Fuminori Yoneyama

Structural Analysis and Characterization of Lacticin Q: a Novel Bacteriocin Belonging to a New Family of Unmodified Bacteriocins of Gram-Positive Bacteria

ABSTRACT Lactococcus lactis QU 5 isolated from corn produces a novel bacteriocin, termed lacticin Q. By acetone precipitation, cation-exchange chromatography, and reverse-phase high-performance liquid chromatography, lacticin Q was purified from the culture supernatant of this organism, and its molecular mass was determined to be 5,926.50 Da by mass spectrometry. Subsequent analyses of amino acid and DNA sequences revealed that lacticin Q comprised 53 amino acid residues and that its N-terminal methionine residue was formylated. In contrast to most bacteriocins produced by gram-positive bacteria, lacticin Q had no N-terminal extensions such as leader or signal sequences. It showed 66% and 48% identity to AucA, a hypothetical protein from Corynebacterium jeikeium plasmid pA501, and aureocin A53, a bacteriocin from Staphylococcus aureus A53, respectively. The characteristics of lacticin Q were determined and compared to those of nisin A. Similar to nisin A, lacticin Q exhibited antibacterial activity against various gram-positive bacteria. Lacticin Q was very stable against heat treatment and changes in pH; in particular, it was stable at alkaline pH values, while nisin A was inactivated. Moreover, lacticin Q induced ATP efflux from a Listeria sp. strain in a shorter time and at a lower concentration than nisin A, indicating that the former affected indicator cells in a different manner from that of the latter. The results described here clarified the fact that lacticin Q belongs to a new family of class II bacteriocins and that it can be employed as an alternative to or in combination with nisin A.

Peptide-Lipid Huge Toroidal Pore, a New Antimicrobial Mechanism Mediated by a Lactococcal Bacteriocin, Lacticin Q

ABSTRACT Lacticin Q is a pore-forming bacteriocin produced by Lactococcus lactis QU 5, and its antimicrobial activity is in the nanomolar range. Lacticin Q induced calcein leakage from negatively charged liposomes. However, no morphological changes in the liposomes were observed by light scattering. Concomitantly with the calcein leakage, lacticin Q was found to translocate from the outer to the inner leaflet of the liposomes, after it initially bound to the membrane within 2 s. Lacticin Q also induced lipid flip-flop. These results reveal that the antimicrobial mechanism of lacticin Q can be described by the toroidal pore model. This is the first report of a bacteriocin of gram-positive bacteria that forms a toroidal pore. From liposomes, lacticin Q leaked fluorescence-labeled dextran with a diameter of 4.6 nm. In addition, lacticin Q caused the leakage of small proteins, such as the green fluorescent protein, from live bacterial cells. There are no other reports of antimicrobial peptides that exhibit protein leakage properties. The proposed pore formation model of lacticin Q is as follows: (i) quick binding to outer membrane leaflets; (ii) the formation of at least 4.6-nm pores, causing protein leakage with lipid flip-flop; and (iii) the migration of lacticin Q molecules from the outer to the inner membrane leaflets. Consequently, we termed the novel pore model in the antimicrobial mechanism of lacticin Q a “huge toroidal pore.”

Characterization and Structure Analysis of a Novel Bacteriocin, Lacticin Z, Produced by Lactococcus lactis QU 14

A novel bacteriocin, lacticin Z, produced by Lactococcus lactis QU 14 isolated from a horse’s intestinal tract was identified. Lacticin Z was purified through a three step procedure comprised of hydrophobic-interaction, cation-exchange chromatography, and reverse-phase HPLC. ESI-TOF MS determined the molecular mass of lacticin Z to be 5,968.9 Da. The primary structure of lacticin Z was found to consist of 53 amino acid residues without any leader sequence or signal peptide. Lacticin Z showed homology to lacticin Q from L. lactis QU 5, aureocin A53 from Staphylococcus aureus A53, and mutacin BHT-B from Streptococcus rattus strain BHT. It exhibited a nanomolar range of MICs against various Gram-positive bacteria, and the activity was completely stable up to 100 °C. Unlike many of other LAB bacteriocins, the stability of lacticin Z was emphasized under alkaline conditions rather than acidic conditions. All the results indicated that lacticin Z belongs to a novel type of bacteriocin.

Lacticin Q, a Lactococcal Bacteriocin, Causes High-Level Membrane Permeability in the Absence of Specific Receptors

ABSTRACT To characterize the mode of action of lacticin Q (LnqQ), its membrane-permeabilizing activity was compared with that of nisin A because of the similar antimicrobial features of these compounds. Lipid II, the receptor for nisin A, was not required for LnqQ activity. LnqQ induced high-level membrane permeability in the absence of specific receptors.

Lactococcal membrane-permeabilizing antimicrobial peptides

A number of lactococcal antimicrobial peptides, bacteriocins have been discovered and characterized. Since Lactococcus spp. are generally regarded as safe bacteria, their bacteriocins are expected for various application uses. Most of lactococcal bacteriocins exert antimicrobial activity via membrane permeabilization. The most studied and prominent bacteriocin, nisin A is characterized in the high activity and has been utilized as food preservatives for more than half a century. Recently, other lactococcal bacteriocins such as lacticin Q were found to have distinguished features for further applications as the next generation to nisin.

Lacticin Q-Mediated Selective Toxicity Depending on Physicochemical Features of Membrane Components

ABSTRACT Lacticin Q, a lactococcal pore-forming bacteriocin, shows activity toward Gram-positive bacteria but not Gram-negative bacteria. Lacticin Q did not induce permeability of the outer membrane of Gram-negative bacteria. Experiments using model membranes containing outer membrane components suggested that lacticin Q binds to the outer membrane of Gram-negative bacteria but is unable to penetrate it. The lack of activity of lacticin Q was attributed to physicochemical features of the outer membrane components.

Biosynthetic characterization and biochemical features of the third natural nisin variant, nisin Q, produced by Lactococcus lactis 61-14.

Aims:  To characterize the genetic and biochemical features of nisin Q.

Identification of enterocin NKR-5-3C, a novel class IIa bacteriocin produced by a multiple bacteriocin producer, Enterococcus faecium NKR-5-3

The structure of enterocin NKR-5-3C, an anti-listerial bacteriocin produced by a multiple bacteriocin producer, Enterococcus faecium NKR-5-3, was determined. Enterocin NKR-5-3C is a novel class IIa bacteriocin that possesses an YGNGL motif sequence and two disulfide bridges in its structure. It is encoded on gene ent53C together with an 18-amino-acid-residue double glycine leader peptide.

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

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 ATP-binding 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.

Lethal hydroxyl radical accumulation by a lactococcal bacteriocin, lacticin Q

ABSTRACT The antimicrobial mechanism of a lactococcal bacteriocin, lacticin Q, can be described by the toroidal pore model without any receptor. However, lacticin Q showed different degrees of activity (selective antimicrobial activity) against Gram-positive bacteria even among related species. The ability of lacticin Q to induce pore formation in liposomes composed of lipids from different indicator strains indicated that its selective antimicrobial activity could not be attributed only to membrane lipid composition. We investigated the accumulation of deleterious hydroxyl radicals after exposure to lacticin Q as a contributing factor to cell death in the indicator strains. When lacticin Q of the same concentration as the MIC or minimum bactericidal concentration was added to the indicator cultures, high levels of hydroxyl radical accumulation were detected. Treatment with hydroxyl radical scavengers, thiourea and 2,2′-bipyridyl, decreased the levels of hydroxyl radical accumulation and recovered cell viability. These results suggest that, with or without pore formation, the final antimicrobial mechanism of lacticin Q is the accumulation of hydroxyl radicals, which varies by strain, resulting in the selective antimicrobial activity of lacticin Q.