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PubMed1997

Isolation, characterization, and complementation of a motility mutant of Spiroplasma citri.

The helical mollicute Spiroplasma citri, when growing on low-agar medium, forms fuzzy colonies with occasional surrounding satellite colonies due to the ability of the spiroplasmal cells to move through the agar matrix. In liquid medium, these helical organisms flex, twist, and rotate rapidly. By using Tn4001 insertion mutagenesis, a motility mutant was isolated on the basis of its nondiffuse, sharp-edged colonies. Dark-field microscopy observations revealed that the organism flexed at a low frequency and had lost the ability to rotate about the helix axis. In this mutant, the transposon was shown to be inserted into an open reading frame encoding a putative polypeptide of 409 amino acids for which no significant homology with known proteins was found. The corresponding gene, named scm1, was recovered from the wild-type strain and introduced into the motility mutant by using the S. citri oriC plasmid pBOT1 as the vector. The appearance of fuzzy colonies and the observation that spiroplasma cells displayed rotatory and flexional movements showed the motile phenotype to be restored in the spiroplasmal transformants. The functional complementation of the motility mutant proves the scm1 gene product to be involved in the motility mechanism of S. citri.

Amino Acid Sequence↗
PubMed1999

Gene disruption through homologous recombination in Spiroplasma citri: an scm1-disrupted motility mutant is pathogenic.

To determine whether homologous recombination could be used to inactivate selected genes in Spiroplasma citri, plasmid constructs were designed to disrupt the motility gene scm1. An internal scm1 gene fragment was inserted into plasmid pKT1, which replicates in Escherichia coli but not in S. citri, and into the S. citri oriC plasmid pBOT1, which replicates in spiroplasma cells as well as in E. coli. Electrotransformation of S. citri with the nonreplicative, recombinant plasmid pKTM1 yielded no transformants. In contrast, spiroplasmal transformants were obtained with the replicative, pBOT1-derived plasmid pCJ32. During passaging of the transformants, the plasmid was found to integrate into the chromosome by homologous recombination either at the oriC region or at the scm1 gene. In the latter case, plasmid integration by a single crossover between the scm1 gene fragment carried by the plasmid and the full-length scm1 gene carried by the chromosome led to a nonmotile phenotype. Transmission of the scm1-disrupted mutant to periwinkle (Catharanthus roseus) plants through injection into the leafhopper vector (Circulifer haematoceps) showed that the motility mutant multiplied in the insects and was efficiently transmitted to plants, in which it induced symptoms similarly to the wild-type S. citri strain. These results suggest that the spiroplasmal motility may not be essential for pathogenicity and that, more broadly, the S. citri oriC plasmids can be considered promising tools for specific gene disruption by promoting homologous recombination in S. citri, a mollicute which probably lacks a functional RecA protein.

Chemokines, C↗
PubMed2003

Mass distribution and spatial organization of the linear bacterial motor of Spiroplasma citri R8A2.

In the simple, helical, wall-less bacterial genus Spiroplasma, chemotaxis and motility are effected by a linear, contractile motor arranged as a flat cytoskeletal ribbon attached to the inner side of the membrane along the shortest helical line. With scanning transmission electron microscopy and diffraction analysis, we determined the hierarchical and spatial organization of the cytoskeleton of Spiroplasma citri R8A2. The structural unit appears to be a fibril, approximately 5 nm wide, composed of dimers of a 59-kDa protein; each ribbon is assembled from seven fibril pairs. The functional unit of the intact ribbon is a pair of aligned fibrils, along which pairs of dimers form tetrameric ring-like repeats. On average, isolated and purified ribbons contain 14 fibrils or seven well-aligned fibril pairs, which are the same structures observed in the intact cell. Scanning transmission electron microscopy mass analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified cytoskeletons indicate that the 59-kDa protein is the only constituent of the ribbons.

Bacterial Proteins↗
PubMed1988

Evaluation of [3H]thymidine uptake method for studying growth of spiroplasmas under various conditions.

[3H]thymidine uptake and colony counts are quantitative and inexpensive methods for studying Spiroplasma growth. Using these techniques, we demonstrated subtle effects on the growth of suckling mouse cataract agent of medium alterations, inoculum size, and freezing of cultures. In addition, suckling mouse cataract agent multiplied more actively under aerobic than under anaerobic conditions. These techniques have wide application for the study of Spiroplasma growth and will be useful for the development of a defined medium.

Anaerobiosis↗
PubMed2005

Detection of DNA of 'Candidatus Mycoplasma haemominutum' and Spiroplasma sp. in unfed ticks collected from vegetation in Japan.

DNA fragments of 'Candidatus Mycoplasma haemominutum', a feline heamobartonella pathogen, were detected from unfed Ixodes ovatus collected from vegetation in Hokkaido, Fukushima and Yamaguchi Prefectures, and unfed Haemaphysalis flava in Yamaguchi Prefecture. This finding suggests that ixodid tick is a possible vector of 'C. Mycoplasma haemominutum'. Spiroplasma DNA was also detected from unfed I. ovatus in Hokkaido, Fukushima and Yamaguchi Prefectures. The analysis of nucleotides sequence suggested that this Spiroplasma was distinct from registered species.

Animals↗
PubMed1992

[Spiroplasma from Tabanidae capable to multiply and to persist in the central nervous system in mice].

Using alternate passages of cultivation in vitro at 37 degrees C and in suckling mice following intracerebral inoculation, a tabanid spiroplasma was demonstrated capable of multiplication and persistence in mice for 6 successive passages, without production of specific antibody. This is the first report of a spiroplasma from a common flying haematophagous arthropod shown to produce persistent infection of a mammal.

Animals↗
PubMed1991

[Experimental infection of Aedes albopictus mosquitoes by a spiroplasma strain isolated from Culex annulus in Taiwan].

Two Aedes albopictus strains are infected by one of the four presently known mosquito spiroplasma strains, the CUAS-1 strain isolated from Culex annulus from Taïwan. An infection by intra-thoracic inoculation leads to a pathogenic effect, dose-dependent, the longevity of mosquitoes being shortened. This pathogenicity is observed at various time after inoculation depending of the mosquito strain. On the other hand, an oral infection has no consequence on the longevity of infected females. However, the replication of spiroplasma has been demonstrated with both infection procedures.

Aedes↗
PubMed1987

Organization and structure of tRNA genes in Spiroplasma melliferum.

To investigate the organization of tRNA genes in the honeybee pathogen, Spiroplasma melliferum (previously referred to as Spiroplasma sp. BC3), total labeled tRNA from S. melliferum was used as a hybridization probe to an EcoRI digest of the genomic DNA. The results show two, or possibly three, strongly hybridizing bands. Comparing the pattern obtained to that of Bacillus subtilis DNA suggests either that the S. melliferum tRNA genes are more extensively clustered or, more likely, that the S. melliferum genome does not encode a full complement of tRNA genes, as has been suggested for other Mollicutes. We screened a library of EcoRI fragments of S. melliferum DNA cloned in pBR322 with total radioactive tRNA as a probe and selected one of the tRNA gene clusters. Subsequent sequence analysis of a portion of the clone showed 10 tRNA genes probably comprising a single operon. Comparison of sequence with a tRNA gene cluster from Mycoplasma mycoides and a portion of a cluster from B. subtilis showed an identical order of tRNA genes and the isoacceptors encoded. Such a striking comparison in gram-positive eubacteria suggests an important function for regulation and co-transcription of these tRNA genes.

Bacillus subtilis↗
PubMed1987

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, not only in Syria but also in Morocco and France (Corsica) from 1978 to 1985. Neoaliturus (Circulifer) haematoceps was the only species found to harbor the spiroplasma in Morocco, Syria and France (Corsica). Leafhoppers of this species were collected in Corsica, raised in Bordeaux and shown to be vectors of S. citri. The search for N. haematoceps in nature has revealed that Salsola kali (Chenopodiacae) is a favored host plant of this leafhopper. This plant has a wide geographical distribution. In Iran, for instance, it is well known, and grows close to sugar beet and citrus-growing areas. In such areas, sugar beet is known to be infected by curly top virus, and citrus by CSD. N. haematoceps is a vector of both of these diseases. N. tenellus, the vector of S. citri in the USA, is present in the Mediterranean area, but in view of its paucity it does not seem to be a major vector there. In Iran, even though less abundant than N. haematoceps, it could play a role in S. citri transmission.

Animals↗
PubMed1987

Cloning and sequencing of the genome of spiroplasma virus 4.

Spiroplasma virus 4 (Sp V4) has a circular single-stranded DNA. The replicative form (RF) of Sp V4 has been purified from infected cells of Spiroplasma melliferum, strain G1, and cloned in Escherichia coli (HB101) using plasmid pBR328 as the vector. The cloned RF was shown to be infectious by transfection. The Sp V4 RF was randomly subcloned in E. coli (TG1) using the M1 3 mp8 RF as the vector and sequenced by the dideoxy chain termination technique. We found that UGA is probably not a termination codon, but codes for tryptophan. Eight open reading frames, including that for the 65,000-dalton capsid protein, have been detected; they involve all three reading frames.

Bacteriophages↗
PubMed1987

Effect of nutrients and metabolic inhibitors on the growth of spiroplasmas.

Glycolytic intermediates replaced glucose as carbohydrate sources in a chemically defined medium CC-494N, whereas keto acids of citric acid cycle intermediates and glyoxylate were supplemented with 10 mM glucose. Glucose-6-phosphate, fructose-1,6-bisphosphate, 3-phosphoglyceraldehyde, and pyruvate supported limited growth for Spiroplasma floricola (23-6), whereas only pyruvate supported SR 3 spiroplasma and S. melliferum (AS 576). alpha-Ketoglutarate at 5 mM or greater, hematin, 2,4-dinitrophenol, sodium fluoride, and arsenite were dramatically inhibitory, whereas acetic anhydride, tricarballylate, acetylene dicarboxylate, and sodium cyanide were mildly inhibitory at 10 mM or lower. Malonate at 50 mM was not inhibitory.

Carbohydrate Metabolism↗
PubMed1987

Spiroplasmas: gene structure and expression.

Upon sequencing of the SpV4 genome, eight putative open reading frames (ORFs) including that for the 65-kilodalton (kDa) capsid protein were detected. They involve all three reading frames. Three promoter sequences were found, as well as a transcription terminator and the initiation site for complementary strand synthesis. Ribosome binding sites and regulatory sequences are closely related to those of Eubacteria. Codon usage analysis showed that A and T terminated codons are preferably used. UAA is the major termination codon. Upon cloning of the full-size SpV4 replicative form, the capsid protein gene could not be expressed in Escherichia coli, whereas the spiralin gene cloned in the same bacterium is expressed. These results suggest that in spiroplasmas, as in Mycoplasma capricolum, UGA is not a termination codon, but very probably codes for tryptophan. Spiralin contains no tryptophan. Hence, its gene contains no UGA codons and can thus be expressed in E. coli. On the other hand, the gene for capsid protein has nine UGA codons and cannot be fully expressed in the bacterium. Our results fully support the bacterial origin of spiroplasmas.

Bacteria↗
PubMed1987

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. Therefore, a DNA hybridization technique was developed. The radioactive DNA probe was prepared either with total S. citri DNA or with the cloned DNA fragment carrying the spiralin gene of S. citri. Used in dot hybridization, the probe was able to detect concentrations of S. citri one 100th the size of those revealed by ELISA.

Animals↗
PubMed1979

Spiroplasma-like inclusions in Creutzfeldt-Jakob disease.

Spiral membranous inclusions were discovered on electron microscopic study of brain biopsy tissues from a 46-year-old man with Creutzfeldt-Jakob disease (CJD). These replicate coiled membranous configurations measured 850 to 1,000 nm in length and 75 to 137.5 nm in width and were located within axoplasm, primarily in presynaptic terminals. These inclusions closely resemble Spiroplasma, a plant pathogen, and the finding of these structures in CJD suggests the concurrence of Spiroplasma infection with a human chronic degenerative brain disease.

Axons↗
PubMed1984

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 have been isolated several times from field-collected Ollarianus strictus (Ball), a leafhopper that, like several other common California leafhoppers, can acquire S. citri from plants under laboratory conditions but apparently cannot transmit it. In California, several plants commonly are doubly infected with S. citri and either the aster yellows agent or a C. tenellus-transmitted agent that causes floral virescence. In the Southwest, S. citri has been found throughout the year in naturally infected perennial and annual plants and in C. tenellus. Its isolation from C. tenellus and from cultivated turnip plants in Washington, from C. tenellus in Utah, and from citrus and other plants in Arizona, indicates a wide geographical distribution in the western U.S.

Animals↗
PubMedDate not supplied

Proposed subgroups of spiroplasmas of high guanine plus cytosine content, group IV.

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

Animals↗
PubMed1982

Viruses of mycoplasmas and spiroplasmas.

Acholeplasmas, mycoplasmas and spiroplasmas of the class Mollicutes have been shown to carry DNA viruses. Morphologically they occur as rods, polyhedrons with short or long tails and as enveloped spheres. These latter resemble enveloped nonlytic animal viruses and are now placed in the new family of bacterial viruses the Plasmaviridae. Viruses of mycoplasmas and spiroplasmas present a suitable experimental model for studies of the virus-cell interactions.

Acholeplasma↗