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PubMed1987

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 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.

Acylation↗

PubMed1984

A Spiroplasma tRNA gene cluster.

Using molecular cloning techniques, a clone containing a 7-kb insert of Spiroplasma species BC3 DNA that hybridized strongly to total labeled Spiroplasma tRNA was identified. Sequence analysis of a portion of this recombinant plasmid identified a cluster of tRNA genes. The gene order was as follows: tRNACys-tRNAArg-tRNAPro-tRNAAla-tRNAMet-tRNAIle and a portion of tRNASer All the genes encode the 3'-terminal CCA and have very high A + T and relatively long intergenic regions. An RNA polymerase promoter site was found upstream of the tRNACys gene. The tRNA gene cluster found in Spiroplasma can be compared with a similar Bacillus subtilis gene cluster, which raises interesting questions concerning gene organization and transcription.

Base Sequence↗

PubMed1984

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 contained a complete ai genome. We propose that the infecting viral DNA circularizes and integrates adjacent to the cryptic prophage in a site-specific recombination event at unique points in both the virus and host genomes.

Bacteriophages↗

PubMedDate not supplied

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 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.

Bacterial Proteins↗

PubMedDate not supplied

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. Introduction of the virus with its host into plants already infected with a severe and potentially lethal strain of S. citri results in a marked suppression of symptoms and a reduction in the number of spiroplasmas.

Bacteriophages↗

PubMedDate not supplied

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.

Cloning, Molecular↗

PubMed1984

Use of cell cultures to study spiroplasma infections.

Invertebrate and mammalian cell cultures have been utilized to study various properties of spiroplasmas. Recent studies have focused on the interaction of Spiroplasma mirum with AG-4676, a cell line derived from the rat eye lens. S. mirum produced cytotoxicity in this cell line, consisting of vacuolization and an increase in the number of binucleated cells. The number of S. mirum organisms attached to the cell increased with increased age of infection. S. mirum does not grow in Dulbecco's modified Earle's medium + 10% fetal bovine serum, the growth medium for AG-4676. It does, however, grow in conditioned medium obtained as supernatant of 1- to 5-day-old AG-4676. This conditioned medium from AG-4676 also supported growth of S. mirum (GT-48), corn stunt spiroplasmas (Rio Grande) and the following unspeciated strains: M-55 (I-6), N-525 (I-7), CN-5 (IX) and AES-1 (X). It did not support S. citri, honeybee BC-3, EA-1 (VIII), MQ-6 (XII) or CC-1 (XIII). The growth promotion titer for S. mirum was 1:8, but this varied with the lot of fetal bovine serum. Biochemical characterization of this growth-promoting factor is in progress.

Adsorption↗

PubMed1984

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. For all of these strains (except CSS, which is similar to S. citri), the location of oxidoreduction sites was affected by the time of incubation in potassium tellurite, and could be observed in different locations of the helix. 277F showed first labeling at the blunt end and a second, strong labeling at the tapered end. The other strains showed labeling all along the helix, but labeling at the blunt end generally appeared first. When Drosophila (Dm-1) cell cultures were infected with S. citri or 277F, the organisms adsorbed to the cells. Observation of infected cells by electron microscopy revealed that S. citri attached to the cells by the blunt end, while 277F attached either by the blunt end or the tapered one. The infection of leafhopper cell cultures (AS-2) with S. citri has been followed by transmission electron microscopy after incorporation of tritiated thymidine in the organism, and an immunocytochemical method has been developed to locate the organisms inside the cells.

Animals↗

PubMed1981

Helical mycoplasmas (spiroplasmas) from Ixodes ticks.

A new spiroplasma isolated from Ixodes pacificus collected in Oregon was serologically and morphologically distinct from known spiroplasmas. The new spiroplasma could also be isolated in tick cell cultures. Discovery of a new fastidious mycoplasma in ticks offers opportunities to explore the possible role of these agents in human and animal diseases.

Animals↗

PubMed1997

Random insertion of transposon Tn4001 in the genome of Spiroplasma citri strain GII3.

Electroporation of Spiroplasma citri strain GII3 with plasmid pMUT containing the Staphylococcus aureus transposon Tn4001 resulted in random insertion of Tn4001 into the spiroplasmal genome. Transformation frequencies reached 10(-8) per colony-forming unit (CFU) when 100 microg of plasmid DNA and 3 x 10(9) S. citri CFU were used. Three other strains of S. citri failed to be transformed under the same conditions. In most cases Tn4001 was randomly inserted in the genome of S. citri strain GII3, without insertion of the carrier plasmid. For most transformed spiroplasmas, Tn4001 was stably maintained in the absence of antibiotic selection for at least 80 bacterial generations, making Tn4001 a potential tool for S. citri mutagenesis.

Anti-Bacterial Agents↗

PubMed2002

Purification and characterization of the major lipoprotein (P28) of Spiroplasma apis.

The plasma membrane of Spiroplasma apis contains a 28-kDa major protein (P28), like other spiroplasmas which also possess a main 26- to 28-kDa membrane polypeptide, called spiralin. In the work described here, we have developed a simple and efficient method for the purification of P28 of this mollicute, a wall-less eubacteria. Proteins were first selectively extracted from the isolated membrane with the mild detergents (i) sodium N-lauroylsarcosinate (Sarkosyl) and (ii) 3-[(3-cholamidopropyl)dimethylamonio]-1-propyl sulfonate (Chaps) and subjected to size-exclusion HPLC in the presence of Chaps. The P28-enriched fraction was thereafter subjected to the second chromatographic step involving cation exchange HPLC in the presence of the same detergent. P28 was purified at the milligram level (yield, 40%). Metabolite labeling with [14C]palmitic acid and chemical analysis of P28 indicated that it is covalently modified by two O-ester-bound fatty acids and one amide-linked chain and contains a S-glycerylcysteine at the N-terminus. By charge-shift electrophoresis, Triton X-114 phase separation, and growth inhibition tests it was shown that P28 is a typical amphiphilic protein exposed, at least partly, at the cell surface. Together, our data provided evidence that P28 is a "classical" lipoprotein (i.e., triacylated) like the members of the spiralin family.

Bacterial Outer Membrane Proteins↗

PubMed1992

Uptake of a fluorescent-labeled fatty acid by spiroplasma floricola cells.

12-(1-pyrene)dodecanoic fatty acid (P12) uptake by Spiroplasma floricola BNR-1 cells was characterized with regard to its kinetics, specificity, metabolism and susceptibility to protein and lipid inhibitors. The uptake process depended on temperature and pH, and exhibited biphasic saturation kinetics with a very low (2.7 microM) and a high (37 microM) apparent Km value. Lauric, myristic, palmitic, stearic and oleic fatty acids did not compete with P12 for transport. The fluorescence of P12 was exclusively recovered in the neutral lipid fraction, suggesting that this fatty acid is not further utilized for phospholipid biosynthesis. Valinomycin, carbonylcyanide m-chlorophenyldrazone (CCCP), dicyclohexylcarbodiimide (DCCD), and pronase strongly reduced P12 uptake by cells, but not by membrane vesicles, affecting the high affinity (low Km) component of the uptake system. Uptake of P12 by cells, as well as by membrane vesicles, was very sensitive to glutaraldehyde, chlorpromazine, phospholipase A21 and ascorbate with FeCl3, which affected the low affinity (high Km) component of a transport system. Digitonin stimulated P12 uptake. We suggest that the incorporation of P12 into spiroplasma cell membrane is a two-step process: a high specificity energy-dependent and protease-sensitive binding to the outer surface of membrane, and a low specificity and energy-independent diffusion and partition into the membrane lipid environment.

Ascorbic Acid↗

PubMed1996

Extensive chromosome aberrations in Spiroplasma citri Strain BR3.

Genetic variations in the plant pathogen, Spiroplasma citri strain BR3, were characterized through physical genome mapping of the original isolate, BR3-3X, and two derivatives, BR3-T and BR3-G, obtained after several years of different maintenance conditions. BR3-T was transmitted from plant to plant via its natural insect vector, the leafhopper Circulifer tenellus, while BR3-G was maintained only in plants by periodic grafting and has lost its ability to be insect transmitted. By pulsed field gel electrophoresis (PFGE) analysis and DNA hybridization, extensive changes in chromosomal DNA restriction patterns relative to the parent, BR3-3X, were observed in both BR3-T and BR3-G, each of which also had a larger genome size than the parent line. Genetic organization was relatively conserved between BR3-T and BR3-3X. In contrast, a large chromosomal inversion and deletions of approximately 10 kb near each of the inversion borders were observed in BR3-G. One of the deletions, which included several possibly functional genes, was closely linked to a SpV1-related transposase gene. The locations of the deletion borders were also determined. The results of this study demonstrated remarkable genome instability of spiroplasmas.

Amino Acid Sequence↗

PubMed1992

Mode of action of the Spiroplasma CpG methylase M.SssI.

The cytosine DNA methylase from the wall-less prokaryote, Spiroplasma strain MQ1 (M.SssI) methylates completely and exclusively CpG-containing sequences, thus showing sequence specificity which is similar to that of mammalian DNA methylases. M.SssI is shown here to methylate duplex DNA processively as judged by kinetic analysis of methylated intermediates. The cytosine DNA methylases, M.HpaII and M.HhaI, from other prokaryotic organisms, appear to methylate in a non-processive manner or with a very low degree of processivity. The Spiroplasma enzyme interacts with duplex DNA irrespective to the presence of CpG sequences in the substrate DNA. The enzyme proceeds along a CpG-containing DNA substrate molecule methylating one strand of DNA at a time.

DNA (Cytosine-5-)-Methyltransferases↗

PubMed1989

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 mutation frequency; liquid holding conditions increase the number of both survivors and mutant colonies. This suggests that, in UV-irradiated S. citri cells maintained under liquid holding conditions, there is no dark reactivation but induction of an error-prone repair system of the SOS type. In S. citri, the error-free light and dark repair systems are inefficient. Results allow the development of a method to select UV-induced mutations usable as markers in genetic studies of Spiroplasma cells.

Dose-Response Relationship, Radiation↗

PubMed2005

The kinky propulsion of Spiroplasma.

Bacteria have evolved many different means of generating movement. In this issue of Cell, Shaevitz et al. (2005) describe the swimming movement of a helical bacterium called Spiroplasma. They discover that Spiroplasma propels itself by generating two temporally distinct kinks that travel the length of the bacterium. These results point to the existence of a contractile apparatus that drives cell movement.

Cell Movement↗

PubMedDate not supplied

Localization and persistence of spiroplasmas in an experimental brain infection in suckling rats.

The GT-48 strain of Spiroplasma mirum was employed in an experimental brain infection in 1-day old suckling rats in order to define the localization and persistence of organisms in brain and spleen and to evaluate the responses of three distinct genetic lines of rats. Rat pups inoculated intracerebrally with 30-300 organisms exhibited peak infections at 15-20 days post-inoculation, while challenge levels of 3-30 organisms showed some persistent infections for as long as 60 days. The responses of three genetic lines of rats to the experimental spiroplasma infection did not differ significantly over a 60-day follow-up period. The acute-phase infection in suckling rats showed a rapidly developing and widespread dissemination of organisms to all major areas of the brain.

Acute Disease↗

PubMedDate not supplied

[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 R.Br.) of the oriental coast and on bush plants. A breeding of this leafhopper was initiated, and we demonstrated that it was able to acquire S. citri from infected periwinkles, multiply the organisms in its body and transmit them to healthy plants. This leafhopper can also be infected after injection of S. citri. The leafhoppers thus inoculated can transmit the spiroplasma very efficiently to healthy plants. This is the first report of the transmission of S. citri by Neoaliturus haematoceps both after acquisition on a diseased plant and after injection.

Animals↗