RESUMEN

Four spiralins were compared by rocket immunoelectrophoresis, quantitative immunoblotting techniques, and the spiroplasma deformation test with the use of antispiralin (polyclonal) monospecific antibodies. This investigation revealed that the spiralins of Spiroplasma citri and S. melliferum are antigenically related and that probably no more than two epitopes simultaneously saturable with antibodies are shared by the two proteins. One at least of these epitopes is accessible to antibodies on the spiroplasma cell surface.

Asunto(s)

RESUMEN

DNA probes are expected to prove a specific, sensitive, rapid and inexpensive means for diagnosis of mycoplasma infections, replacing procedures that depend on cultivation of the fastidious organisms. Probes made up of conserved genes, such as rRNA genes, do offer the advantage of identifying and distinguishing multiple species with a single labeled reagent. The mycoplasmal rRNA gene probe pMC5 was effective in detection and identification of mycoplasmas infecting cell cultures. However, use of pMC5 for detection of spiroplasmas and mycoplasma-like organisms (MLOs) in infected plants was hindered by hybridization of this probe with chloroplast rRNA genes. Moreover, for identifying species and strains by pMC5, a complex hybridization procedure--involving DNA purification, digestion, electrophoresis, and Southern blot hybridization--is required. More specific DNA probes, on the other hand, can identify specific Mollicutes by the much simpler, faster and more sensitive dot blot technique. Thus, a probe made of a cloned Spiroplasma citri plasmid could detect by this technique as little as 10 pg of S. citri DNA (equivalent to about 10(3) organisms) in infected plants and insects. DNA probes specific for Mycoplasma pneumoniae and M. genitalium were selected from genomic libraries and prepared in pUC13 by screening the libraries for inserts hybridizing only with DNA of the specific mycoplasma. The probes, labeled by nick translation with 32P-nucleotides, could detect as little as approximately 100 pg of the specific mycoplasmal DNA by dot blot hybridization. To eliminate radioactivity, the above DNA probes were labeled by biotinylation of sulfonation systems. Dot blot hybridization with these probes showed decreased sensitivity of detection by about one order of magnitude, and some nonspecific background reaction with large quantities of nonhomologous DNAs.

Asunto(s)

RESUMEN

Spiralin, a 28-kDa (kilodalton) polypeptide, is the major antigen of Spiroplasma citri and S. melliferum in which it usually represents more than 20% of total membrane protein. The amino acid compositions of the spiralins purified from both spiroplasma species unambiguously show that these proteins are homologous. In addition, several lines of evidence indicate that such a protein is present in the menbrane of S. apis. A 25-kDa polypeptide antigenically related to S. citri spiralin has also been purified from the membrane of the nonhelical variant ASP-1. The spiralin of S. melliferum B88 has been used as a model for extensive characterization. This antigen binds detergent under nondenaturing conditions, can be incorporated into liposomes, and forms protein micelles upon gentle removal of detergent. Digestion of the micelles with trypsin leads to the precipitation of an insoluble material containing a major polypeptide of 3.9 kDa. The amino acid composition of this fragment is different from that of intact spiralin. It is highly enriched in glycine and serine and, as an insoluble peptide, exhibits an unexpectedly high polarity index (PI = 51.4%). Screening for acyl proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodetection in the membrane of S. melliferum indicates that spiralin is actually acylated. This set of properties is evidence that spiralin is an intrinsic membrane protein and strongly suggests that acylation triggers or facilitates integration of the molecule into the lipid bilayer of the spiroplasma membrane.

Asunto(s)

RESUMEN

The extraction of proteins from the membrane of the mollicute (mycoplasma) Spiroplasma citri by sodium N-dodecyl-N,N-dimethyl-3-amino-1-propane sulfonate (SB12) and sodium N-tetradecyl-N,N-dimethyl-3-amino-1-propane sulfonate (SB14) was studied with electrophoretic methods. The membranes were prepared by osmotic lysis of the cells and depleted of the bulk of extrinsic proteins. It was possible to extract up to 35 and 45% of membrane proteins with SB12 and SB14, respectively. Maximal yield was obtained in both cases with detergent concentrations greater than or equal to 5 mumoles/mg of membrane protein. Spiralin, the major protein in the S. citri membrane, was highly selectively solubilized without the loss of antigenicity, with a yield of about 90% with SB12 and close to 100% with SB14, for a detergent concentration greater than or equal to 0.2 M. The degree of selectivity in favour of spiralin was higher with SB12 (purity approximately equal to 70%) than with SB14 (purity approximately equal to 50%). Treatment of the S. citri membrane with high concentrations of SB12 is a simple and fast procedure for partial purification of spiralin. This example shows that, in some cases, it should be possible to modulate the selectivity of the extraction of membrane proteins simply by varying the relative concentration of detergent.

Asunto(s)

RESUMEN

Spiroplasma floricola (BNR-1), Spiroplasma sp. MQ-1 and S. apis (B-31) grown in media containing horse serum exhibited intense yellow pigmentation. Yellow pigments were not observed in S. citri (R8A2) and Spiroplasma sp. strains BC-3 and PPS-1 grown in the same medium. The reddish-yellow pigment showed up in lipid extracts of both spiroplasma membranes and horse serum. It exhibited the typical features of bilirubin: specific absorption spectrum from 390 to 500 nm with a peak at 453 nm, and a characteristic sequence of color changes on addition of HNO3 to its solution in chloroform. The pigment comigrated with commercial bilirubin from bull gall and stained greenish blue when subjected to mild oxidation by iodine. S. floricola contained 5.4 micrograms bilirubin/mg cell protein or 9.7 micrograms bilirubin/mg membrane protein. High-performance liquid chromatography (HPLC) showed the presence of significant amounts of 5-methylcytosine and very little 6-methyladenine in the DNA of S. floricola, S. apis and Spiroplasma sp. strains PPS-1 and MQ-1. S. citri and Spiroplasma sp. strain BC-3 contained 6-methyladenine and very little, if any, 5-methylcytosine. The methylated cytosine residues in Spiroplasma sp. MQ-1 were almost exclusively located in the sequence CpG, as in eukaryotes.

Asunto(s)

Asunto(s)

RESUMEN

The number and organization of ribosomal RNA (rRNA) genes in the genome of Mycoplasma, Ureaplasma, Acholeplasma and Spiroplasma species were studied by the Southern hybridization technique. Restriction endonuclease-digested DNAs of the organisms were hybridized with nick-translated probes consisting of defined portions of the rrnB rRNA operon of Escherichia coli and with a recombinant plasmid pMC5 constructed of pBR325 and an insert containing M. capricolum genes for 23S, 5S and most of the 16S rRNA gene. The hybridization data indicate the presence of only one or two sets of rRNA genes in the mollicutes tested, a number lower than in eubacteria. The rRNA genes in mollicutes appear to be organized as clusters (acting apparently as operons) in the typical prokaryotic fashion, 5'-16S-23S-5S-3'. Despite the marked sequence homology shared by the rRNA operons of the different mollicutes and of E. coli, the operons are not identical. Thus, there is an EcoRI restriction site in the 16S rRNA genes in only 8 of the 13 species tested. The recombinant plasmid pMC5 has provided a sensitive probe for detection and identification of mollicutes in contaminated cell cultures. The purified DNA of the tested cell culture, or its supernatant fluid, was digested by EcoRI, and Southern blot hybridization of the products was performed with nick-translated pMC5. The probe did not hybridize with eukaryotic DNA. Each of the mollicutes species examinated exhibited a species-specific hybridization pattern. The hybridization tests enabled the identification of the four most prevalent mycoplasma contaminants of cell cultures, M. orale, M. hyorhinis, M. arginini and A. laidlawii. The test is capable of detecting 1 ng of mycoplasmal DNA, roughly equivalent to the DNA content of 10(5) mycoplasmas. The possibility of using this approach for detection and identification of noncultivable mycoplasmas in plant and insect tissues is under investigation.

Asunto(s)

RESUMEN

Lipoglycans , distinguishable from bacterial lipopolysaccharides, are associated with the cytoplasmic membranes of several genera of Mollicutes, namely Acholeplasma, Mycoplasma neurolyticum , Anaeroplasma , and Thermoplasma. Structurally, the lipoglycans are long heteropolysaccharides covalently linked to a lipid. The exact structures of three have been determined. Thermoplasma oligosaccharide is attached to a diglycerol tetraether ; A. granularum to a diacyl glycerol. The lipoglycan from A. axanthum is unique by its possession of glycerol phosphate and galactose phosphate side chains and the occurrence of fatty acids in N-acyl linkages. Only one molecular species of lipoglycan occurs in a given species. These lipoglycans possess a variety of biological activities. The terminal three sugar residues define their antigenic specificity; they attach to specific receptors on mammalian cells; they exhibit pyrogenicity in rabbits and clot Limulus lysate; they stimulate the production of IgM antibody both in vivo and in vitro; they modulate the immune response to T-cell dependent antigens; they exhibit immunosuppressive and immunostimulatory activities.

Asunto(s)

RESUMEN

Spiroplasma strains of group IV were compared by two-dimensional protein analyses on polyacrylamide gels. Although considerable diversity was evident, the assemblages studied were less heterogeneous than the known strains of group I. Two electrophoretic techniques were used to identify spiroplasma proteins that had been used to immunize rabbits. These included monoclonal antibodies prepared against Spiroplasma citri. In the first technique, protein antigens were purified by immunoaffinity chromatography, then identified with SDS-PAGE. In the second technique, spiroplasma proteins were first separated by SDS-PAGE, then antigens were identified by antibody binding to blot-transferred proteins. Finally, two-dimensional protein electrophoresis has been used as a source of immunogens to characterize monospecific antibodies against individual S. citri proteins.

Asunto(s)

RESUMEN

We propose that Group I spiroplasmas be subdivided into seven, rather than four, subgroups. The seven subgroups showed remarkable homogeneity when several representative strains were compared. Hybridization reactions between DNAs of representative strains within subgroups were generally at least 90 percent, and usually at least 80 percent co-migrating cell proteins were found. In addition, when plasmid DNA was excluded, profiles of restricted DNA among strains within subgroups were very similar. In contrast, comparisons between Group I subgroups showed substantial heterogeneity. This heterogeneity was indicated by DNA-DNA hybridization reactions as low as 10-20 percent and only 10-15 percent co-migrating cell proteins. Spiroplasma citri (subgroup I-1), the honeybee spiroplasma (subgroup I-2), and the corn stunt spiroplasma (subgroup I-3) are all pathogenic organisms with more or less limited host ranges. Strains of these three subgroups have been repeatedly isolated from affected hosts. Since strains of subgroups I-2 and I-3 can be clearly differentiated from other Group I subgroups and all other spiroplasmas, the DNA-DNA hybridization reactions of the subgroups do not exceed 70 percent, and because they are important pathogens, we propose (subject to completion of standard requirements for species descriptions) that they be recognized as new species of the genus Spiroplasma.

Asunto(s)

RESUMEN

A plasmid, pM41, has been isolated from the Spiroplasma citri strain M4 (group I-1) and characterized by restriction mapping. Using a 32P-labeled probe specific of the plasmid, we have shown by DNA-DNA hybridization that a plasmid identical to pM41 or a closely related plasmid, is present in several, but not all, S. citri strains. DNA sequences that hybridize to pM41 were also identified in three other spiroplasmas not belonging to the S. citri species. Protein patterns of several S. citri strains have been compared in order to investigate the effect of pM41 on the spiroplasma protein profiles or maps. In fact, the presence of pM41 does not appear to modify the protein pattern.

Asunto(s)

RESUMEN

The plant surface and insect-inhabiting spiroplasmas of group IV, unlike other spiroplasmas, have not been demonstrated to utilize arginine. They require cholesterol for growth, produce spots and films on some media, and do not hydrolize arbutin. Electrophoretic and serological comparisons of strains from North America and Europe indicate the existence of strain differences within group IV. This study provides evidence for the existence of three discrete subgroups, group IV-(1) represented by temperate American strains, group IV-(2) represented by subtropical American strain PPS1, and group IV-(3) represented by Mediterranean and French strains.

Asunto(s)

RESUMEN

Spiroplasmas contain long flexuous fibrils composed of a protein, molecular weight 55,000, which is specific to Spiroplasma and is highly conserved among different species. The protein cannot be detected in other wall-less prokaryotes reported to contain actin-like proteins and is unrelated to eukaryotic cytoskeletal components. Fibrils occur in similar concentrations in helical and nonhelical strains of Spiroplasma citri. Proposals that fibrils are responsible for maintenance of helical cell shape and rotary motility are discussed in the light of these findings. Evidence is presented which suggests that fibrils may be arrayed as one or more bundles in intact cells and a consistent association of these structures with DNA filaments is noted. These observations are discussed in relation to possible models to account for the maintenance of helical morphology and to the segregation of chromosomes during cell division.

Asunto(s)

RESUMEN

Comparison of the lipid composition between members of the Mycoplasmatales reveals a striking diversity of lipid structures, not only between the six genera but among species within the same genus. This is in contrast to nearly all other bacterial groups in which members of the same genus possess essentially the same lipids. There are in fact more similarities between lipids of a given species of mycoplasma and a genus of bacterium than there are between lipids of a given species of mycoplasma and a genus of bacterium than there are between mycoplasma species. Mycoplasmal lipids suggest that these organisms do not represent a phylogenetically related group at all, but are probably degenerative forms of bacteria, particularly gram-positive bacteria, which have lost the ability to synthesize a cell wall.

Asunto(s)

RESUMEN

The proteins of 91 strains of Mycoplasma from 19 established species and three groups of uncertain taxonomic position were separated by two-dimensional polyacrylamide gel electrophoresis. Considerable variations in protein patterns were found among strains from the same species or subspecies; the percentage of matching spots (% of congruence) ranged from 42%-100%, but many proteins were strongly conserved in all strains of a given species. Maps of these conserved proteins could be used to identify strains. It was concluded that (1) large colony-type strains of Mycoplasma mycoides subspecies mycoides are more closely related to M. mycoides subspecies capri than to small colony-type strains of M. mycoides subspecies mycoides; (2) Mycoplasma capricolum, Leach group 7, and the F38 group of mycoplasmas are all related to M. mycoides; (3) the 2D group of strains are not related to either M. mycoides or Mycoplasma bovis, (4) M. bovis and Mycoplasma agalactiae are closely related; (5) apart from a small overlap in pattern between Acholeplasma laidlawii and Acholeplasma granularum, the type strains of seven established species of Acholeplasma each had distinct protein patterns that justified their classification as separate species; and (6) analysis of two-dimensional protein patterns is a valuable method for resolving problems in mycoplasma taxonomy and for identifying strains.

Asunto(s)

RESUMEN

The guanine-plus-cytosine (G + C) content of spiroplasmal DNA was calculated from the melting temperature determined spectrophotometrically and the buoyant density determined by equilibrium density gradient centrifugation in CsCl. Only two ranges of G + C values were found: 25-27 mol% and 29-32 mol%. The DNA of the following spiroplasmas has 25-27 mol% G + C: Spiroplasma citri (serogroup I-1); the spiroplasmas pathogenic to the honeybee (KC3, BC3, and B63; serogroup I-2); the corn stunt strain (E275; serogroup I-3); the tick strain 277F (serogroup I-4); the drosophila strain (serogroup II); and one group of flower spiroplasmas (serogroup III). The DNA of a second group of flower spiroplasmas (serogroup IV) and the SMCA strain (serogroup V) has a G + C content of 29-31 mol/. The classification of flower spiroplasmas into two groups on the basis of G + C content agrees well with the groupings based on serologic and protein analysis. Spiroplasmas isolated from honeybees in Morocco (B13) or froghoppers in Corsica (L89) have 29-31 mol% G + C, a value that corroborates the relatedness of these strains and the flower spiroplasmas of serogroup IV found by serologic analysis. Reannealing experiments between the vivo-labeled DNA of S. citri and unlabeled DNA of other spiroplasmas gave the following percentages of hybridization: 64% with honeybee spiroplasma DNA, 49% with corn stunt spiroplasma DNA, and 19% with tick spiroplasma 277F DNA; no significant hybridization was observed with DNA of any other spiroplasma. The taxonomic position of the tick spiroplasma 277F within serogroup I was confirmed by hybridization experiments involving [3H]DNA of this strain. The value of polyacrylamide gel analysis of DNA fragments produced by the action of EcoRI restriction enzyme on DNAs from various spiroplasmas is also discussed.

Asunto(s)

RESUMEN

Interactions between the ionic detergent Sarkosyl (sodium lauroyl sarcosinate), Mg2+ ions, and the Spiroplasma citri cell membrane were analyzed microscopically and electrophoretically. Studies were performed under conditions where membrane proteins were apparently not released from the membrane by the detergent (molar ratio of MgCl2/Sarkosyl = 0.5). Although the S. citri membrane interfered with the crystallization phenomenon to some extent, the formation of Sarkosyl-Mg2+ crystals occurred regardless to the sequence of addition of the three components. Concomitantly the structure of the membrane disintegrated and membrane components were adsorbed to the crystal surfaces. The membrane protein fraction bound to the crystals was composed of the majority of the putatively intrinsic polypeptides, including the amphiphilic protein spiralin, and several extrinsic polypeptides. The polypeptide compositions of M-bands (crystal fractions loaded with membrane material) prepared from S. citri cells and from isolated S. citri membranes were similar, as shown by sodium dodecyl-sulfate electrophoresis and crossed immunoelectrophoresis. These results show that, the S. citri cell membrane, in contrast to bacterial membranes, is not protected from the effect of Sarkosyl by Mg2+ ions.

Asunto(s)

RESUMEN

The arrangement of the amphiphilic protein spiralin and of the other major polypeptides in the Spiroplasma citri cell membrane was investigated by one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The analyses were performed on untreated membranes for the detection of disulfide bonds and on membranes treated with dimethylsuberimidate and dithiobis(succinimidyl propionate). All membranes were depleted of the bulk of extrinsic proteins. Spiralin monomers and oligomers (mainly dimers) were detected. Almost all the oligomers appeared to be stabilized by intermolecular disulfide bonds. Components D7 (39,000 daltons), D9 (51,000 daltons), D13 (69,000 daltons), D14b (76,000 daltons), D16 (89,000 daltons), and D17 (95,000 daltons), which are the other (presumably intrinsic) main polypeptides of the S. citri membrane, were also involved in homooligomers stabilized by disulfide bonds. However, in contrast to spiralin, larger amounts of D7, D9, and D14b were involved in high-molecular-weight multimers (molecular weight, greater than 400 X 10(3) after cross-linking with dithiobis(succinimidyl propionate). Extensive cross-linking with dimethylsuberimidate showed that spiralin was the polypeptide least readily integrated to large covalent complexes. These results suggest that spiralin probably does not form a two-dimensional network in the S. citri membrane depleted of the bulk of extrinsic proteins.

Asunto(s)

RESUMEN

Ten of twelve spiroplasma strains from different sources carried multiple covalently closed circular duplex deoxyribonucleic acid molecules, as shown by ethidium bromide-cesium chloride gradient centrifugation of cell lysates and examination of resulting bands by electron microscopy and agarose gel electrophoresis. Two to eight size classes per strain, comprising molecules of masses from 1 X 10(6) to 26 X 10(6), were detected. Several size classes of molecules were found in common in different spiroplasma strains. The amount of covalently closed circular deoxyribonucleic acid per strain was as much as 12% of total cellular deoxyribonucleic acid. The presence of sizes of the circular molecules appeared unrelated to either carriage or active production of known spiroplasma viruses, and it is tentatively concluded that they are plasmids rather than genomes or replicative forms of viruses.

Asunto(s)

RESUMEN

Cells of the nonhelical strain of Spiroplasma citri underwent changes of morphology comparable to those which occurred in the normal helical strain. Cells of the nonhelical strain had the same ultrastructural features as helical cells and released long flexible fibrils similar to those seen in other spiroplasmas. Nonhelical organisms showed an increased tendency to aggregate, forming cell clusters of an unusual annular form. The cytoplasmic membrane of the nonhelical strain lacked a single protein present in all helical strains. Loss of helicity associated with the senescence of spiroplasma cells was not accompanied by the disappearance of this protein. Differences in colony morphology were shown to be a consequence of motility, and a technique was developed which facilitated the identification of nonmotile organisms.

Asunto(s)