RESUMEN
Protoplasts of Bacillus megaterium readily reverted to bacillary form in liquid media and when plated in a soft-agar layer onto the surface of appropriate agar media. Three phases of the reversion sequence could be differentiated by phase contrast microscopy: (i) increase in size of the individual protoplasts, (ii) non oriented division of the protoplasts and (iii) outgrowth of the bacillary forms. With time-lapse photomicrography, reversion sequences of single protoplasts were demonstrated.
Asunto(s)
Bacillus megaterium/citología , Protoplastos/citología , Agar , Bacillus megaterium/crecimiento & desarrollo , División Celular , Medios de Cultivo , Protoplastos/crecimiento & desarrolloAsunto(s)
Cristalización , Microtúbulos/ultraestructura , Proteínas/análisis , Protoplastos/ultraestructura , Saccharomyces/ultraestructura , Núcleo Celular , Cristalografía , Cicloheximida/farmacología , Citoplasma , Proteínas Fúngicas/biosíntesis , Técnicas Histológicas , Cuerpos de Inclusión , Microscopía Electrónica , Microscopía de Contraste de Fase , Microtúbulos/análisis , Mitocondrias , Protoplastos/análisis , Protoplastos/crecimiento & desarrollo , Saccharomyces/crecimiento & desarrollo , Proteínas ViralesRESUMEN
The effect of a synthetic glucose analogue, 2-deoxy-2-fluoro-d-glucose (FG) on growth and glucose metabolism of Saccharomyces cerevisiae was studied. The addition of FG (0.005-0.05%) to a 2% glucose medium resulted in reduction of the initial growth rate and, after several hours, in a complete cessation of the culture growth. These two events were due to extensive lysis of the population which continued long after the period when no more growth was recorded. Electron microscope examination of lysed cells showed that the lysis was a consequence of a dissolution of the cell walls. FG inhibited to a similar extent the initial growth rate and the incorporation of radioactivity from labeled glucose into growing population. The inhibition of radioactivity incorporation from glucose by growing protoplasts was much less. The yeast was found to be extremely FG sensitive whenever the synthesis of new cell wall material was involved. All observations imply that FG interferes mainly with the cell wall formation of S. cerevisiae. A comparison of the FG effects on metabolic activity of protoplasts, simultaneous secretion of mannan-proteins into the growth medium, and the formation of glucan fibrils on the surface of protoplasts demonstrated that the cell wall glucan synthesis is the most FG-sensitive process and evidently the growth-limiting factor in intact cells. FG-resistant cells were selected during growth experiments. They exhibited an altered mode of cell division when grown in the presence of FG.
Asunto(s)
Glucosa/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Autólisis , Dióxido de Carbono/biosíntesis , Isótopos de Carbono , División Celular , Pared Celular/metabolismo , Flúor/farmacología , Proteínas Fúngicas/biosíntesis , Glucosa/metabolismo , Manometría , Microscopía Electrónica , Polisacáridos/biosíntesis , Protoplastos/efectos de los fármacos , Protoplastos/crecimiento & desarrollo , Protoplastos/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismoRESUMEN
The physiological differences between Bacillus subtilis (ATCC 6633) cells derived from a glucose-salts-yeast extract (GSY) medium and those of cells from tryptose broth permitted the identification of variables in protoplasting environments which noticeably affected the clarity of mesosomal ribosomes. They were the sucrose and magnesium ion concentrations and the type of buffer used. The environment suitable for conversion of GSY cells to the protoplast state was a 0.02 M tris(hydroxymethyl)aminomethane-hydrochloride buffer, pH 7.2, containing 0.6 M sucrose and 0.03 M MgCl(2). Branched mesosomal tubules and a unique organization of vesicles were detected in thin sections and in negative stains of the specimens. Ribosomes were demonstrable in the extruded structures associated with protoplasts that had been prepared according to four fixation schedules and embedded in either of two epoxy plastics. Adjustments in the fixation schedules improved the clarity of the large bodies of protoplast cytoplasm to a degree equivalent to that of their dangling appendages.
Asunto(s)
Bacillus subtilis/citología , Protoplastos/citología , Ribosomas , Bacillus subtilis/crecimiento & desarrollo , Técnicas Bacteriológicas , Tampones (Química) , Medios de Cultivo , Citoplasma , Resinas Epoxi , Glutaral , Concentración de Iones de Hidrógeno , Magnesio , Microscopía Electrónica , Ácido Fosfotúngstico , Protoplastos/crecimiento & desarrollo , Coloración y Etiquetado , Sacarosa , TrometaminaAsunto(s)
Bacillus subtilis/enzimología , Desoxirribonucleasas/análisis , Ribonucleasas/análisis , Bacteriólisis , Fraccionamiento Celular , Pared Celular/enzimología , Medios de Cultivo , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Microscopía Electrónica , Protoplastos/enzimología , Protoplastos/crecimiento & desarrolloAsunto(s)
Histoplasma/citología , Protoplastos , Agar , Animales , Pared Celular , Medios de Cultivo , Congelación , Gelatina , Hexosas , Histoplasma/crecimiento & desarrollo , Humanos , Sueros Inmunes , Cuerpos de Inclusión , Lípidos , Sulfato de Magnesio , Manitol , Microscopía de Contraste de Fase , Fragilidad Osmótica , Protoplastos/crecimiento & desarrollo , Conejos/inmunología , Regeneración , Esporas Fúngicas/crecimiento & desarrollo , Factores de TiempoAsunto(s)
Bordetella pertussis/citología , Protoplastos/crecimiento & desarrollo , Técnicas Bacteriológicas , Bordetella pertussis/efectos de los fármacos , Bordetella pertussis/crecimiento & desarrollo , Recuento de Células , Medios de Cultivo , Densitometría , Glicina/farmacología , Metamorfosis Biológica , Factores de TiempoAsunto(s)
Hongos/crecimiento & desarrollo , Protoplastos/crecimiento & desarrollo , Regeneración , Pared Celular/crecimiento & desarrollo , Medios de Cultivo , Grabado por Congelación , Gelatina , Aparato de Golgi/fisiología , Microscopía Electrónica , Esporas Fúngicas/crecimiento & desarrollo , Levaduras/crecimiento & desarrolloRESUMEN
Growing "protoplasts" of Streptococcus faecalis were shown to incorporate newly administered radioactive precursors in the same manner as growing intact streptococci. No observable differences could be found between the size of the leucine precursor pools of the two cultures. The extent of turnover of protein and ribonucleic acid in both "protoplast" and streptococcal cultures appeared to be identical. Finally, the absolute rate of macromolecular biosynthesis was found to be equivalent whether determined on the basis of "new" or "old" label incorporation.
Asunto(s)
Enterococcus faecalis/metabolismo , Leucina/metabolismo , Protoplastos/metabolismo , Timidina/metabolismo , Uracilo/metabolismo , Proteínas Bacterianas/biosíntesis , Tampones (Química) , Isótopos de Carbono , Medios de Cultivo , Densitometría , Enterococcus faecalis/crecimiento & desarrollo , Muramidasa , Ósmosis , Protoplastos/crecimiento & desarrollo , ARN Bacteriano/biosíntesis , Estadística como Asunto , Sacarosa , Tritio , TrometaminaAsunto(s)
Hexosas/farmacología , Histoplasma/efectos de los fármacos , Sulfato de Magnesio/farmacología , Protoplastos/crecimiento & desarrollo , Pared Celular , Medios de Cultivo , Citoplasma , Glucosa , Histoplasma/citología , Histoplasma/crecimiento & desarrollo , Microscopía de Contraste de Fase , ÓsmosisAsunto(s)
Bacteriocinas/farmacología , Clostridium perfringens/efectos de los fármacos , Adsorción , Aire , Proteínas Bacterianas/biosíntesis , Técnicas Bacteriológicas , Bacteriófagos/crecimiento & desarrollo , Isótopos de Carbono , Pared Celular/efectos de los fármacos , Citratos , Clostridium perfringens/crecimiento & desarrollo , Clostridium perfringens/metabolismo , Clostridium perfringens/efectos de la radiación , Medios de Cultivo , ADN Bacteriano/biosíntesis , Formas L/crecimiento & desarrollo , Plomo , Microscopía Electrónica , Hidrolisados de Proteína/metabolismo , Protoplastos/crecimiento & desarrollo , ARN Bacteriano/biosíntesis , Efectos de la Radiación , Coloración y Etiquetado , Sacarosa/farmacología , Timidina/metabolismo , Tritio , Tripsina/farmacología , Rayos Ultravioleta , Uridina/metabolismoAsunto(s)
Ascomicetos/citología , Ascomicetos/efectos de los fármacos , Hexosas/farmacología , Sulfato de Magnesio/farmacología , Protoplastos/efectos de los fármacos , Protoplastos/crecimiento & desarrollo , Ascomicetos/crecimiento & desarrollo , Pared Celular , Medios de Cultivo , Diploidia , Glucosa , Haploidia , Microscopía de Contraste de Fase , Protoplastos/citología , Esporas Fúngicas/citología , Esporas Fúngicas/crecimiento & desarrolloAsunto(s)
Pared Celular , Protoplastos/citología , Regeneración , Levaduras/citología , Autorradiografía , División Celular , Membrana Celular , Medios de Cultivo , Enzimas , Grabado por Congelación , Gelatina , Geles , Microscopía Electrónica , Microscopía de Contraste de Fase , Morfogénesis , Polisacáridos/biosíntesis , Protoplastos/crecimiento & desarrollo , Protoplastos/metabolismo , Saccharomyces/citología , Saccharomyces/crecimiento & desarrollo , Saccharomyces/metabolismo , Caracoles , Coloración y Etiquetado , Levaduras/crecimiento & desarrollo , Levaduras/metabolismoAsunto(s)
Bacillus megaterium/metabolismo , Membrana Celular/metabolismo , Glicosaminoglicanos/biosíntesis , Polisacáridos Bacterianos/biosíntesis , Protoplastos/metabolismo , Alanina/metabolismo , Aminoácidos/metabolismo , Autorradiografía , Bacillus megaterium/efectos de los fármacos , Bacitracina/farmacología , Isótopos de Carbono , Cloranfenicol/farmacología , Cloruros/farmacología , Cromatografía en Papel , Ácido Edético/farmacología , Glucosamina/metabolismo , Ácido Clorhídrico/farmacología , Cinética , Sustancias Macromoleculares , Magnesio/farmacología , Muramidasa/farmacología , Azúcares de Nucleósido Difosfato/metabolismo , Presión Osmótica , Penicilinas/farmacología , Péptidos/metabolismo , Ácidos Pimélicos/metabolismo , Protoplastos/efectos de los fármacos , Protoplastos/crecimiento & desarrollo , Temperatura , Factores de Tiempo , Tritio , Trometamina/farmacología , Nucleótidos de Uracilo/metabolismo , Vancomicina/farmacologíaRESUMEN
Osmotically fragile forms of Streptococcus faecalis 9790 were grown in 0.5 m sucrose- or 0.5 m NH(4)Cl-stabilized medium. The "protoplast" cultures exhibit an average growth rate constant of 0.66 to 0.94 mass doublings/hr. In a variety of experiments, turbidity and the net content of protein, ribonucleic acid (RNA) and deoxyribonucleic acid increase at the same rate, indicating balanced macromolecular biosynthesis. A total of two to three mass doublings was obtained, with no evidence of cell division. After osmotic shock, "protoplast" cultures released 93 to 94% of their RNA content in a form not sedimentable at 12,800 x g for 15 min, in contrast to streptococci, which released 7% of their RNA content after the same treatment.
Asunto(s)
Proteínas Bacterianas/biosíntesis , ADN Bacteriano/biosíntesis , Enterococcus faecalis/metabolismo , Protoplastos/metabolismo , ARN Bacteriano/biosíntesis , Cloruro de Amonio , Isótopos de Carbono , Medios de Cultivo , Densitometría , Enterococcus faecalis/crecimiento & desarrollo , Leucina/metabolismo , Muramidasa , Fragilidad Osmótica , Protoplastos/crecimiento & desarrollo , Sacarosa , Timidina/metabolismo , Tritio , Uracilo/metabolismoRESUMEN
Coat-stripped spores suspended in hypertonic solutions and supplied with two essential cations can be converted into viable protoplasts by lysozyme digestion of both cortex and germ cell wall. Calcium ions are necessary to prevent membrane rupture, and magnesium ions are necessary for changes indicative of hydration of the core, particularily the nuclear mass. Since remnant spore coat covered such protoplasts of Bacillus subtilis and the germ cell wall of B. cereus spores is not lysozyme digestible, coatless spores of B. megaterium KM were more useful for these studies. Lysozyme digestion in cation-free environment produced a peculiar semi-refractile spore core free of a cortex but prone to rapid hydration and lytic changes on the addition of cations. Strontium could replace Ca(2+) but Mn(2+) could not replace Mg(2+) in these digestions. When added to the spores, dipicolinic acid and other chelates appeared to compete with the membrane for the calcium needed for stabilization during lysozyme conversion to protoplasts. It is argued that calcium could function to stabilize the inner membrane anionic groups over the anhydrous dipicolinic acid-containing core of resting spores.