Enzymatic characterization of a maltogenic amylase from Lactobacillus gasseri ATCC 33323 expressed in Escherichia coli

A gene corresponding to a maltogenic amylase (MAase) in Lactobacillus gasseri ATCC 33323 (lgma) was cloned and expressed in Escherichia coli. The recombinant LGMA was efficiently purified 24.3-fold by one-step Ni-NTA affinity chromatography. The final yield and specific activity of the purified recombinant LGMA were 68% and 58.7 U/mg, respectively. The purified enzyme exhibited optimal activity for beta-CD hydrolysis at 55 degrees C and pH 5. The relative hydrolytic activities of LGMA to beta-CD, soluble starch or pullulan was 8:1:1.9. The activity of LGMA was strongly inhibited by most metal ions, especially Zn(2+), Fe(2+), Co(2+) and by EDTA. LGMA possessed some unusual properties distinguishable from typical MAases, such as being in a tetrameric form, having hydrolyzing activity towards the alpha-(1,6)-glycosidic linkage and being inhibited by acarbose.     

Analysis of the genome sequence of Lactobacillus gasseri ATCC 33323 reveals the molecular basis of an autochthonous intestinal organism

This study presents the complete genome sequence of Lactobacillus gasseri ATCC 33323, a neotype strain of human origin and a native species found commonly in the gastrointestinal tracts of neonates and adults. The plasmid-free genome was 1,894,360 bp in size and predicted to encode 1,810 genes. The GC content was 35.3%, similar to the GC content of its closest relatives, L. johnsonii NCC 533 (34%) and L. acidophilus NCFM (34%). Two identical copies of the prophage LgaI (40,086 bp), of the Sfi11-like Siphoviridae phage family, were integrated tandomly in the chromosome. A number of unique features were identified in the genome of L. gasseri that were likely acquired by horizontal gene transfer and may contribute to the survival of this bacterium in its ecological niche. L. gasseri encodes two restriction and modification systems, which may limit bacteriophage infection. L. gasseri also encodes an operon for production of heteropolysaccharides of high complexity. A unique alternative sigma factor was present similar to that of B. caccae ATCC 43185, a bacterial species isolated from human feces. In addition, L. gasseri encoded the highest number of putative mucus-binding proteins (14) among lactobacilli sequenced to date. Selected phenotypic characteristics that were compared between ATCC 33323 and other human L. gasseri strains included carbohydrate fermentation patterns, growth and survival in bile, oxalate degradation, and adhesion to intestinal epithelial cells, in vitro. The results from this study indicated high intraspecies variability from a genome encoding traits important for survival and retention in the gastrointestinal tract.     

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%.     

Effects of a Potential Probiotic Strain Lactobacillus gasseri ATCC 33323 on Helicobacter pylori-Induced Inflammatory Response and Gene Expression in Coinfected Gastric Epithelial Cells

In the present study, we aimed to investigate the modulatory effects of a potential probiotic bacterium Lactobacillus gasseri ATCC 33323 on Helicobacter pylori-induced inflammatory response and gene expression in human gastric adenocarcinoma (AGS) cell line. The gastric epithelial cells were coinfected with a collection of H. pylori clinical strains alone or in combination with L. gasseri at a multiplicity of infection (MOI) of 1:100 for each bacterium, and incubated for different time points of 3, 6, and 12 h. IL-8 secretion from coinfected AGS cells after incubation at each time point was measured by an enzyme-linked immunosorbent assay (ELISA). The mRNA expression of IL-8, Bcl-2, β-catenin, integrin α₅, and integrin β₁ genes was determined by quantitative RT-PCR amplification of total RNA extracted from coinfected epithelial cells. L. gasseri significantly (P < 0.05 and P < 0.01) decreased the production of IL-8 in AGS cells coinfected with H. pylori strains at 6 h post-infection. We also detected that L. gasseri significantly (P < 0.05) down-regulated the gene expression level of IL-8 in H. pylori-stimulated AGS cells after 6 and 12 h of coinfection. Similarly, L. gasseri caused a significant decrease (P < 0.05) in mRNA expression of Bcl-2, β-catenin, integrin α₅, and integrin β₁ genes in AGS cells at 3 and 6 h after infection with H. pylori strains as compared with non-infected control cells. In conclusion, our results demonstrated that L. gasseri ameliorates H. pylori-induced inflammation and could be developed as a supplementation to the current treatment regimens administrated against H. pylori infection.

     

Antibacterial activity associated with Lactobacillus gasseri ATCC 9857from the human female genitourinary tract

The 10-fold concentrated spent MRS culture cell-free supernatant concentrate [(cCFS)] of the human female genitourinary tract isolate Lactobacillus gasseri ATCC 9857 was shown to exhibit antibacterial activity towards gram-positive sporogenous and asporogenous fermentative eubacteria in liquid and on solid media under conditions that eliminated the activity of lactic acid (-glycerophosphate) and hydrogen peroxide (catalase). The antibacterial activity of the cCFS was characterized by automated turbidometry (Bioscreen) and non-linear regression analysis (Gompertz model) using MRS broth cultures of the indicator strain L. acidophilus ATCC 11975. It exhibited a bactericidal mode of action, sensitivity to trypsin and proteinase K, partial sensitivity to pepsin and pronase E, partial heat stability at 121 °C for 15 min, and retained significantly more activity following exposure to pH 3.0 and 5.0 compared with pH 7.2 and 9.0. The inhibitory spectrum included a wide range of Lactobacillus species, Bifidobacterium bifidum, B. infantis and B. catenulatum, Lactococcus cremoris, Leuconostoc cremoris, Pediococcus pentosaceus, Bacillus cereus, Clostridium tyrobutyricum, C. pasteurianum, C. sporogenes, Staphylococcus carnosus, and Enterococcus faecalis. Although partial inhibition of Escherichia coli ATCC 25922 by cCFS was observed in liquid medium, inhibition of freshly isolated human uropathogenic E. coli strains could not be demonstrated on TSB agar plates by agar well diffusion. Following partial resolution by gel permeation FPLC on Superose-12, the fractionated cCFS was shown to comprise at least two inhibitory peptides (3.05 and 5.27 kDa) as well as aggregated inhibitory peptide material (21.65, 41.50, 81.20, and 120.90 kDa). The 3.05 kDa peptide, designated Gassericin D, inhibited L. acidophilus strains ATCC 11975 and ACA-DC 241. The 5.27 kDa peptide, designated Gassericin C, inhibited L. gasseri strain UCSC LF221Snb and En. faecalis DPC 3319. The aggregated 21.65 kDa peptide material strongly inhibited L. acidophilus ATCC 11975 and weakly inhibited Listeria inocua DPC 3306. The aggregated 41.50 kDa peptide material strongly inhibited Ba. cereus DPC 3316 and weakly inhibited L. acidophilus ACA-DC 241. The ability of L. gasseri ATCC 9857 to produce bacteriocin-like activity may be of importance in the biopreservation of nutraceuticals and in the management of female genitourinary and gastrointestinal tract infections involving En. faecalis.     

Identification and characterization of novel surface proteins in Lactobacillus johnsonii and Lactobacillus gasseri

We have identified and sequenced the genes encoding the aggregation-promoting factor (APF) protein from six different strains of Lactobacillus johnsonii and Lactobacillus gasseri. Both species harbor two apf genes, apf1 and apf2, which are in the same orientation and encode proteins of 257 to 326 amino acids. Multiple alignments of the deduced amino acid sequences of these apf genes demonstrate a very strong sequence conservation of all of the genes with the exception of their central regions. Northern blot analysis showed that both genes are transcribed, reaching their maximum expression during the exponential phase. Primer extension analysis revealed that apf1 and apf2 harbor a putative promoter sequence that is conserved in all of the genes. Western blot analysis of the LiCl cell extracts showed that APF proteins are located on the cell surface. Intact cells of L. johnsonii revealed the typical cell wall architecture of S-layer-carrying gram-positive eubacteria, which could be selectively removed with LiCl treatment. In addition, the amino acid composition, physical properties, and genetic organization were found to be quite similar to those of S-layer proteins. These results suggest that APF is a novel surface protein of the Lactobacillus acidophilus B-homology group which might belong to an S-layer-like family.     

Identification and cloning of gusA, encoding a new beta-glucuronidase from Lactobacillus gasseri ADH

The gusA gene, encoding a new beta-glucuronidase enzyme, has been cloned from Lactobacillus gasseri ADH. This is the first report of a beta-glucuronidase gene cloned from a bacterial source other than Escherichia coli. A plasmid library of L. gasseri chromosomal DNA was screened for complementation of an E. coli gus mutant. Two overlapping clones that restored beta-glucuronidase activity in the mutant strain were sequenced and revealed three complete and two partial open reading frames. The largest open reading frame, spanning 1,797 bp, encodes a 597-amino-acid protein that shows 39% identity to beta-glucuronidase (GusA) of E. coli K-12 (EC 3.2.1.31). The other two complete open reading frames, which are arranged to be separately transcribed, encode a putative bile salt hydrolase and a putative protein of unknown function with similarities to MerR-type regulatory proteins. Overexpression of GusA was achieved in a beta-glucuronidase-negative L. gasseri strain by expressing the gusA gene, subcloned onto a low-copy-number shuttle vector, from the strong Lactobacillus P6 promoter. GusA was also expressed in E. coli from a pET expression system. Preliminary characterization of the GusA protein from crude cell extracts revealed that the enzyme was active across an acidic pH range and a broad temperature range. An analysis of other lactobacilli identified beta-glucuronidase activity and gusA homologs in other L. gasseri isolates but not in other Lactobacillus species tested.     

The PTS transporters of <Emphasis Type="Italic">Lactobacillus gasseri</Emphasis> ATCC 33323

Background  

Lactobacilli can utilize a variety of carbohydrates which reflects the nutrient availability in their respective environments. A common lactobacilli in the human gastrointestinal tract, Lactobacillus gasseri, was selected for further study. The currently available annotation of the L. gasseri ATCC 33323 genome describes numerous putative genes involved in carbohydrate utilization, yet the specific functions of many of these genes remain unknown.     

Inhibition of food-borne pathogenic bacteria by bacteriocins from Lactobacillus gasseri

Seventy-three strains of the Lactobacillus acidophilus group and a Lact. reuteri isolated from human faeces were examined for production of antimicrobial agents against 16 strains of six species of food-borne enteric pathogenic bacteria. Several strains of Lact. gasseri showed wide inhibitory activity against the tested bacteria. Gassericin A produced by Lact. gasseri LA39 was one of the most widely active bacteriocins. It was bactericidal without causing cell lysis.     

Identification and cloning of a gene encoding tannase (tannin acylhydrolase) from Lactobacillus plantarum ATCC 14917(T)

The gene tanLpl, encoding a novel tannase enzyme (TanLpl), has been cloned from Lactobacillus plantarum ATCC 14917(T). This is the first report of a tannase gene cloned from a bacterial source other than from Staphylococcus lugdunensis, which has been reported elsewhere. The open reading frame of tanLpl, spanning 1410 bp, encoded a 469-amino-acid protein that showed 28.8% identity to the tannase of S. lugdunensis with several commonly conserved sequences. These sequences could not be found in putative tannases reported for other bacteria and fungi. TanLpl was expressed in Escherichia coli DH5alpha from a pGEM-T expression system and purified. SDS-PAGE analysis indicated that purified TanLpl was a monomer polypeptide of approximately 50 kDa in size. Subsequent enzymatic characterization revealed that TanLpl was most active in an alkaline pH range at 40 degrees C, which was quite different from that observed for a fungal tannase of Aspergillus oryzae. In addition, the Michaelis-Menten constant of TanLpl was markedly lower than that of A. oryzae tannase. The evidence suggests that TanLpl should be classified into a novel family of tannases.     

Enhancement of immunoregulatory effects of Lactobacillus gasseri TMC0356 by heat treatment and culture medium

Aims: The aim of this study was to investigate the influence of heat treatment and culture media on the immunoregulatory effects of a probiotic strain, Lactobacillus gasseri TMC0356 (TMC0356). Methods and Results: TMC0356 cultured in deMan–Rogosa–Sharpe and same food grade (FG) media were inactivated with the heat treatment at 70 and 90°C. Viable and heat‐killed TMC0356 were tested for their ability to induce interleukin (IL)‐12 production in the murine macrophage cell line J774.1. These TMC0356 were examined for their resistance to N‐acetylmuramidase. Their morphology was observed by scanning electron microscopy. The heat‐killed TMC0356 significantly induced IL‐12 production in J774.1 cells and exhibited enhanced resistance to N‐acetylmuramidase compared with viable TMC0356. Morphological changes were observed in TMC0356 when cultured in FG medium. Cell morphology and induction of IL‐12 production in J774.1 cells were also associated. Conclusions: These results suggest that heat treatment and culture medium composition modified the immunoregulatory effects of TMC0356 to induce IL‐12 production in macrophages. Significance and Impact of the Study: These results demonstrate that probiotic immunoregulatory effects may be modified by the processing technology of cell preparation.     

Potential of Lactobacillus gasseri isolated from infant faeces to produce bacteriocin

Thirty strains of Lactobacillus gasseri isolated from infant faeces were examined for production of an antimicrobial agent against 10 strains of the seven species of the genus Lactobacillus. Each of the strains inhibited the growth of at least one of the indicator strains. The agent was sensitive to proteolytic enzymes and stable for 20 min at 120°C. It is a bacteriocin designated as gassericin A.     

Purification of Glucoamylase from Lactobacillus amylovorus ATCC 33621

An intracellular glucoamylase (E.C. 3.2.1.3) was purified to homogeneity from Lactobacillus amylovorus on a Fast Protein Liquid Chromatography System (FPLC) with a Mono Q ion-exchanger and two Superose 12 gel filtration columns arranged in series. The enzyme activity was quantified with a specific, chromogenic substrate, p-nitrophenyl-β-maltoside. Preparative gel electrophoresis was then used to further purify active enzyme fractions. Native polyacrylamide gel electrophoresis (Native-PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single protein band of molecular weight 47 kDa. Glucoamylase activity of the purified protein was confirmed by its ability to degrade starch on a 0.025% starch-polyacrylamide gel stained with I₂/KI. Glucoamylase exhibited optimum catalytic activity at pH 6.0 and 45°C, and the enzyme had an isoelectric point near 4.39. The glucoamylase contained high levels of hydrophilic amino acids, comparable to fungal glucoamylases. Received: 12 July 1996 / Accepted: 10 September 1996     

Characterization of glucoamylase from Lactobacillus amylovorus ATCC 33621

Summary An intracellular glucoamylase, purified from Lactobacillus amylovorus, reacted selectively with polysaccharides. Kinetic studies indicated low affinity for maltose and maltotriose (K_m 58 g/ml and 178 g/ml) and higher affinity for starch and dextrin (K_m 0.01 g/ml and 0.02 g/ml). Glucoamylase was inhibited almost 50% by 10 mM glucose. Cu²⁺ and Pb²⁺ inhibited glucoamylase at 1.0 mM but EDTA and other metal chelators had no effect on the enzyme activity. Acarbose and Tris inhibited the enzyme by 84% and 98%, respectively at 1 mM, while iodoacetate and p-chloromecuribenzoic acid inhibited activity by 98% and 78%, respectively at 10 mM. The purified enzyme was thermolabile at temperatures greater than 55°C and thus has potential for application in the brewing industry.     

鼠李糖乳杆菌-格氏乳杆菌联用对阴道致病菌的抑制作用

目的本文通过提取细菌基因组进行16S rDNA PCR扩增和测序,分析菌株的进化树分支,鉴定一株乳酸菌菌株RD-0060并检测RD-0060与已有菌株RD-0046联用的抑菌能力和细胞粘附能力。方法结合现有菌株RD-0046(格氏乳杆菌,Lactobacillus gasseri),采用牛津杯法研究RD-0060单菌、RD-0060和RD-0046联用抑制致病菌的能力。通过共培养细菌和阴道上皮细胞VK2/E6E7,研究RD-0060单菌和RD-0060/RD-0046二联菌粘附能力。结果 RD-0060为鼠李糖乳杆菌(Lactobacillus rhamnosus),具有抑制阿托波菌、阴道加德纳菌和常见好氧型病菌的功能,对阴道上皮细胞也有较强的粘附能力;RD-0060和RD-0046二联菌的抑菌效果和细胞粘附能力比单菌株更强。结论鼠李糖乳杆菌和格氏乳杆菌联用能显著抑制阴道致病菌生长,并且能够大量粘附阴道细胞,而且两菌株联用有协同效果,具有良好的临床应用和开发前景。     

Characterization of the aggregation promoting factor from Lactobacillus gasseri, avaginal isolate

Lactobacillus gasseri 2459, isolated from the human vagina, exhibits a strongautoaggregating phenotype. Filter-sterilized spent supernatants of this strain promote aggregationof Lact. plantarum LL441 and Enterococcus faecalis EF. Aggregation wasabolished upon exposure of the cells to proteases and, in the case of Ent. faecalis , tometaperiodate, which suggests the involvement of cell-surface proteins and glycoproteins,respectively, in the aggregation phenotype. In accordance with this, a 75 kDa surface protein, andpossibly another of approximately 94 kDa, appears in Lact. plantarum LL441 culturesincubated with Lact. gasseri culture supernatants. The diffusible aggregation promotingfactor was purified from stationary phase culture supernatants and determined to be a 2 kDahydrophilic peptide active at pH 3–4 and stable at neutral and acid pH. The activity wasresistant to heat, chymotrypsin, chelating agents, triton X-100 and reducing agents, but sensitiveto other proteases and SDS.     

Isolation and characterization of a new bacteriocin from Lactobacillus gasseri KT7

A bacteriocin-producing Lactobacillus gasseri strain, KT7, was isolated from infant faeces. The supernatant fluid showed inhibitory activity not only against some lactic acid bacteria but also, against some pathogenic and food-spoilage species, including Clostridium, Listeria and Enterococcus. An antimicrobial peptide designated gassericin KT7 was isolated from Lactobacillus gasseri KT7. It was purified to homogeneity by a single four-step procedure: a crude supernatant fluid obtained from early stationary-phase culture in MRS medium was subjected to ammonium sulphate fractionation, CM-Sephadex cation-exchange chromatography, Phenyl-Sepharose hydrophobic chromatography and reverse-phase HPLC chromatography. Gassericin KT7 was sensitive to proteolytic enzymes, resistant to heat, active over a wide range of pH, and migrated as a 4.5-5.0 kDa peptide on SDS-PAGE. The bacteriocin was produced constitutively during exponential growth. It was bactericidal to sensitive cells and the bactericidal effect was not produced by cell lysis. The amino acid composition of the bacteriocin was determined and no modified amino acid was found among the residues identified.     

Mapping of three plasmids from Lactobacillus helveticus ATCC 15009

A 1.4 kb DNA fragment from the chromosomal DNA of Penicillium nalgiovense was isolated which confers proteolytic activity to E. coli DH5α cells when cloned under the control of the E. coli lacZ promoter. The protein was excreted by the cells as was shown by the formation of a clearing zone in skim milk medium. A retransformation of the plasmid carrying the protease gene into P. nalgiovense leads to transformants with both increased and with nearly no proteolytic activity under neutral conditions. Southern blotting experiments revealed that the transforming plasmid had apparently integrated into the homologous locus and thereby inactivated the residual gene.     

Growth and exopolysaccharide production by Lactobacillus helveticus ATCC 15807 in an adenine-supplemented chemically defined medium

AIMS: To analyse the exopolysaccharide (EPS) production by Lactobacillus helveticus ATCC 15807 in a chemically defined medium (CDM) and the effect of nutrients and stress culture conditions on cell growth and EPS formation. METHODS AND RESULTS: Cultures were conducted in CDM: (i) containing essential and nonessential bases and vitamins; (ii) without nonessential bases and vitamins [Simplified CDM (SCDM)]; (iii) SCDM supplemented individually with vitamins and bases. The influence of carbohydrates, pH and osmotic culture conditions on growth and polymer formation was analysed. Adenine and lactose stimulated both growth and EPS production. Constant pH fermentations (4.5 and 6.2) did not improve EPS synthesis while NaCl and glycerol were detrimental for growth and polymer formation. In all media the EPS monomer composition was glucose and galactose (2.5 : 1). CONCLUSIONS: A SCDM containing adenine and lactose was optimal for cell growth and EPS formation by Lact. helveticus ATCC 15807. Controlled pH (6.2 and 4.5) and osmotic stress culture conditions did not improve polymer production. The EPS characteristics were identical in all media. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides a better knowledge on EPS synthesis by Lact. helveticus. A CDM to perform regulation studies on EPS production by Lact. helveticus species is now available.