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PubMed1985

Spiroplasma membrane lipids.

Membranes of six spiroplasma strains belonging to different Spiroplasma species and subgroups were isolated by a combination of osmotic lysis and sonication in the presence of EDTA to block endogenous phospholipase activity. Analysis of membrane lipids showed that in addition to free and esterified cholesterol the spiroplasmas incorporated exogenous phospholipids from the growth medium. Sphingomyelin was preferentially incorporated from phosphatidylcholine-sphingomyelin vesicles or from the serum used to supplement the growth medium. Palmitate was incorporated better than oleate into membrane lipids synthesized by the organisms during growth. The major phospholipid synthesized by the spiroplasmas was phosphatidylglycerol. The positional distribution of the fatty acids in phosphatidylglycerol of Spiroplasma floricola resembled that found in Mycoplasma species, in which the saturated fatty acids prefer position 2 in the glycerol backbone and not position 1 as found in Acholeplasma species and elsewhere in nature. Electron paramagnetic resonance analysis of spin-labeled fatty acids incorporated into S. floricola membranes exhibited homogeneous single-component spectra without immobilized regions. The S. floricola membranes were more rigid than those of Acholeplasma laidlawii and less rigid than those of Mycoplasma gallisepticum.

Cholesterol↗
PubMed1991

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 constructed from the N-terminal amino acid sequence of two peptides obtained after V8 protease hydrolysis of the fibril protein. The probes were used to identify the clones in a genomic DNA library of Spiroplasma citri that contained inserts carrying the probe sequence. Sequencing of a 3.3-kbp fragment yielded the full open reading frame of the fibril protein gene and the start of a second open reading frame of an unknown protein. The fibril protein is composed of 515 amino acids, which have a computed molecular mass of 59 kDa. Northern (RNA) blot hybridization and primer extension experiments showed that transcription of the fibril protein gene starts from a promoter located 100 nucleotides upstream of the initiation codon and stops at a rho-independent type terminator, leading to a 1.7-kbp transcript. Southern blot hybridization of genomic DNA using the fibril protein gene as the probe showed that a single copy of the gene is present in the chromosomes of both S. citri and Spiroplasma melliferum. The genotypic symbol fib is proposed for the spiroplasma fibril protein gene.

Amino Acid Sequence↗
PubMed2006

Absence of Spiroplasma or other bacterial 16s rRNA genes in brain tissue of hamsters with scrapie.

Spiroplasma spp. have been proposed to be the etiological agents of the transmissible spongiform encephalopathies (TSEs). In a blind study, a panel of 20 DNA samples was prepared from the brains of uninfected hamsters or hamsters infected with the 263K strain of scrapie. The brains of the infected hamsters contained > or =10(10) infectious doses/g. The coded panel was searched for bacterial 16S rRNA gene sequences, using primers selective for spiroplasma sequences, primers selective for mollicutes in general, and universal bacterial primers. After 35 PCR cycles, no samples were positive for spiroplasma or any other bacterial DNA, while control Spiroplasma mirum genomic DNA, spiked at 1% of the concentration required to account for the scrapie infectivity present, was readily detected. After 70 PCR cycles, nearly all samples yielded amplified products which were homologous to various bacterial 16S rRNA gene sequences, including those of frequent environmental contaminants. These sequences were seen in uninfected as well as infected samples. Because the concentration of scrapie infectivity was at a known high level, it is very unlikely that a bacterial infection at the same concentration could have escaped detection. We conclude that the infectious agent responsible for TSE disease cannot be a spiroplasma or any other eubacterial species.

Animals↗
PubMed1978

Spiroplasma associated with flowers of the tulip tree (Liriodendron tulipifera L.).

Spiroplasmas were isolated and cultivated from nonsurface-sterilized petals and bracts excised from flowers of Liriodendron tulipifera L. (tulip tree) in Anne Arundel, Charles, and Prince George's Counties in Maryland, and East Lyme County, Connecticut. All isolates grew at 30 and at 37 degrees C. Morphology of colonies on solid agar (1%) medium containing serum differed among isolates: some isolates formed highly diffuse, barely visible colonies; others formed distinct colonies with granular centers surrounded by minute surface or submerged 'satellite' colonies. Cellular morphology and motility of organisms in broth and in agar culture were typical of the spiroplasmas pathogenic in plants and insects. In viscous media containing methylcellulose, spiroplasmas freely suspended in the medium swam, and organisms in contact with glass slide or coverslip "crawled" across the solid surface. Frowth inhibition, metabolic inhibition, and organism deformation tests failed to reveal a serologic relationship between spiroplasma strain 23-6 from tulip tree flowers and spiroplasma strain AS 576 from honey bee.

Agar↗
PubMed2003

Spiroplasma citri, a plant pathogenic molligute: relationships with its two hosts, the plant and the leafhopper vector.

Spiroplasma citri, the type species of the genus Spiroplasma (Spiroplasmataceae, Mollicutes), is restricted to the phloem sieve tubes and transmitted by phloem sap-feeding insects, as is characteristic of the phytopathogenic mollicutes. The spiroplasmas are the only mollicutes showing motility and helical morphology, apparently mediated by a contractile fibrillar cytoskeleton bound to the inner surface of the spiroplasmal membrane. MreB genes, which are involved in cell-shape determination, have been identified in S. citri. Identified genes of other functional groups are those involved in the transmission of S. citri by the leafhoppers and genes coding for lipoproteins, including spiralin, bound to the outer surface of the spiroplasma membrane. S. citri mutants that are unable to use fructose induce only mild and delayed symptoms. Fructose utilization by the sieve tube-restricted wild-type spiroplasmas is postulated to deprive the companion cells of fructose, thereby impairing sucrose loading into the sieve tubes.

Animals↗
PubMed2004

Shaping and moving a spiroplasma.

The Mollicutes (Spiroplasma, Mycoplasma and Acholeplasma) are the most minimal cells known to exist, being the smallest and simplest free-living and self-replicating forms of life. Phylogenetically, the Mollicutes are related to gram-positive bacteria and have evolved, by regressive evolution and genome reduction, from Clostridia. The smallest genome in this group (Mycoplasma genitalium - 5.77 x 10(5) bp) is only twice that of a large virus (e.g., Entomopox viruses). The largest Mollicute genome (Spiroplasma LB12 - 2.2 x 10(6) bp) is only about half that of, e.g., Escherichia coli. Structurally, the Mollicutes lack cell walls and flagella, but have internal cytoskeletons and are motile and chemotactic. Only a cholesterol-containing unit membrane envelops the cells. No analogs to the bacterial chemotactic and motility (che, mot, fla) genes, genes for a two-component signal transduction system, genes associated with gliding, or genomic homologs for the eukaryotic cytoskeleton and motor proteins were found in the Mollicutes. The Spiroplasmas are unique amongst the Mollicutes in having a well-defined basic helical cell geometry. In this respect, the Spiroplasma cell can, essentially, be viewed as a helical dynamic membranal tube (diameter approximately 0.2 microm; equivalent to that of one eukaryotic flagellar axoneme or to a bacterial flagellar bundle). A flat cytoskeletal ribbon of parallel fibrils is attached to the inside of the cellular tube. Both tube and cytoskeleton are mutually coiled into a dynamic helix driven by differential length changes of the fibrils, which function as linear motors. The cytoskeletal ribbon follows the shortest (inner) helical line on the inner surface of the cellular tube. Being helical allows for further analytical reduction and consequent structural quantification of Spiroplasma. Of particular importance is the ability to correlate light and electron microscopy data and to calculate the fibril lengths (and corresponding molecular dimensions) in the helical and nonhelical dynamic states. The structural unit of the contractile cytoskeleton is a approximately 50-Angstrom-wide filament comprised of pairs of the 59-kD fib gene product. The monomers are arranged in pairs with opposite polarities allowing for a approximately 100-Angstrom-long axial repeat. The functional unit of the contractile cytoskeletal ribbon is a fibril comprised of an aligned pair of filaments. Neighboring repeats form a tetrameric ring with a lateral repeat of approximately 100 A. The axial length of the rings may shorten by approximately 40%, driving the changes in the fibril lengths and, consequently, helical dynamics. Local length changes result in helical symmetry breaking and nonreciprocating cell movements allowing for net directional displacement. Flexing allows for changes in swimming direction.

Chemotaxis↗
PubMed2025

Spiroplasma endosymbiont reduction of host lipid synthesis and Stomoxyn-like peptide contribute to trypanosome resistance in the tsetse fly Glossina fuscipes.

Tsetse flies (Glossina spp.) vector African trypanosomes that cause devastating diseases in humans and domestic animals. Within the Glossina genus, species in the Palpalis subgroup exhibit greater resistance to trypanosome infections compared to those in the Morsitans subgroup. Varying microbiota composition and species-specific genetic traits can significantly influence the efficiency of parasite transmission. Notably, infections with the endosymbiotic bacterium Spiroplasma have been documented in several Palpalis subgroup species, including Glossina fuscipes fuscipes (Gff). While Spiroplasma infections in Gff are known to hinder trypanosome transmission, the underlying mechanisms remain unknown. To investigate Spiroplasma-mediated factors affecting Gff vector competence, we conducted high-throughput RNA sequencing of the gut tissue along with functional assays. Our findings reveal elevated oxidative stress in the gut environment in the presence of Spiroplasma, evidenced by increased expression of nitric oxide synthase, which catalyzes the production of trypanocidal nitric oxide. Additionally, we observed impaired lipid biosynthesis leading to a reduction of this important class of nutrients essential for parasite and host physiologies. In contrast, trypanosome infections in Gff's midgut significantly upregulated various immunity-related genes, including a small peptide, Stomoxyn-like, homologous to Stomoxyn first discovered in the stable fly, Stomoxys calcitrans. We observed that the Stomoxyn-like locus is exclusive to the genomes of Palpalis subgroup tsetse species. GffStomoxyn is constitutively expressed in the cardia (proventriculus) and synthetic GffStomoxyn exhibits potent activity against Escherichia coli and bloodstream form of Trypanosoma brucei parasites, while showing no effect against insect stage procyclic forms or tsetse's commensal endosymbiont Sodalis in vitro. Reducing GffStomoxyn levels significantly increased trypanosome infection prevalence, indicating its potential trypanocidal role in vivo. Collectively, our results suggest that the enhanced resistance to trypanosomes observed in Spiroplasma-infected Gff may be due to the reduced lipid availability necessary for parasite metabolic maintenance. Furthermore, GffStomoxyn could play a crucial role in the initial immune response(s) against mammalian parasites early in the infection process in the gut and prevent gut colonization. We discuss the molecular characteristics of GffStomoxyn, its spatial and temporal expression regulation and its microbicidal activity against Trypanosome parasites. Our findings reinforce the nutritional influences of microbiota on host physiology and host-pathogen dynamics.

Animals↗
PubMed1987

New spiroplasmas from insects and flowers: isolation, ecology, and host association.

Eight spiroplasma strains from insects and one from spring flowers failed to react with antisera specific for any of the 11 described spiroplasma groups, with sera directed against spiroplasma Group I subgroups, or with sera directed against two unnumbered groups previously reported to occur in tabanid flies. Strains, all from Maryland, were isolated from the hemolymph of the spotted cucumber beetle Diabrotica undecimpunctata and the lampyrid beetle Ellychnia corrusca, and the guts of the cantharid beetles Cantharis bilineatus and C. carolinus. Other strains were obtained from a tabanid fly, Tabanus gladiator and from the firefly Photuris pennsylvanica in Maryland and from the mosquito Culex tritaeniorhynchus in Taiwan. An isolate from pooled Cicadulina bipunctella leafhoppers in Syria apparently represented a unique group. A single isolate from spring flowers in Oklahoma also appeared to be unrelated to existing groups or subgroups. One-way deformation tests using sera prepared against known beetle and tabanid spiroplasmas showed each of the above strains to be unique. Although these results strongly indicate that the nine strains studied are representatives of unique new spiroplasma groups, the formal designation of new groups awaits fulfillment of recently proposed criteria.

Animals↗
PubMedDate not supplied

Spiroplasma apis, a new species from the honey-bee Apis mellifera.

Two spiroplasma strains (B31 and B39) recovered from diseased honey-bees (Apis mellifera) in southwestern France were similar in biochemical, serological and pathological properties. The organisms grew at 30 degrees C, required cholesterol for growth, fermented glucose, catabolized arginine and produced a film and spot reaction. The two spiroplasmas were serologically indistinguishable but were related to serogroup IV spiroplasmas, which had been previously isolated from flower surfaces and from insects. The isolates were distinct from the three previously established species of Spiroplasma and from other presently known serogroups. The G + C content of the DNA from strain B31 was 30 +/- 1 mol %. Both B31 and B39 strains were associated with a lethal infection ("May disease") of the honey-bee. On the basis of the characterization presented here, it is proposed that these spiroplasmal pathogens of bees and allied strains be classified as a new species, Spiroplasma apis, the type strain of which is B31 (ATCC 33834).

Animals↗
PubMedDate not supplied

Spiroplasma taxonomy and identification of the sex ratio organisms: can they be cultivated?

The spiroplasmas that occur naturally in several species of Drosophila were the first spiroplasmas ever observed, even though their discoverers, D.F. Poulson and B. Sakaguchi, in 1961 described them as being "treponema-like spirochetes." These Drosophila spiroplasmas are transovarially, or maternally, transmitted by infected females whose progenies are composed entirely of females. A more recently discovered Drosophila spiroplasma found in flies originating in Ito, Japan, is also maternally inherited but does not result in the elimination of males from the progeny of infected females. In spite of their early discovery, their high numerical density in the hemolymph of infected females (10(6)-10(7)/microliters), and numerous attempts at in vitro cultivation, they remain prime examples of non-cultivable spiroplasmas. It is the purpose of this paper to recount some of the approaches used in attempts at their cultivation.

Animals↗
PubMed1981

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. aureus. The findings are discussed in relation to the technique used and in respect of the implications for laboratory work.

Disinfectants↗
PubMed2003

Gene content and organization of an 85-kb DNA segment from the genome of the phytopathogenic mollicute Spiroplasma kunkelii.

Spiroplasma kunkelii, the causative agent of corn stunt disease in maize (Zea maysL.), is a helical, cell wall-less prokaryote assigned to the class Mollicutes. As part of a project to sequence the entire S. kunkelii genome, we analyzed an 85-kb DNA segment from the pathogenic strain CR2-3x. This genome segment contains 101 ORFs and two tRNA genes. The majority of the ORFs code for predicted proteins that can be assigned to respective clusters of orthologous groups (COGs). These COGs cover diverse functional categories including genetic information storage and processing, cellular processes, and metabolism. The most notable gene cluster in this genome segment is a super-operon capable of encoding 24 ribosomal proteins. The organization of genes in this operon reflects the unique evolutionary position of the spiroplasma. Gene duplications, domain rearrangements, and frameshift mutations in the segment are interpreted as indicators of phase variation in the spiroplasma. To our knowledge, this is the first analysis of a large genome segment from a plant pathogenic spiroplasma.

Base Sequence↗
PubMed2006

Fitness effects of Wolbachia and Spiroplasma in Drosophila melanogaster.

Maternally inherited endosymbionts that manipulate the reproduction of their insect host are very common. Aside from the reproductive manipulation they produce, the fitness of these symbionts depends in part on the direct impact they have on the female host. Although this parameter has commonly been investigated for single infections, it has much more rarely been established in dual infections. We here establish the direct effect of infection with two different symbionts exhibiting different reproductive manipulation phenotypes, both alone and in combination, in the fruit fly Drosophila melanogaster. This species carries a cytoplasmic incompatibility inducing Wolbachia and a male-killing Spiroplasma, occurring as single or double (co-) infections in natural populations. We assessed direct fitness effects of these bacteria on their host, by comparing larval competitiveness and adult fecundity of uninfected, Wolbachia, Spiroplasma and Wolbachia-Spiroplasma co-infected females. We found no effect of infection status on the fitness of females for both estimates, that is, no evidence of any benefits or costs to either single or co-infection. This leads to the conclusion that both bacteria probably have other sources of benefits to persist in D. melanogaster populations, either by means of their reproductive manipulations (fitness compensation from male death in Spiroplasma infection and cytoplasmic incompatibility in Wolbachia infection) or by positive fitness interactions on other fitness components.

Animals↗
PubMed1983

Creutzfeldt-Jakob disease. Failure to detect spiroplasmas by cultivation and serological tests.

Specimens from confirmed cases of Creutzfeldt-Jakob disease (CJD) were examined for evidence of spiroplasma infection. No spiroplasmas or other mycoplasmas were cultivated from brain tissue of 18 cases and no antibodies to several recognised Spiroplasma spp. were detected in sera from 15 patients. These negative cultural and serological results provide no support for published suggestions that CJD brain contains structures morphologically resembling spiroplasmas.

Antibodies↗
PubMed1987

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 distinct plant pathogenic species of Spiroplasma. The results of Southern blot hybridization indicated that each of the pRA1-associated sequences is present as multiple copies in short, dispersed, and repetitive sequences in the chromosomes of these three strains. None of the sequences was detectable in chromosomal DNA from an additional nine Spiroplasma strains examined.

Chromosome Mapping↗
PubMedDate not supplied

Neurotropic response of Spiroplasma mirum following peripheral inoculation in the rat.

Spiroplasma mirum experimentally produces a persistent brain infection in suckling rats when administered intracerebrally. In order to examine spiroplasma infection induced by a more natural route, suckling rats were inoculated both intraperitoneally and subcutaneously with strain GT-48. No mortality was recorded over a 50-day study period. Rats developed localized alopecia and showed a significant reduction in body weight. Minimal pathological alterations were observed in brain tissues, in addition to cataracts, retinal degeneration and panophthalmitis. Spiroplasmas were recovered from brain and spleen on day 2 following peripheral inoculation with highest titres in the spleen. Spleen titres significantly declined (p less than 0.022) during the 50-day study period. On day 50, brain titres exceeded those in the spleen. The attraction of spiroplasma to central nervous system tissues may be related to its nutritional dependence on sterols.

Animals↗
PubMed1989

Effect of trimethoprim/sulphamethoxazole and hyperbaric oxygen on experimental Spiroplasma mirum encephalitis.

This study was undertaken to determine the susceptibility of experimentally induced Spiroplasma mirum infection in the rat to trimethoprim/sulfamethoxazole (TMP/SMX) in combination with hyperbaric oxygen (HBO). One-day-old Fisher 344 rats were intracerebrally inoculated with the GT-48 strain of S. mirum and were exposed to regimens employed combined antibiotic and HBO treatments. The exclusive use of TMP/SMX produced a significant reduction in mortality (P less than 0.0001) and an absence of clinical signs of infection. HBO in combination with TMP/SMX showed similar effect on mortality and no evident clinical disease. The addition of HBO did result in a significant decrease in spiroplasma brain titres but was no more effective in preventing the spiroplasma-induced fatal microcystic encephalopathy than when the antibiotics were used alone. The exclusive use of HBO produced a catastrophic mortality rate in the spiroplasma-infected rats, which is contrary to the effect of HBO on conventional bacterial infections.

Animals↗
PubMed2006

Isolation of Spiroplasma sp. from an Ixodes tick.

Spiroplasmas are helical mycoplasmas which are found in plants and arthropods, also in ticks. Some Spiroplasma species are incriminated as potential pathogens for vertebrates. During a study on Q fever in North Rhine-Westphalia, an intracellularly growing microorganism could be isolated from a pool of Ixodes ticks. The agent replicated within cytoplasmic vacuoles similar to those of Coxiella burnetii. PCR using coxiellae-, ehrlichiae- or chlamydiae-specific primers showed that agent Z/16 was distinct from these bacteria. In contrast to coxiellae or chlamydiae, the agent could not be stained according to the method of Giménez. Also electron microscopy provided evidence that the isolate Z/16 is different from coxiellae and chlamydiae. Determination of 16S ribosomal RNA gene sequences provided evidence that the isolate Z/16 can be classified as Spiroplasma sp. To our knowledge, this is the first report of an isolation of a Spiroplasma strain using a mammalian cell line. The pathogenic potential of the organism needs further investigation.

Animals↗