Identification of Five Phospho-β-glycosidases from Lactobacillus gasseri ATCC33323T Cultured in Lactose Medium

Lactobacillus gasseri ATCC33323^T has seven putative phospho-β-glycosidase genes. Using column chromatography, we found that this strain cultured in lactose medium expresses five phospho-β-glycosidases (LacG1, LacG2, Pbg1, Pbg2, and Pbg3), where these gene expressions can be suppressed by glucose. To our knowledge, this is the first report indicating that five glycosidases are induced from a single bacterial strain using a single carbon source, lactose.     

Lactobacillus gasseri PA-3, but not L. gasseri OLL2996, reduces the absorption of purine nucleosides in rats

Lactobacillus gasseri PA-3 (PA-3) is a bacterial strain with a strong ability to degrade purine nucleosides. We previously showed that PA-3 incorporates purines in vitro and that oral administration of PA-3 and purines to rats attenuated their absorption of purines. It remains unclear whether these effects of PA-3 depend on bacterial strains. This study therefore compared the abilities of PA-3 and another bacterial strain of L. gasseri, OLL2996, which has shown decreased ability to degrade purine nucleosides in vitro, to incorporate purine nucleosides and to inhibit the absorption of purines fed to rats. Each bacterial strain was incubated in the presence of ¹⁴C-adenosine or ¹⁴C-inosine and the incorporation of each purine was evaluated by measuring their radioactivity. In vivo, rats were fed ¹⁴C-labeled purines along with PA-3 or OLL2996 and the absorption of these ¹⁴C-labeled purines was evaluated by analyzing radioactivity of blood samples. PA-3 incorporated about twice as much ¹⁴C-adenosine and ¹⁴C-inosine as OLL2996. The elevation of radioactivity levels in blood was 10–20% lower in rats treated with PA-3 than in control rats, after feeding with both ¹⁴C-adenosine and ¹⁴C-inosine as purines. In contrast, treatment with OLL2996 did not have statistically significant effects on radioactivity compared with the control group. These results indicate that the magnitude of bacterial inhibition of purine absorption is dependent on bacterial strain, correlating at least partly with the ability to incorporate and degrade purines.     

Isolation and partial characterization of bacteriocins produced by Lactobacillus gasseri JCM 2124

Four antibacterially active peptides (B1 to B4) were purified from the culture broth of L. gasseri JCM 2124. The B2 peptide (gassericin B2) was determined to be 4400 Da by mass spectrometry and partially sequenced. Gassericin B2 did not show any sequence similarities to other known bacteriocins. The B1 and B3 peptides shared identical sequences with two peptides of a two-component bacteriocin from Lactobacillus acidophilus. However, synergistic activity upon complementation of B1 and B3 was not observed. Based on amino acid sequencing and molecular mass, it is suggested that B1 and B4 peptides were derived from B3 (gassericin B3).     

ClpL is essential for induction of thermotolerance and is potentially part of the HrcA regulon in Lactobacillus gasseri

Stress-inducible proteins are likely to contribute to the survival and activity of probiotic bacteria during industrial processes and in the gastrointestinal tract. The recently published genome sequence of probiotic Lactobacillus gasseri ATCC 33323 suggests the presence of ClpC, ClpE, ClpL, and ClpX from the Clp ATPase family of stress proteins. The heat-shock response of L. gasseri was studied using 2-D DIGE. A total of 20 protein spots showing significant (p<0.05) increase in abundance after 30 min heat-shock were identified, including DnaK, GroEL, ClpC, ClpE, and ClpL. To study the physiological role of ClpL, one of the most highly induced proteins during heat-shock, its corresponding gene was inactivated. The DeltaclpL mutant strain had growth characteristics that were indistinguishable from wild-type under several stress conditions. However, in the absence of functional ClpL, L. gasseri exhibited drastically reduced survival at a lethal temperature and was unable to induce thermotolerance. Genome sequences indicate that the expression of clp genes in several Lactobacillus species is regulated by HrcA, instead of CtsR, the conserved clp gene regulator of low G+C Gram-positive bacteria. Electrophoretic mobility shift assays using L. gasseri HrcA protein and clpL upstream fragments revealed, for the first time, a direct interaction between HrcA and the promoter of a clp gene from a Lactobacillus.     

In vitro enzymatic modification of puerarin to puerarin glycosides by maltogenic amylase

Puerarin (daidzein 8-C-glucoside), the most abundant isoflavone in Puerariae radix, is prescribed to treat coronary heart disease, cardiac infarction, problems in ocular blood flow, sudden deafness, and alcoholism. However, puerarin cannot be given by injection due to its low solubility in water. To increase its solubility, puerarin was transglycosylated using various enzymes. Bacillus stearothermophilus maltogenic amylase (BSMA) was the most effective transferase used compared with Thermotoga maritima maltosyl transferase (TMMT), Thermus scotoductus 4-alpha-glucanotransferase (TS4alphaGTase), and Bacillus sp. I-5 cyclodextrin glucanotransferase (BSCGTase). TMMT and TS4alphaGTase lacked acceptor specificity for puerarin, which lacks an O-glucoside linkage between D-glucose and 7-OH-daidzein. The yield exceeded 70% when reacting 1% puerarin (acceptor), 3.0% soluble starch (donor), and 5U/100 microL BSMA at 55 degrees C for 45 min. The two major transfer products of the BSMA reaction were purified using C(18) and GPC chromatography. Their structures were identified as alpha-d-glucosyl-(1-->6)-puerarin and alpha-D-maltosyl-(1-->6)-puerarin using ESI+ TOF MS-MS and 13C NMR spectroscopy. The solubility of the transfer products was 14 and 168 times higher than that of puerarin, respectively.     

Marker-free chromosomal integration of the manganese superoxide dismutase gene (sodA) from Streptococcus thermophilus into Lactobacillus gasseri

A strategy for functional gene replacement in the chromosome of Lactobacillus gasseri is described. The phospho-beta-galactosidase II gene (lacII) was functionally replaced by the manganese superoxide dismutase (MnSOD) gene (sodA) from Streptococcus thermophilus, by adapting the insertional inactivation method described for lactobacilli [Russell, W.M. and Klaenhammer, T.R. 2001 Efficient system for directed integration into the Lactobacillus acidophilus and Lactobacillus gasseri chromosomes via homologous recombination. Appl. Environ. Microbiol. 67, 4361-4364]. L. gasseri carrying the heterologous sodA gene grew on lactose as efficiently as the wild-type parent. An active MnSOD was expressed in the transgenic strain, and the enzyme migrated on PAGE-SOD activity gels to the same position as that of MnSOD from S. thermophilus. The expression of MnSOD from a single copy of sodA integrated in the chromosome of L. gasseri provided enhanced tolerance to hydrogen peroxide, and extended the viability of carbon/energy starved cultures stored at 25 degrees C. This is the first report showing the successful utilization of the pORI plasmids system to generate marker-free gene integration in L. gasseri strains.     

Extracellular secretion of a maltogenic amylase from Lactobacillus gasseri ATCC33323 in Lactococcus lactis MG1363 and its application on the production of branched maltooligosaccharides

A maltogenic amylase gene from Lactobacillus gasseri ATCC33323 (LGMA) was expressed in Lactococcus lactis MG1363 using the P170 expression system. The successful production of recombinant LGMA (rLGMA) was confirmed by the catalytic activity of the enzyme in liquid and solid media. The N-terminal amino acid sequencing analysis of the rLGMA showed that it was Met-Gln-Leu-Ala-Ala-Leu-, which was the same as that of genuine protein, meaning the signal peptide was efficiently cleaved during secretion to the extracellular milieu. The optimal reaction temperature and pH of rLGMA (55 degrees C and pH 5, respectively) and enzymatic hydrolysis patterns on various substrates (beta-cyclodextrin, starch, and pullulan) supported that rLGMA was not only efficiently secreted from the Lactococcus lactis MG1363 but was also functionally active. Finally, the branched maltooligosaccharides were effectively produced from liquefied corn starch, by using rLGMA secreted from Lactococcus lactis, with a yield of 53.1%.     

Lactobacillus gasseri PA-3 utilizes the purines GMP and guanosine and decreases their absorption in rats

Excessive intake of purine-rich foods elevates serum uric acid levels, making it a risk factor for hyperuricemia.

We hypothesized that lactic acid bacteria ingested with food might utilize purines and contribute to their decreased absorption in the intestines, thereby preventing hyperuricemia. We previously reported that Lactobacillus gasseri PA-3 (PA-3) incorporates adenosine/inosine and related purines and that oral ingestion of PA-3 reduced the absorption of these purines in rats. However, it is unclear whether PA-3 also decreases the absorption of other purines, such as guanosine 5′-monophosphate (GMP) and guanosine. This study investigated whether PA-3 incorporates GMP and guanosine and reduces their absorption in rats.

PA-3 incorporated both purines, with ¹⁴C-GMP uptake being greater than that of ¹⁴C-guanosine. Radioactivity in rat blood was significantly lower 30, 45, and 60 minutes after administration of ¹⁴C-GMP plus PA-3 than after administration of ¹⁴C-GMP alone and was significantly lower 15 minutes after administration of ¹⁴C-guanosine plus PA-3 than after administration of ¹⁴C-guanosine alone.

PA-3 incorporates GMP and guanosine in vitro. Oral administration of PA-3 with GMP and guanosine reduces the intestinal absorption of these purines in vivo. These findings, together with those of previous studies, indicate that PA-3 reduces the absorption of major purines contained in foods. PA-3 may also attenuate the excessive absorption of dietary purines in humans, protecting these individuals against hyperuricemia.     

Cloning of a maltogenic alpha-amylase from Bacillus stearothermophilus

Abstract We have cloned and expressed a novel maltogenic alpha-amylase from B. stearothermophilus on plasmid in B. subtilis . Originally the plasmid was very unstable in the absence of selection, but was stabilized due to a spontaneous, copy number reducing mutation. The promoter region and the extension of the gene have been analysed, and a provisional DNA sequence has been determined. The N-terminal of the mature amylase has been determined and shown to be in accordance with signal peptidase processing after a typical Gram-positive signal sequence of 33 amino acids.     

Evaluation of purine utilization by Lactobacillus gasseri strains with potential to decrease the absorption of food-derived purines in the human intestine

Abstract

It is well accepted that frequent and heavy intake of purine-rich foods causes elevation of serum uric acid levels, which is a risk factor of hyperuricemia. Reducing intestinal absorption of dietary purines may attenuate the elevation of serum uric acid levels and exacerbation of hyperuricemia. This reduction may be achieved by the ingestion of lactic acid bacteria that take up purines in the intestine. In this study, we investigated the degree of uptake and utilization of purines of three lactobacilli strains. Among them, Lactobacillus gasseri PA-3 (PA-3) showed the greatest incorporation of ¹⁴C-adenine. PA-3 also incorporated ¹⁴C-adenosine and ¹⁴C-AMP. Additionally, using defined growth medium, PA-3 demonstrated greater proliferation in the presence of these purines than in their absence. Although further investigation is required, ingestion of PA-3 may lower serum uric acid levels by reducing intestinal absorption of purines in humans.     

Preparation and characterization of maltosyl-sucrose isomers produced by transglycosylation of maltogenic amylase from Bacillus stearothermophilus

To develop a new transfer product of sucrose, sucrose was modified to maltosyl-sucrose using the transglycosylation activity of maltogenic amylase from Bacillus stearothermophilus (BSMA). The transglycosylation reaction was conducted with maltotriose and sucrose as the donor and acceptor, respectively. The presence of various sucrose transfer products was confirmed by thin layer chromatography (TLC) and high performance anion exchange chromatography (HPAEC). The sucrose transfer products were isolated by alkali-degradation followed by charcoal column chromatography using 20% (v/v) ethanol, then purified by ion exchange and Biogel P-2 gel permeation chromatographies. The structures of the major transfer products were determined to be 6^G--maltosyl-sucrose (maltosyl-sucrose 1) and 6^F--maltosyl-sucrose (maltosyl-sucrose 2) by LC-MS and ¹³C NMR. The mixture of maltosyl-sucrose 1 and 2 showed low sweetness, high hygroscopicity, low Maillard reactivity, and high acid and heat stability. Furthermore, it had an inhibitory effect on mutansucrase and water-insoluble glucan formation. These results indicated that the mixture of maltosyl-sucrose 1 and 2 is a suitable sugar substitute useful for various food products.     

细菌麦芽糖淀粉酶在枯草芽孢杆菌中的诱导型异源表达

【目的】实现地衣芽孢杆菌麦芽糖淀粉酶在枯草芽孢杆菌中的高效异源表达,并研究该重组酶的酶学性质。【方法】克隆巨大芽孢杆菌木糖异构酶基因的启动子区域及其调控蛋白,构建一个大肠杆菌/芽孢杆菌穿梭型诱导表达质粒,使用该诱导型启动子介导麦芽糖淀粉酶编码基因,实现其在枯草芽孢杆菌中的功能表达。对重组枯草芽孢杆菌的诱导条件进行优化,提高麦芽糖淀粉酶的产量。【结果】获得了诱导表达麦芽糖淀粉酶基因的重组枯草芽孢杆菌菌株。最适诱导温度为45°C,最适诱导剂添加浓度为1%,最适添加诱导剂时间为接种培养9 h后。重组酶蛋白分子量大小为67 k D,对该酶的酶学性质研究发现,以可溶性淀粉为底物,反应生成麦芽糖和葡萄糖,其中麦芽糖含量为60.42%。重组酶最适作用温度为45°C,最适作用p H为6.5,Ca2+、Co2+、EDTA对该重组麦芽糖淀粉酶具有激活作用。【结论】通过木糖诱导表达系统可以实现麦芽糖淀粉酶在枯草芽孢杆菌中的高效诱导型表达,酶活最高可达296.64 U/m L发酵液,在工业上有着较好的应用前景。     

Identification of five phospho-beta-glycosidases from Lactobacillus gasseri ATCC33323T cultured in lactose medium

Lactobacillus gasseri ATCC33323(T) has seven putative phospho-beta-glycosidase genes. Using column chromatography, we found that this strain cultured in lactose medium expresses five phospho-beta-glycosidases (LacG1, LacG2, Pbg1, Pbg2, and Pbg3), where these gene expressions can be suppressed by glucose. To our knowledge, this is the first report indicating that five glycosidases are induced from a single bacterial strain using a single carbon source, lactose.     

Biochemical and crystallographic characterization of a glucansucrase from Lactobacillus reuteri 180

Glucansucrases are large extracellular transglycosidases secreted by lactic acid bacteria. Using sucrose as a substrate they synthesize high molecular mass α-glucans or, in the presence of suitable acceptor molecules, low molecular mass oligosaccharides. Although about 60 glucansucrases have been classified in glycoside hydrolase family GH70, no three-dimensional structure has been reported for any. With the aim of solving the first structure of a GH70 glucansucrase, purification and crystallization experiments were performed with a fully active, 117 kDa N-terminally truncated fragment of glucansucrase GTF180 from Lactobacillus reuteri 180 (residues 742–1772). Crystallization experiments yielded crystals that belong to two different triclinic crystal forms (space group P1) and one orthorhombic crystal form (space group P2₁2₁2₁). Native data sets for both triclinic and the orthorhombic crystals were collected at 1.7 and 2.0 Å resolution, respectively. Enzyme activity assays, pH and temperature optima show comparable values for both the full-length and the N-terminally truncated GTF180.     

Expression of mRNA encoding IFNα in macrophages stimulated with Lactobacillus gasseri

Abstract The ability of Lactobacillus gasseri , a dairy lactic acid bacterium, to induce interferon (IFN) was investigated in murine macrophage cultures. IFN α was substantially induced by some strais of L. gasseri and the titers were the highest at a concentration of 100 μg ml⁻¹ of L. gasseri DSM20243^T. The expression of mRNA encoding IFN α was detected in spleen-macrophages (SP-M θ ) and Peyer's patch-adherent cells stimulated with L. gasseri DSM20243^T. Actinomycin D and cycloheximide added to SP-M θ cultures showed that the mRNA was synthesized by 0.5 h, and that IFN α was produced within 3 to 6 h after the stimulation with L. gasseri DSM20243^T. The results support the notion that dairy products containing L. gasseri can be 'physiologically functional foods'.     

Caseicin 80: purification and characterization of a new bacteriocin from Lactobacillus casei

When grown in complex or synthetic media, Lactobacillus casei B 80 synthesizes a mitomycin C-inducible polypeptide with very specific bactericidal activity against the sensitive strain Lactobacillus casei B 109. The amount of secreted bacteriocin in the culture solution was low, about 1 mg/l. The bacteriocin which we called caseicin 80, was also detectable in cell extracts, although only 2% of the total activity was retained intracellularly. Caseicin 80 was concentrated by ultrafiltration and purified by cation exchange chromatography with Cellulose SE-23 and Superose. The molecular weight was in the range of M _r=40,000–42,000 and the isoelectric point was pH 4.5.     

Detection of Lactobacillus gasseri OLL2716 strain administered with yogurt drink in gastric mucus layer in humans

In animal models and human trials, Lactobacillus gasseri OLL2716 (LG21) strain suppressed Helicobacter pylori colonization in the stomach. The aim of the present study was to clarify whether orally administered LG21 strain can enter the gastric mucus layer. Biopsy samples were taken from the gastric antrum and corpus of two healthy volunteers (H. pylori infected and non-infected) who drank yogurt supplemented with LG21 strains. DNA of LG21 and H. pylori in the mucus layer was detected using the laser-assisted microdissection and non-contact pressure catapulting (LMPC) method and the semi-nested PCR method with primer sets of RNA helicases of superfamily II gene-Insertion sequence for LG21 strain and those of ureA gene for H. pylori. In the volunteer with H. pylori infection, DNA fragments of LG21- and H. pylori-specific regions from both antrum and corpus were amplified, whereas in a non-infected volunteer, only the LG21 DNA from the antrum was amplified. The present study demonstrated that LG21 strains administered through a yogurt drink can enter into the gastric mucus layer. Our novel method may be useful in studying gastric probiotics for H. pylori infection.     

Anti-biofilm potential of Lactobacillus plantarum Y3 culture and its cell-free supernatant against multidrug-resistant uropathogen Escherichia coli U12

Uropathogens develop biofilms on urinary catheters, resulting in persistent and chronic infections that are associated with resistance to antimicrobial therapy. Therefore, the current study was performed to control biofilm-associated urinary tract infections through assaying the anti-biofilm ability of lactic acid bacteria (LAB) against multidrug-resistant (MDR) uropathogens. Twenty LAB were obtained from pickles and fermented dairy products, and screened for their anti-biofilm and antimicrobial effects against MDR Escherichia coli U12 (ECU12). Lactobacillus plantarum Y3 (LPY3) ({"type":"entrez-nucleotide","attrs":{"text":"MT498405","term_id":"1843980272","term_text":"MT498405"}}MT498405), showed the highest inhibitory effect and biofilm production. Pre-coating of a microtitre plate with LPY3 culture was more potent than co-incubation. Pre-coating with LPY3 culture generated a higher anti-biofilm effect with an adherence of 14.5% than cell free supernatant (CFS) (31.2%). Anti-biofilm effect of CFS was heat stable up to 100 °C with higher effect at pH 4–6. Pre-coating urinary catheter with LPY3 culture reduced the CFU/cm² of ECU12 attached to the catheter for up to seven days. Meanwhile, CFS reduced the ECU12 CFU/cm² for up to four days. Scanning electron microscope confirmed the reduction of ECU12 adherence to catheters after treatment with CFS. Therefore, Lactobacillus plantarum can be applied in medical devices as prophylactic agent and as a natural biointervention to treat urinary tract infections.     

Antimicrobial and antiadhesive properties of a biosurfactant isolated from Lactobacillus paracasei ssp. paracasei A20

Aims: The aim of this study was to determine the antimicrobial and antiadhesive properties of a biosurfactant isolated from Lactobacillus paracasei ssp. paracasei A20 against several micro‐organisms, including Gram‐positive and Gram‐negative bacteria, yeasts and filamentous fungi. Methods and Results: Antimicrobial and antiadhesive activities were determined using the microdilution method in 96‐well culture plates. The biosurfactant showed antimicrobial activity against all the micro‐organisms assayed, and for twelve of the eighteen micro‐organisms (including the pathogenic Candida albicans, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus agalactiae), the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were achieved for biosurfactant concentrations between 25 and 50 mg ml^?1. Furthermore, the biosurfactant showed antiadhesive activity against most of the micro‐organisms evaluated. Conclusions: As far as we know, this is the first compilation of data on antimicrobial and antiadhesive activities of biosurfactants obtained from lactobacilli against such a broad group of micro‐organisms. Although the antiadhesive activity of biosurfactants isolated from lactic acid bacteria has been widely reported, their antimicrobial activity is quite unusual and has been described only in a few strains. Significance and Impact of the Study: The results obtained in this study regarding the antimicrobial and antiadhesive properties of this biosurfactant opens future prospects for its use against micro‐organisms responsible for diseases and infections in the urinary, vaginal and gastrointestinal tracts, as well as in the skin, making it a suitable alternative to conventional antibiotics.     

Strain-specific detection by pulsed-field gel electrophoresis of Lactobacillus gasseri TMC0356 in human feces after oral administration of these organisms

The present study aimed to develop an innovative, strain-specific means of identifying the probiotic Lactobacillus gasseri TMC0356 and to determine whether orally administered TMC0356 could be recovered from the human intestine. High molecular weight genomic DNA was isolated from TMC0356 and 14 reference strains of L. gasseri, including the type strain. The DNA samples were digested with the selected rare-cutting restriction endonucleases SmaI, SacII and ApaI and the resulting fragments separated by pulsed-field gel electrophoresis (PFGE) in a size range between 20 to 290 kb. TMC0356 could be distinguished from the other L. gasseri strains on the basis of the SmaI and SacII macrorestriction patterns. Furthermore, L. gasseri strains isolated from the feces of subjects who had ingested TMC0356 were identical to TMC0356 in the SmaI, SacII and ApaI macrorestriction fragments of digested DNA. These results suggest that PFGE of genomic DNA digested with SmaI, SacII, could be a practical means of identification of TMC0356. Furthermore, these results indicate that ingested TMC0356 can survive in, and colonize, the human intestine.