Complete genome sequence of the probiotic Lactobacillus plantarum ST-III

Lactobacillus plantarum strain ST-III, a probiotic strain with several functions, was isolated from kimchi. Here we report the complete genome sequence of ST-III and compared it with two published L. plantarum genomes.     

Identification of prebiotic fructooligosaccharide metabolism in Lactobacillus plantarum WCFS1 through microarrays

Short-chain fructooligosaccharides (scFOS) and other prebiotics are used to selectively stimulate the growth and activity of lactobacilli and bifidobacteria in the colon. However, there is little information on the mechanisms whereby prebiotics exert their specific effects upon such microorganisms. To study the genomic basis of scFOS metabolism in Lactobacillus plantarum WCFS1, two-color microarrays were used to screen for differentially expressed genes when grown on scFOS compared to glucose (control). A significant up-regulation (8- to 60-fold) was observed with a set of only five genes located in a single locus and predicted to encode a sucrose phosphoenolpyruvate transport system (PTS), a beta-fructofuranosidase, a fructokinase, an alpha-glucosidase, and a sucrose operon repressor. Several other genes were slightly overexpressed, including pyruvate dehydrogenase. For the latter, no detectable activity in L. plantarum under various growth conditions has been previously reported. A mannose-PTS likely to encode glucose uptake was 50-fold down-regulated as well as, to a lower extent, other PTSs. Chemical analysis of the different moieties of scFOS that were depleted in the growth medium revealed that the trisaccharide 1-kestose present in scFOS was preferentially utilized, in comparison with the tetrasaccharide nystose and the pentasaccharide fructofuranosylnystose. The main end products of scFOS fermentation were lactate and acetate. This is the first example in lactobacilli of the association of a sucrose PTS and a beta-fructofuranosidase that could be used for scFOS degradation.     

Oxygen utilization by Lactobacillus plantarum

Cell-free extracts of Lactobacillus plantarum contain non-proteinaceous compounds which mimic superoxide dismutase activity. Using the test system in which O ₂ ^– is generated by xanthine oxidase, superoxide dismutase activity is found in cell-free extracts, where proteins are removed by precipitation. This activity is strongly decreased after dialysis of cell-free extracts. Superoxide dismutase activity was also investigated by means of pulse radiolysis. Cell-free extracts of Escherichia coli were also investigated as a comparison, which were known to contain superoxide dismutase. With cell-free extracts of both L. plantarum and E. coli the decay of O ₂ ^– was markedly increased. However, the type of reaction of the O ₂ ^– decay was of first order in the presence of E. coli extracts due to superoxide dismutase(s), and of second order in the presence of L. plantarum extracts, indicating that O ₂ ^– elimination is not an enzymic reaction. Mn²⁺ phosphate(s) might be responsible for the observed elimination of O ₂ ^– . The production of O ₂ ^– is not detectable during NADH-, lactate- or pyruvate oxidase reactions in L. plantarum extracts.     

Heterologous protein secretion by Lactobacillus plantarum using homologous signal peptides

Aims: To test seven selected putative signal peptides from Lactobacillus plantarum WCFS1 in terms of their ability to drive secretion of two model proteins in Lact. plantarum, and to compare the functionality of these signal peptides with that of well‐known heterologous signal peptides (Usp45, M6). Methods and Results: Signal peptide functionality was assessed using a series of modular derivatives of the pSIP vectors for peptide pheromone‐controlled high‐level gene expression in lactobacilli. Several of the constructs with homologous signal peptides yielded similar or higher reporter protein activities than constructs with heterologous signal peptides. Two of the homologous signal peptides (Lp_0373 and Lp_0600) appeared as especially promising candidates for directing secretion, as they were among the best performing with both reporter proteins. Conclusions: We have identified homologous signal peptides for high‐level secretion of heterologous proteins in Lact. plantarum. With the model proteins, some of these performed better than commonly used heterologous signal peptides. Significance and Impact of the Study: The homologous signal peptides tested out, in this study, could be useful in food‐grade systems for secretion of interesting proteins in Lact. plantarum. The constructed modular secretion vectors are easily accessible for rapid signal peptide screening.     

Complete resequencing and reannotation of the Lactobacillus plantarum WCFS1 genome

There is growing interest in the beneficial effects of Lactobacillus plantarum on human health. The genome of L. plantarum WCFS1, first sequenced in 2001, was resequenced using Solexa technology. We identified 116 nucleotide corrections and improved function prediction for nearly 1,200 proteins, with a focus on metabolic functions and cell surface-associated proteins.     

植物乳杆菌细菌素的研究与应用

植物乳杆菌细菌素不仅种类多,产生菌在发酵过程中还可产生良好的保健功效,因此成为研究的热点。本文对植物乳杆菌细菌素的种类、分子结构、抑菌机制及遗传控制做了较为详尽的介绍,并简要介绍了植物乳杆菌细菌素在食品、医药、饲料中的应用,为进一步研究植物乳杆菌细菌素提供了参考。     

Anaerobic l-lactate degradation by Lactobacillus plantarum

Abstract Lactobacillus plantarum strains used as silage inoculants were investigated for their ability to metabolize lactic acid anaerobically after prolonged incubation (7–30 days) when glucose was absent from the medium. When citrate was present in the medium together with glucose during the initial fermentation, the lactic acid produced was degraded. Citrate was concomitantly degraded, resulting in accumulation of formic, acetic and succinic acids along with CO₂. The anaerobic degradation was confirmed by the use of l ¹⁴C(U) labelled lactate. The existence of pyruvate formate lyase in L. plantarum was indicated by using ¹⁴C-labelled pyruvate and HPLC identification of end-products. The 1-¹⁴C-carboxylic acid group of pyruvate was converted to formic acid, and the 3-¹⁴C was found in acetic acid. The key enzyme(s) in this metabolic pathway appears to require anaerobic conditions and induction by citrate.     

Electrotransformation of Lactobacillus plantarum using linearized plasmid DNA

J.K. THOMPSON, K.J. MCCONVILLE, C. MCREYNOLDS AND M.A. COLLINS. 1997. Evidence is presented that linearized plasmid DNA is capable of electrotransforming Lactobacillus plantarum at a frequency 500-fold lower than with the covalently closed circular molecule. When the linearized plasmid was religated prior to transformation the transformation efficiency was < 10-fold higher, suggesting that open circular plasmid was only slightly more efficient in the transformation of Lact. plantarum than linear DNA. This observation has implications for direct cloning into this species since the high background transformation frequency produced by the linear DNA could potentially obscure the recovery of clones. Nevertheless, using positive selection for enhanced chloramphenicol resistance, cloned fragments of Lact. helveticus DNA were obtained using the shuttle vector pGKV110.     

两株母乳源植物乳杆菌的全基因组测序分析

【背景】母乳是一个重要的益生菌筛选库,其中植物乳杆菌是一种用途广泛、适应性强的益生菌。然而不同菌株具有不同的功能,现有的生理生化方法对其潜在益生特性研究十分有限,有必要采用高通量的方法寻找具有种群特异性的优质益生菌。【目的】结合菌株生化特征在全基因组的测序与分析的基础上对两株植物乳杆菌的潜在功能进行预测,并重点找寻与肠液耐受性及细菌素的合成相关的基因,即在基因组的结构上对菌株的表型进行探究。【方法】分离筛选出两株母乳源植物乳杆菌MP55、MP37,并利用Illumina genome analyzer对菌株的全基因组进行测序,采用Prokka软件对细菌基因组进行注释,采用Carbohydrate-active enzymes (CAZy)、Koyto encyclopedia of genes and genomes(KEGG)和Clusters of orthologous genes(COG)数据库对基因组进行功能注释;同时采用Prodigal、RNAmmer等工具对编码序列、核糖体RNA进行预测,并用CGView软件绘制菌株的基因组环形图谱。【结果】通过基因组装得到了两株植物乳杆菌的全基因组信息,植物乳杆菌MP37、MP55基因组大小分别为3 204 421 bp和3 299 180 bp;(G+C)mol%含量分别为44.36%和44.46%;分别包含3 012个和3 101个DNA编码序列,结合菌株生化特征在基因组上找到4个与肠液耐受相关的基因及一段细菌素合成相关基因簇。基因组序列原始数据和拼接结果已提交至"gcMeta"平台。【结论】通过高通量测序分析在基因组水平上揭示了植物乳杆菌MP55、MP37在肠道存活性与抑制病原菌相关的可能机理。植物乳杆菌MP55、MP37是两株潜在的益生菌候选菌株,实验结果为进一步阐明其益生菌特性的功能机制提供了遗传学基础。     

Polyphasic investigation of the diversity within Lactobacillus plantarum related strains revealed two L. plantarum subgroups

The diversity of 140 strains related to Lactobacillus plantarum was investigated using a polyphasic approach combining two molecular techniques: randomly amplified polymorphic DNA fingerprinting (RAPD) and Southern hybridisation with a pyr probe on BglI digests of chromosomal DNA, as well as phenotypic characterization. The RAPD technique allowed us to classify a subset of 60 representative strains into four groups. One group belonged to Lactobacillus paraplantarum, the second to Lactobacillus pentosus and the two remaining groups to L. plantarum (G(L)p1 and G(L)p2). The Southern hybridisation technique (F. Bringel, M.-C. Curk and J.-C. Hubert, Int. J. Syst. Bacteriol. 46: 588-594, 1996) revealed nine groups of profiles (I to IX). Results indicated an excellent convergence between RAPD and hybridisation classifications for more than 93% (56/60) of the strains studied. When we compared the fermentation patterns of the L. plantarum strains, three differences were found. Melezitose fermentation was not fermented by the G(L)p2 RAPD group, unlike the G(L)p1 RAPD group which included L. plantarum type strain NCIMB11974T. Second, alpha-methyl-D-mannoside was fermented by a majority of the strains of the G(L)p1 RAPD group but by none of the strains in the G(L)p2 RAPD group. Third, dulcitol was catabolized by nearly half of the strains of the G(L)p2 RAPD group but by none of the strains in the G(L)p1 RAPD group. Molecular diversity within L. plantarum was confirmed using Southern profiles, PCR amplification and subsequent sequencing of these PCR products. A 773 bp sequence overlapping the pyrDF genes showed high homology: at least 97% identical in L. plantarum strains (V to IX) and 99.9% identical in hybridisation groups VII and VIII. The same G-T transversion which destroyed the pyrF BglI site was found in 11 strains (hybridisation groups VI, VII and VIII). DNA rearrangements were identified downstream from the pyr genes, by PCR amplification and Southern hybridisation profile analysis in three strains of hybridisation groups VIII and IX, two of which also harboured the G-T transversion.     

Comparative genome analysis of Campylobacter jejuni using whole genome DNA microarrays

Whole genome DNA microarrays were constructed and used to investigate genomic diversity in 18 Campylobacter jejuni strains from diverse sources. New algorithms were developed that dynamically determine the boundary between the conserved and variable genes. Seven hypervariable plasticity regions (PR) were identified in the genome (PR1 to PR7) containing 136 genes (50%) of the variable gene pool. When comparisons were made with the sequenced strain NCTC11168, the number of absent or divergent genes ranged from 2.6% (40 genes) to 10.2% (163) and in total 16.3% (269) of the genes were variable. PR1 contains genes important in the utilisation of alternative electron acceptors for respiration and may confer a selective advantage to strains in restricted oxygen environments. PR2, 3 and 7 contain many outer membrane and periplasmic proteins and hypothetical proteins of unknown function that might be linked to phenotypic variation and adaptation to different ecological niches. PR4, 5 and 6 contain genes involved in the production and modification of antigenic surface structures.     

Identification of Antioxidants Produced by Lactobacillus plantarum

We identified two compounds that demonstrated 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity from cultures of Lactobacillus plantarum. Spectroscopic analyses proved these compounds to be L-3-(4-hydroxyphenyl) lactic acid (HPLA) and L-indole-3-lactic acid (ILA). The respective EC₅₀ values for HPLA and ILA were 36.6 ± 4.3 mM and 13.4 ± 1.0 mM.     

Oxygen dependent lactate utilization by Lactobacillus plantarum

Lactobacillus plantarum P5 grew aerobically in rich media at the expense of lactate; no growth was observed in the absence of aeration. The oxygen-dependent growth was accompanied by the conversion of lactate to acetate which accumulated in the growth medium. Utilization of oxygen with lactate as substrate was observed in buffered suspensions of washed whole cells and in cell-free extracts. A pathway which accounts for the generation of adenosine triphosphate during aerobic metabolism of lactate to acetate via pyruvate and acetyl phosphate is proposed. Each of the enzyme activities involved, nicotinamide adenine dinucleotide independent lactic dehydrogenase, nicotinamide adenine dinucleotide dependent lactic dehydrogenase, pyruvate oxidase, acetate kinase and NADH oxidase were demonstrated in cell-free extracts. The production of pyruvate, acetyl phosphate and acetate was demonstrated using cell-free extracts and cofactors for the enzymes of the proposed pathway.Abbreviations MRS Man, Rogosa and Sharpe (1960) medium modified as in Materials and methods - TY Tryptone Yeast Extract broth - OUL Oxygen uptake with lactate as substrate - DCPIP 2,6-Dichlorophenolindophenol - LDH Lactic dehydrogenase     

Lactobacillus casei and Lactobacillus plantarum initiate catabolism of methionine by transamination

AIMS: To study the ability of Lactobacillus casei and Lact. plantarum strains to convert methonine to cheese flavour compounds. METHODS AND RESULTS: Strains were assayed for methionine aminotransferase and lyase activities, and amino acid decarboxylase activity. About 25% of the strains assayed showed methionine aminotransferase activity. The presence of glucose in the reaction mixture increased conversion of methionine to 4-methylthio-2-ketobutanoate (KMBA) and 4-methylthio-2-hydroxybutanoate (HMBA) in all strains. The methionine aminotransferase activity in Lact. plantarum and Lact. casei showed variable specificity for the amino group acceptors glyoxylate, ketoglutarate, oxaloacetate and pyruvate. None of the strains showed methionine lyase or glutamate and methionine decarboxylase activities. CONCLUSION: The presence of amino acid converting enzymes in lactobacilli is strain specific. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work suggest that lactobacilli can be used as adjuncts for flavour formation in cheese manufacture.     

Energy conservation in malolactic fermentation by Lactobacillus plantarum and Lactobacillus sake

A comparably poor growth medium containing 0.1% yeast extract as sole non-defined constituent was developed which allowed good reproducible growth of lactic acid bacteria. Of seven different strains of lactic acid bacteria tested, only Lactobacillus plantarum and Lactobacillus sake were found to catalyze stoichiometric conversion of l-malate to l-lactate and CO₂ concomitant with growth. The specific growth yield of malate fermentation to lactate at pH 5.0 was 2.0 g and 3.7 g per mol with L. plantarum and L. sake, respectively. Growth in batch cultures depended linearly on the malate concentration provided. Malate was decarboxylated nearly exclusively by the cytoplasmically localized malo-lactic enzyme. No other C₄-dicarboxylic acid-decarboxylating enzyme activity could be detected at significant activity in cell-free extracts. In pH-controlled continuous cultures, L. plantarum grew well with glucose as substrate, but not with malate. Addition of lactate to continuous cultures metabolizing glucose or malate decreased cell yields significantly. These results indicate that malo-lactic fermentation by these bacteria can be coupled with energy conservation, and that membrane energetization and ATP synthesis through this metabolic activity are due to malate uptake and/or lactate excretion rather than to an ion-translocating decarboxylase enzyme.     

Sizing of the Lactobacillus plantarum genome and other lactic acid bacteria species by transverse alternating field electrophoresis

Pulsed-field gel electrophoresis (transverse alternating field electrophoresis system), combined with rare cutting endonucleases, was used to size the genome of five strains of lactic acid bacteria belonging to the species Lactobacillus plantarum. The sum of the fragment sizes obtained with SfiI restriction digest yielded a total value of 2700–2905 kb. Moreover, SfiI digestion gives specific patterns which could allow an intraspecific identification of L. plantarum strains. The genome sizes of Pediococcus pentosaceus, Pc. acidilactici, and Carnobacterium divergens have been estimated by this method as being 1200 kb, 1560 kb, and 3200 kb respectively.     

Culture conditions of tannase production by Lactobacillus plantarum

Lactobacillus plantarum produced an extracellular tannase after 24 h growth on minimal medium of amino acids containing 2 g tannic acid l^–1. Enzyme production (6 U ml^–1) was optimal at 37 °C and pH 6 with 2 g glucose l^–1 and 7 g tannic acid l^–1 in absence of O₂.