Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Cloning and Characterization of the Lactococcus lactis subsp. lactis ATCC 7962 pts HI Operon

  • Kim, Tea-Youn (Department of Food Science and Technology, Gyeongsang National University) ;
  • Park, Rae-Jun (Department of Food Science and Technology, Gyeongsang National University) ;
  • Chang, Hae-Choon (Department of Food and Nutrition, Chosun University) ;
  • Chung, Dae-Kyun (Institute and Department of Genetic Engineering, Kyung Hee University) ;
  • Lee, Jong-Hoon (Department of Foods and Biotechnology, Kyunggi University) ;
  • Lee, Hyong-Joo (Department of Food Science and Technology, Seoul National University) ;
  • Kim, Jeong-Hwan (Department of Food Science and Technology, Gyeongsang National University)
  • Published : 2000.12.01
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Abstract

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.

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References

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