD-17 in co-cultivation.