Characterization of the protein conferring immunity to the antimicrobial peptide carnobacteriocin B2 and expression of carnobacteriocins B2 and BM1.
Cloning of a 16-kb DNA fragment from the 61-kb plasmid of Carnobacterium piscicola LV17B into plasmidless C. piscicola LV17C restores the production of the plasmid-encoded carnobacteriocin B2 and the chromosomally-encoded carnobacteriocin BM1 and restores the immune phenotype. This fragment also has sufficient genetic information to allow the expression of carnobacteriocin B2 and its immunity in a heterologous host. The gene locus (cbiB2) responsible for immunity to carnobacteriocin B2 is located downstream of the structural gene for carnobacteriocin B2 and encodes a protein of 111 amino acids (CbiB2). CbiB2 was expressed in Escherichia coli as a fusion of the maltose-binding protein and CbiB2. The fusion protein was purified on an amylose column and cleaved with factor Xa, and pure CbiB2 was isolated by high-performance liquid chromatography. The N-terminal amino acid sequence and mass spectrometry (molecular weight [mean +/- standard error], 12,662.2 +/- 3.4) of the purified protein agree with the information deduced from the nucleotide sequence of cbiB2. Western blot (immunoblot) analysis indicates that the majority of the intracellular pool of this immunity protein is in the cytoplasm and that a smaller proportion is associated with the membrane. CbiB2 confers immunity to carnobacteriocin B2, but not to carnobacteriocin BM1, when it is expressed in homologous or heterologous hosts. No protective effect is observed for sensitive cells growing in the presence of the bacteriocin when the immunity protein is added to the medium. The purified immunity protein does not show significant binding to microtiter plates coated with carnobacteriocin B2 and is not able to inactivate the bacteriocin in solution.
Quadri
L E
LE
Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Canada.
Sailer
M
M
Terebiznik
M R
MR
Roy
K L
KL
Vederas
J C
JC
Stiles
M E
ME
D000595:Amino Acid Sequence; D001419:Bacteria / Q000187:drug effects / Q000235:genetics*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D001430:Bacteriocins / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D001483:Base Sequence; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D013058:Mass Spectrometry; D008566:Membranes / Q000737:chemistry; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010641:Phenotype; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017421:Sequence Analysis; D013347:Subcellular Fractions / Q000737:chemistry
D001426:Bacterial Proteins; D001430:Bacteriocins; C092186:CbiB2 protein, Carnobacterium piscicola; D011993:Recombinant Fusion Proteins; C086963:bacteriocin B2 protein, Carnobacterium piscicola; C086962:bacteriocin BM1 protein, Carnobacterium piscicola