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At least 253 records · Page 14Linked to original sources
PubMed2005

Cryo-electron tomography reveals the cytoskeletal structure of Spiroplasma melliferum.

Evidence has accumulated recently that not only eukaryotes but also bacteria can have a cytoskeleton. We used cryo-electron tomography to study the three-dimensional structure of Spiroplasma melliferum cells in a close-to-native state at approximately 4-nanometer resolution. We showed that these cells possess two types of filaments arranged in three parallel ribbons underneath the cell membrane. These two filamentous structures are built of the fibril protein and possibly the actin-like protein MreB. On the basis of our structural data, we could model the motility modes of these cells and explain how helical Mollicutes can propel themselves by means of coordinated length changes of their cytoskeletal ribbons.

Bacterial Proteins↗
PubMed1988

Chromosomal gene transfer in Spiroplasma citri.

The study of resistance marker rearrangement in Spiroplasma citri mutants provides evidence of transfer of chromosomal information followed by recombination. This is the first report of such a transfer in vivo in the mollicutes--that is, in the smallest self-replicating organisms. The double-resistant phenotypes obtained are stable even without selection pressure. The mechanism of gene transfer is insensitive to deoxyribonuclease, requires contact, and possibly, areas of fusion of the cell membranes; it shares properties with the transfer by protoplast fusion in Gram-positive bacteria. The extensive degenerative evolution of mollicutes has retained, in S. citri, bacterial functions of chromosomal transfer and recombination.

Arsenates↗
PubMed1984

Activity of herbicolin A against Mycoplasma, Acholeplasma, Ureaplasma, and Spiroplasma species.

Herbicolin A, a novel acyl peptide antibiotic, inhibits the growth of the sterol-requiring Mycoplasma, Ureaplasma, and Spiroplasma species, with MICs varying from 1.5 to 100 micrograms/ml. With the exception of Acholeplasma modicum, all of the non-sterol-requiring species of the family Acholeplasmataceae were totally resistant to herbicolin A when tested on serum-containing medium but were inhibited to some extent on medium devoid of serum and any other source of sterol or fatty acids.

Acholeplasma↗
PubMed1996

Insusceptibility of members of the class Mollicutes to rifampin: studies of the Spiroplasma citri RNA polymerase beta-subunit gene.

In order to study the mechanism of insusceptibility of Spiroplasma citri to rifampin, we have cloned and sequenced its rpoB gene, which encodes the beta subunit of RNA polymerase. By comparison of the deduced amino acid sequence with sequences of beta subunits from susceptible and resistant bacteria, it was possible to identify several differences in the so-called Rif region (encompassing rpoB codons 500 to 575 in the Escherichia coli sequence). We constructed a chimeric rpoB gene made of the E. coli rpoB gene in which the Rif region was replaced by the equivalent region from S. citri. E. coli cells harboring this chimeric gene were resistant to rifampin. Subsequent experiments involving site-directed mutagenesis demonstrated that a single amino acid substitution (asparagine at position 526) was able to provide high-level rifampin resistance in E. coli.

Amino Acid Sequence↗
PubMed1978

High concentrations of horse serum inhibit growth of corn stunt spiroplasma.

Corn stunt spiroplasma (CSS) grew faster and achieved higher titers in liquid or agar medium containing 5 or 10 percent horse serum than it did in medium containing 20 percent horse serum. When growth in liquid medium was initiated with a small inoculum, CSS achieved excellent growth in the presence of 5 percent serum but did not grow in medium containing 0 or 20 percent serum. Addition of arginine to liquid or agar medium supplemented with 20 percent serum stimulated CSS growth, but addition to that containing 5 percent serum did not.

Animals↗
PubMed1981

Pathogenicity of Spiroplasma sp. strain SMCA in rabbits: clinical, microbiological, and histological aspects.

Newborn rabbits inoculated intracerebrally with early-passaged broth cultures of Spiroplasma strain SMCA (suckling mouse cataract agent) either died or developed eye disease. Death occurred 4 to 12 days after infection. Rabbits which died showed hemorrhages throughout the brain, especially in areas leading to the optic nerve, and hemorrhages in the liver. SMCA could be reisolated from brain, liver, and eyes. Rabbits with eye disease did not open their eyes during an observation time of 3 to 4 months. Pathological examination revealed marked microophthalmia and severe cataract formation characterized by complete degeneration of lens fibers and liquefaction and mineralization of the lens. Other ocular changes were chronic panophthalmitis with complete destruction of the retina. Neither eye disease nor death could be induced by inoculating SMCA subcutaneously into newborn rabbits. In adult rabbits, no disease occurred after intravenous or subcutaneous injection or after inoculation into the conjunctival sac.

Aging↗
PubMed1983

Characterization of Spiroplasma mirum (suckling mouse cataract agent) in a rabbit lens cell culture.

Spiroplasma mirum (suckling mouse cataract agent) was studied in an epithelial cell line AG-4676, derived from rabbit eye lens. Rabbit eye lens is a natural target tissue of S. mirum infection. The organism grew rapidly in this cell line, reaching titers of 10(7) to 10(9) color change units per ml at 7 days after infection. This is the same level as that achieved in SP-4 medium designed specifically for S. mirum. No lag period was apparent in growth in AG-4676. S. mirum did not grow in Dulbecco minimal essential medium-10% fetal bovine serum, the medium for AG-4676, indicating the need for cells or a cellular product. S. mirum-infected AG-4676 cells exhibited vacuolization and granulation and an increase in polynucleation compared with uninfected controls (36/100 versus 14/100, P less than 0.001). Infection significantly decreased the growth rate of AG-4676, especially late in the growth cycle. In a representative experiment, growth of AG-4676 at 11 days was reduced from 9 X 10(5) to 2 X 10(4) cells by S. mirum infection. S. mirum grew to high titers in conditioned medium of AG-4676, obtained from cell-free supernatants of 1- to 5-day-old AG-4676 cultures. This growth promotion was not due to osmotic conditioning of the medium. Preliminary characterization of this growth promotion substance showed it to be active after 0.22-micron filtration, heating at 56 degrees C for 30 min, freezing and thawing, and dilution at 10(-1) but not 10(-2). AG-4676-propagated S. mirum produced death or cataracts in suckling Wistar rats at the same frequency (55/60, 91.7%) as SP-4-propagated organisms (60/65, 92.3%).

Animals↗
PubMed1976

Lipid composition and lipid metabolism of Spiroplasma citri.

In a horse serum-based medium containing a full complement of fatty acids, cells of Spiroplasma citri were seen to preferentially incorporate palmitic acid. In the same medium, which had a steryl ester-to-sterol ratio of 3.64, a steryl ester-to-sterol ratio of 0.23 was seen in the cells, cholesterol being preferentially incorporated over cholesteryl ester. Like most other mycoplasmas, S. citri was shown to be unable to synthesize fatty acids or esterify cholesterol. The neutral lipids of S. citri grown in a medium containing horse serum consisted of free cholesterol, cholesteryl ester, free fatty acids, triglycerides and diglycerides. All polar lipids were phospholipids, with no glycolipids detected. These phospholipids, which are characteristic of many mycoplasmas, are phosphatidyl glycerol, diphosphatidyl glycerol, and their lyso derivatives. Sphingomyelin was also incorporated when cells were grown on horse serum. A sterol requirement for the growth of S. citri was confirmed using a serum-free medium supplemented with bovine serum albumin, palmitic acid, and various concentrations of sterols dissolved in Tween 80. The addition of palmitic acid stimulated growth but was not essential for growth. S citri was shown to grow best on cholesterol and beta-sitosterol and was able to grow on stigmasterol and ergosterol to a lesser degree. No growth was obtained using mevalonate, deoxycholate, or taurodeoxycholate as an alternative to sterol. S. citri was also able to grow when palmitic acid was replaced with oleic acid, linoleic acid, or linolenic acid. Alterations in the lipid composition of the growth medium and hence in the lipid composition of S. citri induced changes in the characteristic helical morphology of the cells, concurrent with loss of cell viability. Culture, age, and pH were also factors in determining cell morphology and viability.

Cholesterol↗
PubMed1977

Composition and enzyme activities of Spiroplasma citri membranes.

Spiroplasma citri was cultured in three different media that supplied cholesterol and fatty acids from: (i) horse serum, (ii) pleuropneumonia-like organism (PPLO) serum fraction, or (iii) bovine serum albumin-fatty acid-cholesterol. The ability of PPLO serum fraction to support growth varied by lot number. Neither PPLO serum fraction nor the bovine serum albumin medium supported growth as well as the horse serum medium. Analysis of cholesterol, lipid phosphorus, and membrane protein showed the horse serum- and PPLO-grown cells to be indistinguishable, but the bovine serum albumin-grown cells were deficient in lipid phosphorus. The three cultures did not show markedly different fatty acid compositions, but, in all cases, the cultures preferentially incorporated palmitic acid and discriminated against linoleic acid. Cultures grown for different times from logarithmic growth through a degenerative phase showed relatively constant ratios of cholesterol/protein and lipid phosphorus/protein. Fatty acid composition was also relatively constant at the different stages. Adenosine triphosphatase and p-nitrophenyl phosphatase were mainly associated with the membrane, whereas reduced nicotinamide adenine dinucleotide oxidase was either readily removed or not associated with the membrane. The reduced nicotinamide adenine dinucleotide oxidase was inactivated at temperatures above 35 degrees C.

Bacterial Proteins↗
PubMed1978

Effect of external environmental factors on the morphology of Spiroplasma citri.

Spiroplasma citri was examined by electron microscopy for morphological changes when maintained under a variety of conditions. PPLO serum fraction maintained spiral and helical morphology of S. citri at pH values of 8.0, 7.5, and 7.0, but only partially at pH 6.0 and 5.0. The absence of PPLO serum fraction resulted in round, deteriorated cells at all pH values tested. Bovine serum albumin (BSA), Phytone, soluble starch, potato starch, spermine, lipid-extracted PPLO serum fraction, and lipid-extracted BSA could substitute for PPLO serum fraction in maintaining spiral and helical morphology at pH 7.5. At pH 5.0, only BSA, lipid-extracted BSA, and lipid-extracted PPLO serum fraction were effective. Only BSA supported growth of S. citri for more than two transfers, whereas all other substitutes could not support growth longer than two transfers.

Blood↗
PubMed1979

Simplified preparation of mycoplasmas, an acholeplasma, and a spiroplasma for scanning electron microscopy.

A simple, effective procedure was developed for scanning electron microscopic examination of mycoplasmas and similar organisms. Cultivation of several mycoplasmal species, an acholeplasma, and a spiroplasma in broth media in Leighton tubes with cover slips resulted in attachment of the organisms to the cover slips. The attached cells were easily processed for either scanning electron microscopy or light microscopy. By eliminating the need for centrifugation, which was used in previously described techniques, physical stress on the cell is minimized. The effects of different preparative procedures on the morphology of Mycoplasma gallisepticum are described.

Acholeplasma laidlawii↗
PubMed1979

Separation and partial characterization of two deoxyribonucleic acid polymerases from Spiroplasma citri.

The separation and partial characterization of two deoxyribonucleic acid polymerases from Spiroplasma citri have been achieved. The two enzymes had different elution properties on diethylaminoethyl (DEAE) cellulose and differed in their sensitivity to N-ethylmaleimide (NEM), preference for different template-primers, and sedimentation velocity in linear glycerol gradients. The first enzyme activity, ScA, was retained on DEAE-cellulose and was not inhibited by NEM. Activated deoxyribonucleic acid and poly(dA)-oligo(dT12) were the preferred template-primers. Arabinosyl-cytidine triphosphate had no effect. The sedimentation coefficient of ScA was 6.3s. The second activity, ScB, was not retained on DEAE-cellulose and was inhibited by NEM. Poly(dA)-oligo(dT12) was the preferred template-primer, whereas activated DNA was only poorly utilized. ScB was not affected by arabinosyl-cytidine triphosphate, and its sedimentation coefficient was 4.4s. The polymerization activities of the two enzymes were maximum at 37 to 40 degrees C.

Arabinofuranosylcytosine Triphosphate↗
PubMed1980

Purification and preliminary characterization of Spiroplasma fibrils.

Fibrils 3.5 nm in diameter were released from the honeybee spiroplasma (BC3) by treatment with detergents and then purified by isopycnic centrifugation. Purified fibrils were flexuous, of indeterminate length, and had an axial repeat of 8.5 nm. The fibrils were associated in pairs, but in 1 M salt formed aggregates with a marked striated appearance. Pronase completely degraded the fibrils, but trypsin had little effect. The fibrils were composed of a single protein of molecular weight 55,000 which represented about 1% of the total cell protein. A protein of molecular weight 26,000 appeared to be associated with the fibrils. The significance of this in relation to membrane attachment and the possible role of fibrils in maintenance of cell shape and in motility are discussed.

Bacterial Proteins↗
PubMed1981

Electrophoretic analysis of the arrangement of spiralin and other major proteins in isolated Spiroplasma citri cell membranes.

The arrangement of the amphiphilic protein spiralin and of the other major polypeptides in the Spiroplasma citri cell membrane was investigated by one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The analyses were performed on untreated membranes for the detection of disulfide bonds and on membranes treated with dimethylsuberimidate and dithiobis(succinimidyl propionate). All membranes were depleted of the bulk of extrinsic proteins. Spiralin monomers and oligomers (mainly dimers) were detected. Almost all the oligomers appeared to be stabilized by intermolecular disulfide bonds. Components D7 (39,000 daltons), D9 (51,000 daltons), D13 (69,000 daltons), D14b (76,000 daltons), D16 (89,000 daltons), and D17 (95,000 daltons), which are the other (presumably intrinsic) main polypeptides of the S. citri membrane, were also involved in homooligomers stabilized by disulfide bonds. However, in contrast to spiralin, larger amounts of D7, D9, and D14b were involved in high-molecular-weight multimers (molecular weight, greater than 400 X 10(3) after cross-linking with dithiobis(succinimidyl propionate). Extensive cross-linking with dimethylsuberimidate showed that spiralin was the polypeptide least readily integrated to large covalent complexes. These results suggest that spiralin probably does not form a two-dimensional network in the S. citri membrane depleted of the bulk of extrinsic proteins.

Bacterial Outer Membrane Proteins↗
PubMed1983

Characterization and molecular cloning in Escherichia coli of a plasmid from the mollicute Spiroplasma citri.

Two plasmids, pMH1 with 7 kilobase pairs and pM41 with 8 kilobase pairs, were purified from the plant pathogen Spiroplasma citri and characterized by restriction mapping. Upon in vitro DNA recombination with plasmid pBR328 as a vector, we have cloned pMH1 in Escherichia coli. A radioactive probe obtained upon nick translation of the recombinant plasmid was used to further characterize and compare pMH1 and pM41.

Cloning, Molecular↗
PubMed1985

Gene for spiralin, the major membrane protein of the helical mollicute Spiroplasma citri: cloning and expression in Escherichia coli.

A library of cloned Spiroplasma citri genomic sequences was constructed by incorporating HindIII digestion fragments into the plasmid vector pBR328. Immunological screening allowed the identification of a recombinant plasmid containing the gene for spiralin, the major membrane protein of S. citri. The spiralin produced by the Escherichia coli transformant was characterized by immunological detection with monoclonal antibody after Western blotting of two-dimensional (isoelectric focusing and sodium dodecyl sulfate-polyacrylamide) electrophoresis gels and by partial proteolytic mapping. The gene for spiralin occurred within a 6.5-kilobase-pair cloned DNA fragment. Spiralin in E. coli was produced regardless of the orientation of the insert within the pBR328 vector. A spiroplasmal DNA sequence which acted as a promoter in E. coli was cloned along with the structural spiralin gene which is expressed in E. coli from that sequence.

Bacterial Outer Membrane Proteins↗
PubMed1990

Organization and nucleotide sequences of the Spiroplasma citri genes for ribosomal protein S2, elongation factor Ts, spiralin, phosphofructokinase, pyruvate kinase, and an unidentified protein.

The gene for spiralin, the major membrane protein of the helical mollicute Spiroplasma citri, was cloned in Escherichia coli as a 5-kilobase-pair (kbp) DNA fragment. The complete nucleotide sequence of the 5.0-kbp spiroplasmal DNA fragment was determined (GenBank accession no. M31161). The spiralin gene was identified by the size and amino acid composition of its translational product. Besides the spiralin gene, the spiroplasmal DNA fragment was found to contain five additional open reading frames (ORFs). The translational products of four of these ORFs were identified by their amino acid sequence homologies with known proteins: ribosomal protein S2, elongation factor Ts, phosphofructokinase, and pyruvate kinase, respectively encoded by the genes rpsB, tsf, pfk, and pyk. The product of the fifth ORF remains to be identified and was named protein X (X gene). The order of the above genes was tsf--X--spiralin gene--pfk--pyk. These genes were transcribed in one direction, while the gene for ribosomal protein S2 (rpsB) was transcribed in the opposite direction.

Amino Acid Sequence↗