We have characterized a family of repetitive DNA elements with homology to the MgPa cellular adhesion operon of Mycoplasma genitalium, a

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In order to develop a species-specific PCR for the detection of Mycoplasma genitalium, the sequence of 1,490 bases of the 16S rRNA gene was determined for M. genitalium G37 (type strain) and four Danish isolates of M. genitalium. The sequences of the four Danish strains, mutually different with respect to their MgPa gene, were 100% homologous, although they carried a single common base substitution compared to the type strain. Among members of the Mycoplasma pneumoniae phylogenetic cluster, M. genitalium showed the most-prominent homology to the 16S rRNA sequence of M. pneumoniae (98% homology). From regions showing the least homology to the M. pneumoniae 16S rRNA gene sequence, primers were chosen to amplify DNA from M. genitalium only. Two sets of primers were selected for their ability to detect <10 to 50 M. genitalium genome copies without cross-reactions with M. pneumoniae. The performance of these primers was compared to the performance of two pairs of primers amplifying parts of the MgPa adhesin gene; 1,030 randomly selected specimens submitted for Chlamydia trachomatis culture were screened with one of the 16S rRNA gene primer sets. A total of 41 specimens were found to be positive for this gene; 40 of these could be confirmed by one of the MgPa primer sets, whereas the other MgPa primer set detected only 21 positive specimens out of 40. These results indicate that estimates of the prevalence of M. genitalium in various populations using MgPa PCR primers could be incorrectly low if the PCR primers are located in variable regions of the MgPa gene.Two Mycoplasma genitalium strains were originally isolated in 1980 from the urogenital tracts of 2 of 13 men with nongonococcal urethritis (NGU) (27). Despite repeated attempts with conventional culture techniques, no other urogenital isolates have been reported (21, 24). Using a cell-culture-based method, however, we succeeded in isolating four new strains from the urethras of male patients with NGU who were PCR positive for M. genitalium (11). M. genitalium and Mycoplasma pneumoniae share several structural properties, such as the flask shape and the terminal tip-like structure, and a significant antigenic relationship between the two mycoplasma species has hampered diagnostic serology (15; K. Lind, Letter, Lancet ii:1158-1159, 1982).Because traditional diagnostic procedures for M. genitalium such as culture and serology have failed, other methods have been investigated. The development of a DNA probe provided some evidence for the presence of M. genitalium in the male urogenital tract (8), but data indicating that M. genitalium is a potential cause of NGU have only recently been demonstrated by the use of PCR (2, 9, 10, 13, 26).M. genitalium strains recently isolated from Danish patients (11) show a significant degree of sequence diversity of the main adhesin (MgPa) gene. This variability has not yet been sufficiently characterized to determine the presence of conserved regions in the gene so that PCR primers covering all M. genitalium strains can be designed....

The genes coding for outer surface protein OspC from 22 Borrelia burgdorferi strains isolated from patients with Lyme borreliosis were cloned and sequenced. For reference purposes, the 16S rRNA genes from 17 of these strains were sequenced after being cloned. The deduced OspC amino acid sequences were aligned with 12 published OspC sequences and revealed the presence of 48 conserved amino acids. On the basis of the alignment, OspC could be divided into an amino-terminal relatively conserved region and a relatively variable region in the central portion. The distance tree obtained divided the ospC sequences into three groups. The first group contained ospC alleles from all (n ‫؍‬ 13) sensu stricto strains, the second group contained ospC alleles from seven Borrelia afzelii strains, and the third group contained ospC alleles from five B. afzelii and all (n ‫؍‬ 9) Borrelia garinii strains. The ratio of the mean number of synonymous (d S ) and nonsynonymous (d N ) nucleotide substitutions per site calculated for B. burgdorferi sensu stricto, B. garinii, and B. afzelii ospC alleles suggested that the polymorphism of OspC is due to positive selection favoring diversity at the amino acid level in the relatively variable region. On the basis of the comparison of 16S rRNA gene sequences, Borrelia hermsii is more closely related to B. afzelii than to B. burgdorferi sensu stricto and B. garinii. In contrast, the phylogenetic tree obtained for the B. hermsii variable major protein, Vmp33, and 18 OspC amino acid sequences suggested that Vmp33 and OspC from B. burgdorferi sensu stricto strains share a common evolutionary origin.The outer membrane protein OspC is an immunodominant surface protein of the spirochete Borrelia burgdorferi, which causes Lyme borreliosis. The first detectable antibody response to B. burgdorferi consists of immunoglobulin M antibodies to OspC as well as to the 41-kDa flagellin (29). Thus, OspC, like the flagellum (9), may become an important diagnostic antigen.The OspC protein is also considered a vaccine candidate, since active immunization with recombinant OspC protected gerbils (22) and mice (23) against subsequent challenge with the homologous B. burgdorferi strain. Moreover, protection against B. burgdorferi challenge was observed in recent studies involving hamsters immunized with a mutant of strain 297 expressing ospC but lacking OspA and OspB (12). These findings strongly suggest that a Lyme borreliosis vaccine may benefit from inclusion of OspC in conjunction with other antigens.Significant heterogeneity exists in OspC at the immunological level (25,28) and at the genetic level (13,17,25). On the basis of the analysis of the nucleotide sequences from seven B. burgdorferi strains, we found that ospC sequences fall into three groups, each corresponding to one of the genospecies B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii (25).In order to further investigate the evolution of the ospC locus, ospC genes from 22 B. burgdorferi strains were cloned and sequenced and compared...

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