원문정보
초록
영어
Bacteria of the genus Leuconostoc represent a diversified group of heterofermentative organisms commonly found in the fermented foods. Among lactic acid bacteria (LAB), Leuconostoc plays important role for the fermentation of plant materials including kimchi and sauerkraut producing organic acids and carbon dioxide. For the molecular level study and genetic modulation of Leuconostoc, development of effective gene transformation systems is of great necessity.
For construction of plasmid shuttle vectors auto-replicating in Leuconostoc, plasmid-harboring Leuconostoc strains were screened from kimchi. pCB18 had one open reading frame (ORF) potentially encoding replicase protein and pCB42 revealed two ORFs with transposase and DNA-binding protein. Using pCB18 a Leuconostoc-E. coli shuttle vector (pLeuCM) was constructed with pUC19 and the staphylococcal chloramphenicol acetyltransferase (CAT) gene. Also, pLeuCM42 was constructed using pCB42. The autoreplicative Leuconostoc-E. coli shuttle showed a segregational stability in Leuconostoc over 100 generation of cell division under nonselective condition. Gene of β-galactosidase was obtained by PCR amplification from Lb. plantarum. It was subcloned into pLeuCM and pLeuCM42 to construct pLeuCMgal and pLeuCM42gal, and introduced into E. coli and Ln. citreum 95. Successful expression of β-gal in hosts were confirmed by enzyme activity assays. However, the transformant containing
pLeuCMgal lost the plasmids after 50 generations of growth under non-selective medium revealing low segregational stability. The insertion of foreign DNAs in pLeuCM was supposed to result in disturbing plasmid replication and accumulation of ssDNA intermediate. Meanwhile, pLeuCM42 and pLeuCM42gal showed a high segregational stability in Leuconostoc over 100 generation. The high stability of pLeuCM42 is an advantage when a food-grade vector is to be constructed from pLeuCM42 since no antibiotic marker genes are allowed for food-grade vectors. In this respect, pLeuCM42 might be useful for construction of a food-grade vector.
The systems developed and applied in this study would be useful tools for the genetic manipulation of Leuconostoc and they will facilitate the genomic study of the strains.