Mycoplasma genitalium: an efficient strategy to generate genetic variation from a minimal genome

Mycoplasma genitalium, a human pathogen associated with sexually transmitted diseases, is unique in that it has smallest genome of any known free-living organism. The goal of this study was to investigate if and how M. genitalium uses a minimal genome to generate genetic variations. We analysed the sequence variability of the third gene (MG192 or mgpC) of the M. genitalium MgPa adhesion operon, demonstrated that the MG192 gene is highly variable among and within M. genitalium strains in vitro and in vivo, and identified MG192 sequence shifts in the course of in vitro passage of the G37 type strain and in sequential specimens from an M. genitalium-infected patient. In order to establish the origin of the MG192 variants, we examined nine genomic loci containing partial copies of the MgPa operon, known as MgPar sequences. Our analysis suggests that the MG192 sequence variation is achieved by recombination between the MG192 expression site and MgPar sequences via gene cross-over and, possibly, also by gene conversion. It appears plausible that M. genitalium has the ability to generate unlimited variants from its minimized genome, which presumably allows the organism to adapt to diverse environments and/or to evade host defences by antigenic variation.     

Mycoplasma genitalium P140 and P110 cytadhesins are reciprocally stabilized and required for cell adhesion and terminal-organelle development

Mycoplasma genitalium is a human pathogen that mediates cell adhesion by a complex structure known as the attachment organelle. This structure is composed of cytadhesins and cytadherence-associated proteins, but few data are available about the specific role of these proteins in M. genitalium cytadherence. We have deleted by homologous recombination the mg191 and mg192 genes from the MgPa operon encoding the P140 and P110 cytadhesins. Molecular characterization of these mutants has revealed a reciprocal posttranslational stabilization between the two proteins. Loss of either P140 or P110 yields a hemadsorption-negative phenotype and correlates with decreased or increased levels of cytoskeleton-related proteins MG386 and DnaK, respectively. Scanning electron microscopy analysis reveals the absolute requirement of P140 and P110 for the proper development of the attachment organelle. The phenotype described for these mutants resembles that of the spontaneous class I and class II cytadherence-negative mutants [G. R. Mernaugh, S. F. Dallo, S. C. Holt, and J. B. Baseman, Clin. Infect. Dis. 17(Suppl. 1):S69-S78, 1993], whose genetic basis remained undetermined until now. Complementation assays and sequencing analysis demonstrate that class I and class II mutants are the consequence of large deletions affecting the mg192 and mg191-mg192 genes, respectively. These deletions originated from single-recombination events involving sequences of the MgPa operon and the MgPa island located immediately downstream. We also demonstrate the translocation of MgPa sequences to a particular MgPa island by double-crossover events. Based on these observations, we propose that in addition to being a source of antigenic variation, MgPa islands could be also involved in a general phase variation mechanism switching on and off, in a reversible or irreversible way, the adhesion properties of M. genitalium.     

mgpB and mgpC sequence diversity in Mycoplasma genitalium is generated by segmental reciprocal recombination with repetitive chromosomal sequences

Mycoplasma genitalium is associated with sexually transmitted infections in men and women that, if untreated, can persist, suggesting that mechanism(s) exist to facilitate immune evasion. Approximately 4% of the limited M. genitalium genome contains repeat sequences termed MgPar regions that have homology to mgpB and mgpC, which encode antigenic proteins associated with attachment. We have previously shown that mgpB sequences vary within a single strain of M. genitalium in a pattern consistent with recombination between mgpB and MgPar sequences (Iverson-Cabral et al.). In the current study, we show that mgpC heterogeneity similarly occurs within the type strain, G-37(T), cultured in vitro and among cervical specimens collected from a persistently infected woman. In all cases, alternative mgpC sequences are indicative of recombination with MgPar regions. Additionally, the isolation of single-colony M. genitalium clonal variants containing alternative mgpB or mgpC sequences allowed us to demonstrate that mgpB and mgpC heterogeneity is associated with corresponding changes within donor MgPar regions, consistent with reciprocal recombination. Better-defined systems of antigenic variation are typically mediated by unidirectional gene conversion, so the generation of genetic diversity observed in M. genitalium by the mutual exchange of sequences makes this organism unique among bacterial pathogens.     

A survey of the Mycoplasma genitalium genome by using random sequencing.

A total of 508 random clones from five Mycoplasma genitalium genomic libraries were partially sequenced and analyzed. This resulted in the identification of 291 unique contigs. Sequence information from these clones (100,993 nucleotides), representing approximately 17% of this pathogen's genome, was analyzed by comparison to the DNA and protein sequence data bases. The frequency with which clones could be identified, by virtue of possessing homology to another data base entry, was 46%. Sequence analysis indicated the following. (i) The M. genitalium genome contains many genes involved in various metabolic processes. (ii) Repetitive DNA may comprise as much as 4% of this genome. (iii) The MgPa adhesin gene may be the result of horizontal transfer from an unknown origin. (iv) Not all dinucleotide pairs are present in this genome at the expected frequency. (v) This genome potentially encodes approximately 390 proteins and makes very efficient use of its limited amount of DNA. In addition, this study allowed us to estimate the number of genes involved with various cellular functions.     

Screening and identification of the mimic epitope of the adhesion protein of Mycoplasma genitalium

Mycoplasma genitalium adhesion protein (MgPa) is the major adhesion protein of M. genitalium, and its C-terminal domain (amino acid 1075-1444) is the most immunogenic region. However, the exact epitopes of the adhesion protein of M. genitalium are still unclear. We used the purified polyclonal antibody against the recombinant adhesion protein to screen the mimic epitopes of MgPa using a random 12-peptide phage display library. Immunoscreening via the phage display peptide library revealed that 3 motifs (P-S-A-A/V-X-R-F/W-E/S-L-S-P, A-K-I/L-T/Q-X-T-L-X-L, and K-S-L-S-R-X-D-X-I) may represent 3 different mimotopes of MgPa. Results of bioinformatics analysis by MIMOX demonstrated that the key consensus amino acid residues in the aligned mimotopes may be S, A, and F for cluster 1; A, K, I, T, and L for cluster 2; and K, S, L, R, D, and I for cluster 3. Three representative phages could recognize sera from M. genitalium-positive patients to varying degrees, whereas they could not recognize the sera from Mycoplasma pneumoniae -positive patients or the sera from healthy people. These findings will help to clarify the mimic epitopes of MgPa to facilitate diagnosis of the antigen and to understand the antigenic structure of MgPa.     

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.     

An unusual gene containing a dnaJ N-terminal box flanks the putative origin of replication of Mycoplasma genitalium.

Origins of replication are known to be highly conserved among widely divergent microbial species, with the gene order in those regions being dnaA-dnaN-recF-gyrB. On the basis of sequence identities to entries in GenBank, the gene order of a 6-kb fragment of Mycoplasma genitalium DNA was determined to be dnaN-orf311-gyrB-gyrA-serS, which is structurally similar to the ancestral origin of replication. We have directly linked the dnaN gene to the M. genitalium dnaA gene by PCR amplification. However, we found a novel open reading frame, designated orf311, in place of an expected sequence encoding recF. Orf311 contains a DnaJ box motif at its N terminus, but it has no overall homology to any other protein or sequence in the database. We are unable to detect any recF homolog in M. genitalium by hybridization or during a random sequencing survey of the genome.     

The tubulin genes of Trypanosoma cruzi

The organization of the alpha- and beta-tubulin genes in the genome of Trypanosoma cruzi have been analysed by Southern blotting using tubulin probes derived from Trypanosoma brucei. The tubulin array appears to be more complex in this organism than in other members of the same family. Some tubulin genes are tightly clustered in an alternating (alpha-beta)n array with a basic repeat unit length of 4.3 kb. However, other pairs of alternating alpha- and beta-tubulin sequences appear to be physically separated from the basic group. This finding indicates that the tubulin gene cluster present in T. cruzi is less perfectly conserved than in T. brucei. T. (Herpetosoma) rangeli is similar to T. (Schizotrypanum) cruzi in its tubulin gene organization whereas most of these genes are tandemly clustered in the genome of T. (Trypanozoon) evansi, with a basic repeat unit length of 3.6 kb as previously described for T. (Trypanozoon) brucei. Two overlapping recombinant clones containing T. cruzi tubulin sequences have been isolated from a genomic cosmid library of T. cruzi epimastigotes using the T. brucei tubulin probes. Partial sequencing of the T. cruzi beta-tubulin gene has confirmed its identity and shows more than 70% homology with the sea urchin, chicken and T. b. rhodesiense beta-tubulin reported gene sequences. Analysis of tubulin gene organization through the parasite life cycle does not show evidence of major rearrangements within the repeat unit. Several T. cruzi strains and cloned lines whilst sharing the 4.3-kb tubulin repeat unit, exhibited very variable tubulin gene organization with tubulin probes. These striking differences in the organization of this structural gene among T. cruzi strains and cloned lines suggest that the heterogeneity previously reported in parasite populations may be related to a very dynamic, diploid genome.     

Prevalence of novel repeat sequences in and around the P1 operon in the genome of Mycoplasma pneumoniae

The presence of numerous different repetitive elements in the genome of Mycoplasma pneumoniae has been documented by several laboratories. One which we previously identified, denoted as SDC1, has now been further characterized, verified to be distinct from those discussed in previous publications and shown to lack homology to several other species of Mycoplasma when tested under our stringency conditions. As many as eight versions of the SDC1-type repeat, which is more than 400 bp long, are scattered throughout the genome of M. pneumoniae. The prototype for SDC1 is found within a gene encoding a putative 130-kDa membrane-binding protein lying just downstream from the gene encoding the cytadhesin protein P1. In fact, all of the reported M. pneumoniae repetitive elements have at least one representative either within or adjacent to the P1 operon; many if not all of these lie within open reading frames. The function of these repetitive elements is still unclear.     

Strain variation in the katG region of Mycobacterium tuberculosis

Southern blot analysis of chromosomal DNA from clinical isolates of Mycobacterium tuberculosis using cosmid DNA probes revealed extensive strain variation in the katG region of the genome. In addition to deletion of the katG gene itself in some isoniazid-resistant strains, adjacent DNA fragments were missing or altered in a range of drug-sensitive and drug-resistant isolates. A species-specific 2 kb Kpnl fragment located 10 kb upstream of katG in M. tuberculosis H37Rv hybridized to fragments of differing size in different clinical isolates and was characterized in detail. Sequence analysis of this fragment showed that it comprised three tandem copies of a novel 75 bp repeat element flanked by multiple copies of the previously described 10 bp major polymorphic tandem repeat of M. tuberculosis (MPTR). The copy number of the 75 bp repeat was found to vary between strains, allowing application of a poly-merase chain reaction amplification strategy for strain differentiation. These results indicate that the katG region of the M. tuberculosis genome is highly variable and unstable. The presence of repetitive sequences may contribute to instability in this region of the genome.     

Hypervariable DNA fingerprinting in Escherichia coli: minisatellite probe from bacteriophage M13.

Extensive restriction-fragment-length polymorphism was revealed in Escherichia coli strains by using a region of the bacteriophage M13 genome as a DNA hybridization probe. This variation was observed across natural strains, in clinical samples, and to a lesser extent in laboratory strains. The sequence in M13 which revealed this fingerprint pattern was a region of the gene III coat protein, which contains two clusters of a 15-base-pair repeat. Oligonucleotides made to a consensus of these repeats also revealed the fingerprint profile. While this consensus sequence has significant homology to the lambda chi site sequence, an oligonucleotide made of the chi sequence did not reveal polymorphic fingerprint patterns in E. coli. The strain variation revealed by the M13 and M13-derived oligonucleotide probes will be useful for bacterial characterization and should find use in studies of bacterial evolution and population dynamics. The findings raise questions about what these repeated sequences are and why they are so variable.     

Homology modeling, comparative genomics and functional annotation of Mycoplasma genitalium hypothetical protein MG_237

Mycoplasma genitalium is a human pathogen associated with several sexually transmitted diseases. The complete genome of M. genitalium G37 has been sequenced and provides an opportunity to understand the pathogenesis and identification of therapeutic targets. However, complete understanding of bacterial function requires proper annotation of its proteins. The genome of M. genitalium consists of 475 proteins. Among these, 94 are without any known function and are described as 'hypothetical proteins'. We selected MG_237 for sequence and structural analysis using a bioinformatics approach. Primary and secondary structure analysis suggested that MG_237 is a hydrophilic protein containing a significant proportion of alpha helices, and subcellular localization predictions suggested it is a cytoplasmic protein. Homology modeling was used to define the three-dimensional (3D) structure of MG-237. A search for templates revealed that MG_237 shares 63% homology to a hypothetical protein of Mycoplasma pneumoniae, indicating this protein is evolutionary conserved. The refined 3D model was generated using (PS)(2)-v2 sever that incorporates MODELLER. Several quality assessment and validation parameters were computed and indicated that the homology model is reliable. Furthermore, comparative genomics analysis suggested MG_237 as non-homologous protein and involved in four different metabolic pathways. Experimental validation will provide more insight into the actual function of this protein in microbial pathways.     

Molecular comparisons of plasmids isolated from colicinogenic strains of Escherichia coli

The plasmid content of 14 colicinogenic strains of Escherichia coli has been examined. Four strains contained miniplasmids (1.2-2.0 kb). Small plasmids (4-7 kb) were detected in all the strains specifying group A colicins (colicins A, E1, E2, E3 and K; group I plasmids). Larger plasmids (55-130 kb) were detected in seven out of nine strains specifying group B colicins (colicins B, H, Ia, Ib, M, V and S1; group II plasmids). DNA-DNA hybridization with group II plasmids showed a wide variation in the degree of DNA sequence homology among its members. In contrast little (if any) DNA sequence homology was detected with the group I plasmids when the same group II plasmid DNAs were used as hybridization probes. The results of DNA-DNA hybridization studies with the two small group II plasmids (pcolG-CA46 and pcolD-CA23) suggested that these plasmids are equivalent to deleted forms of larger group II plasmids. Our hybridization data thus support the division of colicin plasmids into the two groups (I and II) that have been previously defined from genetic and physiological studies.     

Sequence rearrangement in the AT-rich minisatellite of the novel rice transposable element Basho.

In the process of characterizing a rice wx deletion mutant, an AT-rich minisatellite sequence that consisted of units of approximately 80 bp was detected about 2.3 kb downstream of the wx gene. This AT-rich minisatellite was a multiple-copy element (1 x 10(3) to 2 x 10(3) copies per haploid genome) and interspersed in the rice genome. By BLAST homology search it was indicated that not only the tandem repeat but also both flanking sequences were conserved among copies. According to the characteristics of the termini (5'-CHH ... CTAG-3') and a target site preference for T, this AT-rich minisatellite accompanying the flanking sequences was classified into a novel transposon, Basho. The results of direct amplification of Basho showed that relatively large variation in size existed in the Basho family. We estimate the variation to be generated by not only alteration of the number of units in the minisatellite but also by duplications of larger blocks including the conserved flanking sequences caused by single-strand mispairing (SSM) at noncontiguous repeats. Because the AT-rich minisatellite contained in Basho possessed several motifs of the matrix attachment region (MAR) in its repeat unit, the functional role as MAR in the rice genome was discussed.     

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.