Characterization of the protein conferring immunity to the antimicrobial peptide carnobacteriocin B2 and expression of carnobacteriocins B2 and BM1

Abstract

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.

Effect of Amino Acid Substitutions on the Activity of Carnobacteriocin B2

“…The results were expressed in arbitrary units (AU) of bacteriocin (1 AU is the minimal amount of peptide required to produce a visible clearing on the lawn of the indicator strain; inhibition was recorded as positive if a distinct clearing was observed; the limit of detection was 0.05 AU g Ϫ1 ). C. divergens LV13 transformed with pLQ400i, a plasmid expressing the carnobacteriocin B2 immunity protein that protects the strain against CbnB2 (13), was also challenged with the purified peptides in the same way as C. divergens LV13. No inhibition of the indicator strain was detected when 0.1% trifluoroacetic acid alone was used in the assay.…”

“…This cluster has four other genes required for the wild type Bac ϩ Imm ϩ phenotype that, based on sequence homology, function as (i) an ATP binding cassette-type transporter and (ii) an "accessory protein" of the bacterial general secretion pathway-independent secretion system, (iii) a histidine protein kinase and (iv) a response regulator of the bacterial two component signal transduction system. 3 A fifth gene in the cluster, cbiB2, encodes an immunity protein (111 amino acids) that protects the producer organism against the antimicrobial effect of carnobacteriocin B2 (13).…”

Genetic characterisation and heterologous expression of leucocin C, a class IIa bacteriocin from Leuconostoc carnosum 4010

“…Peptides in subgroup 3 as well as enterocin SE-K4 and carnobacteriocin B2 in subgroup 4 exceptionally have neither the conserved tryptophan at the C-tails, nor the cysteine residues to form the C-terminal disulfide bridge (Table 2). Marrec et al 2000Metivier et al 1998Aymerich et al 1996Henderson et al 1992Marugg et al 1992Tichaczek et al 1994Kalmokoff et al 2001Birri et al 2010Feng 2009Kawamoto et al 2002Bennik et al 1998Yamazaki et al 2005Bhugaloo-Vial et al 1996Jack et al 1996Vaughan et al 2001Fimland et al 2002bHeng et al 2007 Subgroup 2 Kwaadsteniet et al 2006Quadri et al 1995Eguchi et al 2001Diep et al 2006 The YGNGV/L "pediocin box" is marked (bold), the leucine residue (L), odd, and uncertain amino acids (X) are marked as bold and red. Names of the six newly identified class IIa bacteriocins are in red (Rea et al 2011).…”

“…For example, sakacin P immunity protein (sakP-im) from group B is able to protect cells against pediocin, leucocin C and its own bacteriocin sakacin P. All three belong to the same subclass (subgroup 1) of class IIa bacteriocins. However, sakP-im-producing bacteria were susceptive to enterocin A, which is also in the subgroup 1, even though enterocin A immunity protein protected against (Quadri et al 1995). Besides, the function of immunity protein has been found to be strain-dependent (Johnsen et al 2005a).…”

A two-component signal-transduction cascade in Carnobacterium piscicola LV17B: two signaling peptides and one sensor-transmitter

“…The genetic determinants of precarnobacteriocin B2 (cbnB2) and its immunity protein (cbiB2) are located on pCP40, whereas those of precarnobacteriocin BM1 (cbnBM1) and its proposed immunity protein (cbiBM1) are located on the chromosome [19]. The analyzed region of pCP40 contains four genes (cbnKRTD) required for bacteriocin production [17,19] and three additional small ORFs (cbnS, X and Y) (Fig. 1A).…”