Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Sequence Analysis of a Cryptic Plasmid pKW2124 from Weissella cibaria KLC140 and Construction of a Surface Display Vector

  • Kim, Soo Young (Department of Biological Sciences, Konkuk University) ;
  • Oh, Chang Geun (Department of Biological Sciences, Konkuk University) ;
  • Lee, Young Joo (Department of Biological Sciences, Konkuk University) ;
  • Choi, Kyu Ha (Department of Biological Sciences, Konkuk University) ;
  • Shin, Doo Sik (Department of Biological Sciences, Konkuk University) ;
  • Lee, Si Kyung (Department of Applied Biology and Chemistry, Konkuk University) ;
  • Park, Kab Joo (Institute for Basic Sciences, Konkuk University) ;
  • Shin, Hakdong (Department of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University) ;
  • Park, Myeong Soo (Department of Hotel Culinary Arts, Yeonsung University) ;
  • Lee, Ju-Hoon (Department of Food Science and Biotechnology, Kyung Hee University)
  • Received : 2013.01.07
  • Accepted : 2013.01.29
  • Published : 2013.04.28
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Abstract

Plasmid isolation of kimchi-derived Weissella cibaria KLC140 revealed six different plasmids. The smallest plasmid, pKW2124, was DNA sequenced and characterized, showing 2,126 bp with a GC content of 36.39% and five putative open reading frames (ORFs). In silico analysis of these ORFs showed ORF1 encodes a putative replication protein similar to rolling circular replication proteins from other lactic acid bacteria. However, a single-stranded intermediate was not detected when S1 nuclease was treated, suggesting it may follow theta replication. Interestingly, the replication initiation site of this plasmid is 100% identical to other plasmids from lactic acid bacteria, suggesting it may function for replication initiation. To construct a surface layer expression vector, pTSLGFP, slpA encoding the surface layer protein from Lactobacillus acidophilus was PCR amplified and fused with the gfp gene, forming a SLGFP fused gene. The plasmid pKW2124 was cloned into the XbaI site of pUC19, forming an Weissella-E. coli shuttle vector pKUW22. NheI-linearized pTSLGFP was ligated into pKUWCAT containing pKUW22 and the chloramphenicol acetyltransferase gene from pEK104, resulting in an 8.6 kb pKWCSLGFP surface layer expression vector. After transformation of this vector into W. cibaria KLC140, a GFP fluorescence signal was detected on the surface of the transformant, substantiating production of SLGFP fused protein and its secretion. This is the first report for construction of a Weissella surface layer expression vector, which may be useful for surface layer production of beneficial proteins in Weissella.

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References

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