Open Accessgly Gene Cloning and Expression and Purification of Glycinecin A, a Bacteriocin Produced by Xanthomonas campestris pv. glycines 8ra
Author(s) -
Sunggi Heu,
JongNam Oh,
Youngsung Kang,
Sangryeol Ryu,
Somi K. Cho,
Youngsup Cho,
Moonjae Cho
Publication year - 2001
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.67.9.4105-4110.2001
Subject(s) - xanthomonas campestris , bacteriocin , biology , escherichia coli , xanthomonas , recombinant dna , ammonium sulfate precipitation , gene , cloning (programming) , biochemistry , molecular cloning , microbiology and biotechnology , genetics , peptide sequence , bacteria , size exclusion chromatography , enzyme , computer science , programming language
Glycinecin A, a bacteriocin produced by Xanthomonas campestris pv. glycines, inhibits the growth of X. campestris pv. vesicatoria. We have cloned and expressed the genes encoding glycinecin A in Escherichia coli. Recombinant glycinecin A was purified from cell extracts by ammonium sulfate precipitation followed by chromatography on Q-Sepharose, Mono Q (ion exchange), and size exclusion columns. Purified glycinecin A is composed of two polypeptides, is active over a wide pH range (6 to 9), and is stable at temperatures up to 60 degrees C. Glycinecin A is a heterodimer consisting of 39- and 14-kDa subunits, as revealed through size exclusion chromatography and cross-linking analysis. Two genes, glyA and glyB, encoding the 39- and 14-kDa subunits, respectively, were identified based on the N-terminal sequences of the subunits. From the nucleotide sequences of glyA and glyB, we conclude that both genes are translated as bacteriocin precursors that include N-terminal leader sequences. When expressed in E. coli, recombinant glycinecin A was found primarily in cell extracts. In contrast, most glycinecin A from Xanthomonas was found in the culture media. E. coli transformed with either glyA or glyB separately did not show the bacteriocin activity.