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 grampositive 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.Bacteriocins are ribosomally synthesized antimicrobial peptides or proteins that are active against closely related species (17). Those produced by lactic acid bacteria (LAB) have attracted increasing interest in terms of their safety since many LAB are generally regarded as safe (GRAS) (7). Bacteriocins produced by gram-positive bacteria, including LAB, are mainly classified into three classes (18,26). Class I bacteriocins, the so-called lantibiotics, are heat-stable post-translationally modified peptides that contain multiple rings bridged by lanthionine or 3-methyllanthionine residues (22). Class II bacteriocins are small heat-stable nonlantibiotic peptides and are in turn divided into three subgroups (25, 26). Class IIa bacteriocins are Listeria sp.-active peptides with a consensus YGNGVXC sequence at their N termini (10). Class IIb bacteriocins comprise two peptides, both of which are required for complete antibacterial activity. Class IIc bacteriocins are the other class II bacteriocins; this subclass includes enterocin P, which is processed and secreted by the Sec pathway (4), and enterocin L50 (5), which is secreted without a leader sequence. However, some class II bacteriocins can be placed in more than one subclass, while others do not fit easily into any of the subclasses (27).Class III bacteriocins are heat-labile proteins, including millericin B, that hydrolyze specific peptide bonds in the stem and/or interpeptide b...

Bioethanol production from algae is a promising approach that resolves problems associated with biofuel production from land biomass, such as bioethanol-food conflicts and the indirect land use change. However, it presents several technical difficulties because existing ethanologenic microbes can neither degrade alginate, a major component of brown algae, nor assimilate alginate degradation products. We developed an integrated bacterial system for converting alginate to ethanol using a metabolically modified, alginate-assimilating, pit-forming bacterium, Sphingomonas sp. A1 (strain A1). Overexpression of Zymomonas mobilis pdc and adhB was achieved using a strong constitutive expression promoter newly identified in strain A1 and by inserting multiple gene copies using the methylation sensitivity of XbaI. Metabolome analysis revealed by-product accumulation, and its synthesis pathway was blocked by gene disruption. The ethanologenic recombinant strain A1 accumulated 13.0 g L À1 ethanol in 3 d using alginate as the sole carbon source.

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."

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.

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