arginine metabolism by spiroplasma citri. 19767639
effect of external environmental factors on the morphology of spiroplasma citri.spiroplasma citri was examined by electron microscopy for morphological changes when maintained under a variety of conditions. pplo serum fraction maintained spiral and helical morphology of s. citri at ph values of 8.0, 7.5, and 7.0, but only partially at ph 6.0 and 5.0. the absence of pplo serum fraction resulted in round, deteriorated cells at all ph values tested. bovine serum albumin (bsa), phytone, soluble starch, potato starch, spermine, lipid-extracted pplo serum fraction, and lipid-extr ...197824042
separation and partial characterization of two deoxyribonucleic acid polymerases from spiroplasma citri.the separation and partial characterization of two deoxyribonucleic acid polymerases from spiroplasma citri have been achieved. the two enzymes had different elution properties on diethylaminoethyl (deae) cellulose and differed in their sensitivity to n-ethylmaleimide (nem), preference for different template-primers, and sedimentation velocity in linear glycerol gradients. the first enzyme activity, sca, was retained on deae-cellulose and was not inhibited by nem. activated deoxyribonucleic acid ...197942638
an in vitro comparison of some biochemical and biological properties of california and morocco isolates of spiroplasma citri. 197943764
the pathogenicity of mycoplasmas for plants.many plant diseases belonging to the yellows group are believed to be caused by wall-free prokaryotes resembling mycoplasmas, which are spread by leafhopper vectors. im most cases the evidence for mycoplasma aetiology rests upon the finding by electron microscopy of mycoplasma-like bodies in phloem tissue of diseased plants, coupled in some cases with symptom remission following treatment of plants with tetracyclines. the only plant-pathogenic mycoplasmas which have so far been cultured are the ...197944613
spiroplasmas: serological grouping of strains associated with plants and insects.spiroplasma strains from plant and arthropod hosts, and from surfaces of flowers, were classified into three serological groups (designated i, ii, and iii) based on results from growth-inhibition tests. no significant cross reactions were observed among groups. the groupings were confirmed by ring-interface precipitin and microprecipitin tests, using membrane preparations as test antigens, and by organism-deformation tests. serogroup i contained three subgroups: subgroup a (spiroplasma citri str ...197993504
carbohydrate content and enzymic activites in the membrane of spiroplasma citri. 1977142817
composition and enzyme activities of spiroplasma citri membranes.spiroplasma citri was cultured in three different media that supplied cholesterol and fatty acids from: (i) horse serum, (ii) pleuropneumonia-like organism (pplo) serum fraction, or (iii) bovine serum albumin-fatty acid-cholesterol. the ability of pplo serum fraction to support growth varied by lot number. neither pplo serum fraction nor the bovine serum albumin medium supported growth as well as the horse serum medium. analysis of cholesterol, lipid phosphorus, and membrane protein showed the h ...1977191432
comparison of the membrane composition of spiroplasma citri and the corn stunt spiroplasma.components of membranes isolated from spiroplasma citri and corn stunt spiroplasma grown at 28 degrees c were analyzed. on a protein basis, lipid phosphorus was lower and cholesterol was higher in s. citri. only minor differences between the two species were found in fatty acid composition, reduced nicotinamide adenine dinucleotide diaphorase, and adenosine triphosphatase.1979422510
amphilphilic nature of spiralin, the major protein of the spiroplasma citri cell membrane.spiralin could not be solubilized in the absence of detergents, and it was shown by charge-shift crossed immunoelectrophoresis that this protein was capable of binding detergents under nondenaturing conditions. these properties indicate the amphiphilic nature of spiralin, which therefore should be regarded as an intrinsic membrane protein. the efficiency of mild (ionic and neutral) detergents to solubilize spiralin was as follows: deoxycholate greater than lauroyl sarcosinate, cholate, taurochol ...1979500570
[electron microscopical evidence of spiroplasma citri in nucleus of infected testplants (author's transl)].this is the first report for the evidence of mycoplasmas in an organelle of a plant cell. spiroplasma citri was detected by electron microscopy in the nucleus of a phloem parenchyma cell of chrysanthemum carinatum schousboe plants, inoculated experimentally by euscelis plebejus fall.1979549392
solubilization of spiroplasma citri cell membrane proteins with the anionic detergent sodium lauroyl-sarcosinate (sarkosyl).1. up to 90 per cent of the membrane proteins from spiroplasma citri could be solubilized with the anionic detergent sarkosyl (sodium lauroyl-sarcosinate). maximal solubilization was obtained with 6 to 20 mumoles of of detergent per mg of membrane protein. the insoluble residue, comprising about 10 per cent of the membrane protein, contained mainly the protein spiralin, which is quantitatively the major one of this membrane. 2. mg2+ ions completely prevented solubilization of the membrane protei ...1978687677
comparison of lipids from spiroplasma citri and corn stunt spiroplasma.the qualitative lipid composition of spiroplasma citri and corn stunt spiroplasma is identical. small amounts of acylated glucose and steryl glucoside were found.1978711681
mycoplasma-dependent activation of normal lymphocytes: mitogenic potential of mycoplasmas for mouse lymphocytes.nonviable preparations of a wide variety of glucose-utilizing mycoplasma species, including acholeplasma laidlawii and spiroplasma citri, were found to be mitogenic for mouse lymphocytes. particularly strong reactions were obtained with mycoplasma synoviae, m. gallisepticum, m. pneumoniae, s. citri, and a strain of m. fermentans that was previously isolated from a leukemic patient. nonviable preparations of arginine-utilizing mycoplasmas inhibited the uptake of [3h]thymidine by lymphocytes, but ...1977924676
spiroplasmavirus citri 3: propagation, purification, proteins, and nucleic acid.svc3 is a short-tailed polyhedral virus particle morphologically detectable in many spiroplasmas. it was isolated from two different spiroplasmas (spiroplasma citri and the suckling mouse cataract agent) by infecting lawns and broth culture of another strain of spiroplasmavirus citri. virions from either donor strain had a buoyant density of 1.26 grams per cubic centimeter (metrizamide) or 1.45 grams per cubic centimeter (cesium chloride), and contained five proteins and linear double-stranded d ...1977929198
in vitro anti-mycoplasmal activity of amphotericin b methyl ester.the polyene macrolide antibiotic amphotericin b (ab) and its chemically modified derivative amphotericin b methyl ester (ame) were tested for in vitro activity against acholeplasma laidlawii, spiroplasma citri and mycoplasma gallisepticum. both polyene macrolide preparations demonstrated anti-mycoplasmal activity. however, ame was mycoplasmacidal toward all three strains of mycoplasma at levels which previous studies have indicated would be permissible for most cell culture systems, whereas the ...1976950321
[selective solubilization of spiroplasma citri membrane proteins with sodium desoxycholate]. 19751222145
distribution and composition of lipopolysaccharides from mycoplasmas.polymeric carbohydrates containing glycerol and fatty acids were isolated from whole cells and membranes of mycoplasmas by hot aqueous phenol extraction and gel filtration. lipopolysaccharides were found to occur in four species of acholeplasma, two of anaeroplasma, and in mycoplasma neurolyticum. none were detected in spiroplasma citri or in five species of mycoplasma. all lipopolysaccharides contained both neutral and n-acylated amino sugars in ratios varying from 1:1 to 3:1. the neutral sugar ...19761254559
lipid composition and lipid metabolism of spiroplasma a horse serum-based medium containing a full complement of fatty acids, cells of spiroplasma citri were seen to preferentially incorporate palmitic acid. in the same medium, which had a steryl ester-to-sterol ratio of 3.64, a steryl ester-to-sterol ratio of 0.23 was seen in the cells, cholesterol being preferentially incorporated over cholesteryl ester. like most other mycoplasmas, s. citri was shown to be unable to synthesize fatty acids or esterify cholesterol. the neutral lipids of s. citr ...19761254560
cloning and sequencing of is1086, an alcaligenes eutrophus insertion element related to is30 and is4351.a new insertion sequence (is), designated is1086, was isolated from alcaligenes eutrophus ch34 by being trapped in plasmid pjv240, which contains the bacillus subtilis sacb and sacr genes. the 1,106-bp is1086 element contains partially matched (22 of 28 bp) terminal-inverted repeats and a long open reading frame. hybridization data suggest the presence of one copy of is1086 in the strain ch34 heavy-metal resistance plasmid pmol28 and at least two copies in its chromosome. analysis of the is1086 ...19921334071
a physical and genetic map of the spiroplasma citri genome.a physical and genetic map of the spiroplasma citri genome has been constructed using several restriction enzymes and pulsed field gel electrophoresis. a number of genes were subsequently localized on the map by the use of appropriate probes. the genome size of the spiroplasma estimated from restriction fragments is close to 1780 kbp, the largest of all mollicutes studied so far. it contains multisite insertions of spiroplasma virus 1 (spv1) sequences. the physical and genetic map of the s. citr ...19921349741
spiroplasma citri ugg and uga tryptophan codons: sequence of the two tryptophanyl-trnas and organization of the corresponding genes.from the total trnas of spiroplasma citri, we isolated and purified two trna(trp) species by using chromatography on an rpc-5 column followed by denaturing polyacrylamide gel electrophoresis. the sequence of the two trnas, as well as the sequences of the corresponding genes, were determined. one of the two trna(trp) species has a cca anticodon and is able to pair with the universal ugg tryptophan codon, while the second has a u*ca (u* is a modified uridine) anticodon and is able to pair with uga ...19921383193
mutagenesis of a tryptophan codon from tgg to tga in the cat gene does not prevent its expression in the helical mollicute spiroplasma citri. 19921544572
antigenic relatedness between the spiralins of spiroplasma citri and spiroplasma melliferum.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 ...19901697579
first step toward a virus-derived vector for gene cloning and expression in spiroplasmas, organisms which read uga as a tryptophan codon: synthesis of chloramphenicol acetyltransferase in spiroplasma citri.spiroplasmas are wall-less procaryotes in which the uga codon serves not as a stop signal but as a code for the amino acid tryptophan. spiroplasma genes that contain uga codons thus cannot be studied in the usual escherichia coli cloning and expression systems. although this problem can be circumvented by using uga-suppressor strains of e. coli, spiroplasmas themselves would provide a more efficient cloning and expression host. we have now successfully employed the replicative form (rf) of a fil ...19911706702
nucleotide sequence of the spiroplasma citri fibril protein gene.electron microscopic observation of spiroplasmas lysed by detergent (sodium deoxycholate) revealed the release of bundles of fibrils from the cells. individual fibrils are 4 nm in diameter and possess a 9-nm periodicity along their length. these fibrils are thought to function as cytoskeletal structures involved in the shape and motility of spiroplasmas. polyacrylamide gel electrophoresis of density gradient-purified fibrils showed a protein of approximately 55 kda. oligonucleotide probes were c ...19911712358
search for eukaryotic motility proteins in spirochetes: immunological detection of a tektin-like protein in spirochaeta halophila.the serial endosymbiotic theory (set) for the spirochete origin of undulipodia (cilia and eukaryotic flagella) predicts that a greater number of axonemal proteins will show homology to spirochete than to other bacterial proteins. to continue testing, the set proteins associated with eukaryotic motility (tektin, centrin and calmodulin) were sought in spirochaeta halophila. western blot immunological detection techniques (for tektin and centrin) and two-dimensional gel analysis (for calmodulin) we ...19911863720
organization and nucleotide sequences of the spiroplasma citri genes for ribosomal protein s2, elongation factor ts, spiralin, phosphofructokinase, pyruvate kinase, and an unidentified protein.the gene for spiralin, the major membrane protein of the helical mollicute spiroplasma citri, was cloned in escherichia coli as a 5-kilobase-pair (kbp) dna fragment. the complete nucleotide sequence of the 5.0-kbp spiroplasmal dna fragment was determined (genbank accession no. m31161). the spiralin gene was identified by the size and amino acid composition of its translational product. besides the spiralin gene, the spiroplasmal dna fragment was found to contain five additional open reading fram ...19902139649
spiralins of spiroplasma citri and spiroplasma melliferum: amino acid sequences and putative organization in the cell membrane.spiralin is the major membrane protein of the helical mollicute spiroplasma citri. a similar protein occurs in the membrane of spiroplasma melliferum, an organism related to s. citri. the gene encoding spiralin has been sequenced. a restriction fragment of the spiralin gene has been used as a probe to detect the gene encoding s. melliferum spiralin. a 4.6-kilobase-pair clai dna fragment from s. melliferum strongly hybridized with the probe. this fragment was inserted in pbr322 and cloned in esch ...19902211527
complete nucleotide sequence of the genome of spiroplasma citri virus spv1-r8a2 b. 19902320423
a phylogenetic analysis of the mycoplasmas: basis for their classification.small-subunit rrna sequences were determined for almost 50 species of mycoplasmas and their walled relatives, providing the basis for a phylogenetic systematic analysis of these organisms. five groups of mycoplasmas per se were recognized (provisional names are given): the hominis group (which included species such as mycoplasma hominis, mycoplasma lipophilum, mycoplasma pulmonis, and mycoplasma neurolyticum), the pneumoniae group (which included species such as mycoplasma pneumoniae and mycopla ...19892592342
involvement of a spiroplasma citri plasmid in the erythromycin-resistance erythromycin-resistant strain (m4 er-1) was selected from spiroplasma citri m4+. the transfer by transformation of the erythromycin-resistance character to the erythromycin-sensitive s. citri strain r8a2+ was studied. transfer became effective and reproducible when cells were treated with alkali cations plus polyethylene glycol. comparison of the efficiency of transformation of the erythromycin-sensitive strain s. citri r8a2+ by total and extrachromosomal dna purified from the erythromycin-re ...19892623082
spiroplasma species share common dna sequences among their viruses, plasmids and genomes.alkaline-southern-blot analyses showed that a spiroplasma plasmid, pra1, obtained from spiroplasma citri (maroc-r8a2), contained dna sequences that were homologous to spiroplasma type 3 viruses (sv3) obtained from s. citri (maroc-r8a2), s. citri (608) and s. mirum (smca). in addition, pra1 and sv3(608) dna shared common, but not necessarily related, sequences with extrachromosomal dna derived from 11 spiroplasma species or strains. furthermore, sv3(608) had dna homology with the chromosome from ...19872830894
[spiroplasmas and hematophagous arthropods; prospects in tropical pathology].spiroplasmas are original wall-free prokaryotes which exhibit motility and a helical shape although lacking any cell wall or motile apparatus. they are known by phytopathologists because some of them are responsible for severe arthropod-borne plant diseases, such as spiroplasma citri, the etiologic agent of "citrus stubborn disease". other spiroplasmas are pathogenic for a number of insects: drosophila, bees and beetles. more recently spiroplasmas were also isolated from ticks, mosquitoes, horse ...19852873901
lethal and mutation frequency responses of spiroplasma citri cells to uv irradiation.the effect of uv irradiation on viability and mutant colony frequency in the mollicute spiroplasma citri was investigated at 3 phases of growth. the first uv-induced mutants obtained in mollicutes were selected: xylitol-resistant (xylr) and arsenic acid-resistant mutants (arsr). lethal and mutation frequency responses of s. citri cells increase with the age of the cell cultures. in all uv-irradiated populations, light exposure slightly increases the number of survivors and decreases the induced ...19892909865
gene for spiralin, the major membrane protein of the helical mollicute spiroplasma citri: cloning and expression in escherichia coli.a library of cloned spiroplasma citri genomic sequences was constructed by incorporating hindiii digestion fragments into the plasmid vector pbr328. immunological screening allowed the identification of a recombinant plasmid containing the gene for spiralin, the major membrane protein of s. citri. the spiralin produced by the escherichia coli transformant was characterized by immunological detection with monoclonal antibody after western blotting of two-dimensional (isoelectric focusing and sodi ...19852999069
short, interspersed, and repetitive dna sequences in spiroplasma species.small fragments of dna from an 8-kbp plasmid, pra1, from a plant pathogenic strain of spiroplasma citri were shown previously to be present in the chromosomal dna of at least two species of spiroplasma. we describe here the shot-gun cloning of chromosomal dna from s. citri maroc and the identification of two distinct sequences exhibiting homology to pra1. further subcloning experiments provided specific molecular probes for the identification of these two sequences in chromosomal dna from three ...19873039555
chromosomal gene transfer in spiroplasma citri.the study of resistance marker rearrangement in spiroplasma citri mutants provides evidence of transfer of chromosomal information followed by recombination. this is the first report of such a transfer in vivo in the mollicutes--that is, in the smallest self-replicating organisms. the double-resistant phenotypes obtained are stable even without selection pressure. the mechanism of gene transfer is insensitive to deoxyribonuclease, requires contact, and possibly, areas of fusion of the cell membr ...19883261453
localization of spiralin in escherichia coli cells transformed with the recombinant plasmid pesi.the expression of spiralin in the transformant strain hb101 tsp of escherichia coli has been investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page), immunodetection after electrotransfer, and crossed immunoelectrophoresis. the protein has been sought in whole cells, cytoplasm, and plasma and outer membranes of the bacterium. untransformed e. coli cells and spiroplasma citri cells were used as negative and positive references, respectively. contrary to earlier claims ...19873312094
epidemiology of spiroplasma citri in the old world.epidemics of citrus stubborn disease (csd) have been known in the mediterranean citrus-growing areas as early as 1928. one such outbreak of stubborn disease occurred around 1980 in newly established citrus nurseries on the syrian coast. natural transmission of spiroplasma citri could be shown to be involved. enzyme-linked immunosorbent assay (elisa) and culture of s. citri were used to detect the presence of s. citri in leafhoppers. over 50 leafhopper species were submitted to these analyses, no ...19873312105
spiroplasma plasmids.extrachromosomal dna, constituting plasmids or replicative forms of viruses, has been detected in a variety of spiroplasmas, particularly in spiroplasma citri. only a few of the s. citri plasmids were characterized by restriction enzyme mapping, and essentially nothing is known on functions encoded by the plasmids. our studies revealed in s. citri (r8a2) an 8.0-kbp plasmid that differed from previously described plasmids in its restriction map. it was also clonable in pbr322. the plasmid, named ...19873312107
dna probes for detection and identification of mycoplasmas (mollicutes).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 ...19873312110
production of monoclonal antibodies against phloem-limited prokaryotes of plants: a general procedure using extracts from infected periwinkles as immunogen.using spiroplasma citri-infected periwinkle extracts as the source of antigens, together with monoclonal antibody technology, we determined the conditions for mouse immunization and developed a screening assay to detect those hybridomas which produce immunoglobulins specifically directed against the pathogen.19873331292
probable insensitivity of mollicutes to rifampin and characterization of spiroplasmal dna-dependent rna polymerase.the effect of rifampin on five mollicutes (spiroplasma citri, spiroplasma melliferum, spiroplasma apis, acholeplasma laidlawii, and mycoplasma mycoides) was compared with that on escherichia coli. we found that, in contrast to wild-type e. coli, mollicutes were insensitive to rifampin. dna-dependent rna polymerases from s. melliferum and s. apis were purified to the stage where the enzymes were dependent on the addition of exogenous templates for activity. the enzymes were then tested for their ...19863519581
spv3 viruses of drosophila spiroplasmas.the presence of spiroplasmas in the hemolymph of four drosophila willistoni group species has been shown to cause elimination of males from the progeny of infected females. these spiroplasmas, known as sex-ratio organisms (sros), are found in d. equinoxialis (esro), d. nebulosa (nsro), d. paulistorum (psro), and d. willistoni (wsro). in addition, a nonmale-lethal spiroplasma (his) has been found in the hemolymph of d. hydei. only the wsro has been cultivated. each of the drosophila spiroplasmas ...19873667220
amphiphilic properties of spiralin, the major surface antigen of spiroplasmas. a preliminary report.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 ...19873667221
towards monoclonal antibodies against the apple proliferation mycoplasma-like organism.a 6-week-old female balb/c mouse was immunized with a preparation of partially purified sieve tubes of periwinkle (catharantus roseus) plants infected with the apple proliferation mycoplasma-like organism. after fusion of the immunized spleen cells with myeloma cells, the supernatant fluids of the resulting hybridoma cultures were screened for the presence of antibodies in a differential avidin-biotin enzyme-linked immunosorbent assay (elisa) using sieve tube preparations from either healthy or ...19873667236
spiroplasma citri detection by enzyme-linked immunosorbent assay (elisa), culture and dot hybridization.we have used elisa and culture of spiroplasmas in sp4 or m1a medium to detect spiroplasma citri in citrus trees, periwinkles (catharantus roseus) and leafhoppers in iraq and syria. on the basis of hundreds of analyses, we have found that the sensitivity of elisa and culture are of the same order of magnitude. with citrus, both tests are able to detect s. citri in 95% of symptomatic nursery or field trees. the tests are not sensitive enough to reliably detect s. citri in symptomless trees. theref ...19873667239
[the leafhopper neoaliturus haematoceps (mulsant & rey) is a vector of spiroplasma citri in the mediterranean].among the many leafhoppers which we have collected and identified in morocco, turkey, syria and france (corsica), only the species neoaliturus haematoceps was found to be infected with spiroplasma citri. in syria, this leafhopper was encountered on ornamental stock plants (matthiola incana l.) in the oronte valley, and on salsola kali l., a wild chenopodiacae of the semi-arid regions of the coastal area. in corsica, we found it on wild stock plants (matthiola sinuata of the oriental coast ...19863674783
highly selective extraction of spiralin from the spiroplasma citri cell membrane with alkyl-n-sulfobetaines.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 ob ...19854096907
morphology, ultrastructure, and bacteriophage infection of the helical mycoplasma-like organism (spiroplasma citri gen. nov., sp. nov.) cultured from "stubborn" disease of citrus.the mycoplasma-like organism spiroplasma citri gen. nov., sp. nov., isolated from citrus infected with "stubborn" disease and carried in serial cultures in several media, was examined by dark-field microscopy and electron microscopy of negatively-stained and shadowed preparations and of sections. it grows as motile, helical filaments in liquid, but as nonmotile, nonhelical filaments and round bodies in agar cultures. helicity and motility are lost in old broth cultures and upon addition of a var ...19734123916
isolation, chemical composition, and ultrastructural features of the cell membrane of the mycoplasma-like organism spiroplasma citri.thin sections of spiroplasma citri, a mycoplasma-like organism isolated from citrus infected with "stubborn" disease, showed the organisms to be limited by a single trilaminar plasma membrane. an additional outer layer could, however, be frequently seen in freeze-etched preparations of unwashed cells. the organisms were found to be extremely sensitive to lysis by osmotic shock. the cell membrane of s. citri isolated in this way resembled that of mycoplasmas in ultrastructure and gross chemical c ...19734127633
spiroplasmas and acholeplasmas: multiplication in insects.the helical wall-free microorganism, spiroplasma citri, which is associated with citrus stubborn, a disease with no known vector, multiplied in the leafhopper vector of corn stunt but multiplied to higher titer in the vector of aster yellows and decreased the longevity of that insect. acholeplasma laidlawii and a. granularum also multiplied in both leafhoppers.19734796196
spiroplasmas of group i: the spiroplasma citri cluster.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. ...19836089454
integration of a temperate phage infecting spiroplasma citri.a physical map of the genome of a temperate type 3 spiroplasma-virus, ai, has been constructed. host dna has been digested with restriction enzymes, and recombinant dna clones of ai fragments in coliphage m13 vectors have been used as probes to detect viral dna sequences integrated into spiroplasmas. all strains of spiroplasma citri examined contained a deleted form of ai integrated as a cryptic prophage which was unable to confer resistance to ai superinfection. stable ai lysogens also containe ...19846096304
adsorption of proteins from the spiroplasma citri cell membrane by magnesium lauroyl-sarcosinate crystals.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 rega ...19816164403
comparison of the amino acid compositions and antigenic properties of spiralins purified from the plasma membranes of different spiroplasmas.spiralins were purified by agarose-suspension electrophoresis after extraction with detergents from the membranes of the following spiroplasmas: spiroplasma citri c189, s. citri maroc (r8a2), s. citri scaph and the honey-bee spiroplasma b88. the four proteins (molecular mass congruent to 26,000 daltons, as determined by sodium dodecyl sulphate-pore gradient electrophoresis) showed very similar amino acid compositions characterized by the absence of methionine and tryptophan and a high polarity i ...19846201097
analysis of spiroplasma proteins: contribution to the taxonomy of group iv spiroplasmas and the characterization of spiroplasma protein antigens.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 chroma ...19836206657
partial purification and cleavage specificity of a site-specific endonuclease, scini, isolated from spiroplasma citri.a site-specific endonuclease, scini, has been partially purified from the plant pathogen spiroplasma citri. the enzyme recognizes the sequence 5'-g-c-g-c-3' and cleaves between the first g and c. 3'-c-g-c-g-5' scini is an isoschizomer of hhai, but generates dna fragments with 5' rather than 3' single-stranded protrusions.19826276358
guanine-plus-cytosine content, hybridization percentages, and ecori restriction enzyme profiles of spiroplasmal dna.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 ( ...19826289407
characterization and molecular cloning in escherichia coli of a plasmid from the mollicute spiroplasma citri.two plasmids, pmh1 with 7 kilobase pairs and pm41 with 8 kilobase pairs, were purified from the plant pathogen spiroplasma citri and characterized by restriction mapping. upon in vitro dna recombination with plasmid pbr328 as a vector, we have cloned pmh1 in escherichia coli. a radioactive probe obtained upon nick translation of the recombinant plasmid was used to further characterize and compare pmh1 and pm41.19836313631
spiroplasma fibrils.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 sh ...19836382820
the aster yellows controversy: current status.evidence for and against the spiroplasmal etiology of aster yellows (ay) disease is examined. a spiroplasma, serologically identical to spiroplasma citri, was cultivated by some workers from lettuce (lactuca sativa l.) plants claimed to be naturally infected with ay. the isolated spiroplasma was shown to be infectious by injecting macrosteles fascifrons with the cultured organisms and then confining the injected leafhoppers on healthy plants. the reports claiming that a spiroplasma is the etiolo ...19836382826
spiroplasmas in leafhoppers: a review.this review describes the pathway a plant pathogenic mycoplasma or spiroplasma takes in its passage through a leafhopper vector. reference is made to several strains of spiroplasma and acholeplasma, but, in particular, data are presented for spiroplasma citri and the corn stunt spiroplasma. acquisition of the organisms is discussed, together with the different methods of infection (feeding on plants and through membranes or following injection) and the effect they have on the inoculum dose. the ...19836382827
viruses of spiroplasma citri and their possible effects on pathogenicity.strains of spiroplasma citri are persistently infected by viruses which have been separated into three groups on the basis of their morphology. the properties of each group are reviewed. viruses normally only appear in spiroplasma cultures but recently all three types of particle have been identified in cells of a single strain of s. citri within an infected plant. replication of a short-tailed polyhedral virus sp-v3 (ai) appears to be correlated with unusually mild symptom expression. introduct ...19836382829
molecular biology of spiroplasma plasmids.with one exception, all spiroplasma strains examined contained extrachromosomal dna, most of which was in the form of covalently closed circular plasmids. one plasmid, pij2000, carried by spiroplasma citri strain asp-1, was purified and characterized and used to probe for related plasmids in other strains. unsuccessful attempts were made to clone pij2000 into escherichia coli using the vectors pat153 and pbr322. however, spiroplasma chromosomal dna fragments could be cloned without difficulty.19836382830
field ecology of spiroplasma citri in western north america.spiroplasma citri naturally infects plants representing several dicotyledonous families in the southwestern u.s., where it is commonly found in old world brassicaceous weeds and cole crop plants as well as in citrus. of its leafhopper vectors, it is most frequently found in circulifer tenellus (baker). it is rarely found in field-collected scaphytopius nitridus (delong), one of two species of scaphytopius capable of transmission from plants in the laboratory. spiroplasmas presumed to be s. citri ...19846392182
expression of the spiroplasma citri spiralin gene in escherichia coli. use of the recombinant plasmid carrying this gene as a molecular probe.expression in escherichia coli of the cloned spiroplasma citri spiralin gene results in the expression of a 30.5-kda protein serologically related to spiralin. a protein with the same properties is also present in minor amounts in s. citri cells, suggesting that spiralin is first produced as a preprotein containing a signal polypeptide, which is removed during further processing. hybridization experiments have demonstrated that pes1, the recombinant plasmid carrying the spiralin gene, can be use ...19846392183
spiroplasmas and the transfer of genetic material by transformation and transfection.two plasmids, pmh1 with 7 kbp and pm41 with 8 kbp were purified from spiroplasma citri strains mh and m4 respectively. on the basis of guanine + cytosine content and restriction enzyme mapping, the two plasmids are different. the linearized pmh1 plasmid was introduced into escherichia coli plasmid vector pbr328 and could be cloned in e. coli. using radioactive probes specific for each plasmid, we found that pm41 was present in three additional s. citri strains and in three other spiroplasmas not ...19846392186
insect cell cultures in the study of attachment and pathogenicity of spiroplasmas and mycoplasmas.the insect cell lines dm-1 (drosophila melanogaster), as-2 (aceratagallia sanguinolenta) and ac-20 (agallia constricta) were infected with spiroplasmas, mycoplasmas and acholeplasma laidlawii. in dm-1 cultures maintained at 25 degrees c in m1a medium, all strains multiplied except m. hyorhinis and the uncultivable sex-ratio organism. spiroplasma citri r8a2, s. floricola bnr-1 and obmg, s. apis pps-1 and the strains bc-3, corn stunt spiroplasma (css) and 277f produced cytopathogenic effects (cpe) ...19846424527
discovery of a new plant-pathogenic study natural transmission of spiroplasma citri in the mediterranean area, periwinkles (vinca rosea l.) were exposed to natural infection in several locations during the summer of 1983. detection of s. citri in the periwinkles was assayed by elisa and culture of the organisms. some of the periwinkles developed yellows disease symptoms; they contained helical organisms in their sieve tubes, as determined by electron microscopy. a spiroplasma could be cultured from them, but their elisa reactio ...19846511310
arginine aminopeptidase activity of phytopathogenic spiroplasmas.the arginine aminopeptidase activity of arginine-utilizing phytopathogenic spiroplasmas was investigated with arginine beta-naphthylamide substrate using the fluorometric method. hydrolysis of this substrate was demonstrated with broth cultures, washed concentrated whole cells, and cell-free extracts of corn stunt spiroplasma (css) and spiroplasma citri. growing css and s. citri in the presence of 47 mm arginine resulted in a reduction in aminopeptidase activity, indicating that synthesis of the ...19846511313
ultraviolet irradiation mutagenesis and recombination in spiroplasma citri.a method for obtaining and screening uv-induced mutants from spiroplasma citri is described. lethality response curves showed that s. citri is more sensitive to uv irradiation than are other microorganisms. the presence of a shoulder in the lethality response curve showed the existence of systems able to repair uv-induced dna damages. toxic-resistant mutants have been obtained. a uv fluence equal to 10 j/m2 multiplied by 2.5 x 10(3) gave the spontaneous mutation frequency. arsenic acid- and xyli ...19846511358
spiroplasma fibrils.a fundamental question in biology concerns the morphology of spiroplasmas: how does a wall-less microorganism maintain its characteristic morphology as a helical filament? an answer to this question began to form when it was discovered that spiroplasmas treated with any of a number of detergents (sodium deoxycholate, triton x-100, nonidet p-40) release their cytoplasmic contents. if this procedure is performed on a formvar-coated electron microscope grid and the resultant preparation negatively ...19846511359
oxidoreduction sites and relationships of spiroplasmas with insect cells in culture.we have previously shown that spiroplasma citri oxidoreduction sites, as revealed by the reduction of potassium tellurite into electron-dense tellurium crystals detectable by electron microscopy, were located at the blunt end of the organisms. the time of incubation of s. citri in potassium tellurite had no influence on the labeling. we have investigated the presence and location of oxidoreduction sites for other spiroplasmas such as the corn stunt spiroplasma (css), 277f, b88, bnr1 and pps1. fo ...19846511360
synthesis of adenylate nucleotides by mollicutes (mycoplasmas).cultures of the mollicutes (mycoplasma) acholeplasma laidlawii b, acholeplasma morum, mycoplasma bovis, mycoplasma arginini, mycoplasma fermentans and mycoplasma gallisepticum, representing four metabolic groups, were sampled at intervals over a 40 to 50 h period and assayed for the numbers of c.f.u., changes in ph and glucose concentration, and concentrations of atp, adp, amp, lactate and pyruvate. the adenylate energy charge (eca), the mean generation time, and the number of nmol of atp (mg dr ...19836655457
serological classification of spiroplasmas: current concerning serological classification of spiroplasmas are in good agreement, but slightly different numerical designations have been given to existing groups. it is proposed that a standardized system be adopted based on information developed mainly by the irpcm working team on spiroplasmas. the type species (spiroplasma citri) should be redefined to include only the agent of citrus stubborn disease (subgroup i-1). six other subgroups, including three proposed by bové et al. in this volume ...19836679143
a plasmid from s. citri strain m14 hybridizes with extrachromosomal dnas from other spiroplasmas, including corn stunt spiroplasma e275, tick spiroplasma 277f, and coco spiroplasma n525.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 strai ...19836679153
spiroplasma citri-induced lethal wilting of periwinkles is prevented by prior or simultaneous infection of the periwinkle by an mlo.s. citri can be cultured from periwinkles naturally infected in the field and showing typical mycoplasma-like organism (mlo) symptoms such as flower virescence. such plants are undoubtedly infected with both s. citri and with one mlo, the latter being responsible for the symptoms. to verify this hypothesis, the following experiments were carried out. periwinkles were infected with both s. citri and one of three different mlo by graft inoculations. the three mlo were those of phyllody virescence, ...19846712060
characterization of two cryptic plasmids from spiroplasma citri and occurrence of their dna sequences among various spiroplasmas.two plasmids, pmh1 with 7 kilobase pairs (kbp) and pm41 with 8 kbp, were purified from spiroplasma citri strains mh and m4, respectively, and characterized by restriction mapping. upon in vitro dna recombination with plasmid pbr328 as a vector, pmh1 was cloned in escherichia coli. radioactive probes specific of the plasmids were used to investigate the occurrence of pmh1 and pm41 dna sequences among various spiroplasmas. pm41 or a closely related plasmid was found in three other s. citri strains ...19846712061
growth and division of spiroplasma citri: elongation of elementary helices.the smallest viable cell of spiroplasma citri is a two-turn helix (elementary helix). this elementary helix grows into longer parental cells, which then divide by constriction. the helical morphology is conserved during this process. the growth pattern of s. citri membranes has been investigated by different methods of membrane labeling. when labeling is done with specific antibodies, a diffuse growth of the membrane is observed. on the contrary, pulse-labeling of the membrane with tritiated ami ...19846715280
cultivation and partial characterization of spiroplasmas in cell cultures.spiroplasmas were propagated in the drosophila melanogaster cell line dm-1. spiroplasma citri and unidentified strains (corn shunt organism, 277f [tick isolate], powder puff, bnr-1, honey bee, and obmg) grew to 10(8) to 10(9) colony-forming units per ml and could be passaged. cytopathic effect (cpe) varied with the infecting spiroplasma. the honey bee isolate killed dm-1 within 2 to 4 days and produced cpe in four mammalian cells tested. at 25 degrees c, suckling mouse cataract agent produced no ...19826797950
sensitivity of various spiroplasma strains against ethanol, formalin, glutaraldehyde, and phenol.the efficacy of four different disinfectants on spiroplasmas pathogenic for plants, insects and vertebrates was determined using a microtiter technique. the results of the sensitivity testing indicate that spiroplasmas display a considerable resistance in comparison to cell wall bearing organisms. particularly honey bee spiroplasmas proved to be less sensitive to the disinfectants tested, whereas spiroplasma citri and the tick spiroplasmas showed a susceptibility comparable to e. coli and staph. ...19816803475
in vitro susceptibility of spiroplasmas to heavy-metal salts.the susceptibility of six spiroplasma strains to heavy-metal salt was characterized in terms of minimal inhibitory concentrations and minimal biocidal concentrations in broth tube dilution tests. the strains were most susceptible to mercuric chloride and silver nitrate; less susceptible to copper sulfate, cobalt chloride, lead nitrate, and cadmium sulfate; and least susceptible to nickel chloride and zinc sulfate. spiroplasma citri strains maroc r8a2 and c189 were the most susceptible to five of ...19836830208
a third dna polymerase from spiroplasma citri and two other spiroplasmas.recently, two dna polymerases (sca and scb) were isolated and characterized from spiroplasma citri. we now have found a third dna polymerase (scc) not only in s. citri but also in the serologically related honeybee spiroplasma bc3 and the unrelated flower spiroplasma bnr1. enzyme scc is n-ethylmaleimide (nem) sensitive. the three dna polymerases from the honeybee spiroplasma seem to be similar to the respective enzymes of s. citri. however, whereas the nem-resistant enzyme sca from s. citri and ...19827061384
characterization of spiroplasmas by serology.five major serologic groups of spiroplasmas have been distinguished on the basis of serology (as determined in a test of deformation and metabolism inhibition), guanine-plus-cytosine content of dna, and dna-dna hybridization. group i includes strains of spiroplasma citri that form a classical subgroup (serovar) complex of four serologically homogeneous serovars that cross-react with each other at varying levels. the four other serogroups do not cross-react with each other or with members of the ...19827123050
polyacrylamide gel analysis of spiroplasmas proteins and its contribution to the taxonomy of spiroplasmas.representative strains of the known groups and serogroups of spiroplasmas have been compared on the basis of the results of one- and two-dimensional protein analysis on polyacrylamide gels. each of the four subgroups of the spiroplasma citri complex (s. citri and honeybee, corn stunt, and 277f spiroplasmas) has characteristic protein profiles and maps. spiroplasma g1 isolated from flowers has a protein profile closely related to that of the honeybee spiroplasmas (kc3, bc3, as576, b1707, b88, and ...19827123051
dna homology among diverse spiroplasma strains representing several serological groups.deoxyribonucleic acid (dna) homology among 10 strains of spiroplasma associated with plants and insects was assessed by analysis of dna-dna hybrids with single strand specific s1 nuclease. based on dna homology, the spiroplasmas could be divided into three genetically distinct groups (designated i, ii, and iii), corresponding to three separate serogroups described previously. dna sequence homology between the three groups was less than or equal to 5%. based on dna homology, group i could be divi ...19807214225
growth and division of spiroplasmas: morphology of spiroplasma citri during growth in liquid medium.the helical mycoplasma spiroplasma citri was examined by electron microscopy with a newly developed transfer technique which preserves the helical morphology of the organism. the smallest viable cell was found to be a two-turn (elementary) helix. during the logarithmic phase of growth, organisms increased in length and divided by constriction, liberating two-turn elementary helices. the most frequently dividing parental helix was one with approximately four turns, yielding two elementary helices ...19817263615
mycoplasma infections of plants.plants can be infected by two types of wall-less procaryotes, spiroplasmas and mycoplasma-like organisms (mlo), both located intracellularly in the phloem tissues of affected plants. spiroplasmas have been cultured, characterized and shown to be true members of the class mollicutes. mlo have not yet been cultured or characterized; they are thought to be mycoplasma-like on the basis of their ultrastructure as seen in situ, their sensitivity to tetracycline and resistance to penicillin. mycoplasma ...19817287398
morphology and ultrastructure of helical and nonhelical strains of spiroplasma citri.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 prote ...19807380813
electrophoretic analysis of the arrangement of spiralin and other major proteins in isolated spiroplasma citri cell membranes.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 oligo ...19817462152
expression of foreign genes and selection of promoter sequences in acholeplasma laidlawii.the stable maintenance and expression of foreign genes in mollicutes (mycoplasmas) have been difficult to achieve due to the lack of suitable vectors. in this paper we show for the first time that a replicating vector can been used to express foreign genes other than antibiotic resistance genes in acholeplasma laidlawii. plasmids derived from the lactococcal vector pnz18 could introduce and maintain four different genes for many generations in a. laidlawii. one of these, encoding the dominant me ...19957496518
expression in spiroplasma citri of an epitope carried on the g fragment of the cytadhesin p1 gene from mycoplasma pneumoniae.we have previously described the use of the replicative form (rf) of spiroplasma citri virus spv1 as a vector for cloning and expressing foreign genes in s. citri, an organism which reads uga as a tryptophan codon (c. stamburski, j. renaudin, and j.m. bové, j. bacteriol. 173:2225-2230, 1991). we now report cloning and expression in s. citri of the g fragment of cytadhesin p1 gene from mycoplasma pneumoniae. the g fragment was inserted in the spv1 rf downstream of a synthetic ribosome binding sit ...19937683022
identification and characterization of the insertion element is1070 from leuconostoc lactis nz6009.a novel insertion sequence, designated is1070, was identified on the lactose plasmid of leuconostoc lactis nz6009 by nucleotide sequence analysis. the 1027-bp sequence contains partially matched (24 of 28 bp) inverted repeats and has one long open reading frame. the deduced 305-amino-acid sequence demonstrated homology to transposases of is30 from escherichia coli, is4351 from bacteroides fragilis, is1086 from alcaligenes eutrophus, is1161 from streptococcus salivarius, isas2 from aeromonas salm ...19957698675
integrative and free spiroplasma citri oric plasmids: expression of the spiroplasma phoeniceum spiralin in spiroplasma citri.the replication region (oric) of the spiroplasma citri chromosome has been recently sequenced, and a 2-kbp dna fragment was characterized as an autonomously replicating sequence (f. ye, j. renaudin, j. m. bové, and f. laigret, curr. microbiol. 29:23-29, 1994). in the present studies, we have combined this dna fragment, containing the dnaa gene and the flanking dnaa boxes, with a cole1-derived escherichia coli replicon and the tet m determinant, which confers resistance to tetracycline. the recom ...19957751299
cloning and sequencing of the replication origin (oric) of the spiroplasma citri chromosome and construction of autonomously replicating artificial plasmids.a 5.6-kbp fragment of spiroplasma citri dna containing the dnaa gene has been cloned and sequenced. nucleotide sequence analysis shows that this fragment harbors the genes for the replication initiator protein (dnaa), the beta subunit of dna polymerase iii (dnan), and the dna gyrase subunits a and b (gyra and gyrb). the arrangement of these genes, dnaa-dnan-gyrb-gyra, is similar to that found in all gram-positive bacterial genomes studied so far, except that no recf gene was found between dnan a ...19947764984
the role of glu187 in the regulation of phosphofructokinase by bacterial phosphofructokinases, either a glutamic or an aspartic residue is present at position 187, and the mechanism of inhibition by phosphoenolpyruvate seems to be correlated to the nature of residue 187. upon binding phosphoenolpyruvate, only the enzymes with a glu187 would undergo a major allosteric conformational change from an active into an inactive state, whereas the enzymes with an asp187 would only show a simple upward shift in their ph-profile of activity. the phosphofructokinase ...19947904653
the osmotic lysis of spiroplasma cells and its use in enzyme studies.a simple and efficient osmotic lysis method was developed for enzyme studies in spiroplasmas. log phase cells in r2 medium were harvested by centrifugation (19,600 x g for 30 min). wash buffer supplemented with 0.23 m sucrose maintained the helicity of spiroplasma cells during washing. osmotic lysis of spiroplasmas was achieved in h buffer that contained no sucrose. sucrose at concentrations as low as 0.004 m dramatically increased the resistance of the spiroplasmas to osmotic lysis. nadh oxidas ...19947954112
optimization of methods for transfecting spiroplasma citri strain r8a2 hp with the spiroplasma virus spv1 replicative methods for the transfection of bacteria were compared and optimized for use in spiroplasma citri strain hp using the spiroplasma virus spv1 r8a2 b replicative form (rf). these methods included both chemical-mediated protocols [cacl2, rbcl/cacl2, polyethylene glycol (peg)], liposome-mediated transfection, electroporation, freeze/thaw cycling, and natural competence. the best protocols were those which utilized peg or electroporation, yielding transfection frequencies of 1.4 x 10(-4) and 9. ...19938356114
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