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At least 19 recordsLinked to original sources
PubMedDate not supplied

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 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.

Bacterial Proteins↗
PubMedDate not supplied

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 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.

Antibodies, Bacterial↗
PubMed1995

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 recombinant plasmid, named pBOT1, was introduced into S. citri cells, in which it replicated. Plasmid pBOT1 was shuttled from E. coli to S. citri and back to E. coli. In S. citri, replication of pBOT1 did not require the presence of a functional dnaA gene on the plasmid. However, the dnaA box region downstream of the dnaA gene was essential. Upon passaging of the S. citri transformants, the plasmid integrated into the spiroplasmal host chromosome by recombination at the replication origin. The integration process led to duplication of the oriC sequences. In contrast to the integrative pBOT1, plasmid pOT1, which does not contain the E. coli replicon, was stably maintained as a free extrachromosomal element. Plasmid pOT1 was used as a vector to introduce into S. citri the G fragment of the cytadhesin P1 gene of Mycoplasma pneumoniae and the spiralin gene of Spiroplasma phoeniceum. The recombinant plasmids, pOTPG with the G fragment and pOTPS with the spiralin gene, were stably maintained in spiroplasmal transformants. Expression of the heterologous S. phoeniceum spiralin in S. citri was demonstrated by Western immunoblotting.

Adhesins, Bacterial↗
PubMedDate not supplied

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 B63). The flower spiroplasmas fall into two groups on the basis of the mole percentage (mol%) of guanine plus cytosine (G + C) of their deoxyribonucleic acid (DNA) 26 and 30 mol %, respectively) and their serologic properties. The three flower spiroplasmas (OBMG, BNR1, and 23-6) with 26 mol % of G + C have the same basic protein pattern. Although some of the flower spiroplasmas with 30 mol % of G + C have closely related protein patterns, others have more distant profiles. Spiroplasmas B13 and L89, isolated from honeybees and froghoppers, respectively, had DNA with 30 mol % of G + C and could be included with other flower spiroplasmas with similar DNA base composition on the basis of their protein profiles and maps. Individual proteins separated by two-dimensional analysis on polyacrylamide gels have been used for the production of monospecific immunoglobulins, which are useful in taxonomic studies.

Animals↗
PubMed1983

Nutritional requirements of two flower spiroplasmas and honeybee spiroplasma.

A chemically defined medium (CC-494) was used to study the nutritional requirements of three spiroplasmas representing three distinct serogroups: flower spiroplasmas [Spiroplasma floricola and FS (SR-3)] and honeybee spiroplasma [HBS (AS-576)]. Glucose, fructose, and mannose were utilized by all three spiroplasmas. In addition, the honeybee spiroplasma could ferment trehalose, FS (SR-3) could ferment sucrose, and S. floricola could ferment trehalose, sucrose, and raffinose. The three spiroplasmas varied greatly in their requirements of amino acids for growth. S. floricola was the only strain that utilized arginine. HBS (AS-576) required at least one purine and one pyrimidine base (either free base or ribonucleoside) for growth, while both flower spiroplasmas grew with only one base in the medium. Oleic acid, cholesterol, and bovine serum albumin were essential to all three spiroplasmas. Palmitic acid, which was nonessential, promoted growth significantly.

Amino Acids↗
PubMedDate not supplied

Pathogenicity of Spiroplasma apis and other spiroplasmas for honey-bees in southwestern France.

The haemolymph of honey-bees affected by a May disease-like disorder in southwestern France contained numerous spiroplasmas. Further characterization of the organisms and pathogenicity assays showed that the causal agent of the disease was a spiroplasma belonging to group IV. The name Spiroplasma apis was given to the reference strain B31 (ATCC 33834), one of the numerous similar isolates cultured from May-disease-affected bees. Spiroplasma isolates related to S. apis could be grown from the surface of flowers collected within the area visited by bees from the diseased hives. Several other strains belonging to group IV spiroplasmas were also isolated from the surface of flowers growing in southwestern France. In the same area, we also isolated, from pools of apparently healthy honey-bees and from the surface of a tulip tree flower, spiroplasma strains belonging to group I-2. One of these strains was shown to be pathogenic when introduced into adult bees by injection or food ingestion.

Animals↗
PubMed1982

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 that from the BC3 honeybee spiroplasma are retained on DEAE-cellulose and require 0.09 M KCl for elution, the NEM-resistant enzyme A from the flower spiroplasma BNR1 is not retained.

Chromatography, DEAE-Cellulose↗
PubMed1991

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 filamentous spiroplasma virus (SpV1) to clone and express the E. coli-derived chloramphenicol acetyltransferase (CAT) gene in Spiroplasma citri. The CAT gene was inserted in one of the four intergenic regions of the SpV1 RF and introduced into cells by electroporation. Both the RF and the virion DNA produced by the transfected cells contained the CAT gene sequences. Northern blot analysis, primer extension, and S1 mapping showed that transcription of the CAT gene started from a promoter located on the SpV1 RF and was terminated downstream of the CAT gene, still within the viral RF. Expression of the CAT gene was demonstrated by acetylation of chloramphenicol by cell-free extracts from the transfected spiroplasmas.

Base Sequence↗
PubMedDate not supplied

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. 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.

Animals↗
PubMedDate not supplied

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 and also seems to be present as an 8-Kbp plasmid in three spiroplasmas not belonging to the S. citri species. Up to now, pMH1 had been found as a free 7-Kbp plasmid only in the S. citri strain MH. However, DNA sequences corresponding to the entire pMH1 DNA have been found to be integrated in the high-molecular-weight-DNA molecules and perhaps the chromosomal DNA itself of two other S. citri strains. DNA sequences hybridizable with pMH1 DNA have also been found to be integrated into high-molecular-weight-DNA molecules from several spiroplasma strains not belonging to the S. citri species.

Base Sequence↗
PubMed1990

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 Escherichia coli. It was further subcloned in the replicative forms of M13mp18 and M13mp19, and its nucleotide sequence was determined (GenBank accession number M33991). An open reading frame showing 88.6% base sequence homology with the S. citri spiralin gene could be identified and was assumed to be the gene encoding S. melliferum spiralin. The deduced amino acid sequence of the protein had 75% homology with the spiralin sequence. In particular, the two proteins possess a stretch of 20 amino acids which can form an alpha-helix, in which all polar amino acids occupy approximately one-third of the axial projection down the helix. On the basis of these data and published data, we propose a topological model for the structural organization of the spiralin in the cell membrane of spiroplasmas.

Amino Acid Sequence↗
PubMed1990

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 spiroplasma cell surface.

Bacterial Outer Membrane Proteins↗
PubMed1993

Optimization of methods for transfecting Spiroplasma citri strain R8A2 HP with the spiroplasma virus SpV1 replicative form.

Seven 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 9.1 x 10(-4) transfectants/colony-forming unit (CFU), respectively. For both of these protocols, transfection frequencies were higher using CsCl-purified, covalently closed, circular DNA. In the PEG-mediated protocol, Sigma 8000 brand PEG at a final concentration of 44%, and the presence of carrier DNA proved to be optimal with a PEG exposure time of 2 min. Using electroporation, a 1-2 ms pulse of a 6.5 kV/cm electric field was best; washing the host cell pellet prior to electroporation enhanced efficiencies by 50%. Linearization of the DNA resulted in lower transfection efficiencies by either method.

Bacteriophages↗
PubMed1986

The spiroplasma virus 4 replicative form cloned in Escherichia coli transfects spiroplasmas.

The replicative form (RF) of spiroplasma virus 4 (SpV4) has been purified from infected cells of Spiroplasma melliferum strain G1 by alkaline lysis followed by low melting point agarose gel electrophoresis. A partial restriction map has been established. The circular RF was linearized by cutting at the unique ClaI restriction site and has been cloned in Escherichia coli HB101 using the plasmid pBR328 as a vector. The recombinant plasmid was purified by equilibrium centrifugation in ethidium bromide-cesium chloride gradient. After ClaI endonuclease digestion, the inserted SpV4 RF DNA was recovered by low melting point agarose gel electrophoresis and was recircularized by ligation. The cloned SpV4 RF DNA was demonstrated to be infectious by transfection.

Bacteriophages↗
PubMed1979

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.

Bacterial Proteins↗
PubMed1981

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. Influence of pH and temperature on the morphology of the organism was also investigated. In unbuffered medium, growth of the organism produced a significant decrease in pH and a consequent formation of abnormal morphological forms and cell lysis. At 37 degrees C, cell division was inhibited, leading to a progressive disappearance of two-turn helices and an increase in the average length of other helices. Finally, helices were never seen to arise from round bodies at any stage of the growth cycle.

Cell Division↗