Partial Nucleotide Sequence Similarity within Species of Mycoplasma and Acholeplasma

Abstract

333The nucleotide sequence relationships between 18 species of Mycoplasma and 3 species of Acholeplasma were examined by solution DNA hybridization. Radiolabelled DNAs from strains representing 13 Mycoplasma and 2 Acholeplasma species were used as hybridization probes, The mycoplasmas were heterogeneous but displayed a low degree of conserved information of the order of 3 to 5 % of the genome. However, several species within each genus showed 13 to 15 % homology. There was no detectable homology between species from the two genera, and M . pneumoniae and M . neurolyticum appeared to be unrelated to any of the other Mycoplasma species or to each other. These results suggest that it may be possible to isolate genes common to most, if not all, Mycoplasma and Acholeplasma species. I N T R O D U C T I O NThe use of nucleotide sequence homology as a taxonomic tool for bacteria and viruses is accepted practice. Likewise, measurement of nucleic acid hybridization kinetics has become a widely employed technique for detecting viruses in infected and transformed cells (Sharp et al., 1974). Before this technique can be extended to the detection of fastidious microorganisms in tissue a good understanding of the nucleotide sequence relationships within the microbial group is necessary. Suitable micro-organisms for this type of study are the mycoplasmas, especially those closely cell-associated. Although the relationships between various mycoplasmas have been reported (Reich et al., 1966a, b ;Somerson et al., 1967;McGee et al., 1967), the work was not exhaustive and utilized either RNA-DNA hybridization or DNA-DNA hybridization in agar columns, which have limits of accuracy that impede the interpretation of low levels of hybridization.The present study utilized the more quantitative solution hybridization procedure followed by analysis of hybrid formation on hydroxyapatite columns (Britten & Kohne, 1966) in an attempt to provide a base of relationship data upon which to build a molecular detection system. In addition, this study utilized a large number of reciprocal hybridizations with 1251-labelled DNA allowing grouping of the micro-organisms into classes based on low degrees of homology.This study demonstrates that there is little homology among different Mycoplasma and Acholeplasma species. However, there is a partial conservation of nucleotide sequences between several different species that are found in the same or similar hosts. M E T H O D SGrowth of mycoplasmas andpurifcation of DNA. with 10 % (v/v) whole horse serum (Irvine Scientific, Santa Ana, Calif., U.S.A.), 0.5 % (w/v) yeast extract and 100 units penicillin ml-l. Confirmation of growth and the absence of contamination were checked by plating 0.1 ml of the broth culture on PPLO agar supplemented as above. Before harvesting, cultures wereGram-stained to check for contamination. Uncontaminated cultures were centrifuged (40 min, 33 OO0 g) and the pellet was resuspended in 0.15 M-NaCl (1Oml) with sodium dodecyl sulphate (SDS, 1 %, w/v, h a 1 concn), 1 m-EDTA and 100...

Development of a DNA Hybridization Probe for Detection of Mycoplasma Bovis

“…DNA probes for many species of mycoplasmas have been developed. 5,8,12,15,16 Recently, the detection of M. bovis using an oligonucleotide probe was reported. 10 This article describes the development of a genomic DNA probe for detection of M. bovis.…”

“…The 1,150-bp fragment of clone 9-45 was chosen as the most acceptable DNA hybridization probe for M. bovis because of its failure to cross-hybridize with any of the bacteria 16 so it should be possible to find a sequence of suitable size for use as a hybridization probe. The previously reported M. bovis oligonucleotide probe was derived from partial sequences of 16S rRNA and showed some cross-hybridization with M. agalactiae but not with any of the bovine mycoplasmas, including M. arginini.…”

Genomic DNA Analysis of Rodent Mycoplasmas

“…To assess genetic relatedness among prokaryotes, including mycoplasmas, DNA-DNA homology studies have generally been used [3,9,10] . In recent years, with the spread of restriction endonucleases that recognize specific nucleotide sequences, the comparison of DNA cleavage patterns by these enzymes has proved to be a simple, useful method., DNA analysis based on restriction endonuclease cleavage pattern is highly appropriate for mycoplasmas, which have a very small genome, ca.…”