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KMID : 0545120000100060829
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Journal of Microbiology and Biotechnology
2000 Volume.10 No. 6 p.829 ~ p.835
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Cloning and Characterization of the Lactococcus lactis subsp.lactis ATCC 7962 ptsHI Operon
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Kim, Tea Youn
Park, Rae Jun/Chang, Hae Choon/Chung, Dae Kyun/Lee, Jong Hoon/Lee, Hyong Joo/Kim, Jeong Hwan
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Abstract
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The ptsH and ptsl genes of Lactococcus 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 1.3kb PCR product was obtained using a primer set that was hybridized to the internal region of the L. lactis 7962 ptsHI genes and then subcloned into a low-copy number vector, pACYC 184. The 5´ upstream and 3´ downstream regions from the 1.3kb fragment were subsequently cloned 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 ptsHl genes of LL 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 0.3kb transcript corresponding to ptsH and the second, a 2kb transcript corresponding to ptsH and ptsl. The transcription level of ptsH was higher than that of ptsl. 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 ptsl 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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