Phospholipid activation of Lactobacillus plantarum undecaprenyl pyrophosphate synthetase

The ability of a number of phospholipids to stimulate $\text{Lactobacillus}$$\text{plantarum}$ undecaprenyl pyrophosphate synthetase was investigated. The detergent Triton X-100, which is added to stabilize the enzyme during purification and is required for $\text{in}$$\text{vitro}$ activity, was removed with the non-ionic resin XAD-2. The effects of cardiolipin, phosphatidyl ethanolamine, phosphatidyl choline, and phosphatidyl glycerol on the activity of XAD-2 treated undecaprenyl pyrophosphate synthetase were determined. Of the phospholipids studied only cardiolipin stimulated $\text{in}$$\text{vitro}$ enzymic activity as effectively as Triton X-100.     

Lactobacillus plantarum undecaprenyl pyrophosphate synthetase: purification and reaction requirements.

Undecaprenyl pyrophosphate synthetase was partially purified from Lactobacillus plantarum by DEAE-cellulose, hydroxyapatite, and Sephadex G-100 chromatography in Triton X-100. The enzyme has a molecular weight between 53,000 and 60,000. The enzyme demonstrated a fivefold preference for farnesyl pyrophosphate rather than geranyl pyrophosphate as the allylic cosubstrate, whereas dimethylallyl pyrophosphate was not effective as a substrate. Polyprenyl pyrophosphates obtained using either farnesyl or geranyl pyrophosphate as cosubstrate were chromatographically identical. Hydrolysis of these polyprenyl pyrophosphates with either a yeast or liver phosphatase preparation yielded undecaprenol as the major product. Incorporation of radioactive label from mixtures of Δ³-[1-¹⁴C]isopentenyl pyrophosphate and Δ³-2R-[2-³H]isopentenyl pyrophosphate into enzymic product indicated that each isoprene unit added to the allylic pyrophosphate substrate has a cis configuration about the newly formed double bond. The removal of detergent from enzyme solutions resulted in a parallel loss in enzyme activity when analyzed with either farnesyl or geranyl pyrophosphate as cosubstrates. Enzymic activity was restored on addition of Triton X-100 or deoxycholate. The enzyme exhibited a pH-activity profile with optima at pH 7.5 and 10.2. It also demonstrated a divalent cation requirement, with Mg²⁺, Mn²⁺, Zn²⁺, and Co²⁺ exhibiting comparable activities.     

Prenyl transferases from Micrococcus luteus: Characterization of undecaprenyl pyrophosphate synthetase

Three prenyl transferases in Micrococcus luteus were recovered in the soluble fraction following cell disruption. Undecaprenyl pyrophosphate (C₅₅-PP) synthetase chromatographed on DEAE-cellulose independently from geranylgeranyl-PP and octaprenyl-PP synthetases. Further purification of C₅₅-PP synthetase resulted in an approximate 250-fold purification over the crude lysate. The molecular weight of the synthetase was estimated to be between 47,000 and 49,000 by Sephadex G-100 chromatography. The enzyme had a broad specificity toward the allylic pyrophosphate substrate. The reactivities of the allylic substrates increased with chain length, C₁₀ < C₁₅ < C₂₀, except for trans-solanesyl-PP, which was unreactive. Moreover, the enzyme was active on allylic substrates having both cis- and trans-stereochemistry. Although C₅₅-PP and C₅₀-PP were the major products, some shorter chain products were also produced, when t,t-farnesyl pyrophosphate and Δ³sopentenyl pyrophosphate (IPP) were used as substrates. The stereochemistries of the products formed with C₅₅-PP synthetase were established, using [¹⁴C]IPP and 2R-[2-³H] and 2S-[2-³H]IPP. Each new isoprene unit added had a cis-configuration. The enzyme was inactive in the absence of added effectors. It was stimulated by Triton X-100, egg lecithin, and a whole phospholipid extract from M. luteus. Cardiolipin and deoxycholate were poor activators of the enzyme. The product chain length distribution observed with the phospholipid-activated enzyme showed highly favored production of the C₅₅-PP product over the C₅₀-PP product.     

Undecaprenyl pyrophosphate synthetase from Lactobacillus plantarum: a dimeric protein

a++Undecaprenyl pyrophosphate synthetase has been purified from Lactobacillus plantarum. It catalyzes the formation of a C55 polyprenyl pyrophosphate having isoprene residues with cis stereochemistry. The enzyme was shown to be an acidic protein (pI = 5.1), which can be partially purified by preparative gel electrophoresis and Blue-agarose column chromatography. The Km's of the enzyme for its substrates t,t-farnesyl pyrophosphate and isopentenyl pyrophosphate were determined to be 0.13 and 1.92 microM, respectively. The molecular weight of the enzyme was estimated by molecular sieve chromatography and gradient centrifugation to be 56,000 +/- 4000. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the protein was composed of a dimer of 30,000-Da subunits. The enzyme was inactivated by the arginine-specific reagents phenylglyoxal, butanedione and, cyclohexanedione, but this inactivation was not prevented by either of the substrates.     

Photoaffinity labeling of undecaprenyl pyrophosphate synthetase with a farnesyl pyrophosphate analogue

The prenyl transferase undecaprenyl pyrophosphate synthetase was partially purified from the cytosolic fraction of Escherichia coli. Its enzymic products were characterized as a family of cis-polyprenyl phosphates, which ranged in carbon number from C55 to C25. The enzyme is constituted of two subunits of approximately 30,000 molecular weight. A radiolabeled photolabile analogue of t,t-farnesyl pyrophosphate, [3H]2-diazo-3-trifluoropropionyloxy geranyl pyrophosphate, was shown to label Lactobacillus plantarum and E. coli undecaprenyl pyrophosphate synthetase on UV irradiation in the presence of isopentenyl pyrophosphate and divalent cation. The only labeled polypeptide migrated on electrophoresis in a sodium dodecyl sulfate-polyacrylamide gel at a molecular weight of approximately 30,000. No protein was radiolabeled when the natural substrate, t,t-farnesyl pyrophosphate was included in the irradiation mixture. Irradiation in the presence of MgCl2 without isopentenyl pyrophosphate gave less labeling of the polypeptide. Irradiation with only isopentenyl pyrophosphate gave little labeling of the polypeptide. When the enzyme was irradiated with 3H-photoprobe, [14C]isopentenyl pyrophosphate, and MgCl2, the labeled polypeptide gave a ratio of 14C/3H that indicated the product must also bind to the enzyme on irradiation. These results demonstrate the ability to radiolabel the allylic pyrophosphate binding site and possibly product binding site of undecaprenyl pyrophosphate synthetase by a process which is favored when both cosubstrate and divalent cation are present.     

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.     

Properties of a membrane-bound cardiolipin synthetase from Lactobacillus plantarum.

Cardiolipin (CL) synthetase of Lactobacillus plantarum 17-5 catalyzed the stoichiometric conversion of 2 mol of phosphatidylglycerol to 1 mol of CL. The enzyme activity was linear with time for 30 min at 37 C and with protein concentration between 20 and 200 mug of protein per ml. The enzyme was membrane associated, had a pH optimum of 5.1 in phosphate buffer, and was not stimulated by Mg2+, and the activity was not affected by the addition of ethylenediaminetetraacetic acid, cytidine diphosphate diglyceride, or cytidine triphosphate. The reaction was inhibited about 95% by Triton X-100 (0.5% final concentration) and by CL, the end product of the reaction. The activity of this enzyme was studied as a function of growth. The CL synthetase specific activity was highest during the early and midexponential growth phases, as was the cellular content of CL. The results demonstrate a correlation between enzyme-specific activity and lipid content of the cells.     

Characterization of undecaprenol kinase from Lactobacillus plantarum.

A membrane-bound undecaprenol kinase from Lactobacillus has been identified by observing the ATP-dependent phosphorylation of [14C]undercaprenol. The product of this reaction was shown to be [14C]undecaprenyl monophosphate by comparison of its chromatographic mobilities with authentic undecaprenyl monophosphate. It was shown that 32P from [gamma-32P]ATP was incorporated into undecaprenyl monophosphate. The kinase was partially solubilized by a variety of methods utilizing Triton X-100. Both the membrane-associated and solubilized enzymes required Mg2+, Triton X-100 and dimethylsulfoxide for activity. The enzyme preferentially phosphorylated the C34, C50 AND C 55 polyprenols. Geranylgeraniol (C20) and dolichol (C100), however, were utilized only 6% and 13% as well as undecaprenol, respectively. Despite the 8-fold difference in apparent V values, the apparent Km values for dolichol and undecaprenol were both 14 microM. The apparent Km for the nucleotide cosubstrate, ATP, was 2 mM. No other nucleoside triphosphate could substitute for ATP.     

Characterization of Two Virulent Phages of Lactobacillus plantarum

We characterized two Lactobacillus plantarum virulent siphophages, ATCC 8014-B1 (B1) and ATCC 8014-B2 (B2), previously isolated from corn silage and anaerobic sewage sludge, respectively. Phage B2 infected two of the eight L. plantarum strains tested, while phage B1 infected three. Phage adsorption was highly variable depending on the strain used. Phage defense systems were found in at least two L. plantarum strains, LMG9211 and WCSF1. The linear double-stranded DNA genome of the pac-type phage B1 had 38,002 bp, a G+C content of 47.6%, and 60 open reading frames (ORFs). Surprisingly, the phage B1 genome has 97% identity with that of Pediococcus damnosus phage clP1 and 77% identity with that of L. plantarum phage JL-1; these phages were isolated from sewage and cucumber fermentation, respectively. The double-stranded DNA (dsDNA) genome of the cos-type phage B2 had 80,618 bp, a G+C content of 36.9%, and 127 ORFs with similarities to those of Bacillus and Lactobacillus strains as well as phages. Some phage B2 genes were similar to ORFs from L. plantarum phage LP65 of the Myoviridae family. Additionally, 6 tRNAs were found in the phage B2 genome. Protein analysis revealed 13 (phage B1) and 9 (phage B2) structural proteins. To our knowledge, this is the first report describing such high identity between phage genomes infecting different genera of lactic acid bacteria.     

Characterization of Lactobacillus plantarum from wine must by PCR species-specific and RAPD-PCR

AIMS: Physiological and molecular analysis such as PCR species-specific and randomly amplified polymorphic PCR (RAPD-PCR) have been used for typing of Lactobacillus plantarum strains from typical wine must. METHODS AND RESULTS: Phenotypic tests such as API 50CH and evaluation of D-L-lactate production from glucose were used to perform a preliminary characterization of lactobacilli. Furthermore, 18 strains of lactobacilli were analyzed by PCR species-specific oligonucleotides based on short sequences of the recA gene. CONCLUSIONS: Four strains were identified as belonging to the L. plantarum species and were further analysed by RAPD-PCR. The RAPD-PCR profiles were similar in all strains that had positive results for species-specific PCR, suggesting that the four L. plantarum strains were closely related. SIGNIFICANCE AND IMPACT OF THE STUDY: Using PCR species-specific as a preliminary screening test and then RAPD-PCR can be as considered the most reliable method of performing a rapid and correct typing of L. plantarum from wine must.     

Characterization of a cryptic plasmid pM4 from Lactobacillus plantarum M4

A cryptic plasmid from Lactobacillus plantarum M4 isolated from fresh milk, designated as pM4, was sequenced and characterized. It was 3320 bp in length with a G+C content of 38.73 mol%. The plasmid pM4 was predicted to encode three putative ORFs, in which ORF1 shared 99% and 98% homology, respectively, with the Rep proteins of reported plasmids pWCFS101 and pF8801, members of the rolling circle replication (RCR) pC194 family. Sequence analysis revealed a typical pC194 family double strand origin (dso) and a putative single strand origin (sso) located upstream of the rep gene. Mung bean nuclease analysis and Southern hybridization confirmed the presence of single-stranded DNA (ssDNA) intermediates, suggesting that pM4 belongs to the RCR pC194 family. Accumulation of ssDNA in rifampicin-treated strains implied that the host-encoded RNA polymerase was involved in the conversion of ssDNA to double-stranded DNA. Furthermore, the relative copy number of pM4 was estimated to be about 25 in each cell by real-time PCR. The new RCR plasmid would be valuable in constructing cloning vectors for application in the food industry.     

Characterization of antifungal metabolites produced by Lactobacillus plantarum and Lactobacillus coryniformis isolated from rice rinsed water

Molecular Biology Reports - A recent spike in demand for chemical preservative free food has derived the scientific community to develop natural ways of food preservation. Therefore,...     

Heptaprenyl pyrophosphate synthetase from Bacillus subtilis

Heptaprenyl pyrophosphate synthetase was detected in partially purified extracts of Bacillus subtilis. The enzyme catalyzed the synthesis of all-trans C35 prenyl pyrophosphate from isopentenyl pyrophosphate and farnesyl or geranylgeranyl pyrophosphate, but it did not catalyze a reaction between isopentenyl pyrophosphate and either dimethylallyl or geranyl pyrophosphate. The enzyme reaction proceeded with an elimination of 2-pro-R hydrogen of isopentenyl pyrophosphate without accumulation of any prenyl pyrophosphate shorter than C35. The molecular weight of the enzyme was estimated by gel filtration to be 45,000. Michaelis constants for isopentenyl, farnesyl, and geranylgeranyl pyrophosphate were 12.8, 13.3, and 8.3 microM, respectively.     

Farnesyl pyrophosphate synthetase from Bacillus subtilis

Farnesyl pyrophosphate synthetase was detected in extracts of Bacillus subtilis and partially purified by Sephadex G-100, hydroxylapatite, and DEAE-Sephadex chromatography. The enzyme catalyzed the exclusive formation of all-trans farnesyl pyrophosphate from isopentenyl pyrophosphate and either dimethylallyl or geranyl pyrophosphate. Mg2+ was essential for the catalytic activity and Mn2+ was less effective. The enzyme was slightly activated by sulfhydryl reagents. This enzyme was markedly stimulated by K+, NH4+, or detergents such as Triton X-100 and Tween 80, unlike the known farnesyl pyrophosphate synthetases from eucaryotes. The molecular weight of the enzyme was estimated by gel filtration to be 67,000. The Michaelis constants for dimethylallyl and geranyl pyrophosphate were 50 microM and 18 microM, respectively.     

BcrC from Bacillus subtilis acts as an undecaprenyl pyrophosphate phosphatase in bacitracin resistance

Overexpression of the BcrC(Bs) protein, formerly called YwoA, in Escherichia coli or in Bacillus subtilis allows these bacteria to stand higher concentrations of bacitracin. It was suggested that BcrC(Bs) was a membrane-spanning domain of an ATP binding cassette (ABC) transporter involved in bacitracin resistance. However, we hypothesized that this protein has an undecaprenyl pyrophosphate (UPP) phosphatase activity able to compete with bacitracin for UPP. We found that overexpression of a recombinant His6-BcrC(Bs) protein in E. coli (i) increased the resistance of the cells to bacitracin and (ii) increased UPP phosphatase activity in membrane preparations by 600-fold. We solubilized and prepared an electrophoretically pure protein exhibiting a strong UPP phosphatase activity. BcrC(Bs), which belongs to the type 2 phosphatidic acid phosphatase (PAP2) phosphatase superfamily (PF01569), differs totally from the already known BacA UPP phosphatase from E. coli, a member of the PF02673 family of the Protein family (Pfam) database. Thus, BcrC(Bs) and its orthologs form a new class of proteins within the PAP2 phosphatase superfamily, and likely all of them share a UPP phosphatase activity.