• 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).

• 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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• 제9권2호
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• pp.201-205
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• 1999

The effects of growth temperature and a carbon source on the expression of $\beta$-galactosidase gene of Lactococcus lactis ssp. lactis ATCC 7962 (L. lactis 7962) were investigated. At $25^{\circ}C$, L. lactis 7962 had a higher $\beta$-galactosidase activity than cells grown at $30^{\circ}C$ or $37^{\circ}C$, although cells grew most quickly at $37^{\circ}C$ The highest $\beta$-galactosidase activity was observed in cells grown in M17 with lactose (l %) followed by cells grown in a galactose (1 %) medium. L. lactis 7962 exhibited the minimum $\beta$-galactosidase activity in glucose media, indicating catabolite repression. When the cellular levels of $\beta$-galactosidase mRNA were examined using slot blot hybridization, no significant differences were observed between cells grown at $25^{\circ}C$ and cells at $30^{\circ}C$ or $37^{\circ}C$ in the same media. This suggests that the quantity of $\beta$-galactosidase mRNA may not be the reason for the higher $\beta$-galactosidase activities of L. lactis 7962 at $25^{\circ}C$ The level of ccpA (Catabolite Control Protein) transcript remained almost constant during the exponential growth phase irrespective of a carbon sourse.

• 한국미생물·생명공학회지
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• 제27권3호
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• pp.260-265
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• 1999

The structural $\beta$-galactosidase gene (lacZ) from Lactococcus lactis ssp. lactis 7962 was cloned into plamid vector pKF18, which was designated as pKF-gal. Expression of the lacZ from L. lactis 7962 was found to be higher when cells were grown at 3$0^{\circ}C$ than 37$^{\circ}C$. Maximum $\beta$-galactosidase activity was obtained when E. coli/pKF-gal was cultivated for 6hr at 3$0^{\circ}C$ and for 3hr at 37$^{\circ}C$, and L. lactis 7962 was grown for 8hr at 3$0^{\circ}C$. Enzyme induction was achieved by the addition of lactose, galactose, or lactose+IPTG to growing culture. The addition of glucose had no effect on enzyme induction.

• 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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• 제9권4호
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• pp.393-397
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• 1999

The gene (galE) encoding UDP-galactose-4-epimerase, operative in the galactose metabolic pathway, was cloned together with the $\beta$-galactosidase gene (lacZ) from Lactococcus lactis ssp. lactis ATCC7962 (L. lactis 7962). galE was found to have a length of 981 bps and encoded a protein with a molecular mass of 36,209 Da. The deduced amino acid sequence showed a homology with GalE proteins from several other microorganisms. A Northern analysis demonstrated that galE was constitutively expressed by its own promoter. When galactose or lactose was added into medium, the galE transcription was induced by several upstream promoters. The structure of the gal/lac operon of L. lactis 7962 was partially characterized and the gene order around galE was galT-lacA-lacZ-galE-orfX.

• Journal of Microbiology and Biotechnology
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• 제13권1호
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• pp.134-138
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• 2003

The secondary and tertiary structures of ${\beta}$-galactosidase from L. lactis ssp. lactis 7962 were designed using Nnpredict and Sybyl version 6.3. By using site-directed mutagenesis, the mutated enzymes, Tyr-475-phe and Glu-506-Asp, were generated based on the structural modeling of L. lactis ssp. lactis 7962. The enzymes Tyr.-475-Phe and Glu-506-Asp had <$1\%$ of the activity of the native enzyme with ONPG as substrate. The $V_{max}$ values of the mutated enzymes were greatly reduced (1,800~40,000-1314) compared with the value for the native ${\beta}$-galactosidase. However, the $K_m$ values of Tyr-475-Phe and Glu-506-Asp with ONPG, PNPG, PNPF, and PNPA were not significantly different from those of the native enzyme. The results obtained support the suggestion that Tyr-475 and Glu-506 constitute very important parts of the catalytic machinery of the ${\beta}$-galactosidase.

• Preventive Nutrition and Food Science
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• 제5권3호
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• pp.153-159
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• 2000

A 4.4 kb DNA fragment encompassing lacA (galactoside acetyltransferase) and lacZ($\beta$-galactosidase) genes from Lactococus lactis ssp. lactis ATCC 7962 (L. lactis 7962) was introduced ito a Lac strain, Lactococcus lactis ssp. lactis MG1363 (L. lactis MG1363) by using a lactococcal expression vector, pMG36e and expression level of lacZ was examined. Growth rates and $\beta$-galactosidase ($\beta$-gal) activities of MG1363 cells carrying recombinant plasmid, pMLZ3, on M17 broth containing different carbon sources (1%, w/v) were examined. Contrary to the expectations, MG1363 [pMLZ3] grown on lactose showed the lowest enzyme activity (17 units) and cells grown on galactose had the highest $\beta$-gal activity (41 units). Cells grown on glucose had intermediate activity (33 units). These activities are about one tenth of the values observed in L. lactis 7962 where lacZ is present as a single-copy gene in the chromosome. When the cellular concentrations of lacZ transcript were examined using slot blot hybridization, it was found that MG1363[pMLZ3] produced sufficient amounts of transcript. These results indicate that either proteolytic degradation of $\beta$-gal or other regulatory mechanism prevent the translation or accumulation of $\beta$-gal in L. lactis MG1363 cells. In regard to regulation, the presence of the ccpA gene in L. lactis MG1363 was confirmed by Southern blot.

• 한국미생물·생명공학회지
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• 제33권4호
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• pp.241-247
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• 2005

선별마커로써 Lactococcus lactis ssp. lactis ATCC 7962 유래의 $\beta$-galactosidase 유전자를 포함하는 Lactococcus용 셔틀/발현 벡터 pWgall3T를 제조하여 Escherichia coli DH5$\alpha$와 고. Lactis MG1363내로 도입하였다. 이들 형질 전환체들은 X-gal을 포함하는 배지에서 파란색의 표현형을 보임으로써 쉽게 확인할 수 있었다. 또한, L. lactis MG1363 형질전환체로부터 $\beta$-galactosidase 활성을 측정한 결과 기존에 $\beta$-galactosidase를 활성을 지닌 L. lactis ATCC 7962에 비해 glucose를 포함하는 M17배지에서 4배정도 높은 활성을 보임으로써 선별마커로써의 효율성을 나타내었다. pWgal13T는 $\beta$-galactosidase 유전자 외에 L. lactis Wg2유래의 replicon과 외래 유전자의 발현을 위한 L. lactis ssp. cremoris LM0230의 promoter P13C, terminator를 포함하고 있다. 이 벡터의 이용가능성을 확인하기 위하여 외래 유전자 EGFP유전자를 P13C 아래에 삽입하여 E. coli와 L. iactis에서 발현을 확인하였다. 이 연구에서 제조된 Lactococcus용 발현 벡터 pWgal13T는 E. coli와 L. lactis에서 외래 유용 유전자를 생산을 위해 이용 할 수 있을 것이다.

• 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.