Production of polyhydroxybutyrate and alginate from glycerol by Azotobacter vinelandii under nitrogen-free conditions.

Glycerol is an interesting feedstock for biomaterials such as biofuels and bioplastics because of its abundance as a by-product during biodiesel production. Here we demonstrate glycerol metabolism in the nitrogen-fixing species Azotobacter vinelandii through metabolomics and nitrogen-free bacterial production of biopolymers, such as poly-d-3-hydroxybutyrate (PHB) and alginate, from glycerol. Glycerol-3-phosphate was accumulated in A. vinelandii cells grown on glycerol to the exponential phase, and its level drastically decreased in the cells grown to the stationary growth phase. A. vinelandii also overexpressed the glycerol-3-phosphate dehydrogenase gene when it was grown on glycerol. These results indicate that glycerol was first converted to glycerol-3-phosphate by glycerol kinase. Other molecules with industrial interests, such as lactic acid and amino acids including γ-aminobutyric acid, have also been accumulated in the bacterial cells grown on glycerol. Transmission electron microscopy revealed that glycerol-grown A. vinelandii stored PHB within the cells. The PHB production level reached 33% per dry cell weight in nitrogen-free glycerol medium. When grown on glycerol, alginate-overproducing mutants generated through chemical mutagenesis produced 2-fold the amount of alginate from glycerol than the parental wild-type strain. To the best of our knowledge, this is the first report on bacterial production of biopolymers from glycerol without addition of any nitrogen source.

A novel bleb-dependent polysaccharide export system in nitrogen-fixing Azotobacter vinelandii subjected to low nitrogen gas levels.

The alginate biofilm-producing bacterium Azotobacter vinelandii aerobically fixes nitrogen by oxygen-sensitive nitrogenases. Here we investigated the bacterial response to nitrogen/oxygen gas mixtures. A. vinelandii cells were cultured in nitrogen-free minimal media containing gas mixtures differing in their ratios of nitrogen and oxygen. The bacteria did not grow at oxygen concentrations >75% but grew well in the presence of 5% nitrogen/25% oxygen. Growth of wild-type and alginate-deficient strains when cultured with 50% oxygen did not differ substantially, indicating that alginate is not required for the protection of nitrogenases from oxygen damage. In response to decreasing nitrogen levels, A. vinelandii produced greater amounts of alginate, accompanied by the formation of blebs on the cell surface. The encystment of vegetative cells occurred in tandem with the release of blebs and the development of a multilayered exine. Immunoelectron microscopy using anti alginate-antibody revealed that the blebs contained alginate molecules. By contrast, alginate-deficient mutants could not form blebs. Taken together, our data provide evidence for a novel bleb-dependent polysaccharide export system in A. vinelandii that is activated in response to low nitrogen gas levels.

Lethal hydroxyl radical accumulation by a lactococcal bacteriocin, lacticin Q.

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.

A novel bleb-dependent polysaccharide export system in nitrogen-fixing Azotobacter vinelandii subjected to low nitrogen gas levels

The alginate biofilm-producing bacterium Azotobacter vinelandii aerobically fixes nitrogen by oxygensensitive nitrogenases. Here we investigated the bacterial response to nitrogen/oxygen gas mixtures. A. vinelandii cells were cultured in nitrogen-free minimal media containing gas mixtures differing in their ratios of nitrogen and oxygen. The bacteria did not grow at oxygen concentrations >75 % but grew well in the presence of 5 % nitrogen/25 % oxygen. Growth of wild-type and alginate-deficient strains when cultured with 50 % oxygen did not differ substantially, indicating that alginate is not required for the protection of nitrogenases from oxygen damage. In response to decreasing nitrogen levels, A. vinelandii produced greater amounts of alginate, accompanied by the formation of blebs on the cell surface. The encystment of vegetative cells occurred in tandem with the release of blebs and the development of a multilayered exine. Immuno electron microscopy using anti alginate-antibody revealed that the blebs contained alginate molecules. By contrast, alginate-deficient mutants could not form blebs. Taken together, our data provide evidence for a novel bleb dependent polysaccharide export system in A. vinelandii that is activated in response to low nitrogen gas levels (AU)

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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.

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.

Lacticin Q-mediated selective toxicity depending on physicochemical features of membrane components.

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.

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.

Effect of a negatively charged lipid on membrane-lacticin Q interaction and resulting pore formation.

Lacticin Q is an antimicrobial peptide that forms pores on membranes. We investigated effects of negatively charged lipids on the binding and pore formation of lacticin Q with liposomes by surface plasmon resonance analysis and fluorescence dye leakage experiments respectively. Negatively charged lipids accelerated the binding of lacticin Q on the membranes and the resulting pore formation. However, the acceleration was not an essential factor in the killing activity of lacticin Q, since pore-forming activities against electrically neutral and negatively charged liposomes occurred similarly.

Peptide-lipid huge toroidal pore, a new antimicrobial mechanism mediated by a lactococcal bacteriocin, lacticin Q.

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."

Lacticin Q, a lactococcal bacteriocin, causes high-level membrane permeability in the absence of specific receptors.

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.

Complete covalent structure of nisin Q, new natural nisin variant, containing post-translationally modified amino acids.

The third member of the nisin variant, nisin Q, produced by Lactococcus lactis 61-14, is a ribosomally-synthesized antimicrobial peptide, the so-called lantibiotic containing post-translationally modified amino acids such as lanthionine and dehydroalanine. Here, we determined the complete covalent structure of nisin Q, consisting of 34 amino acids, by two-dimensional (1)H nuclear magnetic resonance (NMR) spectroscopy. Sequential assignment of nisin Q containing the unusual amino acids was performed by total correlation spectroscopy (TOCSY) and nuclear Overhauser enhancement spectroscopy (NOESY). The observed long range nuclear Overhauser effect (NOE) in nisin Q indicated assignment of all five sets of lanthionines that intramolecularly bridge residues 3-7, 8-11, 13-19, 23-26, and 25-28. Consequently, the covalent structure of nisin Q was determined to hold the same thioether linkage formation as the other two nisins, but to harbor the four amino acid substitutions, in contrast with nisin A.

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 degrees 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.

Structural analysis and characterization of lacticin Q, a novel bacteriocin belonging to a new family of unmodified bacteriocins of gram-positive bacteria.

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.