Nisin is a lantibiotic produced by strains of Lactococcus lactis subsp. lactis. The target for nisin action is the cytoplasmic membrane of gram‐positive bacteria. To aid understanding of its mode of action, the interaction of nisin with vesicles of differing phospholipid composition were investigated by fluorescence techniques, using a variant of nisin in which the isoleucine at position 30 was replaced by a tryptophan residue. Activity of the site‐directed variant containing tryptophan was established to be similar to that of the wild‐type peptide. Fluorescence experiments showed a blue shift of the emission wavelength maximum in the presence of lipid vesicles, indicating that the tryptophan residue enters a more hydrophobic environment. Quenching experiments with aqueous and membrane‐restricted quenchers (iodide and spin‐labelled lipids, respectively) both confirmed a non‐aqueous environment for the Trp30 residue, and implied that the residue resides between 0.36 nm and 0.52 nm from the centre of the membrane, depending on the lipid identity. The results clearly demonstrate that nisin interacts strongly with the hydrophobic phase of lipid vesicles. This interaction is stronger in the presence of negatively charged lipids suggesting their importance in the functional interaction of nisin with membranes.
Morphological, biochemical and molecular genetic studies were carried out on an unknown non-spore-forming, Gram-positive, rod-shaped bacterium that was isolated from dog faeces. The bacterium grew under strictly anaerobic conditions, was asaccharolytic, and possessed a relatively high G+C content of 61 mol%. Phylogenetic analysis based on comparative 16S rRNA gene sequencing showed that the unidentified bacterium was a member of the family Coriobacteriaceae and represents a hitherto unknown subline within the genus Slackia . Based on the presented findings, a novel species, Slackia faecicanis sp. nov., is described. The type strain of Slackia faecicanis is 5WC12 T (=CCUG 48399 T =CIP 108281 T ).
Abstract Objective —To determine whether feeding activated charcoal, Yucca schidigera , and zinc acetate would ameliorate the frequency and odor characteristics of flatulence in dogs. Design —In vitro screening of active agents followed by a randomized controlled trial. Animals —8 adult dogs. Procedure —A fecal fermentation system was used to assess the effects of activated charcoal, Yucca schidigera , and zinc acetate alone and in combination on total gas production and production of hydrogen sulfide, the primary determinant of flatus malodor in dogs. All 3 agents were subsequently incorporated into edible treats that were fed 30 minutes after the dogs ate their daily rations, and the number, frequency, and odor characteristics of flatulence were measured for 5 hours, using a device that sampled rectal gases and monitored hydrogen sulfide concentrations. Results —Total gas production and number and frequency of flatulence episodes were unaffected by any of the agents. Production of hydrogen sulfide in vitro was significantly reduced by charcoal, Yucca schidigera , and zinc acetate by 71, 38, and 58%, respectively, and was reduced by 86% by the combination of the 3 agents. Consumption of the 3 agents was associated with a significant decrease (86%) in the percentage of flatulence episodes with bad or unbearable odor and a proportional increase in the percentage of episodes of no or only slightly noticeable odor. Conclusions and Clinical Relevance —Results suggest that activated charcoal, Yucca schidigera , and zinc acetate reduce malodor of flatus in dogs by altering the production or availability of hydrogen sulfide in the large intestine. ( J Am Vet Med Assoc 2001;218: 892–896)
Nisin, a 34 residue lantibiotic produced by strains of Lactococcus lactis subsp. lactis, exerts antimicrobial activity against Gram-positive bacteria at the cytoplasmic membrane. The structural aspects of nisin which facilitate membrane interaction and permeabilization have been investigated in planar lipid bilayers and liposomes with proteolytic fragments and site-directed variants. N-Terminal nisin fragments N1-12 and N1-20 had little effect on phospholipid mobility, on macroscopic electrical conductance, or on calcein release from liposomes. By contrast, the I30W nisin A variant induced a time-dependent reduction in lipid mobility, indicative of nisin-membrane surface interactions, as well as a decline in membrane capacitance, rise in conductance, and calcein release from liposomes. In these respects I30W nisin A is similar to native nisin. Charge substitutions were also engineered to generate K12L and H27K nisin A variants, both of which were similar to I30W nisin A with respect to an overall reduction in phospholipid mobility. While the K12L nisin A variant elicited a higher increase in membrane capacitance and electrical conductance than I30W nisin A, the H27K nisin A variant elicited weaker effects. These results point to a substantial role for intramembrane charged residues in controlling ion flow through nisin-doped membranes. Native nisin and variants elicit an enhanced release of calcein from liposomes composed of the negatively-charged phospholipids cardiolipin and phosphatidylserine, compared with phospholipid bearing no net charge, suggesting that an electrostatic attraction encourages the initial nisin-membrane association. The results are discussed in the context of other recently proposed models for nisin action.
A lactococcal expression system was developed which allows the exclusive production of novel nisins encoded by mutated pre-nisin ( nisA ) genes. This system is based on a combination of a specifically constructed host strain and vectors which facilitate the genetic manipulation of the nisA gene. The wild-type chromosomal gene is effectively replaced with a variant nisA gene, by the technique of gene replacement. The recovery of full nisin immunity was employed as a means of directly selecting strains that had acquired an intact nisA gene by the gene replacement process. With this approach the other genes required for pre-nisin maturation are not affected and any alterations to DNA sequences are restricted to only those specific mutations introduced in the nisA gene. The effectiveness of the system was demonstrated by the expression of a number of variant nisA genes leading to the successful production and characterization of nisins containing the substitutions Dha5A, Dha33A, Dha5,33A, H27K, I30W and K12L. The enhanced yields of these engineered nisin molecules, when compared to their production in a plasmid-complementation system, underlines the improvement offered by this gene replacement strategy.
Modified nisin molecules, synthesised by strains of Lactococcus lactis with deliberately mutated nisA genes, have been characterised using Fourier transform ion cyclotron resonance mass spectrometry and electrospray ionisation. The predicted substitutions in the three nisin variants synthesised were first confirmed by precise measurement of the molecular mass (precision ±0.1 Da). Analysis of the lower intensity peaks in the mass spectra showed the presence of some minor components, notably hydrated molecules, in the first two samples. The third sample contained a major hydrated component that could be isolated in pure form by high-performance liquid chromatography. The engineered nisin molecules were further characterised by tandem mass spectrometry using the sustained off-resonance irradiation collisionally-activated decomposition technique. This yielded a number of sequence ions that were compared with those measured in a previous study of nisin A itself. The location of each substituent was deduced from the observed mass shifts of the sequence ions. This permitted definitive confirmation of the predicted substitutions. Out of several possible sites it was confirmed that position 33 contained the additional water molecule in the major hydrated form of one of the nisin variants.
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