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.     

Complete nucleotide sequence of plasmid pST-III from Lactobacillus plantarum ST-III

The complete nucleotide sequence of the 53,560-bp plasmid pST-III from Lactobacillus plantarum ST-III has been determined. The plasmid contains 42 predicted protein-coding sequences, and the functions of 34 coding sequences could be assigned. Homology analysis for the replication protein and the typical features of the origin of replication suggested that pST-III replicates via the theta-type mechanism. Among the predicted genes, we identified a kdp gene cluster (a high-affinity K(+)-transport system) for the first time in the Lactobacillus genus and a system for osmolyte transport. Analysis of the plasmid-encoded functions and the plasmid-cured experiment showed that the genes of pST-III could serve for the niche adaptations of L. plantarum ST-III and make significant contributions to its viability under hyperosmotic conditions. Furthermore, the relative copy number of pST-III was determined to be 6.79±1.55 copies per cell.     

Metabolism of Fructooligosaccharides by Lactobacillus paracasei 1195

Fermentation of fructooligosaccharides (FOS) and other oligosaccharides has been suggested to be an important property for the selection of bacterial strains used as probiotics. However, little information is available on FOS transport and metabolism by lactic acid bacteria and other probiotic bacteria. The objectives of this research were to identify and characterize the FOS transport system of Lactobacillus paracasei 1195. Radiolabeled FOS was synthesized enzymatically from [³H]sucrose and purified by column and thin-layer chromatography, yielding three main products: glucose (G) α-1,2 linked to two, three, or four fructose (F) units (GF₂, GF₃, and GF₄, respectively). FOS hydrolysis activity was detected only in cell extracts prepared from FOS- or sucrose-grown cells and was absent in cell supernatants, indicating that transport must precede hydrolysis. FOS transport assays revealed that the uptake of GF₂ and GF₃ was rapid, whereas little GF₄ uptake occurred. Competition experiments showed that glucose, fructose, and sucrose reduced FOS uptake but that other mono-, di-, and trisaccharides were less inhibitory. When cells were treated with sodium fluoride, iodoacetic acid, or other metabolic inhibitors, FOS transport rates were reduced by up to 60%; however, ionophores that abolished the proton motive force only slightly decreased FOS transport. In contrast, uptake was inhibited by ortho-vanadate, an inhibitor of ATP-binding cassette transport systems. De-energized cells had low intracellular ATP concentrations and had a reduced capacity to accumulate FOS. These results suggest that FOS transport in L. paracasei 1195 is mediated by an ATP-dependent transport system having specificity for a narrow range of substrates.     

Oxygen Metabolism in Lactobacillus plantarum

Lactobacillus plantarum, although able to grow in the presence of oxygen, was found to retain a completely anaerobic metabolism. Thus, L. plantarum did not consume detectable amounts of oxygen and did not contain measureable amounts of those enzyme activities which serve to protect anaerobic cells against the lethality of O(2) (-) and of H(2)O(2). Superoxide dismutase, catalase, and peroxidase appeared to be absent from these cells. L. plantarum was unusually resistant towards hyperbaric oxygen, indicating that it did not reduce oxygen even when exposed to high concentrations of this gas. A photochemical reaction mixture, known to generate O(2) (-), did kill L. plantarum. The lethality was diminished by superoxide dismutase, catalase, or mannitol and was augmented by H(2)O(2). This suggests that the lethal agent generated in the photochemical system was primarily OH., generated from the reaction of O(2) (-) with H(2)O(2).     

Ultrastructure of the Membrane System in Lactobacillus plantarum

Electron microscopic study of Lactobacillus plantarum revealed mesosomes in different stages of maturation and structural relation with other cell organelles. Small, immature mesosomes were bounded by a prominent electron-dense layer with another extremely faint layer on the outside. This corresponds to the appearance of the cytoplasmic membrane. Large mature mesosomes were surrounded by a triple-layered unit membrane having electron-opaque layers of approximately equal density, suggesting that the composition of the boundary membrane alters during development of this structure. Three-dimensional observations derived from serial sections indicated that mesosomes always maintain a connection between the cytoplasmic membrane and the comparable layers of their boundary. The cytoplasmic membrane also consisted of a triple-layered unit membrane, the innermost layer of which was less electron-opaque and was usually hidden by the relatively dense background of the cytoplasm. The innermost layer of the cytoplasmic membrane was most clearly seen in plasmolyzed cells. Only mature mesosomes made distinct contacts with, or were partially immersed in, the nucleoplasm. The boundary of such mesosomes frequently seemed to be discontinuous, suggesting that the mesosome interior was in direct contact with the nucleoplasm. Mesosomes involved in cross-wall formation at a division plane increased in size and passed through a sequence of positions which led ultimately to an association with the nucleoplasms of the daughter cells. The inner surface of the cell wall was lined by a thin, electron-dense layer whose composition and function are unknown. Under the cultural conditions used, this organism regularly contained a polyphosphate granule.     

Mevalonate Metabolism in Supernatant Fractions of Lysates of Lactobacillus plantarum

The supernatant fractions of lysates of Lactobacillus plantarum metabolize mevalonate into lipids. Adenosine triphosphate and uridine, as well as related compounds, and reduced nicotinamide adenine dinucleotide phosphate or reduced nicotinamide adenine dinucleotide stimulate this process. To obtain very active supernatant fractions, the method of lysis is modified to include polyamines during lysozyme treatment of cells and subsequent shocking with citrate buffer.     

植物乳杆菌Lp-2的高密度发酵

高密度培养植物乳杆菌是制作其发酵剂的重要环节。首先,研究了不同的溶氧和pH对植物乳杆菌的分批发酵的影响。在分批发酵的基础上,为进一步提高发酵液中的菌体浓度,进行了补料分批发酵实验。最终通过对蔗糖反馈补料发酵试验对比改造获得了pH反馈补料发酵工艺。此发酵补料工艺可以控制蔗糖残糖量始终处于较低的水平,因此获得了最高的菌体产量。菌体干重达到13.56g/L,较分批培养提高90.05%。     

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 β-fructofuranosidase, a fructokinase, an α-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 β-fructofuranosidase that could be used for scFOS degradation.     

Red Blood Cell Membrane Fluidity in the Etiology of Multiple Sclerosis

Organisms adjust the order, or fluidity, of their cellular membranes in response to changes in their physiochemical environment by adjusting the lipid composition of their membranes. We investigated membrane fluidity using the phospholipid, fatty acid and cholesterol content of red blood cells (RBCs) from multiple sclerosis (MS) patients and correlated this with C-reactive protein (CRP) as well as with the severity of neurological outcome as measured by the Kurtzke Expanded Disability Status Scale (EDSS) and its Functional System Scores. The study group consisted of 31 patients with MS and 30 healthy control subjects. Phospholipids were determined using a colorimetric assay, fatty acids by gas chromatography, cholesterol by an enzymatic assay and CRP by a Beckman nephelometer. Cell membrane fluidity was calculated according to previously established formulae. RBC membrane fluidity as measured by the saturated to polyunsaturated fatty acid ratio was higher in patients than in controls (P = 0.04). The phosphatidylethanolamine saturated to polyunsaturated fatty acid ratio showed highly significant positive correlations with the EDSS and CRP < 5 μg/ml. CRP showed significant inverse correlations with the saturated nature but positive correlations with the ordered-crystalline-phase to liquid-crystalline-phase lipid ratio. In this study we show that membrane fluidity as measured by the relationship between membrane fatty acids, phospholipids and cholesterol is closely interrelated with inflammation and disease outcome in patients with MS. In conclusion, our findings suggest that the membrane lipid composition of patients with MS and, consequently, membrane fluidity are altered, which seems to be influenced by the inflammatory status.     

Citrate metabolism in Lactobacillus casei and Lactobacillus plantarum

Information on the factors influencing citrate metabolism in lactobacilli is limited and could be useful in understanding the growth of lactobacilli in ripening cheese. Citrate was not used as an energy source by either Lactobacillus casei ATCC 393 or Lact. plantarum 1919 and did not affect the growth rate when co-metabolized with glucose or galactose. In growing cells, metabolism of citrate was minimal at pH 6 but significant at pH 4·5 and was greater in cells co-metabolizing galactose than in those co-metabolizing glucose or lactose. In non-growing cells, optimum utilization of citrate also occurred at pH 4·5 and was not increased substantially by the presence of fermentable sugars. In both growing and non-growing cells, acetate and acetoin were the major products of citrate metabolism; pyruvate was also produced by non-growing cells and was transformed to acetoin once the citrate was exhausted. Citrate was metabolized more rapidly than sugar by non-growing cells; the reverse was true of growing cells. Citrate metabolism by Lact. plantarum 1919 and Lact. casei ATCC 393 increased six- and 22-fold, respectively, when the cells were pre-grown on galactose plus citrate than when pre-grown on galactose only. This was probably due to induction of citrate lyase by growth on citrate plus sugar. These results imply that lactobacilli, if present in large enough numbers, can metabolize citrate in ripening cheese in the absence of an energy source.     

Characterization of Rhamnosidases from Lactobacillus plantarum and Lactobacillus acidophilus

Lactobacilli are known to use plant materials as a food source. Many such materials are rich in rhamnose-containing polyphenols, and thus it can be anticipated that lactobacilli will contain rhamnosidases. Therefore, genome sequences of food-grade lactobacilli were screened for putative rhamnosidases. In the genome of Lactobacillus plantarum, two putative rhamnosidase genes (ram1_Lp and ram2_Lp) were identified, while in Lactobacillus acidophilus, one rhamnosidase gene was found (ramA_La). Gene products from all three genes were produced after introduction into Escherichia coli and were then tested for their enzymatic properties. Ram1_Lp, Ram2_Lp, and RamA_La were able to efficiently hydrolyze rutin and other rutinosides, while RamA_La was, in addition, able to cleave naringin, a neohesperidoside. Subsequently, the potential application of Lactobacillus rhamnosidases in food processing was investigated using a single matrix, tomato pulp. Recombinant Ram1_Lp and RamA_La enzymes were shown to remove the rhamnose from rutinosides in this material, but efficient conversion required adjustment of the tomato pulp to pH 6. The potential of Ram1_Lp for fermentation of plant flavonoids was further investigated by expression in the food-grade bacterium Lactococcus lactis. This system was used for fermentation of tomato pulp, with the aim of improving the bioavailability of flavonoids in processed tomato products. While import of flavonoids into L. lactis appeared to be a limiting factor, rhamnose removal was confirmed, indicating that rhamnosidase-producing bacteria may find commercial application, depending on the technological properties of the strains and enzymes.Lactobacilli such as Lactobacillus plantarum have been used for centuries to ferment vegetables such as cabbage, cucumber, and soybean (34). Fruit pulps, for instance, those from tomato, have also been used as a substrate for lactobacilli for the production of probiotic juices (38). Recently, the full genomic sequences of several lactobacilli have become available (1, 22). A number of the plant-based substrates for lactobacilli are rich in rhamnose sugars, which are often conjugated to polyphenols, as in the case of cell wall components and certain flavonoid antioxidants. Utilization of these compounds by lactobacilli would involve α-l-rhamnosidases, which catalyze the hydrolytic release of rhamnose. Plant-pathogenic fungi such as Aspergillus species produce the rhamnosidases when cultured in the presence of naringin, a rhamnosilated flavonoid (24, 26). Bacteria such as Bacillus species have also been shown to use similar enzyme activities for metabolizing bacterial biofilms which contain rhamnose (17, 40).In food processing, rhamnosidases have been applied primarily for debittering of citrus juices. Part of the bitter taste of citrus is caused by naringin (Fig. ​(Fig.1),1), which loses its bitter taste upon removal of the rhamnose (32). More recently, application of rhamnosidases for improving the bioavailability of flavonoids has been described. Human intake of flavonoids has been associated with a reduced risk of coronary heart disease in epidemiological studies (19). Food flavonoids need to be absorbed efficiently from what we eat in order to execute any beneficial function. Absorption occurs primarily in the small intestine (12, 37). Unabsorbed flavonoids will arrive in the colon, where they will be catabolized by the microflora, which is then present in huge quantities. Therefore, it would be desirable for flavonoids to be consumed in a form that is already optimal for absorption in the small intestine prior to their potential degradation. For the flavonoid quercetin, it has been demonstrated that the presence of rhamnoside groups inhibits its absorption about fivefold (20). A number of flavonoids which are present in frequently consumed food commodities, such as tomato and citrus products, often carry rutinoside (6-β-l-rhamnosyl-d-glucose) or neohesperidoside (2-β-l-rhamnosyl-d-glucose) residues (Fig. ​(Fig.1).1). Therefore, removal of the rhamnose groups from such flavonoid rutinosides and neohesperidosides prior to consumption could enhance their intestinal absorption. With this aim, studies were recently carried out toward the application of fungal enzyme preparations as a potential means to selectively remove rhamnoside moieties (16, 30).Open in a separate windowFIG. 1.Chemical structures of rhamnose-containing flavonoids from plants. Relevant carbon atoms in glycoside moieties are numbered. (1) Rutin (quercetin-3-glucoside-1→6-rhamnoside); (2) narirutin (naringenin-7-glucoside-1→6-rhamnoside); (3) naringin (naringenin-7-glucoside-1→2-rhamnoside); (4) p-nitrophenol-rhamnose.In view of the frequent occurrence of lactobacilli on decaying plant material and fermented vegetable substrates, one could anticipate that their genomes carry one or more genes encoding enzymes capable of utilizing rhamnosilated compounds. In the work reported here, we describe the identification of three putative rhamnosidase genes in lactobacillus genomes. We expressed these genes in Escherichia coli and characterized their gene products. The activities of all three lactobacillus rhamnosidases on flavonoids naturally present in tomato pulp were then assessed. One of the L. plantarum genes, which encoded the enzyme with the highest activity and stability in E. coli, was then also expressed in Lactococcus lactis, with the aim of investigating the potential use of such a recombinant organism to improve the bioavailability of fruit flavonoids and thus their efficacy in common foodstuffs.     

Establishment of a Simple Lactobacillus plantarum Cell Consortium for Cellulase-Xylanase Synergistic Interactions

Lactobacillus plantarum is an attractive candidate for bioprocessing of lignocellulosic biomass due to its high metabolic variability, including its ability to ferment both pentoses and hexoses, as well as its high acid tolerance, a quality often utilized in industrial processes. This bacterium grows naturally on biomass; however, it lacks the inherent ability to deconstruct lignocellulosic substrates. As a first step toward engineering lignocellulose-converting lactobacilli, we have introduced genes coding for a GH6 cellulase and a GH11 xylanase from a highly active cellulolytic bacterium into L. plantarum. For this purpose, we employed the recently developed pSIP vectors for efficient secretion of heterologous proteins. Both enzymes were secreted by L. plantarum at levels estimated at 0.33 nM and 3.3 nM, for the cellulase and xylanase, respectively, in culture at an optical density at 600 nm (OD₆₀₀) of 1. Transformed cells demonstrated the ability to degrade individually either cellulose or xylan and wheat straw. When mixed together to form a two-strain cell-based consortium secreting both cellulase and xylanase, they exhibited synergistic activity in the overall release of soluble sugar from wheat straw. This result paves the way toward metabolic harnessing of L. plantarum for novel biorefining applications, such as production of ethanol and polylactic acid directly from plant biomass.     

Regulation of aspartokinase in Lactobacillus plantarum

As a rational approach to the genetic development of a stable lysine overproducing strain of Lactobacillus plantarum for the fermentation of 'ogi', a Nigerian fermented cereal porridge, regulation of lysine biosynthesis in this species was investigated. Spontaneous lysine overproducing mutants of Lact. plantarum were obtained and their aspartokinase activities compared with those of wild-type strains under different conditions. Results showed that aspartokinase activity of Lact. plantarum cell extracts was not inhibited by either lysine, threonine, methionine or combinations of lysine and threonine. Instead, methionine enhanced aspartokinase activity in vitro. Results indicated that lysine biosynthesis in Lact. plantarum could be regulated by lysine via the control of aspartokinase production in a way different to that described for other bacteria.