Heterologous expression of enterocin A, a bacteriocin from Enterococcus faecium, fused to a cellulose-binding domain in Escherichia coli results in a functional protein with inhibitory activity against Listeria.
The genes for the bacteriocins enterocin A and B were isolated from Enterococcus faecium ATB 197a. Using the pET37b(+) vector, the enterocin genes were fused to an Escherichia coli specific export signal sequence, a cellulose-binding domain (CBD(cenA)) and a S-tag under the control of a T7lac promotor. The constructs were subsequently cloned into E. coli host cells. The expression of the recombinant enterocins had different effects on both the host cells and other Gram-positive bacteria. The expression of entA in Esc. coli led to the synthesis and secretion of functional active enterocin A fusion proteins, which were active against some Gram-positive indicator bacteria, but did not influence the viability of the host cells. In contrast, the expression of enterocin B fusion proteins led to a reduced viability of the host cells, indicating a misfolding of the protein or interference with the cellular metabolism of Esc. coli. Indicator strains of Gram-positive bacteria were not inhibited by purified enterocin B fusion proteins. However, recombinant enterocin B displayed inhibitory activity after the proteolytic cleavage of the fused peptides.
Klocke
Michael
M
Department of Bioengineering, Institute of Agricultural Engineering Bornim, Potsdam-Bornim, Germany. mklocke@atb-potsdam.de
Mundt
Kerstin
K
Idler
Frank
F
Jung
Sabrina
S
Backhausen
Jan E
JE
D001430:Bacteriocins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D001709:Biotechnology / Q000379:methods; D002482:Cellulose / Q000378:metabolism; D016984:Enterococcus faecium / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008087:Listeria / Q000187:drug effects*; D008826:Microbial Sensitivity Tests; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*
D001430:Bacteriocins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; C107799:enterocin A; C107801:enterocin B; D002482:Cellulose