Identification of the genes involved in the secretion and self-immunity of lacticin Q, an unmodified leaderless bacteriocin from Lactococcus lactis QU 5

Abstract

Lacticin Q (LnqQ) produced by Lactococcus lactis QU 5 is an unmodified linear bacteriocin, which is synthesized without an N-terminal leader peptide. In vitro synthesis and in vivo expression of LnqQ have revealed the intracellular toxicity of this leaderless peptide, as well as the necessity of a dedicated secretion and self-immunity system of producer cells. Further DNA sequencing and analysis have discovered 11 putative orf genes at the LnqQ locus. None of the orf genes showed similarities to any of the bacteriocin biosynthetic genes characterized to date; however, six orf genes (orf2q-7q), not including the structural gene (lnqQ), were highly conserved at the lacticin Z locus (orf2z-7z), which is a LnqQ homologue produced by L. lactis QU 14. ORF2q (ORF2z), the gene of which is located upstream of the structural gene, is a putative transcriptional regulator, whereas ORF6q and ORF7q (ORF6z and ORF7z) form a putative ATPbinding cassette transporter. The ORF3q-5q (ORF3z-5z) are all predicted to be membrane proteins with no clear functions. Co-expression of LnqQ and ORF3q-7q in a heterologous host allowed the extracellular production of LnqQ; additionally, the expression of ORF3q-7q rendered the host cells immune to LnqQ. This self-immunity was facilitated possibly by two means; firstly, by secreting the active LnqQ peptides, thus reducing the intracellular toxicity, and secondly, by protecting the host cells from extracellularly released LnqQ. This is the first report, to our knowledge, that describes intracellular toxicity of a leaderless bacteriocin and provides a rare example of biosynthetic genes that are required for bacteriocin secretion and immunity.

Screening and Characterization of Novel Bacteriocins from Lactic Acid Bacteria

“…Co-expression of lnqQ and lnqBCDEF in a lactococcal heterologous host allowed the extracellular production of lacticin Q. 42) In addition, expression of lnqBCDEF rendered host cells immune to lacticin Q, and the expression of lnqQ alone revealed the intracellular toxicity of this leaderless peptide. Leaderless bacteriocins such as lacticin Q have intracellular toxicity, since they have no leader sequence to make bacteriocins inactive.…”

Bifunctional Gene Cluster lnqBCDEF Mediates Bacteriocin Production and Immunity with Differential Genetic Requirements

“…LnqD, the largest ORF (432 aa) in the lnqQ locus, has some homology to the membrane protein (486 aa) of Lactobacillus buchneri (25% identity) and the YdbT homolog protein (484 aa) of Leuconostoc pseudomesenteroides (23% identity). Previous work has revealed that a set of lnqBCDEF confers the secretion and self-immunity of LnqQ; however, the role of each gene product has not been elucidated (4). Thus, in this study, comprehensive gene disruption was carried out to determine the necessity of each gene and to deduce its role in LnqQ biosynthesis.…”

“…The genes necessary for bacteriocin production and immunity are located near the structural gene and are generally arranged as an operon (1,2). Lacticin Q (LnqQ), produced by Lactococcus lactis QU 5 (3), is an unmodified leaderless bacteriocin that has a unique gene cluster (lnqBCDEF) following the structural gene (lnqQ) (4). As predicted in silico, lnqEF encodes an ATP-binding cassette (ABC) transporter with LnqE as the membrane-spanning domain and LnqF as the cytoplasmic ATPase domain.…”

“…Culture conditions for the strains listed, DNA manipulation, and other molecular cloning techniques were as described previously (4,(7)(8)(9)). An inverse PCR technique (11) was used to create an in-frame deletion of the gene(s) of interest from the template plasmid, pLNQ, which contains lnqQBCDEF under the control of lactococcal promoter P 32 (4). In one case, primers invF1 and invR1 (Table 2) were used to amplify the entire plasmid (pLNQ) without the lnqB region.…”

Mutations near the cleavage site of enterocin NKR-5-3B prepeptide reveal new insights into its biosynthesis

“…Assessment of possible intracellular accumulation of the Ent53B precursor peptide was done using the previously reported method with slight modifications [31,32]. Briefly, recombinant strains showing negative Ent53B bioactivity were precultivated in GM17 containing 10 µg ml À1 cm at 30 C overnight and sub-cultured into fresh medium containing the same antibiotic with the same culture conditions for 7 h. Cells were then harvested by centrifugation, washed with 50 mM Tris-HCl (pH 7.4), and resuspended in 500 µl of the same buffer.…”