Intraspecies Genetic Relatedness among Strains of Acholeplasma laidlawii and of Acholeplasma axanthum by Nucleic Acid Hybridization

Stephens, Edward B.; Aulakh, Gurmit S.; Rose, David L.; Tully, Joseph G.; Barile, Michael F.

doi:10.1099/00221287-129-6-1929

Cited by 14 publications

(16 citation statements)

References 22 publications

(33 reference statements)

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“…The two distinct tRNA gene PCR profiles may indicate the existence of two different genomic groups for this species. This has also been indicated by earlier pulsedfield gel electrophoresis results showing different genome sizes for two A. laidlawii strains (32) and especially by nucleic acid hybridization studies that demonstrated extensive genomic variation between different strains (42).…”

Section: Discussionmentioning

confidence: 66%

Evaluation of tRNA Gene PCR for Identification of Mollicutes

et al. 2005

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We evaluated the applicability of tRNA gene PCR in combination with fluorescent capillary electrophoresis with an ABI310 genetic analyzer (Applied Biosystems, Calif.) for the identification of different mollicute species. A total of 103 strains and DNA extracts of 30 different species belonging to the genera Acholeplasma, Mycoplasma, and Ureaplasma were studied. Reproducible peak profiles were generated for all samples, except for one M. genitalium isolate, the three M. gallisepticum isolates, and 8 of the 24 Ureaplasma cultures, where no amplification could be obtained. Clustering revealed numerous discrepancies compared to the identifications that had been previously obtained by means of biochemical and serological tests. Final identification was obtained by 16S rRNA gene amplification followed by sequence analysis and/or restriction digestion. This confirmed the identification obtained by tRNA gene PCR in all cases. Seven samples yielded an unexpected tRNA gene PCR profile. Sequence analysis of the 16S rRNA genes showed that six of these samples were mixed and that one had a unique sequence that did not match any of the published sequences, pointing to the existence of a not-yet-described species. In conclusion, we found tRNA gene PCR to be a rapid and discriminatory method to correctly identify a large collection of different species of the class of Mollicutes and to recognize not-yet-described groups.Having no cell wall, Mollicutes form a special class of bacteria. Their small, compact genomes evolved from AT-rich, gram-positive bacteria by means of genome reduction. At the same time, they developed innovative mechanisms to survive as parasitic organisms in a wide variety of host environments. To date, eight genera belonging to the class Mollicutes have been described, and within these genera, up to 200 species, mostly of the genus Mycoplasma, are acknowledged. This variety of species is associated with several taxonomic ambiguities (15,17,18), and a correct identification may be very difficult for numerous reasons. First, a number of Mollicutes, especially the plant-pathogenic spiroplasmas, have not been cultivated, while others require very complex media. As a result, for some species, only a limited number of isolates exist, and these are often not easily accessible. Second, as more sequences and better isolation media become available, more species are continuously being discovered. Finally, for some species, limited data and very few reports are published, especially for less-virulent and nonvirulent species.To correctly differentiate all these species, a universal and fast identification technique would be extremely useful. Some promising methods have already been described (see, e.g., reference 25), but they do not yield digitized data, making exchange between laboratories difficult. An optimized tRNA gene PCR technique, originally described by Welsh and McClelland (27,48), has been shown to be useful for correct and reproducible identification of very diverse bacterial species when combined with hig...

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Section: Discussionmentioning

confidence: 66%

Evaluation of tRNA Gene PCR for Identification of Mollicutes

et al. 2005

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“…DNAs used as probes for these tests were prepared by nick translation as described above and were denatured by heating in boiling water for 10 min. To remove residual double-stranded DNA, the denatured probe solution was passed through a hydroxyapatite column at 56"C, and single-stranded DNA was eluted with 0.12 M phosphate buffer (pH 6.8) and stored at 4°C (23). The unlabeled DNAs tested for hybridization were sheared by sonication (model W350; Heat Systems Ultrasonics, Inc., Plainview, N.Y.) to a fragment size of approximately 400 to 600 nucleotides, as determined by their electrophoretic migration in 0.8% agarose gel, with an Hue111 digest of QX174 DNA as a size marker.…”

Section: Methodsmentioning

confidence: 99%

Common Deoxyribonucleic Acid Sequences in Mycoplasma genitalium and Mycoplasma pneumoniae Genomes

Yogev

Razin

1986

International Journal of Systematic Bacteriology

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Southern blot hybridization of digested deoxyribonucleic acids (DNAs) of Mycoplasma ,genitalium, M . pneumoniae, and M . gallisepticum with probes made of total DNA of any one of these mycoplasmas revealed hybridization bands additional to those containing ribosomal ribonucleic acid (rRNA) gene sequences, which were identifiable by specific rRNA gene probes. The number and intensity of these additional bands were most pronounced with the M . genitalium-M. pneumoniae pair, which shares major antigenic determinants. Hybridization with M . pulmonis and Spiroplasma citri DNAs as probes revealed only the rRNA gene bands in digested DNAs of M . genitalium, M . pneumoniae, and M . gallisepticum. DNA-DNA hybridization tests in solution, with M . genitalium and M . pneumoniae DNA as probes, corroborated the Southern blotting data by showing the highest homology values between M . genitalium and M . pneumoniae (6.5 to 8.1 %), lower with M . gallisepticum (3.5 to 4.0%), and lowest with S. citri (0.5 to 0.9%). The new hybridization approach provides visual evidence for genetic relatedness between M . genitalium and M . pneumoniae and may facilitate the identification and cloning of genomic DNA segments carrying genes shared by two or more mycoplasmas.The new methodology of molecular genetics has provided useful tools for establishing genetic relatedness among bacteria, thus influencing bacterial taxonomy and phylogeny in a most profound way. Methods based on comparison of deoxyribonucleic acid (DNA) cleavage patterns by restriction endonucleases (21) and on hybridization patterns of cleaved DNA with ribosomal ribonucleic acid (rRNA) gene probes (17, 18; S. Razin and D. Yogev, J. Pediatr. Infect. Dis., in press) have recently been added to the longestablished DNA base composition and DNA homology tests (11). In the present communication, we propose another method for assessment of genetic relatedness between bacteria, based on detection of common genomic DNA sequences, as revealed by Southern blot hybridization of digested DNA of one organism with nick-translated total DNA of another organism. Mycoplasma genitalium and M. pneumoniae were selected as major test organisms, since the basis for the similarities exhibited by these two human mycoplasmas has become an important issue in current mycoplasma research (5, 7, 10, 12, 13, 24).The recently discovered M . genitalium, isolated from the urethras of nongonococcal urethritis patients (25), shows a remarkable resemblance to the well-established human pathogen M . pneumoniae in cell shape, possession of a tip structure, complex nutritional requirements, adherence ability (4, 14, 26), and antigenic structure (5, 7, 10, 12-14, 24). Nevertheless, genomic analysis indicates that these two organisms represent two distinct species by currently accepted criteria (11). Thus, the guanine plus cytosine content of the M . genitalium genome is 32.4 5 1.0 mol% (26), compared with the value of 38.6 to 40.8 mol% for the M . pneumoniae genome (20). The cleavage patterns of the DNAs of the two m...

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“…Because both mycoplasmas and mitochondria synthesize DNA by using DNA polymerase gamma rather than DNA polymerase alpha, the use of aphidicolin, which inhibits the action of DNA polymerase alpha, ensures that only my/ mtDNA will be labeled (20,22). Figure 1 depicts restriction of the DNA isolated from "'C-thymidine-labeled, mycoplasma-infected cultured cells in the presence (9).…”

Section: Resultsmentioning

confidence: 99%

“…For example, antigenic variation and genomic shifting have been seen, and some patho-VOL. 173,1991 genic mycoplasmas have been shown to lose antigenic, cytadsorption, and other properties upon passage in broth (8,22). Thus, a cell culture system that is between in vivo systems, with their associated complexities, and axenic systems is ideal as long as one can overcome the problems of host nuclear and mtDNA contamination of the myDNA under study.…”

Section: Discussionmentioning

confidence: 99%

Analysis of Mycoplasma hyorhinis DNA in the presence of host cells without growing the mycoplasma axenically

et al. 1991

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Mycoplasma hyorhinis coisolates with the mitochondria of the cells in which it is carried as an infection. Since both mitochondria and mycoplasmas synthesize DNA by using the prokaryotic DNA polymerase gamma, the use of aphidicolln, which inhibits eukaryotic DNA polymerase alpha, allows for selective synthesis of mycoplasmal and mitochondrial DNA. The restriction patterns of mitochondria and mycoplasmas can easily be differentiated from each other in mixtures of both DNAs. Thus, it is possible to study the molecular biology of this noncultivable mycoplasma in situ rather than after growth in artificial media, with its potential genetic consequences during adjustment to axenic growth.

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Intraspecies Genetic Relatedness among Strains of Acholeplasma laidlawii and of Acholeplasma axanthum by Nucleic Acid Hybridization

Cited by 14 publications

References 22 publications

Evaluation of tRNA Gene PCR for Identification of Mollicutes

Evaluation of tRNA Gene PCR for Identification of Mollicutes

Common Deoxyribonucleic Acid Sequences in Mycoplasma genitalium and Mycoplasma pneumoniae Genomes

Analysis of Mycoplasma hyorhinis DNA in the presence of host cells without growing the mycoplasma axenically

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