• 한국미생물·생명공학회지
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• 제26권6호
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• pp.562-565
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• 1998

Nisin-producing and nisin resistant L. lactis subsp. lactis ATCC7962 harbored six plasmids. To find a plasmid containing a nisin resistant gene, these plasmids were transformed into L lactis LM0230 of plasmid-free and nisin sensitive strain. After screening on nisin selection media containing nisin (150 $\mu\textrm{g}$/$m\ell$), several nisin resistant transformants were obtained and the level of nisin resistance was very similar to that of wild type L lactis subsp. lactis ATCC7962. A 26.5 kb plasmid, named as pCS100, which confers resistance to nisin, was identified in transformants. The pCS100 was digested with EcoRI and Southern blot hybridization was done with nisI probe to localize the nisin resistant gene. A 4 kb EcoRI fragment showed a strong positive signal, and it was cloned into pBluescript for the potential selection marker.

• Journal of Microbiology and Biotechnology
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• 제6권6호
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• pp.386-390
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• 1996

The ${\beta}$-galactosidase gene from Lactococcus lactis subsp. lactis ATCC 7962 was cloned and its enzymatic properties were characterized, with a view to assessing its potential use as a selection marker in the food-grade cloning vector. Chromosomal DNA from L. lactis subsp. lactis 7962 was cleaved with PstI and ligated into pBR322 for transformation into Escherichia coli TGl. Transformants showing ${\beta}$-galactosidase activity possessed the pBR322 plasmid containing a 10 kilobase (kb) PstI fragment and this plasmid was named pCKL11. The cloned ${\beta}$-galactosidase gene came from the chromosomal DNA of L. lactis subsp. lactis 7962 was confirmed by Southern hybridization. A restriction map of pCKL11 was constructed from the cleavage of both pCKL11 and the purified 10kb insert fraqment. The. optimum pH of the ${\beta}$-galactosidase determined with the E. coli harboring the pCKL11 was 7.0. The optimum temperature was $50^{\circ}C$, while the pI of the enzyme was 7.4. These values were the same as those of the enzyme from the parent strain.

• Journal of Microbiology and Biotechnology
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• 제3권4호
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• pp.217-223
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• 1993

To investigate the nature and location of the nisin-resistance determinant of Lactococcus lactis subsp. lactis 7962 (L. lactis 7962), a total plasmid DNA prepared from L. lactis 7962, a nisin producer, was used to transform L. lactis subsp. lactis LM0230, a plasmid-free and nisin-sensitive strain, by protoplast mediated transformation procedures. All of the nisin-resistant transformants acquired the ability to utilize sucrose at the same time, confirming the close linkage between these two determinants in L. lactis 7962. The plasmid DNA profiles of a few selected nisin-resistant transformants were examined by agarose gel electrophoresis. No common plasmid was found among the transformants and some small plasmids previously not present in L. lactis 7962 were detected. These transformants were named as L. lactis KL1, KL2, KL3, KL4, or KL5, respectively based on their plasmid profiles. Growth curves of all transformants were similar to that of L. lactis LM0230, but different from that of L. lactis 7962. L. lactis KL5 showed the highest level of resistance to nisin, growing up to 1, 200 IU nisin/ml after 40 hr incubation. Some nisin-sensitive derivatives of KL1 or KL2 were obtained by plasmid curing experiments. The plasmid DNA profiles of the nisin-sensitive KL1 derivatives were apparently the same as that of the KL1. All of the nisin-sensitive KL2 derivatives were plasmid-free, but a nisin-resistant strain with no apparent plasmid was also obtained. These results indicate that the nisin-resistance of the $Nis^r$ transformants is presumably mediated by the chromosomally located gene(s) rather than plasmid-encoded gene(s).

• 한국식품영양과학회지
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• 제31권1호
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• pp.75-80
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• 2002

된장 제조과정 중 불쾌취와 점질물을 생성하는 부패균인 B. subtilis의 생육억제를 위해 nisin을 생성하는 L. lactis subsp. lactis ATCC 7962, ATCC 11454, IFO 12007을 이용하여 유산발효를 수행하여 pH변화, B. subtilis의 생육저해도등을 검토하였다. 증자콩에서 nisin 생성유산균들의 생육특성을 살펴본 결과, 세 가지의 균주 모두가 증자대두 1g 당 $10^{6}$ CFU 접종하여 24시간 이내에 $10^{9}$ CFU로 급격하게 자라므로 영양요구성이 복잡한 유산균이 증자대두에서 다른 영양소의 첨가없이도 잘 생육함을 확인하였다. 증자콩에서 7962와 11454는 생육에 따라 pH가 급격히 저하되어 된장발효용 종균으로 사용할 수 없었다. 증자한 콩 중에 잘 증식하고 유산발효 후 pH 변화가 완만한 12007을 된장발효의 종균으로 전혀 문제점 없어 사용시 부패균인 B. subtilis의 생육이 효과적으로 저해됨이 확인되었다. 생성된 nisin은 황국균이 생성하는 protease에 의해 분해되며 콩 속에 다량으로 존재하던 유산균은 사입시 첨가되는 식염으로 불화성화되거나 사멸하여 된장의 산패를 막아준다. 그리고 8% 식염을 첨가하여 된장 제조시, 즉 저염 된장 제조시 B. subtilis의 생육이 효과적으로 억제되고 다수로 존재하던 유산균은 담금 후 점차 줄어들어 시간경과 후에는 관찰되지 않았다. 8%함유 식염된장의 숙성 중 pH 변화 역시 12% 함유 식염된장과 유사한 값을 나타내었다.

• Journal of Microbiology and Biotechnology
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• 제10권6호
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• pp.829-835
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• 2000

The ptsH and ptsI genes of Lactococus lactis subsp. lactis ATCC 7962 (L. lactis 7962), encoding the general proteins of phosphotransferase system (PTS) components, HPr and enzyme I, respectively, were cloned and characterized. A 1.3 kb PCR product was obtained using a primer set that was hybridized to the internal region of the L. lactis 7962 pts HI genes and then subcloned into a low-copy number vector, pACYC184. The 5' upstream and 3' downstream region from the 1.3 kb fragment were subsequently clone using the chromosome walking method. The complete ptsHI operon was constructed and the nucleotide sequences determined. Two ORFs corresponding to HPr (88 amino acids) and enzyme I (575 amino acids) were located. The ptsHI genes of L. lactis 7962 showed a very high homology (84-90%) with those genes from other Gram-positive bacteria. A primer extension analysis showed that the transcription started at either one of two adjacent bases upstream of the start codon. Using a Northern analysis, two transcripts were detected; the first, a 0.3 kb transcript corresponding to ptsH and the second, a 2 kb transcript corresponding to ptsH and ptsI. The transcription level of ptsH was higher than that of ptsI. The concentration of the ptsH transcript in cells grown on glucose was similar to that in cells grown on lactose, yet higher than that in cells grown on galactose. The ptsI transcript was scarcely detected in cell grown on lactose or galactose. The ptsI transcript was scarcely detected in cells grown on lactose or galactose. The results of a sequence analysis and Northern blot confirmed that the ptsH and ptsI genes of L. lactis 7962 were arranged in an operon like other known ptsHI genes and the expression of the ptsHI genes was regulated at the transcriptional level in response to the carbon source.

• Journal of Microbiology and Biotechnology
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• 제12권1호
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• pp.157-161
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• 2002

A lactic acid bacteria with ${\beta}$-gal activity was isolated from Kimchi, a traditional fermented vegetable food in Korea. The isolate was identified as a Lactococcus lactis strain and named L. lactis A2. The gene encoding ${\beta}$-gal of L. lactis A2 was cloned as a 5.8 kb PstI fragment. DNA sequencing identified the complete lacA (galactoside acetyltransferase)-lacZ (${\beta}$-galactosidase) genes together with the 3' part of upstream galT (galactose-1-phosphate uridyltransferase), and the 5'region of downstream galE (UDP-galactose-4-epimerase) genes. L. lactis A2 had the same gal/lac operon structure as in L. lactis subsp. lactis 7962. Other genes of the Leloir pathway are most likely to be located in the 5'upstream of the 5.8 kb fragment on the A2 chromosome. Sequences downstream of galE were different from those of L. lactis subsp. lactis 7962.