Identification of a ribonuclease H gene in both Mycoplasma genitalium and Mycoplasma pneumoniae by a new method for exhaustive identification of ORFs in the complete genome sequences.
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Exhaustive identification of open reading frames in complete genome sequences is a difficult task. It is possible that important genes are missed. In our efforts to reanalyze the intergenic regions of Mycoplasma genitalium and Mycoplasma pneumoniae, we have newly identified a number of new open reading frames (ORFs) in both M. genitalium and M. pneumoniae. The most significant identification was that of a ribonuclease H enzyme in both species which until now has not been identified or assumed absent and interpreted as such. In this paper we discuss the biological importance of RNase H and its evolutionary implication. We also stress the usefulness of our method for identifying new ORFs by reanalyzing intergenic regions of existing ORFs in complete genome sequences. (+info)
Indirect enzyme-linked immunosorbent assay for detection of immunoglobulin G reactive with a recombinant protein expressed from the gene encoding the 116-kilodalton protein of Mycoplasma pneumoniae.
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Serology remains the method of choice for laboratory diagnosis of Mycoplasma pneumoniae infection. Currently available serological tests employ complex cellular fractions of M. pneumoniae as antigen. To improve the specificity of M. pneumoniae diagnosis, a recombinant protein was assessed as a serodiagnostic reagent. A panel of recombinant proteins were expressed from a cloned M. pneumoniae gene that encodes a 116-kDa surface protein antigen. The recombinant proteins were assessed for reactivity with patient sera and the most antigenic was further assessed for its serodiagnostic potential by indirect enzyme-linked immunosorbent assay (ELISA). The ELISA based on the recombinant protein was equivalent in sensitivity to the commercial test (Serodia Myco II; Fujirebio Inc.) to which it was compared. Southern and Western blotting data suggested that the recombinant protein derived from the 116-kDa protein of M. pneumoniae could provide a species-specific diagnostic tool, although further assessment is required. (+info)
Comparative study of overlapping genes in the genomes of Mycoplasma genitalium and Mycoplasma pneumoniae.
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Overlapping genes are defined, in this paper, as a pair of adjacent genes whose coding regions are partly overlapping. We systematically analyzed all overlapping genes in the genomes of two closely related species: Mycoplasma genitalium and Mycoplasma pneumoniae. Careful comparisons were made for homologous genes that are overlapped in one species but not in the other. This comparative analysis allows us to propose a model of how overlapping genes emerged in the course of evolution. It was found that overlapping genes were generated primarily due to the loss of a stop codon in either gene, in many cases, the absence of which resulted in elongation of the 3' end of the gene's coding region. More specifically, the loss of the stop codon took place as a result of the following events: deletion of the stop codon (64.4%), point mutation at the stop codon (4.4%), and frame shift at the end of the coding region (6.7%). Overlapping genes, in a sense, can be thought of as the results of evolutionary pressure to minimize genome size. However, our analysis indicates that many overlapping genes, at least in the genomes of M.genitalium and M.pneumoniae, are due to incidental elongation of the coding regions. (+info)
Incidence of upper respiratory tract Mycoplasma pneumoniae infections among outpatients in Rhone-Alpes, France, during five successive winter periods.
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In this prospective study, nasal swab samples from patients with acute respiratory infections were evaluated for the presence of Mycoplasma pneumoniae. This PCR-plus-hybridization-based detection was associated with the detection of other viral agents. During the five winter surveillance periods, 3,897 samples were collected by 75 medical practitioners participating in the Groupe Regional d'Observation de la Grippe surveillance network in Rhone-Alpes (France). M. pneumoniae was detected in 283 samples (7.3%); its rate of detection ranged from 10.1 to 2.0% over the five periods, and it was the second most frequently isolated pathogen during the survey, after influenza A. Three high-prevalence winters were observed, yielding an early winter peak of M. pneumoniae infection which was observed in all age groups. No statistically significant difference was detected between rates of infections in the different age groups, but M. pneumoniae infection was significantly related to lower respiratory tract infection during periods of high prevalence. This study defined the frequency of M. pneumoniae detection from nasal swab specimens in patients with acute respiratory infections, confirming its high prevalence and the presence of large outbreaks due to this pathogen. (+info)
Application of a nested polymerase chain reaction assay to detect Mycoplasma hyopneumoniae from nasal swabs.
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The porcine respiratory disease complex (PRDC) is an increasingly important cause of decreased swine productivity and is characterized by slow growth, decreased feed efficiency, anorexia, cough, and dyspnea. Mycoplasma hyopneumoniae is among the most prevalent and important infectious agents associated with PRDC. Understanding of mycoplasmal pneumonia has been hindered by inadequate diagnostic methods. Many of the currently available tests are relatively insensitive or nonspecific when used in a diagnostic laboratory setting or are too costly or difficult for routine diagnostic use. Several polymerase chain reaction (PCR) assays have been described, but they are not sensitive enough to detect the microorganisms in live pigs, from either nasal or tracheal swabs. A nested PCR using 2 species-specific sets of primers from the 16S ribosomal DNA gave positive results with as little as 80 microorganisms and did not cross-react with other mycoplasma species or with other microorganisms commonly found in the respiratory tract of pigs. This assay was better suited for detection of M. hyopneumoniae from nasal swabs than was conventional PCR. Nasal swab samples were taken at different time periods following experimental challenge of 10 susceptible pigs. Only 2 of the 55 swabs examined gave a positive result with conventional PCR, whereas 30 of the 55 swabs gave a positive result using the nested PCR. Twenty of 40 (50%) nasal swabs from pigs experiencing a respiratory disease outbreak where M. hyopneumoniae had been diagnosed also gave a positive result with the nested PCR. To confirm that the amplified product was specific, 4 nested PCR products were purified, sequences were determined and aligned, and they were confirmed to be from M. hyopneumoniae. (+info)
Colonization of the respiratory and genital tracts of female mice with Mycoplasma pneumoniae and protection afforded to the genital tract by prior respiratory colonization.
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Mycoplasma pneumoniae, strain MY 12763, colonized the throats of 6 BALB/c female mice for at least 18 days after intranasal inoculation. The same strain colonized, in high titre, the vagina of 9 of 10 progesterone-treated BALB/c mice for at least 35 days, but none of 10 oestradiol-treated mice. Mice were less susceptible to genital-tract colonization with the multiple broth-passed FH strain of M. pneumoniae, only 3 of 10 becoming colonized. The 6 mice inoculated intranasally with strain MY 12763 were immune to genital-tract colonization with the same strain, whereas 10 mice without respiratory-tract colonization were susceptible. Protection of the genital tract in this way was at least as effective as that afforded by previous genital-tract colonization. In a further experiment, 26 immunocompetent BALB/c mice colonized previously in the respiratory tract were resistant to vaginal colonization, whereas 20 BALB/c nude mice, similarly colonized in the respiratory tract, were susceptible in the vagina, illustrating the importance of cell-mediated immunity. The possible relevance of the findings to the human situation is discussed. (+info)
Identification and complementation of frameshift mutations associated with loss of cytadherence in Mycoplasma pneumoniae.
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Mycoplasma pneumoniae cytadherence is mediated by a specialized, polar attachment organelle. Certain spontaneously arising cytadherence mutants (designated class I) lack HMW2, fail to localize the adhesin protein P1 to the attachment organelle, and exhibit accelerated turnover of proteins HMW1, HMW3, and P65. Insertional inactivation of hmw2 by Tn4001 results in a phenotype nearly identical to that of the class I mutants, suggesting that the latter may result from a defect in hmw2. In this study, the recombinant wild-type hmw2 allele successfully complemented a class I mutant when introduced by transposon delivery. Synthesis of recombinant HMW2 at wild-type levels resulted in reacquisition of hemadsorption and normal levels of HMW1, HMW3, and P65. Low-level production of HMW2 in some transformants resulted in only an intermediate capacity to hemadsorb. Furthermore, full restoration of HMW1 and P65, but not that of HMW3, was directly proportional to the amount of recombinant HMW2 produced, reflecting the importance of proper stoichiometry for certain cytadherence-associated proteins. The recombinant class I hmw2 allele did not restore cytadherence, consistent with a defect in hmw2 in this mutant. A frameshift was discovered in different oligoadenine tracts in hmw2 from two independent class I mutants. Finally, protein P28 is thought to be the product of internal translation initiation in hmw2. A transposon excision-deletion mutant produced a truncated HMW2 but no P28, consistent with this conclusion. However, this deletion mutant was hemadsorption positive, indicating that P28 may not be required for cytadherence. (+info)
Transcriptional analysis of the hmw gene cluster of Mycoplasma pneumoniae.
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Mycoplasma pneumoniae adherence to host cells is a multifactorial process that requires the cytadhesin P1 and additional accessory proteins. The hmw gene cluster consists of the genes p30, hmw3, and hmw1, the products of which are known to be essential for cytadherence, the rpsD gene, and six open reading frames of unknown function. Putative transcriptional terminators flank this locus, raising the possibility that these genes are expressed as a single transcriptional unit. However, S1 nuclease protection and primer extension experiments identified probable transcriptional start sites upstream of the p32, p21, p50, and rpsD genes. Each was preceded at the appropriate spacing by the -10-like sequence TTAAAATT, but the -35 regions were not conserved. Analysis of the M. pneumoniae genome sequence indicated that this promoter-like sequence is found upstream of only a limited number of open reading frames, including the genes for P65 and P200, which are structurally related to HMW1 and HMW3. Promoter deletion studies demonstrated that the promoter-like region upstream of p21 was necessary for the expression of p30 and an hmw3-cat fusion in M. pneumoniae, while deletion of the promoter-like region upstream of p32 had no apparent effect. Analysis by reverse transcription-PCR confirmed transcriptional linkage of all the open reading frames in the hmw gene cluster. Taken together, these findings suggest that the genes of this locus constitute an operon expressed from overlapping transcripts. (+info)