RESUMEN
Artificial generation of oxygen superoxide radicals in actively growing cultures of Mycobacterium tuberculosis, Myc. smegmatis, and Corynebacterium ammoniagenes is followed by accumulation in the bacterial cells of substantial amounts of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcDP) - an intermediate of the non-mevalonate pathway of isoprenoid biosynthesis (MEP) - most possibly due to the interaction of the oxygen radicals with the 4Fe-4S group in the active center and inhibition of the enzyme (E)-4-oxy-3-methylbut-2-enyl diphosphate synthase (IspG). Cadmium ions known to inhibit IspG enzyme in chloroplasts (Rivasseau, C., Seemann, M., Boisson, A. M., Streb, P., Gout, E., Douce, R., Rohmer, M., and Bligny, R. (2009) Plant Cell Environ., 32, 82-92), when added to culture of Myc. smegmatis, substantially increase accumulation of MEcDP induced by oxidative stress with no accumulation of other organic phosphate intermediates in the cell. Corynebacterium ammoniagenes'', well-known for its ability to synthesize large amounts of MEcDP, was also shown to accumulate this unique cyclodiphosphate in actively growing culture when NO at low concentration is artificially generated in the medium. A possible role of the MEP-pathway of isoprenoid biosynthesis and a role of its central intermediate MEcDP in bacterial response to nitrosative and oxidative stress is discussed.
Asunto(s)
Corynebacterium/metabolismo , Difosfatos/metabolismo , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/metabolismo , Estrés Oxidativo , Especies de Nitrógeno Reactivo/metabolismo , Superóxidos/metabolismo , Terpenos/metabolismo , Vías Biosintéticas , Ácido Mevalónico/metabolismoRESUMEN
We have found that transition of actively dividing Mycobacterium smegmatis cells into the dormant "nonculturable" state is accompanied by increase in the protein/lipid ratio and disappearance of one of the main lipid components of the mycobacterial cells, trehalose monomycolate. In this case, oleic acid is accumulated in the culture medium due to its secretion by the mycobacterial cells. Addition of lipids of different classes to "nonculturable" M. smegmatis cells induces their resuscitation. The lipid reactivating effect is evidently caused by the presence of fatty acids in their composition, because free fatty acids also exhibited reactivation effect. Oleic acid in concentration of 0.05-3 µg/ml exhibited maximal effect, and that allows us to draw a conclusion concerning its signal role in the transition of dormant cells into active state.
Asunto(s)
Lípidos/fisiología , Mycobacterium smegmatis/metabolismo , Cromatografía en Capa Delgada , Factores Cordón/análisis , Cromatografía de Gases y Espectrometría de Masas , Lípidos/análisis , Ácido Oléico/farmacología , Fosfatidilcolinas/farmacologíaRESUMEN
2-C-Methyl-D-erythritol-2,4-cyclopyrophosphate (MEC), an intermediate of the biosynthesis of isoprenoid compounds in bacteria, was found to be capable of exerting a resuscitating effect on resting Mycobacterium smegmatis cells. The introduction of an additional copy of the ispE gene encoding cytidyl-methylerythritol kinase, an enzyme involved in MEC synthesis in M. smegmatis, resulted in the emergence of a capacity for spontaneous reactivation of "nonculturable" M. smegmatis cells, which is not characteristic of the wild-type cells of this species. The involvement of MEC in the transition from the "nonculturable" state to the state of active growth is indicative of a previously unknown function of MEC, assumed to consist in regulation of the bacterial genome activity.
Asunto(s)
Eritritol/fisiología , Mycobacterium smegmatis/crecimiento & desarrollo , Medios de Cultivo , Eritritol/análogos & derivados , Eritritol/química , Genes Bacterianos/genética , Mycobacterium smegmatis/genética , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Terpenos/metabolismo , Transformación BacterianaRESUMEN
To date, the possible existence of "nonculturable" (NC) but potentially viable forms has been shown for some bacteria. NC mycobacteria have attracted particular interest due to the assumption that the latent form of tuberculosis is associated with the conversion of its causative agent, Mycobacterium tuberculosis, into the NC state. A number of approaches have been developed to obtain NC forms of mycobacteria, but the mechanisms of transition into or from this state have been insufficiently studied. This review considers cell-cell communications involved in the formation and reactivation of NC forms of the bacteria M. smegmatis and M. tuberculosis. Special attention has been paid to the secreted Rpf family proteins, which belong to peptidoglycan hydrolases and participate in the resuscitation of NC mycobacteria.
Asunto(s)
Mycobacterium/fisiología , Proteínas Bacterianas/fisiología , Medios de Cultivo , Citocinas/fisiología , Mycobacterium/crecimiento & desarrollo , Mycobacterium/metabolismo , Infecciones por Mycobacterium/microbiología , Transducción de Señal/fisiologíaRESUMEN
Diagnostic methods for detecting forms and strains of Helicobacter pylori isolated from biopsy specimens of gastric mucosa in 28 patients with duodenal ulcers and evaluation of its eradication were compared. Biopsy specimens from all patients were tested for the presence of H. pylori by the urease test, histological method, and PCR with species-specific primers before and after treatment. H. pylori infection was detected in all patients before treatment, the mean titer of serum IgG being 36.7+/-16.6 U/ml. Biopsy specimens positive for H. pylori in PCR were subjected to restriction analysis of specific PCR-amplified genes or their fragments. The fingerprint analysis gave electrophoregrams of restriction products amplified fragment of flaA gene of H. pylori in 7 patients. Differences in restriction maps indicate the presence of 5 H. pylori strains in the studied samples.
Asunto(s)
Úlcera Duodenal/diagnóstico , Úlcera Duodenal/microbiología , Infecciones por Helicobacter/diagnóstico , Infecciones por Helicobacter/tratamiento farmacológico , Helicobacter pylori/aislamiento & purificación , Ranitidina/análogos & derivados , Adolescente , Adulto , Anciano , Anticuerpos Antibacterianos/sangre , Bismuto/uso terapéutico , Dermatoglifia del ADN , Úlcera Duodenal/tratamiento farmacológico , Úlcera Duodenal/patología , Femenino , Flagelina/genética , Mucosa Gástrica/microbiología , Mucosa Gástrica/patología , Infecciones por Helicobacter/patología , Helicobacter pylori/genética , Helicobacter pylori/inmunología , Humanos , Inmunoglobulina G/sangre , Masculino , Persona de Mediana Edad , Omeprazol/uso terapéutico , Reacción en Cadena de la Polimerasa , Ranitidina/uso terapéutico , Recurrencia , Mapeo Restrictivo , Especificidad de la Especie , Factores de Tiempo , Ureasa/sangreRESUMEN
Two membrane antigens were found by cross immunoelectrophoresis in the cell walls of Bacillus brevis var. G.-B., R form, which started to synthesize gramicidin S (20 mg per 1 ml of cultural broth). The cell wall contained no membrane components in cells at the beginning of the logarithmic growth phase. The protein with a molecular mass of 100 kDa is a component of the cell wall outer layer. The protein is not digested by trypsin or pronase when it comprises the cell walls of cells synthesizing gramicidin S. In the preparation of isolated cell walls, this protein becomes susceptible to the action of the above proteases only when the peptidoglycan layer is broken down by lysozyme. Electron microscopy of cells treated with proteases and shadowed with a metal revealed that many cells lacked the cytoplasm. Therefore, the outer layer of B. brevis R cell wall contains small regions susceptible to the action of protease along with regions composed of the 100 kDa protein and resistant to these enzymes. It is possible that the small regions contain membrane components.
Asunto(s)
Bacillus/análisis , Gramicidina/biosíntesis , Antígenos Bacterianos/análisis , Antígenos de Superficie/análisis , Bacillus/inmunología , Bacillus/metabolismo , Proteínas Bacterianas/análisis , Pared Celular/análisis , Pared Celular/efectos de los fármacos , Pared Celular/inmunología , Contrainmunoelectroforesis/métodos , Micrococcus/efectos de los fármacos , Péptido Hidrolasas/farmacología , Fosfolipasas/aislamiento & purificación , Fosfolipasas/farmacologíaRESUMEN
The morphology of cells and cell walls was studied in the Bacillus brevis G.-B. R form during its growth and gramicidin S accumulation in it. The membrane apparatus became more complicated and certain other morphological changes were detected in the cells with aging. The cell wall was rather complex even in young cells and consisted of three electron-dense layers where the external and internal layers had an ordered structure. Only the external layer underwent some modifications in the course of growth and these coincided in time with the beginning of intensive gramicidine S biosynthesis. However, the three-layer structure of the cell wall and the ordered organization of the external and internal layers remained unchanged. A preparation of cell walls and preparations of their external and internal layers were isolated from cells synthesizing gramicidine S in the amount of 20 micrograms/ml of the cultural broth. An acid protein having the molecular mass of 100 kD was shown to be the major component of the external layer according to the data of electrophoresis in PAAG with SDS. The middle layer was sensitive to lysozyme, did not have a ordered structure on electron micrographs, and consisted mainly of peptidoglycan.
Asunto(s)
Bacillus/ultraestructura , Pared Celular/ultraestructura , Gramicidina/biosíntesis , Aminoácidos/metabolismo , Bacillus/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Pared Celular/metabolismo , Técnicas In Vitro , Microscopía Electrónica , Peptidoglicano/metabolismoRESUMEN
The effect of a preparation obtained by butanol extraction of the culture fluid of B. cereus and Ps. carboxydoflava and previously termed an autoregulatory factor d1 on the respiration chain within intact bacterial cells and isolated membranes was investigated. In comparable concentrations this factor d1 inhibits the endogenous respiration of B. cereus and M. lysodeikticus as well as oxidase and dehydrogenase activities of isolated membranes from these bacteria and E. coli. The factor-induced decrease of the cell respiration rate is independent from disorders of the cell permeability osmotic barrier for respiration substrates. The factor d1 shows a higher effect at acidic pH values. It is concluded that the above preparation has membrane-active properties. It is also suggested that in the bacterial cell its target is the inner surface of the cytoplasmic membrane.
Asunto(s)
Bacillus cereus/fisiología , Membrana Celular/efectos de los fármacos , Medios de Cultivo/farmacología , Homeostasis/efectos de los fármacos , Pseudomonas/fisiología , Bacillus cereus/efectos de los fármacos , Medios de Cultivo/aislamiento & purificación , Escherichia coli/efectos de los fármacos , Micrococcus/efectos de los fármacos , Oxígeno/metabolismoRESUMEN
The culture of Bacillus brevis var. G-B R-form was grown in the presence of beta-phenyl-beta-alanine, the inhibitor of gramicidin S synthesis, is characterized by enhanced endogenous respiration and the DPI-reductase activity as compared to the culture synthezising antibiotic. The increased synthesis of the antibiotic in the region of the culture transition from the logarithmic growth phase to the linear one is associated with a decrease in the number of viable cells despite the fact that the culture on the whole does not die but continues to grow. The membranes prepared from young gramicidin S-free cells and from the cells enriched with the antibiotic possess identical electron micrograph images, IR spectra and protein sets as determined by polyacrylamide gel electrophoresis in a Na-DS system. However, in young cell membranes NADH and succinate dehydrogenase are insensitive to gramicidin S and only malate dehydrogenase is inhibited by this antibiotic. In aged cell membranes the activities of all mentioned dehydrogenases are suppressed. Malate dehydrogenase from young cells is weakly inhibited by thyrotrycin obtained from Bac. brevis ATCC 10068; succinate dehydrogenase is entirely insensitive to this antibiotic, while NADH-dehydrogenase is almost completely inhibited by it. The specificity of action on the respiratory chain of peptide antibiotics synthesized by the cells of one strain of Bac. brevis is suggestive of a possible regulatory role of these peptides in the metabolism of the producent. Hence the accumulation of gramicidin S which is adsorbed on the membrane and destroys the respiratory chain function to the cause of the low rate of oxygen uptake by the culture of Bac. brevis var. G-B R-form and of the low activities of DPI-reductases.
Asunto(s)
Alanina/farmacología , Bacillus/metabolismo , Gramicidina/biosíntesis , beta-Alanina/farmacología , Bacillus/efectos de los fármacos , Bacillus/crecimiento & desarrollo , Membrana Celular/enzimología , Cinética , Malato Deshidrogenasa/antagonistas & inhibidores , NADH Deshidrogenasa/antagonistas & inhibidores , Consumo de Oxígeno , Succinato Deshidrogenasa/antagonistas & inhibidores , beta-Alanina/análogos & derivadosRESUMEN
It was shown that malate dehydrogenase of isolated membranes of the gramicidin S producer Bacillus brevis var. G.-B. (R.-form) is completely inhibited by the antibiotic (approximately 200 mkg/mg of protein). Succinate and NADH dehydrogenases at concentration up to 1 mg per mg of protein are insensitive to it, while corresponding oxidases are inhibited by the antibiotic not more than by 65 -- 75% apparently due to partial damage of the terminal parts of the respiratory chain. The respiration of the producer intact cells is inhibited by exogenous gramicidin S by not more than 55 -- 60%, while the respiration of antibiotic-sensitive cells of M.lysodeikticus is inhibited completely. It was shown that phosphatidyl ethanolamine (50%), phosphatidyl glycerol (15% and diphosphatidyl glycerol (25%) are the major phospholipid components of the membranes of the given strain of Bac. brevis. It was assumed that the resistance of Bac. brevis cells to gramicidin S is partly due to the constant ratio of the charged and amphoteric phospholipids. Using 31P-NMR spectroscopy, the kinetics of free phosphoric compounds in the cells and cell extracts of Bac. brevis during culture growth and gramicidin S synthesis were studied. The content of carbohydrate monophosphate, remained unaffected, while that of nucleoside di- and triphosphates and dinucleotides was low and at definite density and gramicidin S content (above 100 mkg/ml) fell down below the resolution capacity of the method employed. Evidence for gramicidin S localization of the Bac. brevis membrane and possible causes for the manifestation of the NADH dehydrogenase activity at a certain stage of culture growth are discussed.
Asunto(s)
Bacillus/enzimología , Reductasas del Citocromo/metabolismo , Gramicidina/fisiología , Malato Deshidrogenasa/metabolismo , NADH Deshidrogenasa/metabolismo , Succinato Deshidrogenasa/metabolismo , Membrana Celular/enzimología , Cinética , Espectroscopía de Resonancia Magnética , Lípidos de la Membrana/fisiología , Fosfolípidos/fisiologíaRESUMEN
The study on the possibility of eliminating gramicidin S from the bacterial cells which had adsorbed it showed that a part of the labeled antibiotic bound by the bacteria may be washed out with buffer or salines. When the cells which had adsorbed gramicidin S were treated with lecithin emulsion, a significant part of the bound antibiotic was transferred to the lecithin liposomes. This turned the gramicidin S effect to the cells: significant but not complete reduction of the membrane barrier properties and dehydrogenase reactivation. Elimination of gramicidin S also reduced the colony forming capacity in a part of the cells.
Asunto(s)
Bacterias/efectos de los fármacos , Gramicidina/farmacología , Adsorción , Bacillus/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Emulsiones , Escherichia coli/efectos de los fármacos , Liposomas/farmacología , Micrococcus/efectos de los fármacos , Fosfatidilcolinas/farmacología , Staphylococcus aureus/efectos de los fármacosRESUMEN
The effects of various metall-containing proteins (plastocyanin, plantacyanin, azurine and cytochromes of the f type) on the activity of photosystem I of chloroplasts, treated with polyene antibiotics, were studied. The inhibiting effect of the polyenes, surgumycin and philipin, was completely removed by an addition of copper-containing protein plastocyanin. No similar effect was exerted by other Cu-containing proteins--azurine and plantacyanin. The cytochromes of the f type isolated from the green algae chlorella, blue-green algae spiruline and aphanezomenone, having different electrophoretic properties, restored the activity of photosystem I of chloroplasts incubated with antibiotics in a different degree. Acid cytochrome f of chlorella restored the activity by 80--100%; less acid cytochrome f from spiruline-only by 50%. The least restoring effect was exerted by aphanezomenone cytochrome, which possesses some basic properties. The chloroplasts treatment with surgumycin did not affect the isolation of the terminal enzyme of the chloroplast electron-transporting chain of ferredoxin--NADP--reductase. Possible environment of plastocyanin in the chloroplast membrane and the mechanism of photosystem I restoration are discussed.