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Defining the structure and receptor binding domain of the leaderless bacteriocin LsbB.

ABSTRACT: LsbB is a class II leaderless lactococcal bacteriocin of 30 amino acids. In the present work, the structure and function relationship of LsbB was assessed. Structure determination by NMR spectroscopy showed that LsbB has an N-terminal ?-helix, whereas the C-terminal of the molecule remains unstructured. To define the receptor binding domain of LsbB, a competition assay was performed in which a systematic collection of truncated peptides of various lengths covering different parts of LsbB was used to inhibit the antimicrobial activity of LsbB. The results indicate that the outmost eight-amino acid sequence at the C-terminal end is likely to contain the receptor binding domain because only truncated fragments from this region could antagonize the antimicrobial activity of LsbB. Furthermore, alanine substitution revealed that the tryptophan in position 25 (Trp(25)) is crucial for the blocking activity of the truncated peptides, as well as for the antimicrobial activity of the full-length bacteriocin. LsbB shares significant sequence homology with five other leaderless bacteriocins, especially at their C-terminal halves where all contain a conserved KXXXGXXPWE motif, suggesting that they might recognize the same receptor as LsbB. This notion was supported by the fact that truncated peptides with sequences derived from the C-terminal regions of two LsbB-related bacteriocins inhibited the activity of LsbB, in the same manner as found with the truncated version of LsbB. Taken together, these structure-function studies provide strong evidence that the receptor-binding parts of LsbB and sequence-related bacteriocins are located in their C-terminal halves.

SUBMITTER: Ovchinnikov KV

PROVIDER: S-EPMC4156037 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Defining the structure and receptor binding domain of the leaderless bacteriocin LsbB.

Ovchinnikov Kirill V KV Kristiansen Per E PE Uzelac Gordana G Topisirovic Ljubisa L Kojic Milan M Nissen-Meyer Jon J Nes Ingolf F IF Diep Dzung B DB

The Journal of biological chemistry 20140703 34

LsbB is a class II leaderless lactococcal bacteriocin of 30 amino acids. In the present work, the structure and function relationship of LsbB was assessed. Structure determination by NMR spectroscopy showed that LsbB has an N-terminal α-helix, whereas the C-terminal of the molecule remains unstructured. To define the receptor binding domain of LsbB, a competition assay was performed in which a systematic collection of truncated peptides of various lengths covering different parts of LsbB was use ...[more]

PMID: 24993828

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Project description:Lytic bacteriophages are expected as effective tools to control infectious bacteria in human and pathogenic or spoilage bacteria in foods. Leaderless bacteriocins (LLBs) are simple bacteriocins produced by Gram-positive bacteria. LLBs do not possess an N-terminal leader peptide in the precursor, which means that they are active immediately after translation. In this study, we constructed a novel antimicrobial agent, an LLB-producing phage (LLB-phage), by genetic engineering to introduce the LLB structural gene into the lytic phage genome. To this end, lnqQ (structure gene of an LLB, lacticin Q) and trxA, an essential gene for T7 phage genome replication, were integrated in tandem into T7 phage genome using homologous recombination in Escherichia coli host strain. The recombinant lnqQ-T7 phage was isolated by a screening method using ΔtrxA host strain. lnqQ-T7 phage formed a clear halo in agar plates containing both E. coli and lacticin Q-susceptible Bacillus coagulans, indicating that lnqQ-T7 phage could produce a significant amount of lacticin Q. Lacticin Q production did not exert a significant effect on the lytic cycle of T7 phage. In fact, the production of lacticin Q enhanced T7 phage lytic activity and helped to prevent the emergence of bacterial populations resistant against this phage. These results serve as a proof of principle for LLB-phages. There are different types of LLBs and phages, meaning that in the future, it may be possible to produce any number of LLB-phages which can be designed to efficiently control different types of bacterial contamination in different settings. IMPORTANCE We demonstrated that we could combine LLB and phage to construct promising novel antimicrobial agents, LLB-phage. The first LLB-phage, lnqQ-T7 phage, can control the growth of both the Gram-negative host strain and neighboring Gram-positive bacteria while preventing the emergence of phage resistance in the host strain. There are several different types of LLBs and phages, suggesting that we may be able to design a battery of LLB-phages by selecting novel combinations of LLBs and phages. These constructs could be tailored to control various bacterial contaminations and infectious diseases.

Dataset Information

Defining the structure and receptor binding domain of the leaderless bacteriocin LsbB.

Publications

Defining the structure and receptor binding domain of the leaderless bacteriocin LsbB.

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