Detection and identification of mycoplasmas by amplification of rDNA

Alignment of published 16S rRNA sequences allowed the definition of a pair of oligonucleotides suitable for polymerase chain reaction (PCR). Using this pair of PCR primers, several mycoplasmas including the four human parasites Mycoplasma genitalium, M. hominis, M. salivarium and M. orale were detected. This DNA amplification was restricted to species of the genus Mycoplasma while no cross-reaction was observed with DNA from other bacteria and eukaryotic cells. Subsequent analysis of amplified products by either specific oligonucleotide hybridization or dideoxy sequencing specified the identity of the detected mycoplasmas. This method offers a highly discriminating and sensitive assay for the direct detection and identification of these microorganisms without the need for prior cultivation.     

PCR和分离培养同时检测儿童呼吸道感染的支原体

为了解广州市儿童呼吸道支原体感染情况,用一条共同的上游引物,二条特异性的下游引物建立的ＰＣＲ方法能同时扩增ＭＰ的６９１ｂｐ和ＭＧ的４３８ｂｐ粘附因子基因片段,但不会扩增其他支原体和细菌的ＤＮＡ。     

Electrophoretic and immunologic comparative analysis of Mycoplasma pneumoniae and Mycoplasma genitalium proteins

The Mycoplasma pneumoniae FH strain routinely used in our laboratory for over 25 years as antigen in serological tests, 2 reference M. pneumoniae strains from ATCC (29342 and M129) and 3 isolates of M. pneumoniae obtained in 1995 from pneumonia patients were compared by SDS-PAGE, complement fixation test (CFT) and by Western-immunoblotting against human and rabbit serum samples with high level of mycoplasmal antibodies. On SDS-PAGE all M. pneumoniae strains showed the same number of 23 polypeptides on the gel with identical molecular weights. The same strains on immunoblotting against human and rabbit serum samples showed six bands: 170, 89, 75, 55, 38 and 33 kDa with the strongest antibody staining in 170-(P1 protein) and 89-kDa bands. Because of its known antigenic relationships Mycoplasma genitalium was used for comparison. The pattern of M. genitalium proteins on SDS-PAGE was similar to pattern of M. pneumoniae but distinguishable. On immunoblotting six proteins of M. genitalium (135, 127, 110, 95, 75 and 45 kDa) reacted with human and rabbits immunoglobulins for M. pneumoniae antigens. Furthermore in complement fixation test both antigens, prepared from M. pneumoniae and M. genitalium, reacted as well with human and rabbit immunoglobulins for M. pneumoniae and with rabbit immunoglobulins for M. genitalium. These cross-reactions observed in serological techniques could give false positive results in routine diagnosis of M. pneumoniae infections. In such situations showing on immunoblott of presence in tested serum sample of antibodies to 170- and 89 kDa proteins could confirm M. pneumoniae infection.     

Identification of a small RNA within the pdh gene cluster of Mycoplasma pneumoniae and Mycoplasma genitalium

A highly abundant and heterogeneous small RNA about 205 to 210 bases long named MP200 RNA has been identified in Mycoplasma pneumoniae. It was localized on the genome within a 319-bp-long intergenic space of the pyruvate dehydrogenase (pdh) gene cluster. A database search at the DNA level revealed the highest similarity to a sequence located within the pdh gene cluster of Mycoplasma genitalium that was also shown to be transcribed into two abundant, but smaller RNAs than the ones in Mycoplasma pneumoniae. The RNAs from both M. pneumoniae and M. genitalium have the potential to code for cysteine-rich 29- and 23-amino-acid-long peptides, but so far, these peptides have not been identified experimentally in bacterial protein extracts.     

The RuvA homologues from Mycoplasma genitalium and Mycoplasma pneumoniae exhibit unique functional characteristics

The DNA recombination and repair machineries of Mycoplasma genitalium and Mycoplasma pneumoniae differ considerably from those of gram-positive and gram-negative bacteria. Most notably, M. pneumoniae is unable to express a functional RecU Holliday junction (HJ) resolvase. In addition, the RuvB homologues from both M. pneumoniae and M. genitalium only exhibit DNA helicase activity but not HJ branch migration activity in vitro. To identify a putative role of the RuvA homologues of these mycoplasmas in DNA recombination, both proteins (RuvA(Mpn) and RuvA(Mge), respectively) were studied for their ability to bind DNA and to interact with RuvB and RecU. In spite of a high level of sequence conservation between RuvA(Mpn) and RuvA(Mge) (68.8% identity), substantial differences were found between these proteins in their activities. First, RuvA(Mge) was found to preferentially bind to HJs, whereas RuvA(Mpn) displayed similar affinities for both HJs and single-stranded DNA. Second, while RuvA(Mpn) is able to form two distinct complexes with HJs, RuvA(Mge) only produced a single HJ complex. Third, RuvA(Mge) stimulated the DNA helicase and ATPase activities of RuvB(Mge), whereas RuvA(Mpn) did not augment RuvB activity. Finally, while both RuvA(Mge) and RecU(Mge) efficiently bind to HJs, they did not compete with each other for HJ binding, but formed stable complexes with HJs over a wide protein concentration range. This interaction, however, resulted in inhibition of the HJ resolution activity of RecU(Mge).     

DNA methylation and Mycoplasma genomes

DNA methylation is one of the many hypotheses proposed to explain the observed deficiency in CpG dinucleotides in a variety of genomes covering a wide taxonomic distribution. Recent studies challenged the methylation hypothesis on empirical grounds. First, it cannot explain why the Mycoplasma genitalium genome exhibits strong CpG deficiency without DNA methylation. Second, it cannot explain the great variation in CpG deficiency between M. genitalium and M. pneumoniae that also does not have CpG-specific methyltransferase genes. I analyzed the genomic sequences of these Mycoplasma species together with the recently sequenced genomes of M. pulmonis, Ureaplasma urealyticum, and Staphylococcus aureus, and found the results fully compatible with the methylation hypothesis. In particular, I present compelling empirical evidence to support the following scenario. The common ancestor of the three Mycoplasma species has CpG-specific methyltransferases, and has evolved strong CpG deficiency as a result of the specific DNA methylation. Subsequently, this ancestral genome diverged into M. pulmonis and the common ancestor of M. pneumoniae and M. genitalium. M. pulmonis has retained methyltransferases and exhibits the strongest CpG deficiency. The common ancestor lost the methyltransferase gene and then diverged into M. genitalium and M. pneumoniae. M. genitalium and M. pneumoniae, after losing methylation activities, began to regain CpG dinucleotides through random mutation. M. genitalium evolved more slowly than M. pneumoniae, gained relatively fewer CpG dinucleotides, and is more CpG-deficient.     

Isolation and characterization of transposon Tn4001-generated, cytadherence-deficient transformants of Mycoplasma pneumoniae and Mycoplasma genitalium

Abstract Cytadherence and subsequent parasitism of host cells by the human pathogens, Mycoplasma pneumoniae and Mycoplasma genitalium , are mediated by adhesins and adherence-related accessory proteins. In this report we demonstrate the use of transposon Tn 4001 to generate Tn-induced transformants displaying cytadherence-deficient characteristics. Mycoplasma pneumoniae Tn-generated transformant, designated 8R, lacked the high-molecular weight adherence-accessory proteins HMW1/4 and was deficient in hemadsorption and cytadherence capabilities. In transformant 8R, Tn 4001 was not localized in or near the hmw 1 gene or in the upstream adhesin (p30/hmw3) locus, suggesting an alternate site associated with the regulation of hmw 1 gene expression. Sequence analysis identified the transposon insertion site at the crl locus previously reported, although the protein characteristics of transformant 8R differed from the earlier described transformants. The M. genitalium Tn-transformant, designated G26, was also defective in hemadsorption and cytadherence. However, transformant G26 synthesized adhesins P140 and P32 suggesting that Tn 4001 transposed into a new gene or site previously unlinked to cytadherence, namely ORF MG032. This study demonstrates the utility of Tn 4001 mutagenesis for both M. pneumoniae and M. genitalium which, in the latter case, has special relevance in light of the recent complete characterization of its continuous total genomic sequence.     

Internalization and intracellular survival of Mycoplasma pneumoniae by non-phagocytic cells

Current theory holds that mycoplasmas remain attached to the surface of epithelial cells although some mycoplasmas have evolved mechanisms for entering host cells that are not naturally phagocytic. The ability of Mycoplasma pneumoniae strain M129 to invade and survive within host cells was studied using a HeLa cell line and a human lung carcinoma cell line (A549). The invasion process into the eukaryotic cells was studied qualitatively by confocal laser scanning microscopy and quantitatively by the gentamicin resistance assay. Internalization was found with A549 cells but not with HeLa cells. Internalization was dependent on the duration of the infection and on temperature. The organism, detected in the cytoplasm and perinuclear regions, survived within the host cells for prolonged periods of time. The intracellular location of M. pneumoniae is obviously a privileged niche, well protected from the immune system and from the action of many antibiotics and may explain the pathogenic potential of this organism.     

Relationship of Mycoplasma pneumoniae to other human Mycoplasma species studied by gel diffusion

Taylor-Robinson, David (National Institute of Allergy and Infectious Diseases, Bethesda, Md.), Otakar Sobeslavsky, and Robert M. Chanock. Relationship of Mycoplasma pneumoniae to other human Mycoplasma species studied by gel diffusion. J. Bacteriol. 90:1432-1437. 1965.-Conditions are presented for the production of four lines of precipitate between Mycoplasma pneumoniae antigen and homologous hyperimmune rabbit serum in double diffusion in agar. The specificity of the reaction was shown by the fact that M. pneumoniae antigen did not react with antisera to the other human mycoplasma species, nor did M. pneumoniae antiserum produce lines with antigens prepared from the other human mycoplasmas. In addition, there was no reduction in the number or intensity of precipitation lines after absorption of M. pneumoniae antiserum with heterotypic mycoplasma antigens, or after absorption of heterotypic mycoplasma antisera with M. pneumoniae antigen. These findings indicate that, of the human mycoplasma species so far studied, M. pneumoniae is antigenically the most distinct.     

Molecular characterization of the P1-like adhesin gene from Mycoplasma pirum.

A DNA fragment has been isolated from the genome of Mycoplasma pirum by use of a genetic probe derived from the conserved region within the genes for the major adhesins of Mycoplasma genitalium and Mycoplasma pneumoniae. A gene encoding an adhesin-like polypeptide was localized, and sequence analysis indicated a G + C content of only 28%, with A- and T-rich codons being preferentially used. A total of 91% of positions 3 were either A or T. The deduced polypeptide is 1,144 amino acids long (126 kDa) and shows 26% identity with the adhesins of M. genitalium and M. pneumoniae. Other features in common with these two membrane proteins include a similar hydropathic profile and a proline-rich C terminus. Antibodies were prepared by using as an immunogen a peptide derived from the C terminus of the M. pirum adhesin-like polypeptide and were found to recognize on immunoblots a 126-kDa polypeptide from an M. pirum cellular extract. The characterization of the adhesin-like gene is a first step toward a better understanding of the mechanisms allowing this human mycoplasma to attach to host cells.     

Comparison of glycoproteins from ten human Mycoplasma species.

Multiple bands of glycoprotein, rare in procaryotes, were detected in ten human Mycoplasma species by staining with periodic acid-Schiff (PAS) reagent after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). A major contaminant formed in Hayflick medium (H medium), corresponding to an apparent molecular weight of about 80 kD, was eliminated by using the organisms grown in PPLO broth supplemented with PPLO serum fraction (P medium), except that M. genitalium and M. pneumoniae were grown in H medium as monolayers on the glass surface. The comparison of glycoproteins among ten human Mycoplasma species indicated that their profiles were shown to be species-specific. However, those of M. buccale and M. faucium were very similar, and M. pneumoniae and M. genitalium seemed to be related.     

Mycoplasma genitalium and Mycoplasma pneumoniae share a 67 kilodalton protein as a main cross-reactive antigen

It was demonstrated that a 67 kilodalton (kDa) protein of Mycoplasma pneumoniae is a main cross-reactive antigen with similar molecular weight protein of Mycoplasma genitalium by Western blot analysis using monoclonal antibody to 67 kDa protein of M. pneumoniae and hyperimmune rabbit sera directed against each mycoplasma strain.     

Functional characterization of the RuvB homologs from Mycoplasma pneumoniae and Mycoplasma genitalium

Homologous recombination between repeated DNA elements in the genomes of Mycoplasma species has been hypothesized to be a crucial causal factor in sequence variation of antigenic proteins at the bacterial surface. To investigate this notion, studies were initiated to identify and characterize the proteins that form part of the homologous DNA recombination machinery in Mycoplasma pneumoniae as well as Mycoplasma genitalium. Among the most likely participants of this machinery are homologs of the Holliday junction migration motor protein RuvB. In both M. pneumoniae and M. genitalium, genes have been identified that have the capacity to encode RuvB homologs (MPN536 and MG359, respectively). Here, the characteristics of the MPN536- and MG359-encoded proteins (the RuvB proteins from M. pneumoniae strain FH [RuvB(FH)] and M. genitalium [RuvB(Mge)], respectively) are described. Both RuvB(FH) and RuvB(Mge) were found to have ATPase activity and to bind DNA. In addition, both proteins displayed divalent cation- and ATP-dependent DNA helicase activity on partially double-stranded DNA substrates. The helicase activity of RuvB(Mge), however, was significantly lower than that of RuvB(FH). Interestingly, we found RuvB(FH) to be expressed exclusively by subtype 2 strains of M. pneumoniae. In strains belonging to the other major subtype (subtype 1), a version of the protein is expressed (the RuvB protein from M. pneumoniae strain M129 [RuvB(M129)]) that differs from RuvB(FH) in a single amino acid residue (at position 140). In contrast to RuvB(FH), RuvB(M129) displayed only marginal levels of DNA-unwinding activity. These results demonstrate that M. pneumoniae strains (as well as closely related Mycoplasma spp.) can differ significantly in the function of components of their DNA recombination and repair machinery.     

The evolution of Mycoplasma genitalium

Mycoplasma genitalium is the smallest microorganism capable of self-replication. With its small genome, M. genitalium is the best representative of a minimal cell. The comparison of genome evolution among the three urogenital mycoplasmas, M. genitalium, M. hominis, and Ureaplasma parvum, not only indicated that they share a core genome of ~250 protein-encoding genes that correspond to their basic cell metabolism, but also showed a striking difference in their energy-generating pathways. M. genitalium is a sexually transmitted organism associated with nongonococcal urethritis in men and several inflammatory reproductive tract syndromes in women, such as cervicitis, pelvic inflammatory disease, and infertility. The treatment of M. genitalium infections has not yet been standardized. Macrolides are recommended, especially single-dose azithromycin; tetracyclines are responsible for a large number of therapeutic failures without any acquired resistance demonstrated. Acquired resistance to macrolides and fluoroquinolones leading to therapeutic failure has been described.     

Demonstration of membrane association and surface location of Mycoplasma pneumoniae antigens using monoclonal antibodies

We examined 10 monoclonal antibodies (mAbs) directed against Mycoplasma pneumoniae proteins of 200, 170, 67, 46 and 42 kDa, and one mAb directed against a glycolipid component. The membrane association of the antigens reacting with our mAbs was investigated, in particular by phase-fractionation involving use of the detergent Triton X-114. The 170 kDa protein was shown to be membrane-associated, and surface exposure of this antigen was demonstrated by its disappearance from SDS-PAGE patterns after treatment of intact mycoplasmas with proteolytic enzymes. Cross-reactions with protein antigens of Mycoplasma genitalium were also shown. A mAb directed against a component of a lipid extract, prepared by the method used for preparation of the antigen used in the complement fixation (CF) test for serological diagnosis of M. pneumoniae infection, reacted with one major and a few minor bands in thin-layer chromatography (TLC) of the crude extract. The glycolipid character of this major antigen was demonstrated by treatment of the extract with sodium periodate, and by development of the TLC with orcinol/ferric chloride. These reactive bands were the same as those detected by the use of polyclonal mouse antiserum and a human convalescent serum, a result showing that the CF antigen contains a glycolipid moiety reacting with our mAb. The surface exposure of this antigen was demonstrated by binding of mAbs to intact cells.     

Susceptibility of newly established mouse strain MPS to Mycoplasma pneumoniae infection

A newly established mouse strain, MPS, which is more sensitive to Mycoplasma pulmonis than ICR, ddY and other mouse strains was examined for its susceptibility to Mycoplasma pneumoniae. In experimental infections with M. pneumoniae, it was observed that M. pneumoniae attached to tracheas of MPS mice, and M. pneumoniae cells were isolated from tracheas and lungs of MPS mice even after four weeks of infection, while no mycoplasmas were isolated from ICR and ddY mice after one week of infection. Specific antibodies against M. pneumoniae were also observed by the Western blotting in the sera of MPS mice infected with M. pneumoniae. Although any lung lesion could not be observed in this work, this newly established mouse strain MPS may be useful for experiments of M. pneumoniae infection, especially for the analysis of strain differences in susceptibility to M. pneumoniae infection.     

Genetic differentiation by nucleic Acid homology I. Relationships among Mycoplasma species of man

Reich, Paul R. (National Institutes of Health, Bethesda, Md.), Norman L. Somerson, Carol J. Hybner, Robert M. Chanock, and Sherman M. Weissman. Genetic differentiation by nucleic acid homology. I. Relationships among Mycoplasma species of man. J. Bacteriol. 92:302-310. 1966.-Genetic relatedness among human mycoplasmas was evaluated by measuring the amount of nucleic acid hybrid retained on a membrane filter. Hybrids were formed from deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) derived from representative strains of seven serologically distinct human Mycoplasma species. The results indicate that serologically distinct human Mycoplasma species can also be distinguished by the homology techniques. Low-level cross-reactivity was observed among nucleic acids derived from the seven species. Genetic heterogeneity was demonstrated among three strains of M. salivarium and between two strains of M. orale type 2. In contrast, comparison of three strains and three passage levels of M. pneumoniae revealed them to be indistinguishable. M. pneumoniae appears to be the most distinct of all human mycoplasmas, as shown by both homology and the high buoyant density value of its DNA. Nucleic acids from mycoplasmas which had identical buoyant densities were in some cases differentiable. Mycoplasmas with different DNA buoyant densities were invariably distinguishable by the homology technique.     

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

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.