RESUMEN
Strains of Mycoplasma mycoides subsp. mycoides have been divided into small colony (SC) and large colony (LC) types (Cottew & Yeats, 1978). The protein patterns of representative strains of these two types and of M. mycoides subsp. capri were compared by a high resolution, two-dimensional gel electrophoretic method. The results suggest that the LC strains are more closely related to M. mycoides subsp. capri than to the SC strains of M. mycoides subsp. mycoides.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Mycoplasma mycoides/clasificación , Electroforesis en Gel de Almidón , Focalización Isoeléctrica , Mycoplasma mycoides/metabolismo , Especificidad de la EspecieRESUMEN
The high resolution, two-dimensional electrophoresis system for the separation of proteins described by O'Farrell, (O'Farrell, P.H. (1975) J. Biol. Chem. 250, 4007--4021) has been modified for the separation of Acholeplasma laidlawii proteins. Reproducible protein patterns have been obtained from A. laidlawii cell, membrane and soluble protein preparations. The isoelectric focusing of membrane proteins was greatly improved by removing the bulk of the membrane lipid before solubilizing the protein. A. laidlawii peripheral membrane proteins were removed from the membrane by low ionic strength washing and by treatment with EDTA. The effect of an exhaustive EDTA treatment and a rapid, warm EDTA treatment were compared. By comparing the protein patterns obtained in these ways it was possible to distinguish two separate groups of peripheral membrane proteins and one integral membrane protein group. The peripheral membrane proteins which were removed from the membrane at low ionic strength (group I) were also insoluble in Triton X-100, whereas additional peripheral membrane proteins extractable by subsequent EDTA treatment (group II) were soluble in Triton X-100. Exterior-facing membrane proteins were distinguished from the interior-facing ones by lactoperoxidase-catalyzed iodination of intact cells and membranes. Group I peripheral membrane proteins faced the cell interior whereas group II proteins faced the cell exterior. We counted approximately 320 individual whole cell proteins. Of these, about 140 were membrane associated and a maximum of 40 proteins were iodinated after iodinating intact cells. A. laidlawii was also grown in the presence of NaH232PO4 and whole cell proteins were separated by two-dimensional gel electrophoresis. One membrane protein and two soluble proteins were labelled.
Asunto(s)
Acholeplasma laidlawii/ultraestructura , Proteínas de la Membrana/metabolismo , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Membrana Celular/ultraestructura , Ácido Edético , Electroforesis en Gel de Poliacrilamida , Proteínas de la Membrana/aislamiento & purificación , Polietilenglicoles , SolubilidadRESUMEN
The rho-form of Mycoplasma contains a striated, axial fiber and associated terminal structure. The presence of this organelle was correlated with the synthesis of two proteins, A and B, of molecular weights of approximately 85,000 and 26,000, respectively, each accounting for about 10% of the total cell protein. Their amino acid compositions showed them to have distinct polypeptide chains. After osmotic lysis of rho-form cells the organelles disappeared; protein A accompanied the membrane fraction, whereas protein B was partly released in soluble form. After lysis by Nonidet P-40 in a medium composed of 4 M glycerol, 50 mM phosphate, and 10 mM MgSO4 at pH 6 (GPM-6), the organelles were preserved and released with ultrastructure unchanged. Protein A was recovered in the soluble fraction and protein B in the particulate (crude fiber) fraction. Treatment of the crude fiber fraction with 0.5 M NaCl in GPM-6 or with a solution containing 4 M glycerol, 10 mM morpholinoethanesulfonate, and 1 mM ethylenediaminetetraacetate at pH 7.0 caused the fibers to disassemble into subunits. By subsequent changes in the ionic conditions and temperature it was possible to cause the subunits to reassemble into ordered aggregates having the same ultrastructure as the native rho-fibers. The optimum temperature for reassembly in the presence of 4 M glycerol was 37 C, the optimum pH was 6.5 to 7.0, and the presence of Mg-2+, replaceable by Ca-2+, SR-2+, or Ba-2+, was essential. Protein B was the only protein detected in the purified, reconsituted fibers.
Asunto(s)
Proteínas Bacterianas , Mycoplasma/ultraestructura , Organoides/ultraestructura , Adenosina Trifosfato , Aminoácidos/análisis , Proteínas Bacterianas/análisis , Proteínas Bacterianas/metabolismo , Bacteriólisis , Fraccionamiento Celular , Cromatografía en Gel , Medios de Cultivo , Digitonina/farmacología , Electroforesis en Gel de Poliacrilamida , Glicerol , Guanosina Trifosfato , Concentración de Iones de Hidrógeno , Magnesio , Peso Molecular , Morfogénesis , Mycoplasma/análisis , Organoides/análisis , Ósmosis , Tensoactivos/farmacología , TemperaturaRESUMEN
The ultrastructure of a variant (rho) form of Mycoplasma is described. The rho-forms are characterized by dark-ground light microscopy as relatively rigid, unbranched, filamentous organisms with discoidal swellings, and by electron microscopy by the presence of an intracytoplasmic axial fiber extending throughout the length of the cell and associated with a terminal structure of characteristic appearance. In negatively stained preparations the fiber presents a pattern of transverse light and dark major bands, the dark band being divided by a central minor light band. The periodicity of the banding varies from 12.0 to 14.5 nm, and the width of the fiber varies from 40 to 120 nm. The fiber appears to be composed of fibrils aligned parallel to the long axis. The evidence indicates that the fiber contains protein and is devoid of nucleic acid. rho-Forms were commonly found in Mycoplasma strains derived from goats and occasionally in bovine strains. They may have a wider distribution, as the growth medium was shown to be important both for the expression of the rho-character and for the selection of the rho-variant. The functional significance, if any, of the fiber and the terminal structure is unknown.