Voltage-dependent depolarization of bacterial membranes and artificial lipid bilayers by the peptide antibiotic nisin

The peptide antibiotic nisin is shown to disrupt valinomycin-induced potassium diffusion potentials imposed on intact cells of Staphylococcus cohnii 22. Membrane depolarization occurred rapidly at high diffusion potentials while at low potentials nisin-induced depolarization was slower suggesting that nisin requires a membrane potential for activity. This assumption was proven in experiments with planar lipid bilayers (black lipid membranes). Macroscopic conductivity measurements indicated a voltage-dependent action of nisin. The potential must have a trans-negative orientation with respect to the addition of nisin (added to the cis-side) and a sufficient magnitude (ca. -100 mV). With intact cells the threshold potential was lower (-50 to -80 mV at pH 7.5 and below -50 mV at pH 5.5). Single channel recordings resolved transient multistate pores, strongly resembling those introduced by melittin into artificial bilayers. The pores had diameters in the range of 0.2–1 nm, and lifetimes of few to several hundred milliseconds. The results indicate that nisin has to be regarded as a membrane-depolarizing agent which acts in a voltage-dependent fashion.Abbreviations BLM Black lipid membranes - CCCP carbonyl cyanide m-chlorophenylhydrazone - DOPC dioleoyl phosphatidylcholine - PS phosphatidylserine - TPP⁺ tetraphenylphosphonium cation     

Influence of the staphylococcinlike peptide Pep 5 on membrane potential of bacterial cells and cytoplasmic membrane vesicles.

The staphylococcinlike peptide Pep 5 rapidly abolished the membrane potential of bacterial cells; active transport of amino acids by cytoplasmic membrane vesicles was inhibited and preaccumulated amino acids were released upon the addition of Pep 5. Artificial asolectin vesicles were not impaired by the peptide. It is concluded that the cytoplasmic membrane is the primary target of Pep 5.     

Induction of autolysis of staphylococci by the basic peptide antibiotics Pep 5 and nisin and their influence on the activity of autolytic enzymes

Pep 5 and nisin are cationic bactericidal peptides which were shown to induce autolysis in Staphylococcus cohnii 22. In contrast to nisin, Pep 5 induced lysis could be stimulated in the presence of glucose. Addition of lipoteichoic acids (LTA) (d-alanine:phosphorus=0.475:1) inhibited all effects of Pep 5 on susceptible cells in a molar ratio LTA:Pep 5 of 10:1. Treatment of S. cohnii 22 with Pep 5 or nisin for 20 min and subsequent washing with 2.5 M NaCl released autolysin activity. Crude preparations of the hydrolyzing enzymes produced free amino groups as well as polysaccharide fragments from the murein backbone, suggesting the presence of a muramidase or glucosamidase, and endopeptidase or amidase. Both enzyme activities were inhibited by lipoteichoic acid; they could be fully reactivated by addition of Pep 5 in sufficient concentrations. The velocity of hydrolysis was not influenced by nisin, whereas it was doubled in presence of Pep 5. The results are discussed in view of a possible mechanism of induction of lysis by Pep 5 and nisin.Abbreviations A.U. arbitrary unit - CCCP carbonylcyanide-m-chlorophenyl hydrazone - DNase deoxyribonuclease - CYG casein yeast extract glucose - IT initial turbidity - LTA lipoteichoic acid - RNase ribonuclease - TSB Tryptone Soy Broth     

The biosynthesis of the lantibiotics epidermin,gallidermin, Pep5 and epilancin K7

Lantibiotics are antibiotic peptides that contain the rare thioether amino acids lanthionine and/or methyllanthionine. Epidermin, Pep5 and epilancin K7 are produced by Staphylococcus epidermidis whereas gallidermin (6L-epidermin) was isolated from the closely related species Staphylococcus gallinarum. The biosynthesis of all four lantibiotics proceeds from structural genes which code for prepeptides that are enzymatically modified to give the mature peptides. The genes involved in biosynthesis, processing, export etc. are found in gene clusters adjacent to the structural genes and code for transporters, immunity functions, regulatory proteins and the modification enzymes LanB, LanC and LanD, which catalyze the biosynthesis of the rare amino acids. LanB and LanC are responsible for the dehydration of the serine and threonine residues to give dehydroalanine and dehydrobutyrine and subsequent addition of cysteine SH-groups to the dehydro amino acids which results in the thioether rings. EpiD, the only LanD enzyme known so far, catalyzes the oxidative decarboxylation of the C-terminal cysteine of epidermin which gives the C-terminal S-aminovinylcysteine after addition of a dehydroalanine residue.Abbreviations Dha 2,3-didehydroalanine - Dhb 2,3-didehydrobutyrine - Lan lanthionine - Melan methyllanthionine     

The Wadi Natrun: Chemical composition and microbial mass developments in alkaline brines of Eutrophic Desert Lakes

Six lakes of the Wadi Natrun, Egypt, were studied with respect to the chemical composition of their brines and the occurrence of microbial mass developments. All investigated lakes showed pH values of approximately 11 and a total salt content of generally more than 30%. The main components were sulfate, carbonate, chloride, sodium, and minor amounts of potassium. Only traces of magnesium and calcium were present, but unusually high concentrations of organic carbon compounds, nitrogen compounds, and phosphate were found. Mass developments of phototrophic sulfur bacteria, halobacteria, cyanobacteria, and green algae were observed. The functions of complete nitrogen and sulfur cycles in the alkaline brines are discussed. The properties of the lakes and their ecology are compared with data on the Dead Sea and Great Salt Lake, Utah.     

Repeated oral administration of capsaicin increases anxiety-like behaviours with prolonged stress-response in rats

This study was conducted to examine the psycho-emotional effects of repeated oral exposure to capsaicin, the principal active component of chili peppers. Each rat received 1 mL of 0.02% capsaicin into its oral cavity daily, and was subjected to behavioural tests following 10 daily administrations of capsaicin. Stereotypy counts and rostral grooming were significantly increased, and caudal grooming decreased, in capsaicin-treated rats during the ambulatory activity test. In elevated plus maze test, not only the time spent in open arms but also the percent arm entry into open arms was reduced in capsaicin-treated rats compared with control rats. In forced swim test, although swimming duration was decreased, struggling increased in the capsaicin group, immobility duration did not differ between the groups. Repeated oral capsaicin did not affect the basal levels of plasma corticosterone; however, the stress-induced elevation of plasma corticosterone was prolonged in capsaicin treated rats. Oral capsaicin exposure significantly increased c-Fos expression not only in the nucleus tractus of solitarius but also in the paraventricular nucleus. Results suggest that repeated oral exposure to capsaicin increases anxiety-like behaviours in rats, and dysfunction of the hypothalamic-pituitary-adrenal axis may play a role in its pathophysiology.     

Mode of action of the antimicrobial peptide aureocin A53 from Staphylococcus aureus

We investigated the mode of action of aureocin A53 on living bacterial cells and model membranes. Aureocin A53 acted bactericidally against Staphylococcus simulans 22, with >90% of the cells killed within a few minutes. Cell death was followed by lysis, as indicated by a clearing of the cell suspension and Gram staining. Aureocin A53 rapidly dissipated the membrane potential and simultaneously stopped biosynthesis of DNA, polysaccharides, and protein. Aureocin A53 induced a rapid release of preaccumulated glutamate and Rb(+). Experiments on model membranes demonstrated that aureocin A53 provoked significant leakage of carboxyfluorescein (CF) exclusively from acidic liposomes but only at relatively high concentrations (0.5 to 8 mol%). Thus, the bactericidal activity of aureocin A53 derives from membrane permeation via generalized membrane destruction rather than by formation of discrete pores within membranes. Tryptophan emission fluorescence spectroscopy demonstrated interaction of aureocin A53 with both acidic and neutral membranes, as indicated by similar blue shifts. Since there was no significant aureocin A53-induced CF leakage from neutral liposomes, its appears that the peptide does interact with neutral lipids without provoking membrane damage.     

Intercellular communication in the rat anterior pituitary gland: an in vivo and in vitro study

The concept of "stimulus-secretion coupling" suggested by Douglas and co-workers to explain the events related to monamine discharge by the adrenal medulla (5, 7) may be applied to other endocrine tissues, such as adrenal cortex (36), pancreatic islets (4), and magnocellular hypothalamic neurons (6), which exhibit a similar ion-dependent process of hormone elaboration. In addition, they share another feature, that of joining neighbor cells via membrane junctions (12, 26, and Fletcher, unpublished observation). Given this, and the reports that hormone secretion by the pars distalis also involves a secretagogue-induced decrease in membrane bioelectric potential accompanied by a rise in cellular [Ca++] (27, 34, 41), it was appropriate to test the possibility that cells of the anterior pituitary gland are united by junctions.     

Specific binding of nisin to the peptidoglycan precursor lipid II combines pore formation and inhibition of cell wall biosynthesis for potent antibiotic activity

Unlike numerous pore-forming amphiphilic peptide antibiotics, the lantibiotic nisin is active in nanomolar concentrations, which results from its ability to use the lipid-bound cell wall precursor lipid II as a docking molecule for subsequent pore formation. Here we use genetically engineered nisin variants to identify the structural requirements for the interaction of the peptide with lipid II. Mutations affecting the conformation of the N-terminal part of nisin comprising rings A through C, e.g. [S3T]nisin, led to reduced binding and increased the peptide concentration necessary for pore formation. The binding constant for the S3T mutant was 0.043 x 10(7) m(-1) compared with 2 x 10(7) m(-1) for the wild-type peptide, and the minimum concentration for pore formation increased from the 1 nm to the 50 nm range. In contrast, peptides mutated in the flexible hinge region, e.g. [DeltaN20/DeltaM21]nisin, were completely inactive in the pore formation assay, but were reduced to some extent in their in vivo activity. We found the remaining in vivo activity to result from the unaltered capacity of the mutated peptide to bind to lipid II and thus to inhibit its incorporation into the peptidoglycan network. Therefore, through interaction with the membrane-bound cell wall precursor lipid II, nisin inhibits peptidoglycan synthesis and forms highly specific pores. The combination of two killing mechanisms in one molecule potentiates antibiotic activity and results in nanomolar MIC values, a strategy that may well be worth considering for the construction of novel antibiotics.     

A key to the adult Costa Rican "helicopter" damselflies (Odonata: Pseudostigmatidae) with notes on their phenology and life zone preferences

We present a key to the Costa Rican species of Pseudostigmatidae, comprising three genera with the following species: Megaloprepus caerulatus, Mecistogaster linearis, M. modesta, M. ornata and Pseudostigma aberrans. Pseudostigma accedens, which may occur in the region, is also included. For each species we give a brief account of morphology, phenology and life zone preferences, including distributional maps based on more than 270 records. These are not all of the known specimens from the area, but a high enough number to give a relatively good picture of the distribution and status of the species. We found M. caerulatus to be active during the first half of the year in seasonal, tropical semi-dry lowland forest and tropical moist forest at mid-elevation, but like M. linearis, M. caerulatus was active all year round in non-seasonal, tropical wet lowland forest and tropical moist forest at mid-elevation. Mecistogaster modesta also flew year round in non-seasonal, tropical wet lowland forest and tropical moist evergreen forest at mid-elevation, and likewise in seasonal and non-seasonal, tropical premontane moist forest. Only a few findings, however, have been made of M. modesta in seasonal, tropical semi-dry deciduous forest and seasonal, tropical moist evergreen forest. Mecistogaster ornata was missing entirely from non-seasonal, tropical wet lowland forest and non-seasonal, tropical moist forest at mid-elevation, while this species was active year round in seasonal, tropical dry lowland forest and tropical semi-dry forest, as well as in seasonal, tropical moist evergreen forest and tropical premontane moist forest, both at mid-elevation. Pseudostigma aberrans has so far been found too few times in Costa Rica for any indication of flight time preference.