Study of the adaptation resistance in bacteria to membrane active polypeptide antibiotics

Variants of Micrococcus lysodeikticus resistant to 100 micrograms/ml of gramicidin S with preserved resistance in subcultures on media without the antibiotic were isolated as a result of prolonged adaptation on a solid medium with increasing concentrations of gramicidin. The sensitive and resistant cells did not differ by their ability to bind gramicidin. Under the antibiotic effect permeability of the cytoplasmic membranes of the intact cells in the sensitive bacteria appeared to be impaired to a greater extent than that of the membranes of the cells in the resistant variant. Comparison of the lytic activity of gramicidin and its derivatives with respect to the protoplasts prepared with the cells of the initial and resistant variants of M. lysodeikticus revealed much higher resistance of the resistant variant protoplasts to the membrane-disorganizing effect of the preparations. Malate dehydrogenase and NADH-oxidase in the membrane preparations of the resistant variant cells differed from analogous enzymes from the membranes of the initial strain by the levels of their activity and sensitivity to gramicidin. It is likely that during adaptation of M. lysodeikticus to gramicidin significant changes in the cell cytoplasmic membranes occurred.     

Effects of light adaptation on the purple membrane structure of Halobacterium halobium.

Absorption, circular dichroism and optical rotatory dispersion of the bacteriorhodopsin containing purple membrane form Halobacterium halobium were studied in regard to the structural stability of this membrane during the photoisomerization of the retinal of the bacteriorhodopsin from the 13-cis to the all-trans configuration. The following conclusions were reached: (a) the macromolecular structure (protein-protein interaction which may result in the possible exciton interaction of the retinal pi-pi* (NV1) transition moments and protein-lipid interaction) are not significantly altered, (b) possibilities of delocalized conformation changes of the apoprotein involving secondary and/or tertiary structure can be ruled out, (c) localized secondary structure conformation changes of the apoprotein must be limited to the involvement of no more than one or two amino acid residues and localized tertiary structure conformation changes of the apoprotein must be limited to a very short segment of the protein chain containing only a few aromatic amino acid residues, and (d) the interaction between the apoprotein and retinal seems to be relatively more pronounced when the retinal is in the all-trans form than the 13-cis from and also the apoprotein seems to impose a more pronounced dissymmetric constraint on the retinal in the all-trans form than in the 13-cis form.     

C-ring cleavage of flavonoids by human intestinal bacteria

Four hitherto undescribed Clostridium strains capable of cleaving the C ring of quercetin, kaempferol, and naringenin at C-3-C-4 were isolated from the fecal flora of humans. None of the strains cleaved catechin. C-ring fission occurred when the substrate was either in solution or in suspension. Mixed cultures of flavonoid-hydrolyzing bacteria, flavonoid-cleaving bacteria, and Escherichia coli, which was used to provide the anaerobic environment, rapidly metabolized rutin to 3,4-dihydroxyphenylacetic acid, indicating that the intestinal half-life of the biologically active aglycone is short. The cleaving strains shared many phenotypic characteristics, including their inability to ferment sugars, but they differed sufficiently to indicate that they represent different species.     

Impact of mutation rate on the adaptation of gut bacteria

To study the role of mutator bacteria in the evolution of bacterial populations, we followed the impact of the mutation rate of Escherichia coli strains in the colonisation of the gut of axenic mice and the evolution of the mutation rate of bacterial populations living in the gut. We show that mutator bacteria have an advantage during the colonization. This adaptive advantage comes from their ability to generate adaptive mutations faster than wild type strains, mutations that allow their maintenance in the ecosystem. However, while mutator bacteria are becoming specialised to the environment they are living in, they accumulate mutations that may be deleterious or lethal in secondary environments. By following the evolution of the mutation rate of bacterial populations living in the gut of mice receiving antibiotics, we show that this therapy selects not only for antibiotic resistant mutants but also for mutator alleles that enhance mutation rates and are responsible for the appearance of the resistance. The costs of a high mutation rate, due to the accumulation of mutations, is seen in environments where changes are recurrent. In an ever-changing situation where every change is new, mutator bacteria might help the evolution of bacterial populations.     

Hypermutation and stress adaptation in bacteria

Hypermutability is a phenotype characterized by a moderate to high elevation of spontaneous mutation rates and could result from DNA replication errors, defects in error correction mechanisms and many other causes. The elevated mutation rates are helpful to organisms to adapt to sudden and unforeseen threats to survival. At the same time hypermutability also leads to the generation of many deleterious mutations which offset its adaptive value and therefore disadvantageous. Nevertheless, it is very common in nature, especially among clinical isolates of pathogens. Hypermutability is inherited by indirect (second order) selection along with the beneficial mutations generated. At large population sizes and high mutation rates many cells in the population could concurrently acquire beneficial mutations of varying adaptive (fitness) values. These lineages compete with the ancestral cells and also among themselves for fixation. The one with the ‘fittest’ mutation gets fixed ultimately while the others are lost. This has been called ‘clonal interference’ which puts a speed limit on adaptation. The original clonal interference hypothesis has been modified recently. Nonheritable (transient) hypermtability conferring significant adaptive benefits also occur during stress response although its molecular basis remains controversial. The adaptive benefits of heritable hypermutability are discussed with emphasis on host–pathogen interactions.     

Transposable elements and adaptation of host bacteria

A transposable element (TE) is a mobile sequence present in the genome of an organism. TEs can cause lethal mutations by inserting into essential, genes, promoting deletions or leaving short sequences upon excision. They therefore may be gradually eliminated from mixed populations of haploid micro-organisms such asEscherichia coli if they cannot balance this mutation load. Horizontal transmission between cells is known to occur and promote the transfer of TEs, but at rates often too low to compensate for the burden to their hosts. Therefore, alternative mechanisms should be found by these elements to earn their keep in the cells. Several theories have been suggested to explain their long-term maintenance in prokaryotic genomes, but little molecular evidence has been experimentally obtained. In this paper, the permanence of transposable elements in bacterial populations is discussed in terms of costs or benefits for the element and for the host. It is observed that, in all studies yet reported, the elements do not behave in their host as selfish DNA but as a co-operative component for the evolution of the couple.     

Influence of the culture medium on antibiotic susceptibility testing of food-associated lactic acid bacteria with the agar overlay disc diffusion method

AIMS: To investigate the influence of the culture medium on antibiotic susceptibility testing of food-associated lactic acid bacteria (LAB) with the agar overlay disc diffusion (DD) method. METHOD: The antibiotic resistance profile of 39 food-associated lactobacilli and enterococci was determined with the agar overlay DD method using a defined medium (i.e. Iso-sensitest agar; ISA) or an undefined medium (i.e. de Man, Rogosa, Sharpe or MRS agar). RESULTS: The study revealed that ampicillin discs and, although to a lesser extent, also tetracycline discs consistently produced larger zones on MRS medium compared to ISA medium. For the antibiotics gentamicin, bacitracin and erythromycin, the radius of the inhibition zones produced on MRS medium was significantly smaller in relation to ISA. For categorizing LAB isolates into resistant, intermediate and susceptible groups, it was demonstrated that major errors can occur in determining bacitracin and gentamicin resistance if MRS medium instead of ISA medium is used. On the other hand, the performance of both media was found to be equivalent for testing tetracycline resistance. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Despite the fact that MRS medium generally supports the growth of lactic acid bacteria much better than the nutrient-poor ISA medium, the present study clearly demonstrates that both media are not compatible in susceptibility testing against various classes of antibiotics. These results may stimulate future discussions on a generally recommended DD method for susceptibility testing of food LAB strains.     

Variability of pressure adaptation in deep sea bacteria

Isolations of pressure-adapted deep sea bacteria from depths of 1,400 to 5,100 m resulted in a variety of psychrophilic barotolerant and barophilic strains. Growth rates determined at different pressures indicated a gradual transition between the two types of pressure-adapted isolates. The presence of barotolerant bacteria in deep water, sustained by sinking particulate matter, causes the nonbarophilic response of natural populations, i.e., increased growth after decompression. With increasing pressure-adaptation in barophilic isolates the maximum growth rates at optimum pressures decrease. Thus, the observed general slow-down of microbial activity in the deep sea takes effect regardless of the common occurrence of psychrophilic and barophilic bacteria. The highest degree of barophilism was observed in isolates from nutrient-rich habitats such as intestinal tracts of deep sea animals or decaying carcasses. Detailed studies with an isolate, growing barophilically on a complex as well as a single-carbon-source medium, showed that (1) culturing at pressures lower than optimal for growth resulted in the formation of cell filaments, (2) growth was unaffected by repeated compression/decompression cycles and (3) no perceptible differences in the distribution of radiolabeled carbon from an amino acid mixture occurred in cells grown at, below and above the pressure optimal for growth.Dedicated to Professor Dr. Hans G. Schlegel on the occasion of his 60th birthday in recognition of his broad microbiological interests and in special appreciation of his lasting support for the Marine Microbiology Course at the Stazione Zoologica (Naples, Italy) now for almost 25 yearsNon-standard abbreviations. The traditional use of atm as a unit of pressure (=10 m of water column, =1.013 bar, =101.3 kN/m²) is retained here in view of the important relation between water depth and hydrostatic pressure in the present study. Due to the compression of seawater and the geographic variability of gravity, there is a progressive deviation of the actual pressure with depth amounting to +4.9 atm at 5,000 m and a latitude of 30°. EPC, cell counts obtained by epifluorescence microscopy. PY, peptone yeast extract medium     

Modulation of liposomal membrane fluidity by flavonoids and isoflavonoids

The polyphenolic structures of flavonoids and isoflavonoids confer them with the ability to scavenge free radicals and to chelate transition metals, a basis for their potent antioxidant abilities. Another possible contributory mechanism toward their antioxidant activities is their ability to stabilize membranes by decreasing membrane fluidity. In this study, the effects of representative flavonoids, isoflavonoids, and their metabolites on membrane fluidity and their preferential localization in the membrane were investigated using large unilamellar vesicles (LUVs) as the membrane models. These results were compared with those of cholesterol and alpha-tocopherol. Changes in fluorescence anisotropy values for a series of n-(9-anthroyloxy) fatty acid probes (n = 6, 12, 16) upon addition of the test compounds were used to monitor alterations in membrane fluidity at graded depths in lipid bilayer. The results of the study suggest that the flavonoids and isoflavonoids, similar to cholesterol and alpha-tocopherol, partition into the hydrophobic core of the membrane and cause a dramatic decrease in lipid fluidity in this region of the membrane. Localization of flavonoids and isoflavonoids into the membrane interiors and their resulting restrictions on fluidity of membrane components could sterically hinder diffusion of free radicals and thereby decrease the kinetics of free radical reactions.     

Effects of thermoradiation on bacteria.

A 60Co source was used to determine the effects of thermoradiation on Achromobacter aquamarinus, Staphylococcus aureus, and vegetative and spore cells of Bacillus subtilis var. globigii. The rate of inactivation of these cultures, except vegetative-cell populations of B. subtilis, was exponential and in direct proportion to temperature. The D10 (dose that inactivates 90% of the microbial population) value for A. aquamarinus was 8.0 Krad at 25 degrees C and 4.9 Krad at 35 degrees C. For S. aureus, D10 was 9.8 and 5.3 Krad at 35 and 45 degrees C, respectively. Vegetative cells of B. subtilis demonstrated a rapid initial inactivation followed by a steady but decreased exponential rate. The D10 at 25 degrees C was 10.3 Krad, but at 35 and 45 degrees C this value was 6.2 and 3.8 Krad, respectively. Between 0 and 95 Krad, survival curves for B. subtilis spores at 75 degrees C showed slight inactivation, increasing in rat at and above 85 degrees C. The D10 values for spores at 85 and 90 degrees C were 129 and 92 Krad, respectively. Significant synergism between heat and irradiation was noted at 35 degrees C for A. aquamarinus and 45 degrees C for S. aureus. The presence of 0.1 mM cysteine in suspending media afforded protection to both cultures at these critical temperatures. On the other hand, cysteine sensitized B. subtilis spores at radiation doses greater than 100 Krad. The combined effect of heat and irradiation was more destructive to bacteria than either method alone.     

Mechanism of action of food-associated polycyclic aromatic hydrocarbon carcinogens

The polycyclic aromatic hydrocarbon carcinogens are formed in the inefficient combustion of organic matter and contaminate foods through direct deposition from the atmosphere or during cooking or smoking of foods. These potent carcinogens and mutagens require metabolism to dihydrodiol epoxide metabolites in order to express their biological activities. In vitro studies show that these reactive metabolites can react with the bases in DNA with different specificities depending upon the hydrocarbon from which they are derived. Thus, the more potent carcinogens react more extensively with adenine residues in DNA than do the less potent carcinogens, with the result that mutation at A . T base pairs is enhanced for the more potent carcinogens. In the past few years, considerable clarification of the mechanism of metabolic activation have been achieved and the focus for the immediate future is expected to be on how the reactive metabolites actually bring about biological responses.     

Effects of behavioral adaptation on a predator-prey model

The Volterra-Lotka predator-prey equations are modified so that the predator's ability to utilize the prey varies in proportion to the average number of encounters between the two species in the past. The behavior of this adaptive system is then described in terms of three parameters — the carrying capacity of the prey, the relative death rate of the predator, and the predator's memoryspan. The most stable situation is shown to occur when the carrying capacity of the prey is large, the predator's death rate is close to zero, and the predator is able to adapt quickly to changing levels of prey density.     

Influence of salt concentration on membrane lipids of halophilic bacteria

Abstract A review of salt-dependent changes in membrane lipid composition of halotolerant, moderately halophilic, and extremely halophilic bacteria is presented. The biosynthetic and regulatory mechanisms underlying the observed changes are discussed. Possible implications for the evolution of extreme halophiles and other Archaebacteria are also discussed.