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PubMed1981

Antibiotic sensitivities in vitro of diverse spiroplasma strains associated with plants and insects.

Fifteen spiroplasma strains, representing five serological subgroups classified in three distinct serogroups, and four strains unassigned to serogroups were examined for sensitivity to antibiotics. The data contribute to the characterization of spiroplasmas and enlarge comparisons between plant pathogenic strains and strains that are evidently a part of the normal epiphytic microflora.

Journal Article↗
PubMedDate not supplied

Polypeptide cartography of Spiroplasma taiwanense.

Spiroplasma taiwanense is the first member of the Class Mollicutes to be subjected to polypeptide cartography using computerized image analysis. The small genome size characteristic of this group was shown to code for low numbers of polypeptides when compared to other bacterial species. Silver-stained two-dimensional electrophoresis gels, following separation by either isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (ISO-DALT) or nonequilibrium pH gradient electrophoresis (NEPHGE), were used to create databases from 10 and 6 gels, respectively, for each technique and produced, respectively, 263 and 287 replicated spots. Polypeptides were mapped with respect to molecular mass and glyceraldehyde-3-phosphate dehydrogenase carbamylation standards. Of interest was the unexpectedly high percentage (50.2%) of the total normalised optical intensity associated with all 263 spots detected by ISO-DALT electrophoresis, having been contributed by just 29 dominant protein spots. These 29 polypeptides are to be given priority in microsequencing and microanalysis aimed at their identification.

Bacterial Proteins↗
PubMed1997

Proteome analysis of Spiroplasma melliferum (A56) and protein characterisation across species boundaries.

Spiroplasma melliferum (Class: Mollicutes) is a wall-less, helical bacterium with a genome of approximately 1460 kbp encoding 800-1000 gene-products. A two-dimensional electrophoresis gel reference map of S. melliferum was produced by Phoretix 2-D gel software analysis of eight high quality gels. The reference map showed 456 silver-stained and replicated protein spots. 156 proteins (34% of visible protein spots) from S. melliferum were further characterised by one, or a combination, of the following: amino acid analysis, peptide-mass fingerprinting via matrix assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry, and N-terminal protein microsequencing. Proteins with close relationship to those previously determined from other species were identified across species barriers. Thus, this study represents the first larger-scale analysis of a proteome based upon the attribution of predominantly 'unique numerical parameters' for protein characterisation across species boundaries, as opposed to a sequence-based approach. This approach allowed all database entries to be screened for homology, as is currently the case for studies based on nucleic acid or protein sequence information. Several proteins studied from this organism were identified as hypothetical, or having no close homolog already present in the databases. Gene-products from major families such as glycolysis, translation, transcription, cellular processes, energy metabolism and protein synthesis were identified. Several gene-products characterised in S. melliferum were not previously found in studies of the entire Mycoplasma genitalium and Mycoplasma pneumoniae (both closely related Mollicutes) genomes. The presence of such gene-products in S. melliferum is discussed in terms of genome size as compared with the smallest known free-living organisms. Finally, the levels of expression of S. melliferum gene-products were determined with respect to total optical intensity associated with all visible proteins expressed in exponentially grown cells.

Amino Acid Sequence↗
PubMed1997

Characterisation of basic proteins from Spiroplasma melliferum using novel immobilised pH gradients.

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has become the method of choice for efficient separation of complex protein mixtures. Previously, analysis of the Spiroplasma melliferum proteome (protein complement of a genome) has been performed with pH 3-10 and narrow range pH 4-7 IPG gel strips. We report here on the use of novel 18 cm basic (pH 6-11) immobilised pH gradients (IPG) to increase the resolution of protein spots visible within 2-D gels. These gradients were synthesised to emulate the gradient of commercially available IPG gel strips in a 5 cm region of overlap so as to attempt construction of a more complete map of cellular protein expression. Approximately 50 additional gene products were detected from S. melliferum that were not previously well-resolved or visible using wide-range pH 3-10 IPG gel strips. Twenty-seven of these were electrotransferred to polyvinylidene difluoride (PVDF) membrane and analysed by N-terminal protein microsequencing. Protein spots with an initial peak yield of as little as 100 femtomoles (fm) were sequenced to 5-10 amino acid residues, demonstrating the importance of improved sample handling procedures and analytical technologies. Many essential metabolic enzymes were shown to have basic pI, including: glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, carbamate kinase and lactate dehydrogenase. A very basic protein (pI approximately 11.0) was identified as uridylate kinase, an enzyme indirectly associated with pyrimidine biosynthesis and thought be absent in some members of the bacterial class Mollicutes. The advent of novel basic (pH 6-11) IPGs has allowed the visualisation of a significantly greater percentage of the 'functional proteome', that portion of the total protein complement of a genome actively translated within a specific time frame, on 2-D electrophoresis gels. This will aid in the characterisation of translated gene products in conjunction with genome sequencing initiatives.

Amino Acid Sequence↗
PubMed1998

Development and use of an established cell line of the leafhopper Circulifer tenellus to characterize Spiroplasma citri-vector interactions.

A continuous cell line of embryonic origin from the leafhopper Circulifer tenellus, CT 1, was established using a protocol modified from thrips cell culture. The line was used to develop an in vitro model to examine the mode of entry of the plant pathogenic mollicute Spiroplasma citri into insect host cells. Confluent monolayers were achieved in 5-6 months using a simple medium developed for maintaining established leafhopper cell lines. The newly established CT 1 line, and that of another leafhopper, Nephotettix cincticeps, were exposed to S. citri and examined by electron microscopy. S. citri was found to cytadhere and to be present in apparent invaginations of the host cell membranes of cell lines of both leafhopper species, supporting the hypothesis that this pathogen enters its insect host via endocytosis.

Animals↗
PubMed1994

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 and gyrB. Several DnaA-box consensus sequences were found upstream of dnaA and in the dnaA-dnaN intergenic region. The dnaA region with the flanking DnaA-boxes and the tetracycline resistance determinant, tetM, were linked into a circular recombinant DNA. This DNA was able to replicate autonomously when introduced by electroporation into S. citri cells. These experiments show that the dnaA region with the DnaA-boxes is the origin of replication of S. citri and can be used to construct gene vectors.

Amino Acid Sequence↗
PubMed1986

Malignant transformation of NIH-3T3 and CV-1 cells by a helical mycoplasma, Spiroplasma mirum, strain SMCA.

A helical mycoplasma, Spiroplasma mirum strain SMCA, produced malignant transformation in mouse NIH 3T3 cells and monkey kidney CV-1 cells. The transformed cells exhibited morphological changes consistent with the transformed phenotype, grew in soft agar and produced tumors in athymic and BALB/c mice. Transmission electron microscopy revealed structures morphologically similar to mycoplasmas present in the cytoplasm of transformed but not untransformed 3T3 cells. The time of inoculation of S. mirum SMCA to 3T3 cells and the passage level of 3T3 cells affected transformation.

Animals↗
PubMedDate not supplied

Chemical analysis of processing of spiralin, the major lipoprotein of Spiroplasma melliferum.

The plasma membrane of Spiroplasma melliferum contains a major membrane-associated lipoprotein called spiralin. In this study, the processing pathway of spiralin was investigated by chemical analysis of the purified protein and by using [35S]cysteine, [35S]methionine, [14C]myristic acid (14C-14:0), [14C]palmitic acid (14C-16:0), and globomycin. SDS-PAGE analysis of membrane proteins showed the leader peptide cleavage of prospiralin and provided evidence for an apparent selectivity in the acylation: the unprocessed protein was labelled with 14C-16:0 only (O-ester-linked acyl chains), and the mature form with both 14C-labelled fatty acids (O-ester-linked + amide-linked chains). Chemical analysis of the purified protein revealed that spiralin contains S-glycerylcysteine and is covalently modified with two O-ester-linked acyl chains and one amide-linked fatty acid chain. However, a specific selectivity in the O- and the N-acylations was not confirmed; palmitate and stearate were the major components. The amounts of O-ester- and amide-linked acyl chains, the resistance to Edman degradation and the presence of S-glycerylcysteine together indicate that spiralin is a "classical" lipoprotein (i.e. is triacylated) and is probably processed by a mechanism similar to that described for gram-negative eubacteria. On the basis of these findings, a biogenesis pathway for spiralin is proposed.

Amino Acids↗
PubMed1998

Gene organization and transcriptional analysis of the Spiroplasma citri rpsB/tsf/x operon.

The nucleotide sequence of a 6863-bp Spiroplasma citri DNA fragment comprising the spiralin gene was determined. Sequence analysis revealed eight putative ORFs that encode ribosomal protein S2, elongation factor Ts, spiralin, 6-phosphofructokinase, pyruvate kinase, and three unidentified proteins (A, B, and X). The gene organization reported here is different from that previously published. Northern blot analysis of rpsB, tsf, and x transcripts indicates that these genes are organized into a single transcriptional unit (operon). However, the detection of an additional transcript corresponding to the rpsB gene alone suggests that a transcriptional mechanism should occur in the 3' region of the rpsB gene, allowing a conditional transcription termination.

Bacterial Proteins↗
PubMed2004

Predicted ATP-binding cassette systems in the phytopathogenic mollicute Spiroplasma kunkelii.

Spiroplasma kunkelii is a cell wall-free, helical, and motile mycoplasma-like organism that causes corn stunt disease in maize. The bacterium has a compact genome with a gene set approaching the minimal complement necessary for cellular life and pathogenesis. A set of 21 ATP-binding cassette (ABC) domains was identified during the annotation of a draft S. kunkelii genome sequence. These 21 ABC domains are present in 18 predicted proteins, and are components of 16 functional systems, which account for 5% of the protein coding capacity of the S. kunkelii genome. Of the 16 systems, 11 are membrane-bound transporters, and two are cytosolic systems involved in DNA repair and the oxidative stress response; the genes for the remaining three hypothetical systems harbor nonsense and/or frameshift mutations, so their functional status is doubtful. Assembly of the 11 multicomponent transporters, and comparisons with other known systems permitted functional predictions for the S. kunkelii ABC transporter systems. These transporters convey a wide variety of substrates, and are critical for nutrient uptake, multidrug resistance, and perhaps virulence. Our findings provide a framework for functional characterization of the ABC systems in S. kunkelii.

ATP-Binding Cassette Transporters↗
PubMed1977

Purification and characterization of spiralin, the main protein of the Spiroplasma citri membrane.

The membrane proteins from Spiroplasma citri have been resolved into 16 components by SDS-polyacrylamide gel electrophoresis. By this technique it was also shown that the molecular weights of these proteins ranged from 13000 to 160 000. One of the proteins, which had an apparent molecular weight of 26 000 was the most abundant and represented more than 22% of total membrane protein. We have designated this protein spiralin. None of the proteins contained carbohydrate. Spiralin has been isolated by a procedure which involves removal of some membrane proteins with the neutral detergent Tween 20, selective solubilization of the Tween residue in DOC and fractionation of the DOC-soluble material by agarose-suspension electrophoresis. The homogeneity of spiralin was demonstrated by analytical polyacrylamide gel electrophoresis under different conditions and by crossed immunoelectrophoresis. Spiralin appeared to bind less DOC than the other membrane proteins of S. citri. This observation does not imply, however, that the binding of DOC to spiralin is weak. Spiralin was neither soluble in detergent-free buffers nor in Tween 20, which indicated that it is an intrinsic membrane protein. The amino-acid composition of spiralin was quite different from that of the membrane. Spiralin lacked methionine, histidine and tryptophan, and had a low content of glycine, leucine, tyrosine and phenylalanine, but a high content of threonine, alanine and valine.

Alanine↗
PubMed1987

Transmembrane proton-motive potential of Spiroplasma floricola.

In Spiroplasma floricola, the transmembrane proton-motive potential delta p was studied. It is composed of a transmembrane electric potential difference, delta psi, and a transmembrane proton gradient, delta pH, according to delta p = delta psi - (Z.delta pH). Using a potential-sensitive carbocyanine dye and 5,5'-dimethyl[2-14C]oxazolidine-2,4-dione as probes, delta psi and delta pH were measured at different [H+] of the medium, and delta p was calculated to be remarkably constant at -123 mV +/- 16% over a wide range of external pH values. Inhibition experiments indicated that it is generated by a membrane-bound, electrogenic, proton-translocating ATPase.

Bacterial Proteins↗
PubMed1988

Membrane surface potential of Spiroplasma floricola.

Anionic charges, cytochemically identified as lipid phosphate groups, cover the outer membrane surface of Spiroplasma floricola. They induce a negative membrane surface potential which affects the distribution of ions, including protons. Accordingly, the pH at the interface differs from the bulk pH. By using the fluorescent lipoid pH indicator 4-heptadecyl-7-hydroxycoumarin, the pH at the membrane surface was determined. From the difference of the bulk and the interfacial pH the membrane surface potential of S. floricola was calculated to be phi = -118 mV.

Electrophysiology↗
PubMed1989

Involvement of a Spiroplasma citri plasmid in the erythromycin-resistance transfer.

An 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-resistant strain M4 Er-1 showed that the plasmid pM42 was able to transfer the erythromycin-resistance. pM42 was mapped with restriction endonucleases and found to be related to the pMH1 plasmid previously isolated from S. citri MH. Hybridization analysis of DNA from sensitive and resistant strains has shown that a sequence from pM42, analogous to a sequence from pMH1, was integrated at a specific locus in the chromosome of the erythromycin-resistant cells, i.e., of the transformed R8A2 cells and of the spontaneous mutant M4 Er-1 strain.

DNA↗
PubMed1996

The unique organization of the rpoB region of Spiroplasma citri: a restriction and modification system gene is adjacent to rpoB.

A 6.5-kb DNA fragment containing the gene (rpoB) encoding the RNA polymerase (RNAP) beta subunit, from the mollicute Spiroplasma citri (Sc), was cloned and sequenced. The classical eubacterial organization, with the genes (rplK, A, J and L) encoding ribosomal proteins L11, L1, L10 and L12 located immediately upstream from rpoB, was not found in the Sc DNA. Instead, an open reading frame (hsdS) potentially encoding a component of a type I restriction and modification system was identified upstream from rpoB, and sequences showing similarities with insertion elements were found between hsdS and rpoB.

Amino Acid Sequence↗
PubMed2005

Spiroplasma swim by a processive change in body helicity.

Microscopic organisms must rely on very different strategies than their macroscopic counterparts to swim through liquid. To date, the best understood method for prokaryotic swimming employs the rotation of flagella. Here, we show that Spiroplasma, tiny helical bacteria that infect plants and insects, use a very different approach. By measuring cell kinematics during free swimming, we find that propulsion is generated by the propagation of kink pairs down the length of the cell body. A processive change in the helicity of the body creates these waves and enables directional movement.

Methylcellulose↗
PubMed2004

Identification and characterization of traE genes of Spiroplasma kunkelii.

Four traE homologs, designated traE1, traE2, traE3 and traE4, were identified and amplified from the genome of the leafhopper-transmitted corn stunt pathogen Spiroplasma kunkelii and were predicted to encode membrane-bound adenine tri-phosphatases (ATPases). Deduced proteins of all traE genes have 62.3% to 89.9% similarity to the conserved VirB4 domain that is frequently a component of type IV secretory pathways involved in intracellular trafficking and secretion of DNA and proteins. In phylogenetic analysis, TraE homologs of S. kunkelii, Mycoplasma pulmonis and Mycoplasma fermentans cluster together and are more similar to TraE proteins of Gram-positive bacteria than to those of Gram-negative bacteria, thereby resembling the 16S rRNA phylogeny. Gene traE2 was most conserved whereas the presence of the three other traE genes varied among S. kunkelii strains, M2, CS-2B, FL-80 and PU8-17. Further, traE1 and traE2 appeared to be located on the chromosome, and traE3 and traE4 genes on plasmids of S. kunkelii strain M2. Transcripts of the spiralin gene and traE2 genes were detected on Northern blots containing total ribonucleic acids (RNA) of S. kunkelii cultures and S. kunkelii-infected plants and insects, in which traE2 appeared to be of a larger transcription unit. Full-length expression products of the other traE genes were not detected. S. kunkelii traE genes could be involved in S. kunkelii cell morphogenesis, adhesion and DNA recombination.

Adenosine Triphosphatases↗
PubMed2003

Study on experimental infections of Spiroplasma from the Chinese mitten crab in crayfish, mice and embryonated chickens.

Tremor disease (TD) of the Chinese mitten crab Eriocheir sinensi has become a serious disease in the Chinese freshwater culture industry in recent years. The agent, belonging to Spiroplasma, was purified from yolk and allantoic fluids of chicken embryos and was then inoculated into the body of crayfish, the abdominal cavity of ICR mice and the allantoid of chicken embryos, respectively. Thirty-eight days after the inoculation, the mice and crayfish were dissected and their tissues sampled and observed with both the light and electron microscope. No infection was detected in mouse or crawfish tissue. But when the different tissues from the inoculated embryonated chickens were tested, the agent was detected only in the brain of embryonated chickens. This indicated that the agent represented a neurotropic characteristic in embryonated chickens, just as it had in the crab. This infective characteristic may be due to the development and maturation of host immunity.

Animals↗