Avermectin modulation of GABA binding to membranes of rat brain, brine shrimp and a fungus, Mucor miehei.

High affinity [3H]GABA (gamma-aminobutyric acid) binding sensitive to muscimol and bicuculline was detected in membranes derived from rat brain and brine shrimp. Avermectin stimulated this GABA binding with maximum stimulation seen in these membranes at 400 and 40-80 ng/ml, respectively. This avermectin stimulation of GABA binding was Cl--dependent, bicuculline and picrotoxin-sensitive and was associated with an increase in Bm but not Kd of the systems. The membranes from Mucor miehei also exhibited high affinity [3H]GABA binding that was insensitive to classical neuronal GABA receptor agonists/antagonists and other agents. This novel GABA receptor was sensitive to Na+ and extremely sensitive to low levels of avermectin (apparent Ki 20-40 ng/ml). This inhibition of GABA binding by avermectin was associated with a decrease in affinity (increase in Kd) and an increase in concentration of receptors (Bm). It is possible that these GABA receptors might play crucial roles in control of cell metabolism and that avermectin can prevent growth of this organism via interference in the receptor activity.

Involvement of outer membrane proteins in freeze--thaw resistance of Escherichia coli.

Two families of Escherichia coli mutants with altered outer membrane protein components were examined for sensitivity to freezing and thawing and other stresses. A mutant unable to make the lipoprotein (lpo) was extremely sensitive to freezing and thawing in water or saline and to challenge with detergent, while the mutant unable to make the porin proteins (ompB) was more resistant than the isogenic wild type; strains unable to make the tsx and ompA proteins were slightly more sensitive to the stresses. Similarly, the lpo deficient strain exhibited more and the ompB less wall and membrane damage than the wild-type strains. Little difference in the extent of wall damage, but more membrane damage, was seen for the two tsx and the ompA strains when compared with the wild-type strain. The roles of the specific proteins in determining sensitivity to freeze-thaw are discussed.

Inhibition of chitin metabolism by avermectin in susceptible organisms.

Avermectin inhibits Mucor miehei and Artemia salina chitin synthesis and to a degree DNA synthesis in the former. The antibiotic interferes with chitin turnover in brine shrimp and inhibits Streptomyces antibioticus chitinase activity in vitro. In light of the proposed mode of action of avermectin and the anomolies in the literature, it is proposed that avermectin can kill susceptible organisms not only by a neurotoxic mechanism but also by inhibiting chitin turnover and synthesis at low concentration and thus the molting/ecdysis process.

Cryosensitivity of Escherichia coli and the involvement of cyclopropane fatty acids.

Strains of Escherichia coli proficient and deficient in cylopropane fatty acid synthesis were compared for fatty acid content, cryosensitivity, presence of freeze-thaw-induced wall and membrane damage, resistance to detergent-stimulated lysis and tolerance to salt and detergents during growth. The mutant populations synthesized much less cyclopropane fatty acids and were more resistant than the wild type to freezing and thawing in saline only, exhibiting less viability loss and less wall and membrane damage. While the resistance of the mutants to NaCl was unaltered, their detergent resistance was decreased under both growth and non-growth conditions. Although these physiological changes were associated with a lower cyclopropane fatty acid content in the mutant strains, it is proposed that the responses were due to the altered membrane fluidity of the mutants due to changes in their unsaturated fatty acid content.

Cyclic AMP and cyclic GMP control of synthesis of constitutive enzymes in Escherichia coli.

Escherichia coli was grown in chemostat culture under glycerol-limited and ammonium-limited conditions at growth rates between 0.1 and 0.5 h-1. At steady state, the concentrations of cyclic AMP and cyclic GMP and the activities of four constitutive enzymes (glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, NADH oxidase and cyclic phosphodiesterase) were determined in the organism. Addition of exogenous cyclic AMP, cyclic GMP or phencyclidine perturbed the steady state and caused inhibition or stimulation of synthesis of phosphodiesterase and isocitrate dehydrogenase. A novel hypothesis is proposed to account for the ability of bacteria to regulate the synthesis of constitutive enzymes with cyclic nucleotides and possibly other small molecules.

Role of DNA repair genes and an R plasmid in conferring cryoresistance on Pseudomonas aeruginosa.

The resistance of Pseudomonas aeruginosa wild-type, uvr, pol and rec strains to ultraviolet (u.v.) light, X-rays and freezing and thawing was determined. An R plasmid, pPL1, which increased resistance of the wild-type uvr, and pol but not rec strains to u.v. light, increased the resistance of only rec and pol mutants to X-rays and freezing and thawing. These findings reinforce the idea of DNA as a target in the organism for freeze-thaw stress and suggest that freeze-thaw-induced DNA damage might be similar to that produced by X-rays but different from that produced by u.v. light.

Effect of defined lipopolysaccharide core defects on resistance of Salmonella typhimurium to freezing and thawing and other stresses.

A family of mutants of Salmonella typhimurium with altered lipopolysaccharide (LPS) core chain lengths were assessed for sensitivity to freeze-thaw and other stresses. Deep rough strains with decreased chain length in the LPS core were more susceptible to novobiocin, polymyxin B, bacitracin, and sodium lauryl sulfate during growth, to ethylenediaminetetraacetic acid and sodium lauryl sulfate in resting suspension, and to slow and rapid freeze-thaw in water and saline, and these strains exhibited more outer membrane damage than the wild type or less rough strains. Variations in the LPS chain length did not dramatically affect the sensitivity of the strains to tetracycline, neomycin, or NaCl in growth conditions or the degree of freeze-thaw-induced cytoplasmic membrane damage. The deeper rough isogenic strains incorporated larger quantities of less-stable LPS and less protein into the outer membrane than did the wild type or less rough mutants, indicating that the mutations affected outer membrane synthesis or organization or both. Nikaido's model of the role of LPS and protein in determining the resistance of gram-negative bacteria to low-molecular-weight hydrophobic antibiotics is discussed in relation to the stress of freeze-thaw.

Transient loss of plasmid-mediated mercuric ion resistance after stress in Pseudomonas aeruginosa.

After freezing and thawing, Pseudomonas aeruginosa harboring a drug resistance plasmid (Hg2+r, Strr), became acutely sensitive to mercuric ions but not to streptomycin in the plating medium, whereas its sensitivity to both agents became more pronounced indicating a synergistic effect. This freeze-thaw-induced sensitivity was transient and capable of being repaired to a simple salts medium. Transient outer and cytoplasmic membrane damage was also observed in frozen and thawed preparations. From kinetics studies, repair of cytoplasmic membrane damage superseded repair of outer membrane damage and damage measured by mercuric ions and mercuric ions plus streptomycin. Osmotically shocked cells were also sensitive to mercuric ions, mercuric ions plus streptomycin, and sodium lauryl sulfate, but not to sodium chloride or streptomycin alone. This sensitivity was again transient and capable of repair in the same simple salts medium. Active transport of a non-metabolizable amino acid, alpha-amino isobutyric acid, was sensitive to mercuric ions and became more so after freezing and thawing. A freeze-thaw-resistant mercuric ion-dependent reduced nicotinamide adenine dinucleotide phosphate oxidoreductase was localized in the cytoplasm of this organism. This enzyme and an intact outer membrane appear to be required for mercuric ion resistance in this strain.

Characterization of 3': 5' -cyclic AMP phosphodiesterase in Klebsiella aerogenes and its role in substrate-accelerated death.

Cyclic AMP phosphodiesterase in Klebsiella aerogenes is a soluble cytoplasmic enzyme with an apparent Km of 0.9 mM and a pH optimum of 7.0. It was inhibited by EDTA, Mg2+ and other metal ions. The enzyme activity was inhibited or activated by some nucleotides but not by any metabolite except pyruvate. It was inhibited by the methylxanthines, caffeine, theophylline and methylisobutylxanthine. During starvation or substrate-accelerated death, the enzyme activity remained essentially constant. It is postulated that during substrate-accelerated death the enzyme acts as a drain on the cellular cyclic AMP levels. The cyclic nucleotide concentrations during substrate-accelerated death are proposed to be controlled directly by adenylate cyclase.

ATP hydrolysis in a marine bacterium.

The membrane-bound adenosine triphosphatase of marine pseudomonad B-16, when solubilized, is able to rebind to depleted membrane residues of the bacterium and to those of Escherichia coli.

Relationship of cytochrome content to the sensitivity of bacteria to NaCl on freezing and thawing.

Eight species of bacteria representing rod, coccus, gram-positive, and gram-negative forms were tested for their sensitivity to sodium chloride during freezing and thawing. Six of the eight species tested were salt-sensitive, though to different degrees, while Lactobacillus casei and Streptococcus faecalis were resistant. Escherichia coli grown anaerobically exhibited only 38% of the salt sensitivity of aerobically grown cells. Analysis of cytochrome pigments in the organisms revealed that the six sensitive organisms all contained these pigments but in varying amounts, while the two resistant ones were devoid of them. Anaerobically grown E. coli contained 50% of the cytochromes of aerobically grown cells. A relationship between cytochrome content of the organisms and salt sensitivity during freezing and thawing was demonstrated with a correlation coefficient of 0.76 (P less than 0.05); the higher the cytochrome content, the more salt-sensitive the organism. This indicated that 58% of the salt sensitivity was due to the cytochrome content. Using a model organism E. coli, the effect of salt during freezing and thawing on the respiratory activity was examined. Freezing and thawing in water or saline decreased the respiration by whole cells of substrates expected to be NAD-linked while NADH-stimulated respiration was increased. In cell-free extracts derived from unfrozen cells or those frozen and thawed in water or saline, the respiration of ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) was constant. The respiration of NADH, succinate, and lactate in cell-free extracts derived from cells frozen and thawed in saline was reduced compared with those extracts derived from unfrozen cells or cells frozen and thawed in water. Studies with E. coli showed that the decreased respiratory activity caused by disruptions in the electron-transport chain could not account for the salt sensitivity on freezing and thawing. More likely, salt sensitivity is related to the presence of bonds between cytochromes and other membrane components which are disrupted by sodium chloride on freezing and thawing. This would then result in loss of membrane integrity and function.

The effect of cooling and warming rates on the survival of a variety of bacteria.

Cooling and warming rates affect bacterial survival profoundly with all bacteria tested (Azotobacter chroococcum, Klebsiella aerogenes, Salmonella typhimurium, Pseudomonas aeruginosa, Streptococcus faecalis) behaving similarly. Most bacteria were sensitive to salt on freezing and thawing. Viabilities of population frozen and thawed in saline were always less (by at least 20%) than comparable ones frozen in water alone. Under these conditions, S. faecalis was resistant to the presence of sodium chloride; viabilities of populations frozen in the presence or absence of sodium chloride were always less than 5% different. The implications of these results in light of the organisms' shape, Gram stain, and respiration are discussed.

The survival of Escherichia coli from freeze-thaw damage: the relative importance of wall and membrane damage.

When Escherichia coli is frozen rapidly in saline and thawed slowly, survival is very low; however, the inclusion of 3% glycerol or 1% Tween 80 in the saline freezing menstruum results in near complete survival. The release of material from, and penetration of, substances into the cell indicate that both membrane and wall damage occur during freezing and thawing. Glycerol, under these conditions, is able to reduce severely both the damage to the wall and membrane, whereas Tween 80 prevents only membrane damage. This indicates that freezing and thawing in saline results in membrane damage which is lethal to the cell whereas wall damage which occurs is not detrimental to cell survival.

The survival of Escherichia coli from freeze-thaw damage: permeability barrier damage and viability.

The effect of cooling rate and subsequent warming rate on survival of lactose-limited Escherichia coli was investigated. As previously reported, in the slow cooling rate range, a peak of survival was noted at 8 degrees C/min with survival decreasing as the cooling rate was increased or decreased from this value. Minimal survival was noted at 100 degrees C/min; increasing the cooling rate above 100 degrees C/min increased survival. At cooling rates greater than 200 degrees C/min, the survival became dependent on subsequent warming rates. Permeability damage, as measured by release of UV-absorbing material, potassium and beta-galactosidase, and increased accessibility of glucose-6-phosphate dehydrogenase to its substrates, was dependent on the cooling rate when cells were frozen in either water or saline. For cooling rates less than about 8 degrees C/min, there was minimal permeability damage to cells frozen in water. However, at rates greater than this value, damage and viability were related; the lower the viability the more the damage to the permeability barrier. The relationship was strengthened by the observations that protectants which increased survival reduced damage as well and that at ultrarapid cooling rates where survivals were dependent on warming rates, the extent, of damage was likewise dependent on the warming rate. Saline frozen cells were damaged by freezing and thawing more than comparable water-frozen cells over the whole cooling rate range. At cooling rates less than 8 degrees C/min, frozen in water, permeability damage of cells frozen in saline increased as the cooling rate decreased. As the cooling rate was increased from 8 degrees C/min, the damage increased as viability decreased. The relevance of these findings to the two-factor hypothesis of cell death is discussed.