gly gene cloning and expression and purification of glycinecin A, a bacteriocin produced by Xanthomonas campestris pv. glycines 8ra.
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.
Heu
S
S
Department of Agricultural Biology, College of Agriculture and Life Sciences, Seoul National University, Suwon 441-744, Korea.
Oh
J
J
Kang
Y
Y
Ryu
S
S
Cho
S K
SK
Cho
Y
Y
Cho
M
M
D001430:Bacteriocins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D019281:Dimerization; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D017422:Sequence Analysis, DNA; D013696:Temperature; D016959:Xanthomonas campestris / Q000235:genetics / Q000378:metabolism*
D001430:Bacteriocins; C439290:glycinecin A