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PubMed1993

Mycoplasmalike organisms (MLOs) and distinguish them from other MLOs.

DNA of 10 lines of rice yellow dwarf (RYD) mycoplasmalike organisms (MLOs) from Japan, the Phillippines, and Thailand hybridized with four probes containing chromosomal and six probes containing extrachromosomal DNA of a Tochigi (Japan) line of RYD MLO. One chromosomal probe (RYD9) and all six extrachromosomal probes hybridized with various other MLOs (sugarcane white leaf, onion yellows, cineraria witches'-broom, Japanese hornwort witches'-broom, water dropwort wiches'-broom, gentian witches'-broom, udo dwarf, tsuwabuki witches'-broom, pelargonium witches's-broom, peach western-X, and pear decline). DNA from the culturable mollicutes Spiroplasma kunkelii, Spiroplasma citri, Mycoplasma hominis, and Mycoplasma orale did not hybridize with RYD MLO probes. The extrachromosomal DNAs hybridizing with the probes showed variations in electrophoretic behavior.

Base Sequence↗
PubMed1992

[Mollicute Acholeplasma florum possesses a gene of phosphoenolpyruvate sugar phosphotransferase system and it uses UGA as tryptophan codon].

Acholeplasmas are mollicutes which do not require sterol for growth and do not possess a phosphoenolpyruvate-dependent sugar-phosphotransferase system. In contrast to spiroplasmas, mycoplasmas and ureaplasmas, they utilize UGA as a stop codon and not as a tryptophan codon. Acholeplasma florum, because of its metabolic properties and its close phylogenetic relationship to members of the genera Spiroplasma and Mycoplasma, does not seem to be an acholeplasma senso stricto. Here, we present molecular data to support this hypothesis. We have detected a gene coding for one of the components of the phosphotransferase system (PTS) in A. florum. In addition we demonstrate that the organism uses UGA as a tryptophan codon and not as a stop codon. These findings offer strong evidence, along with an earlier phylogenetic proposal, that A. florum is not a member of the genus Acholeplasma.

Acholeplasma↗
PubMedDate not supplied

Some biological features of mollicutes.

Mycoplasmas are a bacterial group that is classified in the Mollicute class which includes Mycoplasmas, Spiroplasmas and Acholeplasmas. One hundred and seventy six species have been described in this group. Mycoplasmas are the smallest self living prokaryotes, they do not have a bacterial wall, their genomic size ranges from 577 to 2220 bpk, they are nutritional exigent so it is hard to culture them, but the development of molecular biology techniques has let us detect more mycoplasmas in different hosts. Mycoplasmas have been associated to acute and chronic diseases mainly in animals and humans while spiroplasmas have been found in arthropods, plants and flowers producing or not damage. Some recent studies have shown the role of some structural components of Mycoplasmas in pathogenesis, such as cytoskeleton proteins and adhesins, and the influence of some genetic characteristics on the development of an infectious disease.

Tenericutes↗
PubMed1995

Cross-species identification of proteins separated by two-dimensional gel electrophoresis using matrix-assisted laser desorption ionisation/time-of-flight mass spectrometry and amino acid composition.

Amino acid analysis and matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry were used to identify nine of twelve proteins originally separated by two-dimensional electrophoresis and derived from an organism poorly defined at the molecular level (Spiroplasma melliferum). Two of three unidentified proteins appeared to be novel. The percentage amino acid composition and the molecular mass of peptide fragments generated by tryptic digestion were used to search the PIR/SWISS-PROT and MOWSE databases respectively. Lists of candidate proteins were independently generated and ranked from data obtained by both methods. A putative identification was allocated when a single candidate protein appeared in both lists of computer-generated rankings. Results were verified using N-terminal protein microsequencing. The combined use of amino acid composition and MALDI-TOF mass spectrometry allowed a high degree of confidence to be placed in such identifications because they were based upon homologous data sets of at least 20 parameters (16 amino acids and 4-10 tryptic digest fragments). A further two parameters, estimated M(r) and, to a lesser extent, pI, were also used to reinforce this measure of confidence. Ranking of candidate proteins by one method alone could lead to false identification. Both techniques can process large numbers of samples rapidly. In light of the increasing number of entries in both gene and protein databases, this approach is likely to become an essential first step for the characterisation of proteins, particularly across species boundaries.

Amino Acid Sequence↗
PubMed2000

Use of an internal control in a nested-PCR assay for Mycoplasma hyopneumoniae detection and quantification in tracheobronchiolar washings from pigs.

We have previously reported a nested PCR assay for the detection of Mycoplasma hyopneumoniae directly in tracheobronchiolar washings from living pigs in field conditions. Here, we describe the construction and use of an internal control to monitor the presence of PCR inhibitors. A PCR modified target DNA was constructed by insertion of a small DNA fragment into the M. hyopneumoniae specific DNA target. We have demonstrated that the internal control failed to be amplified in only three tracheobronchiolar washings samples out of the 362 tested. This control molecule was inserted in a Spiroplasma citri derived plasmid vector and introduced into S. citri cells by electroporation. After a few passages we ensured that the recombinant plasmid became inserted into the genome of S. citri. PCR amplification of the DNA of this transformed S. citri strain using nested PCR primers led to amplification of a 900-bp fragment which can be discriminated from the M. hyopneumoniae PCR product 700 bp. The S. citri transformants with the integrated internal control were added to the tracheobronchiolar washings prior to PCR and used as an internal control to check the efficiency of sample processing, and to demonstrate the presence of inhibitors. Furthermore, we have been able to estimate the number of mycoplasma cells in the tracheobronchiolar washings. Quantitation was performed by comparing the PCR signal intensity of the specific M. hyopneumoniae template with known concentrations of the S. citri competitor. The titer in tracheobronchiolar washings ranged approximatively from 10(4)to 10(8)M. hyopneumoniae cells per ml of clinical specimen. Quantitative PCR can be a useful tool for monitoring the progression of M. hyopneumoniae in the disease process.

Animals↗
PubMed2006

Rickettsial agents from parasitic dermanyssoidea (Acari: Mesostigmata).

Mites are often overlooked as vectors of pathogens, but have been shown to harbor and transmit rickettsial agents such as Rickettsia akari and Orientia tsutsugamushi. We screened DNA extracts from 27 mites representing 25 species of dermanyssoids for rickettsial agents such as Anaplasma, Bartonella, Rickettsia, and Wolbachia by PCR amplification and sequencing. DNA from Anaplasma spp., a novel Bartonella sp., Spiroplasma sp., Wolbachia sp., and an unclassified Rickettsiales were detected in mites. These could represent mite-borne bacterial agents, bacterial DNA from blood meals, or novel endosymbionts of mites.

Anaplasma↗
PubMed1985

Rapid and simple identification of mycoplasmas by immunobinding.

A simple and rapid method of species identification of mycoplasmas by immunobinding assay is described. Small amounts of antigen of supernatant from cell cultures, broth cultures or clinical specimens were spotted onto nitrocellulose paper. This was followed by application of specific anti-mycoplasma antisera. After incubation, an enzyme-conjugated antiserum against the first antiserum was applied. A positive reaction was indicated by the development of intense blue color reaction when substrate was added. This method identified mycoplasma species with monoclonal and polyclonal antibodies. It detected 9.3 X 10(3) - 7.5 X 10(4) CFU/ml of organisms depending on mycoplasma species. For identification of mycoplasma, ureaplasma, acholeplasma and spiroplasma species, this assay is useful and rapid compared with other serological methods. In limited studies, the method correlated with microbiological assay of clinical specimens for Mycoplasma pneumoniae.

Animals↗
PubMed1991

Fusion of mycoplasmas: the formation of cell hybrids.

Poly(ethylene glycol) (PEG 8000) can induce cell-cell fusion of Mycoplasma capricolum cells, and it can promote the formation of intergeneric hybrids of various Mycoplasma, Acholeplasma and Spiroplasma species. The extent of fusion was quantitatively evaluated by following the dequenching of octadecylrhodamine fluorescent label incorporated into donor cell membranes after their incubation with recipient cells. The results of dequenching experiments were confirmed by electron microscopy, as well as by angle light-scattering measurements. Fusion appeared to require the presence of Mg2+, but was completely inhibited by either 0.1% glutaraldehyde or 100 microM chlorpromazine, and was partially suppressed by proteolytic enzymes, carbonyl cyanide-m-chlorophenylhydrazone, or thiol reagents.

Acholeplasma laidlawii↗
PubMed2006

Mapping 70S ribosomes in intact cells by cryoelectron tomography and pattern recognition.

Cryoelectron tomography (CET) combines the potential of three-dimensional (3D) imaging with a close-to-life preservation of biological samples. It allows the examination of large and stochastically variable structures, such as organelles or whole cells. At the current resolution it becomes possible to visualize large macromolecular complexes in their functional cellular environments. Pattern recognition methods can be used for a systematic interpretation of the tomograms; target molecules are identified and located based on their structural signature and their correspondence with a template. Here, we demonstrate that such an approach can be used to map 70S ribosomes in an intact prokaryotic cell (Spiroplasma melliferum) with high fidelity, in spite of the low signal-to-noise ratio (SNR) of the tomograms. At a resolution of 4.7 nm the average generated from the 236 ribosomes found in a tomogram is in good agreement with high resolution structures of isolated ribosomes as obtained by X-ray crystallography or cryoelectron microscopy. Under the conditions of the experiment (logarithmic growth phase) the ribosomes are evenly distributed throughout the cytosol, occupying approximately 5% of the cellular volume. A subset of about 15% is found in close proximity to and with a distinct orientation with respect to the plasma membrane. This study represents a first step towards generating a more comprehensive cellular atlas of macromolecular complexes.

Algorithms↗
PubMed2003

The motility of mollicutes.

Recent experiments show that the conformation of filament proteins play a role in the motility and morphology of many different types of bacteria. Conformational changes in the protein subunits may produce forces to drive propulsion and cell division. Here we present a molecular mechanism by which these forces can drive cell motion. Coupling of a biochemical cycle, such as ATP hydrolysis, to the dynamics of elastic filaments enable elastic filaments to propagate deformations that generate propulsive forces. We demonstrate this possibility for two classes of wall-less bacteria called mollicutes: the swimming of helical-shaped Spiroplasma, and the gliding motility of Mycoplasma.

Bacterial Physiological Phenomena↗
PubMed1994

The role of Glu187 in the regulation of phosphofructokinase by phosphoenolpyruvate.

In bacterial phosphofructokinases, either a glutamic or an aspartic residue is present at position 187, and the mechanism of inhibition by phosphoenolpyruvate seems to be correlated to the nature of residue 187. Upon binding phosphoenolpyruvate, only the enzymes with a Glu187 would undergo a major allosteric conformational change from an active into an inactive state, whereas the enzymes with an Asp187 would only show a simple upward shift in their pH-profile of activity. The phosphofructokinase from Spiroplasma citri, which has an Asp187, has been purified and its properties follow this pattern. The behaviour of mutants of the enzyme from Escherichia coli in which Glu187 is replaced by either aspartate or leucine confirms the importance of residue 187. The major allosteric transition of E. coli phosphofructokinase is abolished by the substitution Glu187-->Asp, suggesting that a glutamate at position 187 is necessary (but not sufficient) for the protein to undergo the change from the active into the inactive state induced by phosphenolpyruvate. In addition, the presence of an acidic residue, aspartate or glutamate, at position 187 is required (but not sufficient) for the binding of ADP (or GDP). This requirement of a negative charge for ADP binding could explain the striking conservation of an aspartate residue at position 187 in all the eukaryotic phosphofructokinases.

Amino Acid Sequence↗
PubMed1997

Malate/lactate dehydrogenase in mollicutes: evidence for a multienzyme protein.

The malate (MDH) and lactate (LDH) dehydrogenases belong to the homologous class of 2-ketoacid dehydrogenases. The specificity for their respective substrates depends on residues differing at two or three regions within each molecule. Theoretical peptide-mass fingerprinting and PROSITE analysis of nine MDH and six LDH molecules were used to describe conserved sites related to function. A unique LDH is described which probably also confers MDH activity within the 580 kbp genome of Mycoplasma genitalium (class: Mollicutes). A single hydrophilic arginine residue was found in the active site of the M. genitalium LDH enzyme, differing from an hydrophobic residue normally present in these molecules. The effect of this residue may be to alter active site substrate specificity, allowing the enzyme to perform two closely related tasks. Evidence for a single gene affording dual enzymatic function is discussed in terms of genome size reduction in the simplest of free-living organisms. Since Mollicutes are thought to lack enzymes of the tricarboxylic acid cycle that would otherwise bind and interact with MDH in bacterial species possessing this pathway, active site modification of M. genitalium LDH is the sole requirement for MDH activity of this molecule. The closely related helical Mollicute, Spiroplasma melliferum, was shown to possess two distinct gene products for MDH/LDH activity.

Amino Acid Sequence↗
PubMedDate not supplied

Molecular and biological features of mollicutes (mycoplasmas).

The small size of the mollicute genome considerably restricts the amount of genetic information available to the organisms. This is reflected in the relatively small number of cell proteins synthesized, the lack of many biosynthetic pathways and the marked dependence on exogenous nutrients for growth. The protein synthesizing machinery of mollicutes resembles that of eubacteria and is sensitive to the same antibiotics, except for rifampicin, to which RNA polymerases of mollicutes appear resistant. The mollicute ribosomes are built of 50 S and 30 S subunits and contain about 50 different proteins and 5 S, 16 S and 23 S rRNA, as in eubacteria. However, the 5 S rRNA in mollicutes appears shorter (107-112 nucleotides) than in eubacteria (116-120 nucleotides). We hybridized restriction endonuclease-digested DNA from a variety of Mycoplasma, Ureaplasma, Acholeplasma and Spiroplasma species with nick-translated probes consisting of defined portions of the rrnB rRNA operon of Escherichia coli and the rRNA operon of M. capricolum. The results suggest the presence of only one or two sets (operons) of rRNA genes in the genome of Mollicutes, a number falling considerably below that of the eubacteria examined so far but resembling that found in archaebacteria. Our data also indicate a marked nucleotide sequence homology along the rrnB rRNA operon of E. coli and the rRNA operons of the various mollicutes, indicating that the rRNA genes in mollicutes are linked in the classical prokaryotic fashion 16 S-23 S-5 S. Each mollicute appeared to possess, on its genome, different flanking sequences adjacent to the rRNA operon(s), resulting in species-specific hybridization patterns.(ABSTRACT TRUNCATED AT 250 WORDS)

Acholeplasma↗
PubMed2000

Multiple causes of male-killing in a single sample of the two-spot ladybird, Adalia bipunctata (Coleoptera: coccinellidae) from Moscow.

Thirty-six matrilines from a single Muscovite sample of Adalia bipunctata were assayed, using appropriate primers, for presence of the four male-killing symbionts known to infect this species of ladybird. All four, a Rickettsia, a Spiroplasma and two different strains of Wolbachia, were found to be present. Vertical transmission efficiencies were assessed from F1 and F2 families from each of the matrilines, and were found to differ significantly between symbionts. Potential explanations of the presence of four different male-killing symbionts within a single population, are considered.

Animals↗
PubMed1999

Invasion of one insect species, Adalia bipunctata, by two different male-killing bacteria.

Male-killing bacteria, which are inherited through the female line and kill male progeny only, are known from five different orders of insect. Our knowledge of the incidence of these elements has stemmed from discovery of their phenotype in different species. Our estimate of the frequency with which insects have been invaded by these elements therefore depends on each observation of the male-killing phenotype within a species being associated with a single microorganism. We here record an example of a single insect species being infected with two taxonomically distinct male-killing bacteria. Western European populations of the two-spot ladybird, Adalia bipunctata, have previously been shown to bear a male-killing Rickettsia. However, we here show that the majority of the male-killing lines tested from Central and Eastern Europe do not bear this bacterium. Rather, 16S rDNA sequence analysis suggests male-killing is associated with a member of the genus Spiroplasma. We discuss this conclusion in relation to the evolutionary genetics of male-killing bacteria, and the evolution of male-killing behaviour in the eubacteria.

Animals↗
PubMed2003

The antibiotic activity of cationic linear amphipathic peptides: lessons from the action of leucine/lysine copolymers on bacteria of the class Mollicutes.

Peptides composed of leucyl and lysyl residues ('LK peptides') with different compositions and sequences were compared for their antibacterial activities using cell wall-less bacteria of the class Mollicutes (acholeplasmas, mycoplasmas and spiroplasmas) as targets. The antibacterial activity of the amphipathic alpha-helical peptides varied with their size, 15 residues being the optimal length, independent of the membrane hydrophobic core thickness and the amount of cholesterol. The 15-residue ideally amphipathic alpha helix with a +5 positive net charge (KLLKLLLKLLLKLLK) had the strongest antibacterial activity, similar to that of melittin. In contrast, scrambled peptides devoid of amphipathy and the less hydrophobic beta-sheeted peptides [(LK)nK], even those 15-residue long, were far less potent than the helical ones. Furthermore, the growth inhibitory activity of the peptides was correlated with their ability to abolish membrane potential. These data are fully consistent with a predominantly flat orientation of LK peptides at the lipid/water interface and strongly supports that these peptides and probably the linear polycationic amphipathic defence peptides act on bacterial membranes in four main steps according to the 'carpet' model: (a) interfacial partitioning with accumulation of monomers on the target membrane (limiting step); (b) peptide structural changes (conformation, aggregation, and orientation) induced by interactions with the lipid bilayer (as already shown with liposomes and erythrocytes); (c) plasma membrane permeabilization/depolarization via a detergent-like effect; and (d) rapid bacterial cell death if the extent of depolarization is maintained above a critical threshold.

Anti-Bacterial Agents↗
PubMed1980

Phylogenetic analysis of the mycoplasmas.

The phylogenetic relationships between the mycoplasmas and bacteria have been established from a comparative analysis of their 16S rRNA oligonucleotide catalogs. The genera Mycoplasma, Spiroplasma, and Acholeplasma arose by degenerative evolution, as a deep branch of the subline of clostridial ancestry that led to Bacillus and Lactobacillus. Thermoplasma has no specific relationship to the other mycoplasmas; it belongs with the archaebacteria.

Acholeplasma↗