Bacteriocin curvacin-A

Curvacin A is a bacteriocin produced by Lactobacillus curvatus LTH1174 which is a potential starter organism for the production of fermented dry sausages. This peptide inhibits the growth of the opportunistic food pathogens Listeria monocytogenes and Enterococcus faecalis and thus, curvacin A may enable better performance of a starter and improvement of the hygienic status of meat products. Oligonucleotides were constructed deduced from the peptide sequence and used for the identification of the curvacin A structural gene curA on a 60 kb plasmid of L. curvatus LTH1174. Plasmid-cured derivatives of this strain were unable to produce curvacin A but were still resistant to the bacteriocin. CurA was cloned into Escherichia coli NM554 and its nucleotide sequence was determined. Sequencing revealed the presence of an additional open reading frame of 51 amino acids with unknown function. A promoter was detected upstream of curA by primer extension. Both reading frames form a single transcript. Curvacin A is synthesised as a prepeptide of 59 amino acids which is proteolytically processed to the mature bacteriocin of 41 amino acids.

To analyze the orientation in target cell membranes of the pediocin-like bacteriocin (antimicrobial peptide) curvacin A, 55 variants were generated by site-directed mutagenesis and their potencies against four different target cells determined. The result suggest that the somewhat hydrophilic short central helix (residues 19 to 24), along with the N-terminal beta-sheet-like structure (residues 1 to 16), inserts in the interface region of the target cell membrane, with Ala22 close to the hydrophobic core of the membrane. The following hinge region, with Gly28 as an important residue, may then form a turn wherein Gly28 becomes positioned near the border between the interface and the hydrophobic regions, thus permitting the longer and more-hydrophobic C-terminal helix (residues 29 to 41) to insert into the hydrophobic core of the membrane. This helix contains three glycine residues (G33, G37, and G40) that form a putative helix-helix-interacting GxxxGxxG motif. The replacement of any of these glycines with a larger residue was very detrimental, suggesting their possible involvement in helix-helix interactions with a membrane-embedded receptor protein.

Lactobacillus curvatus LTH 1174, a strain originating in fermented sausage, produces the antilisterial bacteriocin curvacin A. Its biokinetics of cell growth and bacteriocin production as a function of various concentrations of salt (sodium chloride) were investigated in vitro during laboratory fermentations using modified MRS medium. A model was set up to describe the effects of different NaCl concentrations on microbial behavior. Both cell growth and bacteriocin activity were affected by changes in the salt concentration. Sodium chloride clearly slowed down the growth of L. curvatus LTH 1174, but more importantly, it had a detrimental effect on specific curvacin A production (k(B)) and hence on overall bacteriocin activity. Even a low salt concentration (2%, wt/vol) decreased bacteriocin production, while growth was unaffected at this concentration. The inhibitory effect of NaCl was mainly due to its role as an a(w)-lowering agent. Further, it was clear that salt interfered with bacteriocin induction. Additionally, when 6% (wt/vol) sodium chloride was added, the minimum biomass concentration necessary to start the production of curvacin A (X(B)) was 0.90 g (cell dry mass) per liter. Addition of the cell-free culture supernatant or a protein solution as a source of induction factor resulted in a decrease in X(B), an increase in k(B), and hence an increase in the maximum attainable bacteriocin activity.