• Journal of Microbiology and Biotechnology
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• v.19 no.10
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• pp.1176-1183
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• 2009

Lactobacillus crispatus (L. crispatus) ZJ001 is highly adhesive to epithelial cells and expresses S-layer proteins. In this study, S-S-layer layer genes were sequenced and expressed in E. coli to characterize the function of proteins with this particular strain. L. crispatus ZJ001 harbored two S-layer genes slpA and slpB, and only slpA gene was expressed in the bacterium, as revealed by RT-PCR and immunoassays. The mature SlpA showed 47% amino acid sequence identity to SlpB. The SlpA and SlpB of L. crispatus ZJ001 were highly homologous at the C-terminal region to other Lactobacillus S-layer proteins, but were substantially variable at N-terminal and middle regions. Electron microscopic analysis indicated that His-slpA expressed in E. coli was able to form a sheet-like structure similar to the natural S-layer, but His-slpB formed as disc-like structures. In the cell binding experiments, HeLa cells were able to bind to both recombinant His-slpA and His-slpB proteins to the extent similar to the natural S-layer. The cell binding domains remain mostly in the N-terminal regions in SlpA and SlpB, as shown by high binding of truncated peptides SlpA2-228 and SlpB2-249. Our results indicated that SlpA was active and high binding to HeLa cells, and that the slpA gene could be targeted to display foreign proteins on the bacterial surface of ZJ001 as a potential mucosal vaccine vector.

• Journal of Microbiology and Biotechnology
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• v.21 no.8
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• pp.822-829
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• 2011

Evidence shows that probiotic bacteria can undergo substantial structural and morphological changes in response to environmental stresses, including antibiotics. Therefore, this study investigated the effects of penicillin G (0.015, 0.03, and 0.06 mg/l) on the morphology and adhesion of Lactobacillus acidophilus ATCC 4356, including the colony morphotype, biofilm production, hydrophobicity, $H_2_O2$ formation, S-layer structure, and slpA gene expression. Whereas only smooth colonies grew in the presence of penicillin, rough and smooth colony types were observed in the control group. L. acidophilus ATCC 4356 was found to be hydrophobic under normal conditions, yet its hydrophobicity decreased in the presence of the antibiotic. No biofilm was produced by the bacterium, despite testing a variety of different culture conditions; however, treatment with penicillin G (0.015-0.06 mg/l) significantly decreased its production of $H_2_O_2$ formation and altered the S-layer protein structure and slpA gene expression. The S-protein expression decreased with 0.015 mg/l penicillin G, yet increased with 0.03 and 0.06 mg/l penicillin G. In addition, the slpA gene expression decreased in the presence of 0.015 mg/l of the antibiotic. In conclusion, penicillin G was able to alter the S-layer protein production, slpA gene expression, and certain physicochemical properties of Lactobacillus acidophilus ATCC 4356.

• Journal of Microbiology and Biotechnology
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• v.20 no.4
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• pp.749-756
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• 2010

In many conditions, bacterial surface properties are changed as a result of variation in the growth medium and conditions. This study examined the influence of bile salt concentrations (0-0.1%) on colony morphotype, hydrophobicity, $H_2O_2$ concentration, S-layer protein production, and slpA gene expression in Lactobacillus acidophilus ATCC 4356. It was observed that two types of colonies (R and S) were in the control group and the stress condition. When the bile level increased in the medium, the amount of S type was more than the R type. A stepwise increment in the bile concentration resulted in a stepwise decline in the maximum growth rate. The results showed that hydrophobicity was increased in 0.01%-0.02% bile, but it was decreased in 0.1% bile. Treatment by bile (0.01%-0.1%) profoundly decreased $H_2O_2$ formation. S-Layer protein and slpA gene expression were also altered by the stress condition. S-Protein expression was increased in the stress condition. The slpA gene expression increased in 0.01%-0.05% bile and it decreased in 0.1% bile. However, we found that different bile salt concentrations influenced the morphology and some surface properties of L. acidophilus ATCC 4356. These changes were very different in the 0.1% bile. It appears that the bacteria respond abruptly to 0.1% bile.

• Microbiology and Biotechnology Letters
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• v.35 no.4
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• pp.352-356
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• 2007

A Lactobacillus sp. has been isolated from infant feces and characterized according to its physiological properties and identified as Lactobacillus acidophilus KLA1012. A gene coding surface layer protein (SLP) has been cloned and the sequence has been determined. The nucleotide sequence of slpA was 1,338 bp in size and was identical to that of L. acidophilus ATCC 4356 (100%). Amino acid sequence of SLP-A was deduced from the nucleotide sequence and it had signal sequence at N-terminal, consisting of positively charged amino acid mainly lysine. slpA was cloned and heterologously expressed in E. coli M15 and the 45.2 kDa surface-layer protein band was examined by SDS-PAGE and confirmed by Western blotting using polyclonal antibody against L. acidophilus KLA 1012 SLP-A protein.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.202-205
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• 2000

A gene coding $endo-{\beta}$,-1, 4 glucanase from Actinomyces sp. KNG40 and phytase from Hansenula polymorpha were cloned into Esherichia coli JM101 by using E. coli/Lactobacillus shuttle vector pNZ3004 and pNZ123. The plasmid p3PS(1-4) and p123(1-4) have slpA promoter and slpA signal sequence. So, I constructed expression vectors, p3PS(1-4)Endo, phy and p123(1-4)Endo, phy. These constructed vector was transformed in target host Lactobacillus gasseri and reutri. These transformed host expressed endoglucanase and phytase as extracellular fraction. In the enzyme activity of the same vector, host L, gasseri was higher activity than L. reuteri. This indicates that L. gasseri recongnize promoter and signal sequence very well.

• Journal of Microbiology and Biotechnology
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• v.21 no.4
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• pp.421-429
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• 2011

For precise identification of a Lactobacillus K1 isolate, LC-MS/MS analysis of the putative surface layer protein was performed. The results obtained from LTQ-FT-ICR mass spectrometry confirmed that the analyzed protein spot is the surface layer protein originating from Lb. helveticus species. Moreover, the identified protein has the highest similarity with the surface layer protein from Lb. helveticus R0052. To evaluate the proteomic study, multilocus sequence analysis of selected housekeeping gene sequences was performed. Combination of 16S rRNA sequencing with partial sequences for the genes encoding the RNA polymerase alpha subunit (rpoA), phenylalanyl-tRNA synthase alpha subunit (pheS), translational elongation factor Tu (tuf), and Hsp60 chaperonins (groEL) also allowed to classify the analyzed isolate as Lb. helveticus. Further classification at the strain level was achieved by sequencing of the slp gene. This gene showed 99.8% identity with the corresponding slp gene of Lb. helveticus R0052, which is in good agreement with data obtained by nano-HPLC coupled to an LTQ-FT-ICR mass spectrometer. Finally, LC-MS/MS analysis of surface layer proteins extracted from three other Lactobacillus strains proved that the proposed method is the appropriate molecular tool for the identification of S-layer-possessing lactobacilli at the species and even strain levels.

• Microbiology and Biotechnology Letters
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• v.29 no.1
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• pp.1-11
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• 2001

Lactic acid bacteria (LAB) are widely used for various food fermentation. With the recent advances in modern biotechnology, a variety of bio-products with the high economic values have been produced using microorganisms. For molecular cloning and expression studies on the gene of interest, E. coli has been widely used mainly because vector systems are fully developed. Most plasmid vectors currently used for E, coli carry antibiotic-resistant markers. As it is generally believed that the antibiotic resistance markers are potentially transferred to other bacteria, application of the plasmid vectors carrying antibiotic resistance genes as selection markers should be avoided, especially for human consump-tion. By contrast, as LAB have some desirable traits such that the they are GRAS(generally recognized as safe), able to secrete gene products out of cell, and their low protease activities, they are regarded as an ideal organism for the genetic manipulation, including cloning and expression of homologous and heterologous genes. However, the vec-tor systems established for LAB are stil insufficient to over-produce gene products, stably, limiting the use of these organisms for industrial applications. For a past decade, the two popular plasmid vectors, pAM$\beta$1 of Streptococcus faecalis and pGK12 theB. subtilis-E. coli shuttle vector derived from pWV01 of Lactococcus lactis ssp. cremoris wg 2, were most widely used to construct efficient chimeric vectors to be stably maintained in many industrial strains of LAB. Currently, non-antibiotic markers such as nisin resistance($Nis^{r}$ ) are explored for selecting recombi-nant clone. In addition, a gene encoding S-layer protein, slp/A, on bacterial cell wall was successfully recombined with the proper LAB vectors LAB vectors for excretion of the heterologous gene product from LAB Many food-grade host vec-tor systems were successfully developed, which allowed stable integration of multiple plasmid copies in the vec-mosome of LAB. More recently, an integration vector system based on the site-specific integration apparatus of temperate lactococcal bacteriophage, containing the integrase gene(int) and phage attachment site(attP), was pub-lished. In conclusion, when various vector system, which are maintain stably and expressed strongly in LAB, are developed, lost of such food products as enzymes, pharmaceuticals, bioactive food ingredients for human consump-tion would be produced at a full scale in LAB.

• Food Science of Animal Resources
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• v.32 no.3
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• pp.364-368
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• 2012

Bovine lactoferrin is well known as biological activator in defense mechanism related some cells. In this study, we was investigated about the immune modulator as a role of lactoferrin through the transcriptional regulation of genes associated with hypersensitivity such as allergy, athma and inflammatory disease. Effects of inflammatory reaction of bovine lactoferrin was carried out by RT-PCR analysis from isolated total RNA treated with lactoferrin 0, 10, 50, 100, 500 ${\mu}g/mL$ and PMA 100 ng/mL. The expression of the TYROBP, PITPNA, IL-10, SLP1, DC-stamp and ICAM-1 mRNA were increased by synergy effect of bovine lactoferrin and PMA. The results of RT-PCR showed that bovine lactoferrin and PMA had an effect of immune modulator by enhancement of TYROBP, PITPNA, SLP1, DC-stamp, IL-10 and ICAM-1 gene transcription in U937, Mutz-3 and NK92 cells, respectively. Bovine lactoferrin showed a potential of biological function which could be used for industrial applications as a material of food and pharmaceutical.

• Journal of Microbiology and Biotechnology
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• v.23 no.4
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• pp.545-554
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• 2013

Plasmid isolation of kimchi-derived Weissella cibaria KLC140 revealed six different plasmids. The smallest plasmid, pKW2124, was DNA sequenced and characterized, showing 2,126 bp with a GC content of 36.39% and five putative open reading frames (ORFs). In silico analysis of these ORFs showed ORF1 encodes a putative replication protein similar to rolling circular replication proteins from other lactic acid bacteria. However, a single-stranded intermediate was not detected when S1 nuclease was treated, suggesting it may follow theta replication. Interestingly, the replication initiation site of this plasmid is 100% identical to other plasmids from lactic acid bacteria, suggesting it may function for replication initiation. To construct a surface layer expression vector, pTSLGFP, slpA encoding the surface layer protein from Lactobacillus acidophilus was PCR amplified and fused with the gfp gene, forming a SLGFP fused gene. The plasmid pKW2124 was cloned into the XbaI site of pUC19, forming an Weissella-E. coli shuttle vector pKUW22. NheI-linearized pTSLGFP was ligated into pKUWCAT containing pKUW22 and the chloramphenicol acetyltransferase gene from pEK104, resulting in an 8.6 kb pKWCSLGFP surface layer expression vector. After transformation of this vector into W. cibaria KLC140, a GFP fluorescence signal was detected on the surface of the transformant, substantiating production of SLGFP fused protein and its secretion. This is the first report for construction of a Weissella surface layer expression vector, which may be useful for surface layer production of beneficial proteins in Weissella.

• Journal of Acupuncture Research
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• v.20 no.5
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• pp.50-62
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• 2003

Objective: Bee Venom(BV) has been used for various kinds of inflammatory or painful conditions in Oriental Medicine clinics, and there publishes reports on its therapeutic effects and the probable mechanism of those therapeutic effects, where CDs and cytokines plays important role. This study investigated the influences of bee venom on the expressions of CDs and cytokines of HMC cell line Methods: In this study we analysed the expression profile of HMC cell line treated with BV of 10-2ug/ml in relation to that of HMC cell line treated with vehicle by way of CD/cytokine microarray hybridization with 342 genes on it. Results: There were no upregulated genes by more than 3 fold, while there showed some downregulated genes by less than 1/3 fold as follows: colony stimulating factor 2, CD122, IL-7, CD112, TNF-alpha, CD138, CD166, TGFbetaR2, CD42b, CD62L, CD111, interleukin 10 receptor alpha, colony stimulating factor 1(macrophage), CD38 antigen(p45), CD121a, CD33 antigen(gp67), colony stimulating factor 1 receptor, B cell linker protein (SLP65) mRNA, CD94, alanyl(membrane) aminopeptidase, immunoglobulin(CD79A) binding protein 1, CD205, CD241, CD207, CDw121b, integrin alpha L(CD11a), integrin beta 1(CD29), CD91, CD42b. Conclusions: Bee venom treatment induced downregulation of some CDs or cytokines including $TNF-{\alpha}$. IL-1R with its possible implication in an antiinflammatory action of BV. Further research on expression profile changes induced by BV treatment is expected.