Phylogenetic relationships of three porcine mycoplasmas, Mycoplasma hyopneumoniae, Mycoplasma flocculare, and Mycoplasma hyorhinis, and complete 16S rRNA sequence of M. flocculare.

The nucleotide sequence of the 16S rRNA gene of Mycoplasma flocculare was determined and was compared with the sequence of a related porcine mycoplasma, Mycoplasma hyopneumoniae. While the overall level of DNA-DNA homology was approximately 11%, sequence alignment of the two 16S rRNA genes yielded a homology value of more than 95%, emphasizing the highly conserved nature of the 16S rRNA gene. Multiple sequence alignments with other mollicutes indicated that M. flocculare, M. hyopneumoniae, and Mycoplasma hyorhinis form a subcluster within the fermentans phylogroup, and this subcluster is distinct from the Mycoplasma pneumoniae phylogroup. Thus, the three mycoplasmas isolated from porcine respiratory systems exhibit phylogenetic similarities.     

Novel arrangement of rRNA genes in Mycoplasma gallisepticum: separation of the 16S gene of one set from the 23S and 5S genes.

Large restriction fragments from the DNA of Mycoplasma gallisepticum S6 and PG31, which were prepared by digestion with BglI, BssHII, SmaI, or XhoI and which were separated by pulsed-field electrophoresis, were hybridized with probes containing most, or different parts, of an rRNA operon of Mycoplasma capricolum. The results showed that the genomes contained three widely separated rRNA loci. One locus contained genes for all three rRNA species and another contained 23S and probably 5S rRNA genes, whereas the third appeared to have only a 16S rRNA gene.     

Genetic relationships among mycoplasmas based on the 16S-23S rRNA spacer sequence

The nucleotide sequences of the spacer regions between the 16S and 23S rRNA genes of 20 Mycoplasma species were determined following amplification by PCR. Although the spacer regions lacked spacer tRNA genes, they contained the box B and box A sequences in this order from the 5' terminus. The sequence alignment indicated that the 20 species were divided into four clusters, the M. pneumoniae, M. hominis, M. hyorhinis and M. fermentans clusters, and a single floating species, M. hyopneumoniae.     

The ribosomal genes of Mycoplasma capricolum

The nucleotide sequence of 5S rRNA from Mycoplasma capricolum is more similar to that of the gram-positive bacteria than that of the gram-negative bacteria. The presence of two copies of rRNA genes in M. capricolum genome has been demonstrated. The two different rRNA gene clusters have been cloned in E. coli plasmid vectors and analyzed for the rRNA gene organizations, demonstrating that the gene arrangement is in the order of 16S, 23S, and 5S rDNA. The ribosomes of M. capricolum contain about 30 species of proteins in 50S and 20 in 30S subunits. The number and size of the ribosomal proteins are not significantly different from those of other eubacterial ribosomes.     

Genome maps of Campylobacter jejuni and Campylobacter coli.

Little information concerning the genome of either Campylobacter jejuni or Campylobacter coli is available. Therefore, we constructed genomic maps of C. jejuni UA580 and C. coli UA417 by using pulsed-field gel electrophoresis. The genome sizes of C. jejuni and C. coli strains are approximately 1.7 Mb, as determined by SalI and SmaI digestion (N. Chang and D. E. Taylor, J. Bacteriol. 172:5211-5217, 1990). The genomes of both species are represented by single circular DNA molecules, and maps were constructed by partial restriction digestion and hybridization of DNA fragments extracted from low-melting-point agarose gels. Homologous DNA probes, encoding the flaAB and 16S rRNA genes, as well as heterologous DNA probes from Escherichia coli, Bacillus subtilis, and Haemophilus influenzae, were used to identify the locations of particular genes. C. jejuni and C. coli contain three copies of the 16S and 23S rRNA genes. However, they are not located together within an operon but show a distinct split in at least two of their three copies. The positions of various housekeeping genes in both C. jejuni UA580 and C. coli UA417 have been determined, and there appears to be some conservation of gene arrangement between the two species.     

Ribosomal RNA genes in Mycoplasma

Using Southern blotting analysis with labelled mycoplasmal ribosomal RNA as probe, two fragments (1 Kb and 5 Kb) were detected in an EcoR I digest of Mycoplasma capricolum DNA. This analysis revealed that the 5 Kb fragment carries both 16S rRNA sequences and the entire 23S rRNA gene of this mycoplasma. The 1 Kb fragment contains 16S rRNA sequences only. The 5 Kb EcoR I fragment has been cloned and used to characterize the structure of rRNA cistrons in various Mycoplasma strains. These experiments clearly demonstrate a substantial homology of Mycoplasma capricolum rRNA sequences with the E. coli rRNA cistron on one hand, and with Mycoplasma mycoides subsp. capri and Acholeplasma laidlawii on the other hand. This analysis also reveals two rRNA cistrons in Mycoplasma mycoides subsp. capri and Acholeplasma laidlawii whereas one rRNA cistron is present in Mycoplasma capricolum.     

The number of ribosomal RNA genes in Mycoplasma capricolum

Summary We have examined the number of rRNA genes in Mycoplasma capricolum (KID) by hybridization of BglII-, EcoRI- and XbaI-digests of DNA to [3-³²P] 16S, 23S and 5S rRNAs according to the Southern procedure (1975). All the restriction gels gave two radioactive bands with three kinds of rRNA. Furthermore, band positions were indistinguishable from one another when 16S, 23S and 5S rRNAs were used as probes, indicating that each band contains sequences corresponding to the 3-termini of 16S, 23S and 5S rRNAs. It is thus concluded that Mycoplasma capricolum chromosome carries at least two sets of genes for 16S, 23S and 5S rRNAs.     

Characterization of the rRNA genes of Ehrlichia chaffeensis and Anaplasma phagocytophila

The rRNA genes of Ehrlichia chaffeensis and Anaplasma phagocytophila have been analyzed. The 16S rRNA genes were previously characterized for both of these agents. Southern hybridization was used to show that there are single copies of both the 16S and 23S rRNA genes in the genomes of each organism, and that the 16S rRNA genes were upstream from the 23S rRNA genes by at least 16 and 11 Kb for E. chaffeensis and A. phagocytophila, respectively. PCR amplification and gene walking was used to sequence the 23S and 5S rRNA genes, and show that these genes are contiguous and are likely expressed as a single operon. The level of homology between the E. chaffeensis and A. phagocytophila 23S and 5S rRNA genes, and 23S-5S spacers, was 91.8, 81.5, and 40%, respectively. To confirm the hybridization data, genome walking was used to sequence downstream of the 16S rRNA genes, and although no tRNA genes were identified, open reading frames encoding homologues of the Escherichia coli succinate dehydrogenase, subunit C, were found in both E. chaffeensis and A. phagocytophila. Phylogenetic analysis using the 23S rRNA gene suggests that reorganization of the phylum Proteobacteria by division of the class Alphaproteobacteria into two separate subclasses, may be appropriate.     

Mycoplasmas (Mollicutes) have a low number of rRNA genes.

DNA from Mycoplasma, Ureaplasma, Acholeplasma, and Spiroplasma species digested by restriction endonucleases was hybridized with probes consisting of portions of the rrnB rRNA operon of Escherichia coli and the rRNA operon of Mycoplasma capricolum. The results indicate the presence of only one or two sets of rRNA genes in the genome of Mollicutes linked in the procaryotic fashion, 16S-23S-5S.     

Analysis of distribution indicates diverse functions of simple sequence repeats in Mycoplasma genomes

Simple sequence repeats (SSRs) composed of extensive tandem iterations of a single nucleotide or a short oligonucleotide are rare in most bacterial genomes, but they are common among Mycoplasma. Some of these repeats act as contingency loci in association with families of surface antigens. By contraction or expansion during replication, these SSRs increase genetic variance of the population and facilitate avoidance of the immune response of the host. Occurrence and distribution of SSRs are analyzed in complete genomes of 11 Mycoplasma and 3 related Mollicutes in order to gain insights into functional and evolutionary diversity of the SSRs in Mycoplasma. The results revealed an unexpected variety of SSRs with respect to their distribution and composition and suggest that it is unlikely that all SSRs function as contingency loci or recombination hot spots. Various types of SSRs are most abundant in Mycoplasma hyopneumoniae, whereas Mycoplasma penetrans, Mycoplasma mobile, and Mycoplasma synoviae do not contain unusually long SSRs. Mycoplasma hyopneumoniae and Mycoplasma pulmonis feature abundant short adenine and thymine runs periodically spaced at 11 and 12 bp, respectively, which likely affect the supercoiling propensities of the DNA molecule. Physiological roles of long adenine and thymine runs in M. hyopneumoniae appear independent of location upstream or downstream of genes, unlike contingency loci that are typically located in protein-coding regions or upstream regulatory regions. Comparisons among 3 M. hyopneumoniae strains suggest that the adenine and thymine runs are rarely involved in genome rearrangements. The results indicate that the SSRs in the Mycoplasma genomes play diverse roles, including modulating gene expression as contingency loci, facilitating genome rearrangements via recombination, affecting protein structure and possibly protein-protein interactions, and contributing to the organization of the DNA molecule in the cell.     

Detection and discrimination of Mycoplasma pneumoniae and Mycoplasma genitalium by the in vitro DNA amplification.

By using the primers designed on the bases of the sequences of the 16S rRNA genes of Mycoplasma pneumoniae and Mycoplasma genitalium, respectively, specific and sensitive in vitro DNA amplification assay system for the detection and discrimination of these two mycoplasmas was established. The detection limit of the assay was 100 cells for M. pneumoniae and 1,000 cells for M. genitalium. Neither other human mycoplasmas nor oral bacteria existing in human saliva showed any cross-reactions with these primers.     

Organization of the ribosomal RNA genes in Mycoplasma hyopneumoniae: the 5S rRNA gene is separated from the 16S and 23S rRNA genes

Summary In order to study the organization of the ribosomal RNA genes of Mycoplasma hyopneumoniae the rRNA genes were cloned in phage vectors EMBL3 and EMBL4. By subcloning the restriction fragments into various plasmids and analysing the resulting clones by Southern and Northern blot hybridization, a restriction map of the rRNA genes was generated and the organization of the rRNA genes was determined. The results show that the genes for the 16S and 23S rRNAs are closely spaced and occur only once in the genome, whereas the 5S rRNA gene is separated from the other two genes by more than 4 kb.     

Physical mapping of the ribosomal RNA genes of Mycoplasma capricolum.

Physical mapping of the rRNA genes of Mycoplasma capricolum was done by digestion of the mycoplasmal DNA with EcoRI, PstI and BglII and hybridization with nick-translated probes consisting of defined portions of the rrnB ribosomal RNA operon of Escherichia coli. The results indicate that the rRNA genes in the chromosome of M. capricolum are arranged in two clusters, each organized in the order 5'-16S-23S-5S-3', resembling the order of the genes in the rrnB operon, with no large spacer regions separating the genes in each cluster.     

Species-specific PCR for identification of common contaminant mollicutes in cell culture.

Mycoplasma arginini, M. fermentans, M. hyorhinis, M. orale, and Acholeplasma laidlawii are the members of the class Mollicutes most commonly found in contaminated cell cultures. Previous studies have shown that the published PCR primer pairs designed to detect mollicutes in cell cultures are not entirely specific. The 16S rRNA gene, the 16S-23S rRNA intergenic spacer region, and the 5' end of the 23S rRNA gene, as a whole, are promising targets for design of mollicute species-specific primer pairs. We analyzed the 16S rRNA genes, the 16S-23S rRNA intergenic spacer regions, and the 5' end of the 23S rRNA genes of these mollicutes and developed PCR methods for species identification based on these regions. Using high melting temperatures, we developed a rapid-cycle PCR for detection and identification of contaminant mollicutes. Previously published, putative mollicute-specific primers amplified DNA from 73 contaminated cell lines, but the presence of mollicutes was confirmed by species-specific PCR in only 60. Sequences of the remaining 13 amplicons were identified as those of gram-positive bacterial species. Species-specific PCR primers are needed to confirm the presence of mollicutes in specimens and for identification, if required.