• Journal of Pharmaceutical Investigation
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• 제35권2호
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• pp.75-80
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• 2005

A promising application of Lactococcus lactis is its use as live vehicles for production and delivery of heterologous proteins of vaccines and therapeutic substances. Because L. lactis has GRAS ('generally regarded as safe') status, we tested whether L. lactis could function as the carrier of the Ll protein of human papillomavirus (HPV) type 16. The RNA level expression of Ll gene was detected in L. Lactis. The Ll protein was expressed in L. lactis with Ll gene. The growth of strains L. lactis with an empty plasmid (pAMJ328) and L. lactis with Ll-encoding plasmid (pAMJ328-Ll) was slightly decreased in comparison with the growth of strains L. lactis (wild type). However, all the three strains of L. lactis maintained the ability to ferment sugars primarily into lactic acid, indicating that Ll protein did not affect the biochemical property of L. lactis. These results suggest that L. lactis, capable of carrying Ll protein, might be further developed as a biocompatible oral protein delivery system.

• Preventive Nutrition and Food Science
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• 제9권4호
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• pp.324-329
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• 2004

In order to investigate the molecular mechanism of $\gamma$-aminobutyric acid (GABA) production in lactic acid bacteria, we cloned a glutamate decarboxylase (GAD) gene from Lactobacillus plantarum using polymerase chain reaction (PCR). One PCR product DNA was obtained and inserted into a TA cloning vector with a T7 promoter. The recombinant plasmid was used to transform E. coli. The insertion of the product was con­firmed by EcoRI digestion of the plasmid purified from the transformed E. coli. Nucleotide sequence analysis showed that the insert is a full-length Lactobacillus plantarum GAD and that the sequence is $100\%$ and $72\%$ identical to the regions of Lactobacillus plantarum GAD and Lactococcus lactis GAD sequences deposited in GenBank, accession nos: NP786643 and NP267446, respectively. The amino acid sequence deduced from the cloned Lactobacillus plantarum GAD gene showed $100\%$ and $68\%$ identities to the GAD sequences deduced from the genes of the NP786643 and NP267446, respectively. To express the GAD protein in E. coli, an expression vector with the GAD gene (pkk/GAD) was constructed and used to transform the UT481 E. coli strain and the expression was confirmed by analyzing the enzyme activity. The Lactobacillus plantarum GAD gene obtained may facilitate the study of the molecular mechanisms regulating GABA metabolism in lactic acid bacteria.

• Journal of Microbiology and Biotechnology
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• 제23권4호
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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 Microbiology
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• 제43권3호
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• pp.251-256
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• 2005

In this study, we assessed the susceptibility of 12 Lactobacillus strains, all of which had been isolated from the gastrointestinal tracts of chicken, to three antibiotics (chloramphenicol, erythromycin and tetracycline) used commonly as selective markers in transformation studies of lactic acid bacteria. Among these strains, $17\%,\;58\%,\;and\;25\%$ were found to exhibit a high degree of resistance to $200\;{\mu}g/ml$ of tetracycline, erythromycin, and chloramphenicol, respectively. Seven of the 12 Lactobacillus strains exhibiting resistance to at least $50\;{\mu}g/ml$ of chloramphenicol or erythromycin, and five strains exhibiting resistance to at least $50\;{\mu}g/ml$ of tetracycline, were subsequently subjected to plasmid curing with chemical curing agents, such as novobiocin, acriflavin, SDS, and ethidium bromide. In no cases did the antibiotic resistance of these strains prove to be curable, with the exception of the erythromycin resistance exhibited by five Lactobacillus strains (L. acidophilus I16 and I26, L. fermentum I24 and C17, and L. brevis C10). Analysis of the plasmid profiles of these five cured derivatives revealed that all of the derivatives, except for L. acidophilus I16, possessed profiles similar to those of wild-type strains. The curing of L. acidophilus I16 was accompanied by the loss of 4.4 kb, 6.1 kb, and 11.5 kb plasmids.

• Preventive Nutrition and Food Science
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• 제2권2호
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• pp.101-108
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• 1997

A lactic acid bacterium LAB3113, isolated from traditionally fermented Kimchi was found to produce bacteriocin whose activity was very specific toward lactobacilli and not effective against any other bacteria. Lactobacilli affected by the inhibitory substance included Lactobacillus delbrueckii-lactis, L. johnsonii, L. gsseri, and L. curvatus. Based upon biochemical and physiological characteristics, LAB3113 was classified as Lactococcus lactis, and its bacteriocin was named as lactococcin K3113. Lactococcus lactis. LAB3113 produced bacteriocin at th early stage of growth and the concentration of the bacteriocin did not decrease even after alt stationalry phase. Optimal temperature of bacteriocin production was $25^{\circ}C$ at the initial pH 7.0. Partially purified lactococcin K3113 was completely inactivated by protease, but not affected by lipase, lysozyme and RNase. The bacteriocin was very heat-stable even after autoclaving for 20 min. It was also stable in pH changes, an was not affected by th presence of solvents. lacotococcin K3113 appeared to act in bactericidal mode against L. delbrueckii-lactis ATCC4797. Molecular weight of lactococcin K3113 was calibrated as 10,500 dal by SDS-PAGE an activity staining. Lactococcus lactis LAB3113 had four residential plasmids of 3.7kb, 11.2kb, 15.5kb, and 48kh in molecular sizes. Plasmid profile analysis of mutant strain revealed that 15.5 kb plasmid was re-sponsible for the production of lactococcin K3113 and its immunity to the bacteriocin.

• 미생물학회지
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• 제36권2호
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• pp.109-111
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• 2000

Lactobacillus casei ssp. casei NCIB 4114 균주로부터 3,5kb의 플라스미드를 분리하여, 이 플라스미드를 함유하는 Escherichia coli-Lactobacillus 셔틀 백터(shuttle vector)들을 만들었다. tu틀 벡터들은 모두 electroporation에 의해 성공적으로 형질전환 되었다. Electroporation의 최적조건은 벡터DNA 1$\mu$g당 $2{\times}10^5$ 형질전환체의 효율이었고, 이들 벡터의 성공적 도입은 이들 벡터의 유산균에서의 음식등급 벡터로의 사용 가능성을 제시하였다.

• 한국식품영양과학회지
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• 제26권6호
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• pp.1228-1236
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• 1997

Lactobacillus strains were isolated from volunteer's feces, including from newly-born infants and adults in their 20's, by using differential MRS-BPB plates. Total 56 presumptive Lactobacillus strains were isolated and the bacteriocin productions by the isolates were examined by agar diffusion method. Six bacteriocin-producing strains were confirmed. Among them, two isolates, HU-1 and H22-3, showed the most outstanding antimicrobial activities, which were not affected by pH adjustments or catalase treatments of culture. HU-1 was originated from a two-years old boy and H22-3 was originated from a newly-born infant. HU-1 and H22-3 had the same morphology(short rod) when examined by scanning electron microscope, and the same biochemical traits including growth temperature range, salt tolerance and sugar-fermenting abilities. But the growth-inhibition spectrum and plasmid profiles of HU-1 and H22-3 were different. Both strains inhibited the growth of various Gram (+) microorganisms including Listeria monocytogenes. Micrococcus luteus, and Staphylococcus aureus in addition to many species of lactic acid bacteria, indicating the production of broad-spectrum bacteriocins. Bacteriocins produced by HU-1 and H22-3 were stable up to 90℃, 15 min heat treatments. Their activities were not affected by pepsin or trypsin treatments but destroyed by proteinaseK or pronase treatments.

• Journal of Microbiology and Biotechnology
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• 제17권7호
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• pp.1213-1216
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• 2007

The vector pCW5 with plasmid pC7, originally isolated in Lactobacillus paraplantarum C7 derived from kimchi, was constructed using a p32 strong promoter, the pC7 replicon, and green fluorescent protein (GFP) as the reporter. The constructed vector was transformed into E. coli and Leuconostoc mesenteroides, and GFP expression detected using a Western blot analysis. GFP fluorescence was recognized in E. coli and Leuconostoc mesenteroides using a confocal microscope. In addition, GFP fluorescence was also clearly detected in several industrially important lactic acid bacteria (LAB), including Lactobacillus bulgaricus, Lactobacillus paraplantarum, and Lactobacillus plantarum. Thus, pCW5 was shown to be effective for Leuconostoc mesenteroides when using GFP as the reporter, and it can also be used as a broad-host-range vector for other lactic acid bacteria.

• 한국미생물·생명공학회지
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• 제18권3호
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• pp.215-220
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• 1990

L.casei YIT 9018균주에 N-methyl-N'-nitro-N-nitrosoguanidine을 처리하여 thermoinducible mutants를 분리하였고, 이 균주를 $42^{\circ}C$에서 30분 동안 열처리하여 prophage가 cured된 균주 L.casei HYM 1213과 L.casei HYM 4024를 분리하였다. Prophage cured strain L.case HYM 1213과 L.casei HYM 4024는 temperate phage $\phi$Fsw에 대해 지시균으로서 능력을 가지고 있었으며, 어떤 온도에서도 temperate phage $\phi$FSW가 검출되지 않았다. 이들의 생리적 특성을 모균주인 L.caseiYIT 9018과 비교한 결과 L.casei HYM 1213 균주는 균의 증식, pH변화, 산생성능력, 당 발효능력에서 모균주와 유사한 것으로 나타났으나, L.casei HYM 4024균주는 pH변화와 산생성능력이 모균주에 비해 미약한 것으로 나타났다. Plasmid DNA는 두 균주 모두 모균주와 동일한 size를 보여주여, prophage가 cured되었어도 plasmid DNA에는 손실을 가져오지 않았음을 알 수 있었다.

• 한국미생물·생명공학회지
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• 제50권1호
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• pp.63-70
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• 2022

Two small cryptic plasmids, pHG1 and pHG2, were isolated from Levilactobacillus zymae (formerly Lactobacillus zymae) GU240 and characterized. pHG1 is 1,814 bp in size with a GC content of 37.4% and contains two open reading frames. orf1 can potentially encode a protein of 101 amino acids (aa) with 99% identity with the copy number control protein of Lacticaseibacillus paracasei. orf2 can potentially encode a protein of 230 aa with 99% identity with a replication protein from multiple species. Six inverted repeats (IR I-VI) and six direct repeats (DR I-VI) were found in pHG1. pHG2 is 2,864 bp in size, with a GC content of 39.6%. pHG2 has two orfs. orf1 might encode a protein with 99% identity with the TrsL transmembrane protein. orf2 might encode a protein with 99% identity with plasmid recombination proteins from lactic acid bacteria. Both pHG1 and pHG2 may be useful as frames for constructing lactic acid bacteria-Escherichia coli shuttle vectors.