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PubMed2002

Characterization of P40, a cytadhesin of Mycoplasma agalactiae.

An immunodominant protein, P40, of Mycoplasma agalactiae was analyzed genetically and functionally. The gene encoding P40 was cloned from type strain PG2, sequenced, submitted to point mutagenesis in order to convert mycoplasma-specific TGA(Trp) codon to the universal TGG(Trp) codon, and subsequently expressed in Escherichia coli. Nucleotide sequence-derived amino acid sequence comparisons revealed a similarity of P40 to the adhesin P50 of Mycoplasma hominis and to protein P89 of Spiroplasma citri, which is expected to be involved in adhesion. The amino acid sequence of P40 revealed a recognition site for a signal peptidase and strong antigenic and hydrophilic motifs in the C-terminal domain. Triton X-114 phase partitioning confirmed that P40 is a membrane protein. Fab fragments of antibodies directed against recombinant purified P40 significantly inhibited adherence of M. agalactiae strains PG2 to lamb joint synovial cells LSM 192. Sera taken sequentially from sheep infected with PG2 revealed that P40 induced a strong and persistent immune response that gave strong signals on immunoblots containing recombinant P40 even 3 months after infection. The gene encoding P40 was present in a single copy in all of the 26 field strains of M. agalactiae analyzed and was not detected in closely related mycoplasma species. P40 was expressed as a protein with an apparent molecular mass of 37 kDa on sodium dodecyl sulfate-acrylamide gels by all M. agalactiae strains except for serotype C strains, which showed nonsense mutations in their p40 genes.

Adhesins, Bacterial↗
PubMed1992

Cloning and sequencing of IS1086, an Alcaligenes eutrophus insertion element related to IS30 and IS4351.

A new insertion sequence (IS), designated IS1086, was isolated from Alcaligenes eutrophus CH34 by being trapped in plasmid pJV240, which contains the Bacillus subtilis sacB and sacR genes. The 1,106-bp IS1086 element contains partially matched (22 of 28 bp) terminal-inverted repeats and a long open reading frame. Hybridization data suggest the presence of one copy of IS1086 in the strain CH34 heavy-metal resistance plasmid pMOL28 and at least two copies in its chromosome. Analysis of the IS1086 nucleotide sequence revealed striking homology with two other IS elements, IS30 and IS4351, suggesting that they are three close members in a family of phylogenetically related insertion sequences. One open reading frame of the Spiroplasma citri phage SpV1-R8A2 B was also found to be related to this IS family but to a lesser extent. Comparison of the G+C contents of IS30 and IS1086 revealed that they conform to their respective hosts (46 versus 50% for IS30 and Escherichia coli and 64.5% for IS1086 and A. eutrophus). The pressure on the AT/GC ratio led to a very different codon usage in these two closely related IS elements. Results suggesting that IS1086 transposition might be activated by some forms of stress are discussed.

Alcaligenes↗
PubMed1993

Cloning, sequencing, and expression in Escherichia coli of the gene coding for phosphofructokinase in Lactobacillus bulgaricus.

A fragment of 1,185 bp containing the gene coding for phosphofructokinase (ATP:D-fructose-6-phosphate-1-phosphotransferase; EC 2.7.1.11) in Lactobacillus bulgaricus has been cloned, sequenced, and expressed in Escherichia coli. The amino acid sequence of this enzyme was homologous to those of the ATP-dependent phosphofructokinases from E. coli, Thermus thermophilus, Spiroplasma citri, and Bacillus stearothermophilus, suggesting that these enzymes have closely related structures despite their different regulatory properties. The recombinant protein had the same structural and functional properties as did the original enzyme. The 3' end of the 1,185-bp fragment showed the presence of an open reading frame corresponding to the N-terminal amino acid sequence of the pyruvate kinase from L. bulgaricus. This gene organization, the same as that in S. citri (C. Chevalier, C. Saillard, and J. M. Bové, J. Bacteriol. 172:2693-2703, 1990) and B. stearothermophilus (D. Walker, W. N. Chia, and H. Muirhead, J. Mol. Biol. 228:265-276, 1992; H. Sakai and T. Ohta, Eur. J. Biochem. 311:851-859, 1993) but different from that in E. coli (H. W. Hellinga and P. R. Evans, Eur. J. Biochem. 149:363-373, 1985), indicated that the same transcription unit apparently contained the genes for phosphofructokinase and pyruvate kinase, the two key enzymes of glycolysis. The possibility that these genes could be transcribed at the same time suggested that in L. bulgaricus, the coordinated regulation of phosphofructokinase and pyruvate kinase occurs at the levels of both biosynthesis and enzymatic activity.

Amino Acid Sequence↗
PubMed2004

Spreading factors of Mycoplasma alligatoris, a flesh-eating mycoplasma.

Mycoplasma alligatoris causes lethal invasive disease of alligators and caimans. A homolog of the nagH gene, encoding a hyaluronidase secreted by Clostridium perfringens, and a C. perfringens hyaluronidase nagI or nagK pseudogene were discovered in the M. alligatoris genome. The nagH gene was detected by PCR in the closest relative of M. alligatoris, Mycoplasma crocodyli, but not in 40 other species representing the Mycoplasma hominis, Mycoplasma pneumoniae, and Spiroplasma phylogenetic clusters. The hyaluronidase activity in the cellular fraction of M. alligatoris and M. crocodyli SP4 broth cultures was equivalent to 10(-16) U of Streptomyces hyalurolyticus hyaluronidase CFU(-1). Negligible activity was present in the cell-free supernatant fraction. No chondroitinase activity was detected. There is also a novel homolog of the nanI gene, which encodes a sialidase secreted by C. perfringens, in the M. alligatoris genome. The signature YRIP and SXDXGXTW motifs and catalytic residues of the clostridial sialidase are conserved in the mycoplasmal gene, but the leader sequence necessary for its secretion by C. perfringens is absent. The gene was not detected by PCR in any other mycoplasma. Potent cell-associated sialidase activity was present in M. alligatoris colonies on agar but not in the cell-free supernatants of broth cultures or in M. crocodyli. The presence of hyaluronidase and sialidase in M. alligatoris is consistent with the rapid invasiveness and necrotizing effects of this organism, and the lack of sialidase in M. crocodyli is consistent with its comparatively attenuated virulence. This genetic and biochemical evidence suggests that the spreading factors hyaluronidase and sialidase, a combination unprecedented in mycoplasmas, are the basis of the virulence of M. alligatoris.

Alligators and Crocodiles↗
PubMed1997

Insertion sequence IST3091 of Thiobacillus ferrooxidans.

An insertion sequence, designated as IST3091, was located adjacent to the putative origin of replication region of plasmid pTFI91 of Thiobacillus ferrooxidans TFI-91. The DNA sequence of the transposase gene of IST3091 revealed similarity with that of IS30, IS1086, IS4351, and the integrase gene of SpV1-R8A2 B (a bacteriophage of Spiroplasma citri). The sequence of IST3091 is 1063 bp long with partially matched 30-bp terminal inverted repeats. Several restriction fragments of plasmid pTFI91 of T. ferrooxidans containing the IST3091 element were cloned into the vector pHSG398. The hybrid plasmids (pBTL) were transformed into Escherichia coli NK7379 containing a miniF plasmid, which was devoid of transposable elements. The transposition function of the IST3091 element was confirmed by mobilizing hybrid plasmids via conjugation from transformed E. coli NK7379 (donor) to E. coli M8820 (recipient). The presence of the transposed element in transconjugants was detected by polymerase chain reaction amplification.

Amino Acid Sequence↗
PubMed2004

Molecules of the bacterial cytoskeleton.

The structural elucidation of clear but distant homologs of actin and tubulin in bacteria and GFP labeling of these proteins promises to reinvigorate the field of prokaryotic cell biology. FtsZ (the tubulin homolog) and MreB/ParM (the actin homologs) are indispensable for cellular tasks that require the cell to accurately position molecules, similar to the function of the eukaryotic cytoskeleton. FtsZ is the organizing molecule of bacterial cell division and forms a filamentous ring around the middle of the cell. Many molecules, including MinCDE, SulA, ZipA, and FtsA, assist with this process directly. Recently, genes much more similar to tubulin than to FtsZ have been identified in Verrucomicrobia. MreB forms helices underneath the inner membrane and probably defines the shape of the cell by positioning transmembrane and periplasmic cell wall-synthesizing enzymes. Currently, no interacting proteins are known for MreB and its relatives that help these proteins polymerize or depolymerize at certain times and places inside the cell. It is anticipated that MreB-interacting proteins exist in analogy to the large number of actin binding proteins in eukaryotes. ParM (a plasmid-borne actin homolog) is directly involved in pushing certain single-copy plasmids to the opposite poles by ParR/parC-assisted polymerization into double-helical filaments, much like the filaments formed by actin, F-actin. Mollicutes seem to have developed special systems for cell shape determination and motility, such as the fibril protein in Spiroplasma.

Actins↗
PubMed2007

Regulation of carbon metabolism in the mollicutes and its relation to virulence.

The mollicutes are cell wall-less bacteria that live in close association with their eukaryotic hosts. Their genomes are strongly reduced and so are their metabolic capabilities. A survey of the available genome sequences reveals that the mollicutes are capable of utilizing sugars as source of carbon and energy via glycolysis. The pentose phosphate pathway is incomplete in these bacteria, and genes encoding enzymes of the tricarboxylic acid cycle are absent from the genomes. Sugars are transported by the phosphotransferase system. As in related bacteria, the phosphotransferase system does also seem to play a regulatory role in the mollicutes as can be concluded from the functionality of the regulatory HPr kinase/phosphorylase. In Mycoplasma pneumoniae, the activity of HPr kinase is triggered in the presence of glycerol. This carbon source may be important for the mollicutes since it is available in epithelial tissues and its metabolism results in the formation of hydrogen peroxide, the major virulence factor of several mollicutes. In plant-pathogenic mollicutes such as Spiroplasma citri, the regulation of carbon metabolism is crucial in the adaptation to life in plant tissues or the insect vectors. Thus, carbon metabolism seems to be intimately linked to pathogenicity in the mollicutes.

Bacterial Proteins↗
PubMed1996

Inactivation of the very strong HCMV immediate early promoter by DNA CpG methylation in vitro.

The influence of DNA methylation in vitro on the activity of the very strong human cytomegalovirus (HCMV) major immediate early (IE) modulator/enhancer/promoter region was investigated by transient transfection experiments of premonocytic HL-60 cells. While sequence-specific methylation of the major IE enhancer and/or modulator with the cytosine methyl-transferases FnuDII, HhaI and HaeIII had no significant effect, the promoter activity was completely repressed by methylation of the cytosine in 5'-CpG sites with the Spiroplasma methyltransferase SssI. Addition of TNF-alpha or PMA which are strong stimulators of HCMV major IE enhancer/promoter activity in premonocytic HL-60 cells had no effect on repression. Inactivation of the IE enhancer/promoter via methylation by M.SssI could be partially alleviated by co-transfection with an excess of untranscribable highly methylated DNA. These results indicate that a methyl-CpG binding factor is involved as mediator in the inhibitory effect of HCMV enhancer/promoter methylation. Taken together, the HCMV major IE enhancer/ promoter has been shown to be susceptible to transcriptional inactivation by methylation of the cytosines in CpG dinucleotides, a process that is proposed to play a modulatory role in viral latency.

Antigens, Viral↗
PubMed2002

Management of corn leafhopper (Homoptera: Cicadellidae) and corn stunt disease in sweet corn using reflective mulch.

Plastic reflective mulches significantly reduced populations of corn leafhopper, Dalbulus maidis (DeLong & Wolcott), adults and the incidence of corn stunt disease caused by Spiroplasma kunkelii (CSS) in late planted sweet corn (Zea mays L.). The reflective mulches were more effective than were either foliar or soil applied insecticides in managing both the leafhopper and the pathogen it transmits. Yields of marketable ears were 1.5 to 2 times greater in reflective mulch plots than from fallow plots. This was due to larger ears (individual ear weight and length) rather than an increase in the number of ears. The use of reflective mulches provides an alternative strategy to insecticides in the management of both D. maidis and corn stunt disease. Such a strategy may prove useful to growers in Latin America and to limited resource growers and organic growers in the United States who wish to grow corn without the use of insecticides.

Animals↗
PubMed2004

Development of monoclonal antibodies for detection of necrotizing hepatopancreatitis in penaeid shrimp.

Monoclonal antibodies (MAbs) were produced against necrotizing hepatopancreatitis bacteria (NHP-B) of penaeid shrimp. The MAbs tested in dot-immunoblot (D-IB) assays were capable of detecting the NHP-B in hepatopancreas samples collected from moribund juvenile Litopenaeus vannamei during an experimentally induced NHP-B infection. The MAbs were also screened by immunohistochemistry (IHC) using case submissions that were determined to be infected not only by histology, but also polymerase chain reaction (PCR) and in situ hybridization (ISH) assays using specific digoxigenin (DIG)-labeled probes on histological sections prepared from naturally infected shrimp. Two of the MAbs were chosen for development of detection methods for NHP. The MAbs were tested using IHC methods on Davidson's alcohol-formalin-acetic acid (AFA) fixed tissue sections and identified NHP-B infected cells and tissues in a pattern similar to that seen with DIG-labeled NHP-specific gene probes. None of the MAbs reacted with tissue from specific pathogen-free (SPF) shrimp or with shrimp tissues infected with a rickettsia-like bacteria, Vibrio sp., Campylobacter sp., and Spiroplasma sp. The MAbs were found to be negative against these other organisms, demonstrating that they are species specific and useful for rapid diagnostic detection of NHP-B.

Animals↗
PubMed2004

Echigo-1: a panencephalopathic strain of Creutzfeldt-Jakob disease. II. Ultrastructural studies in hamsters.

In this and a companion paper we present immunohistochemical and ultrastructural data on hamsters infected with the Echigo-1 strain of Creutzfeldt-Jakob disease. Ultrastructurally, two types of vacuoles were readily discriminated in the brain: the grey matter vacuoles of spongiform change and intramyelin vacuoles. The vacuoles were always membrane-bound; the membranes were single or double. The axons were entirely missing from the plane of the sections or, if visible, were shrunken and attached to the innermost layer of the myelin. It was noteworthy that some vacuoles indented cell bodies or processes and thus were reminiscent of the intraneuronal vacuoles typical for natural scrapie, BSE and CWD in ungulates and cervids but not of the vacuoles encountered in rodent models of scrapie and CJD. We also noticed vacuoles distending myelinated fibres in which the axons had become dystrophic. Some axons underwent Wallerian degeneration while others met the criteria for dystrophic neuritis. Both alterations existed in the same areas. Typical dystrophic neurites contained abnormal subcellular organelles, mainly electron-dense lysosomal inclusions. Other neurites contained numerous multi-vesicular bodies and autophagic vacuoles. Nuclear paracrystalline rod-like inclusions were occasionally visible in neurons while other inclusions comprised spiroplasma-like inclusions in synaptic boutons. The robust cellular reactions consisted of reactive astrocytes and macrophages filled with cellular debris. It is of note that complex autophagic vacuoles were observed in the cytoplasm of neurons.

Animals↗
PubMed1990

[Detection of mycoplasmas in eye swab specimens of cattle].

In the present investigation 262 conjunctival swabs were taken from 178 cattle and examined for mycoplasmas. The isolation was possible from 111 swabs. Mycoplasmas were found in eyes with clinical symptoms of IBK (in 64 of 148 swabs investigated = 43.2%) as well as in healthy eyes (in 47 of 114 swabs investigated = 41.2%). Consequently a correlation between clinical findings and isolation of mycoplasmas could not be observed. Unfortunately 60 of 111 isolates could not be subcultivated after storage at -20 degrees C. Using the indirect immunofluorescence test 41 of the 42 surviving isolates were identified as M. bovoculi which before has not been isolated in the Federal Republic of Germany. One isolate was determined as A. laidlawii. The 17 M. bovoculi strains investigated for their biochemical reactions showed the same characteristics like the reference strain M. bovoculi M 165/69. In repeating examinations mycoplasmas could be isolated 5 times after one month and 14 times after 6 months. Cattle younger than 2 years were more often infected with mycoplasmas (62.5%) than older animals (19.4%). No difference, however, could be observed in the clinical manifestations of IBK between younger and older animals. Mycoplasmas were more frequently isolated in autumn (43.6%) than in spring (21.4%) and summer (29.3%). In most of the animals examined both eyes were colonized by mycoplasmas. No spiroplasmas could be detected in the 262 conjunctival swabs investigated.

Animals↗
PubMed1987

Immunochemical evidence for an active (F1-F0)-ATPase in mycoplasmas.

In all Acholeplasma, Mycoplasma and Spiroplasma species tested, a protein capable of reacting with antibodies prepared against the beta subunit of the proton-ATPase complex from yeast, chloroplasts and Escherichia coli was detected. The reactive protein of M. gallisepticum was found to be catalytically active, suggesting that mycoplasmas, as other bacteria, possess a proton-translocating ATPase. Characterization of the ATPase activity of M. gallisepticum indicates that this organism also possesses a Na+-stimulated ATPase activity that differs from the proton-ATPase in its pH profile and its resistance to dicyclohexylcarbodiimide (DCCD).

Antibodies, Bacterial↗
PubMed1987

Towards monoclonal antibodies against the apple proliferation mycoplasma-like organism.

A 6-week-old female BALB/c mouse was immunized with a preparation of partially purified sieve tubes of periwinkle (Catharantus roseus) plants infected with the apple proliferation mycoplasma-like organism. After fusion of the immunized spleen cells with myeloma cells, the supernatant fluids of the resulting hybridoma cultures were screened for the presence of antibodies in a differential avidin-biotin enzyme-linked immunosorbent assay (ELISA) using sieve tube preparations from either healthy or infected periwinkles as antigens. One hybridoma clone, giving a negative ELISA with the sieve tube preparation from healthy plants and a positive ELISA with that infected ones, was selected. This hybridoma also gave a negative ELISA when tested against sieve tube preparations from periwinkles affected by citrus greening or Spiroplasma citri, but a positive ELISA when tested against phyllody-affected sieve-tube preparations.

Antibodies, Bacterial↗
PubMedDate not supplied

In vitro studies of mycoplasma-like organisms.

We determined whether the western X mycoplasma (WXM) isolated from Colladonus montanus could be maintained in vitro by ultrathin sections or by assay of infectivity. Large spherical or small electron-dense bodies like those found in intact infected cells were observed in some media. Infectivity of WXM can be maintained for 28 days in cultured salivary glands in a newly developed medium, and for 281 days (seven passages) in modified AcTc, for 231 days (eight passages) in modified PC, 107 days (one passage) in spiroplasma medium, and 52 days (one passage) in modified GITC medium extracts. However, there is no evidence that WXM multiplied in any medium.

Animals↗
PubMedDate not supplied

New laboratory techniques for isolation of Mycoplasma pneumoniae.

The SP-4 culture medium, developed originally for newly isolated plant and insect mycoplasmas (spiroplasmas), has markedly improved the recovery of Mycoplasma pneumoniae from human clinical materials. This medium, in combination with a direct fluorescent antibody test, can enhance the recovery and identification of the organism by 30-40 percent over conventional culture procedures. Although these modifications are a clear improvement in diagnostic techniques for M. pneumoniae, the time required for growth and identification of the agent is still a major disadvantage for rapid clinical diagnosis. Thus, there remains a critical need for techniques that can specifically identify the major antigens (or other components) of the organism within the first week of the infection.

Agar↗
PubMed1984

Deletions, duplications and rearrangements in mycoplasma ribosomal RNA gene sequences.

Mycoplasma genomes lack several entire ribosomal RNA gene sets, and the remaining RNA genes lack some nucleotides. Analysis of mycoplasma 5S RNAs utilizing generalized models for prokaryotic 5S RNA secondary structure allows us to specify the molecular locations and identify many of the nucleotides missing from mycoplasma 5S RNAs and to detect the presence of unusual structural features. The most extensive alterations occur in the 5S RNAs from M. mycoides subsp. capri and M. capricolum, which have a similar pattern. Each of these 5S RNAs, as well as the Spiroplasma BC3 5S RNA, has a concentration of deletions in helix V, and they also share several unusual structures. We also noted the occurrence in mycoplasma 5S RNAs of numerous repeated sequences. The genetic process of unequal crossing-over is suggested as the explanation for the repeated sequences observed in mycoplasma RNAs.

Base Sequence↗
PubMed1981

Laboratory diagnosis of Mycoplasma pneumoniae infections.

A culture medium developed for newly isolated plant and insect mycoplasmas (spiroplasmas) has markedly improved the recovery of Mycoplasma pneumoniae from human clinical materials. This medium (SP-4), in combination with a direct fluorescent antibody test, can improve the recovery and identification of the organism by 30 to 40% over conventional culture procedures. Although these improvements in isolation of the organism may lead to new information on the epidemiology of the disease, the most organism may lead to new information on the epidemiology of the disease, the most rapid diagnosis of clinical M. pneumoniae infections in man is still dependent upon accurate measurement of antibody rises in response to the organism. Recent reports of a number of new serological techniques suggest that further improvements in the serological diagnosis of M. pneumoniae infections are possible.

Antibodies, Bacterial↗