Surface localized glyceraldehyde-3-phosphate dehydrogenase of Mycoplasma genitalium binds mucin

Mycoplasma genitalium is the smallest known self-replicating cell. It was first isolated from urethral specimens in individuals with non-gonococcal urethritis and, more recently, from respiratory and synovial sites. Our laboratory has been interested in defining the mechanisms by which M. genitalium adheres to and colonizes host cell surfaces. In order to determine potential targets of adherence, we examined the interaction of M. genitalium with a primary component of the mucosal epithelial lining, mucin (Mn). Three Mn-binding proteins (MnBPs) of M. genitalium were isolated by affinity chromatography. One of these proteins was identified by N-terminal sequencing as glyceraldehyde-3-phosphate dehy-drogenase (GAPDH). Antiserum raised against recombinant GAPDH blocked binding of intact biosynthetically labelled mycoplasmas to mucin by approximately 70%. Whole cell radioimmunoprecipitation indicated that GAPDH was surface-accessible and surface localization of GAPDH was further verified by membrane fractionation and immunoelectron microscopy. The role of GAPDH as an adhesin to Mn not only provides insights into the organism's mechanisms of adherence and colonization but also into its ability to maximize its limited genome.     

Identification of a ribonuclease H gene in both Mycoplasma genitalium and Mycoplasma pneumoniae by a new method for exhaustive identification of ORFs in the complete genome sequences

Exhaustive identification of open reading frames in complete genome sequences is a difficult task. It is possible that important genes are missed. In our efforts to reanalyze the intergenic regions of Mycoplasma genitalium and Mycoplasma pneumoniae, we have newly identified a number of new open reading frames (ORFs) in both M. genitalium and M. pneumoniae. The most significant identification was that of a ribonuclease H enzyme in both species which until now has not been identified or assumed absent and interpreted as such. In this paper we discuss the biological importance of RNase H and its evolutionary implication. We also stress the usefulness of our method for identifying new ORFs by reanalyzing intergenic regions of existing ORFs in complete genome sequences.     

Mycoplasma genitalium: an emerging cause of sexually transmitted disease in women

Mycoplasma genitalium is an emerging sexually transmitted pathogen implicated in urethritis in men and several inflammatory reproductive tract syndromes in women including cervicitis, pelvic inflammatory disease (PID), and infertility. This comprehensive review critically examines epidemiologic studies of M. genitalium infections in women with the goal of assessing the associations with reproductive tract disease and enhancing awareness of this emerging pathogen. Over 27,000 women from 48 published reports have been screened for M. genitalium urogenital infection in high- or low-risk populations worldwide with an overall prevalence of 7.3% and 2.0%, respectively. M. genitalium was present in the general population at rates between those of Chlamydia trachomatis and Neisseria gonorrhoeae. Considering more than 20 studies of lower tract inflammation, M. genitalium has been positively associated with urethritis, vaginal discharge, and microscopic signs of cervicitis and/or mucopurulent cervical discharge in seven of 14 studies. A consistent case definition of cervicitis is lacking and will be required for comprehensive understanding of these associations. Importantly, evidence for M. genitalium PID and infertility are quite convincing and indicate that a significant proportion of upper tract inflammation may be attributed to this elusive pathogen. Collectively, M. genitalium is highly prevalent in high- and low-risk populations, and should be considered an etiologic agent of select reproductive tract disease syndromes in women.     

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.     

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.     

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.     

Genetic variation in the complete MgPa operon and its repetitive chromosomal elements in clinical strains of Mycoplasma genitalium

Mycoplasma genitalium has been increasingly recognized as an important microbe not only because of its significant association with human genital tract diseases but also because of its utility as a model for studying the minimum set of genes necessary to sustain life. Despite its small genome, 4.7% of the total genome sequence is devoted to making the MgPa adhesin operon and its nine chromosomal repetitive elements (termed MgPars). The MgPa operon, along with 9 MgPars, is believed to play an important role in pathogenesis of M. genitalium infection and has also served as the main target for development of diagnostic tools. However, genetic variation in the complete MgPa operon and MgPars among clinical strains of M. genitalium has not been addressed. In this study we examined the genetic variation in the complete MgPa operon (approximately 8.5 kb) and full or partial MgPar sequences (0.4-2.6 kb) in 15 geographically diverse strains of M. genitalium. Extensive variation was present in four repeat regions of the MgPa operon (with homology to MgPars) among and within strains while the non-repeat regions (without homology to MgPars) showed low-level variation among strains and no variation within strains. MgPars showed significant variation among strains but were highly homogeneous within strains, supporting gene conversion as the likely recombination mechanism. When applying our sequence data to evaluate published MgPa operon-based diagnostic PCR assays and genotyping systems, we found that 11 of 19 primers contain up to 19 variable nucleotides and that the target for one of two typing systems is located in a hypervariable repeat region, suggesting the likelihood of false results with some of these assays. This study not only provides new insights into the role of the MgPa operon in the pathogenesis of M. genitalium infection but has important implications for the development of diagnostic tools.     

The minimal genome paradox

The concept of a 'minimal genome' has appeared as an attempt to answer the question what the minimum number of genes or minimum amount of DNA to support life is. Since bacteria are cells bearing the smallest genomes, it has been generally accepted that the minimal genome must belong to a bacterial species. Currently the most popular chromosome in studies on a minimal genome belongs to Mycoplasma genitalium, a parasite bacterium whose total genetic material is as small as approximately 580 kb. However, the problem is how we define life, and thus also a minimal genome. M. genitalium is a parasite and requires substances provided by its host. Therefore, if a genome of a parasite can be considered as a minimal genome, why not to consider genomes of bacteriophages? Going further, bacterial plasmids could be considered as minimal genomes. The smallest known DNA region playing the function of the origin of replication, which is sufficient for plasmid survival in natural habitats, is as short as 32 base pairs. However, such a small DNA molecule could not form a circular form and be replicated by cellular enzymes. These facts may lead to an ostensibly paradoxical conclusion that the size of a minimal genome is restricted by the physical size of a DNA molecule able to replicate rather, than by the amount of genetic information.     

Identification of a small RNA within the pdh gene cluster of Mycoplasma pneumoniae and Mycoplasma genitalium

A highly abundant and heterogeneous small RNA about 205 to 210 bases long named MP200 RNA has been identified in Mycoplasma pneumoniae. It was localized on the genome within a 319-bp-long intergenic space of the pyruvate dehydrogenase (pdh) gene cluster. A database search at the DNA level revealed the highest similarity to a sequence located within the pdh gene cluster of Mycoplasma genitalium that was also shown to be transcribed into two abundant, but smaller RNAs than the ones in Mycoplasma pneumoniae. The RNAs from both M. pneumoniae and M. genitalium have the potential to code for cysteine-rich 29- and 23-amino-acid-long peptides, but so far, these peptides have not been identified experimentally in bacterial protein extracts.     

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 novel translation initiation region from Mycoplasma genitalium that functions in Escherichia coli.

The tuf gene of Mycoplasma genitalium uses a signal other than a Shine-Dalgarno sequence to promote translation initiation. We have inserted the translation initiation region of this gene in front of the Escherichia coli lacZ gene and shown that it is recognized by the translational machinery of E. coli; the signal operates in vivo at roughly the same efficiency as a synthetic Shine-Dalgarno sequence. The M. genitalium sequence was also used to replace the native translation initiation region of the cat gene. When assayed in E. coli, the M. genitalium sequence is equivalent to a Shine-Dalgarno sequence in stimulating translation of this mRNA also. Site-directed mutagenesis enabled us to identify some of the bases that comprise the functional sequence. We propose that the sequence UUAACAACAU functions as a ribosome binding site by annealing to nucleotides 1082-1093 of the E. coli 16S rRNA. The activity of this sequence is enhanced when it is present in the loop of a stem-and-loop structure. Additional sequences both upstream and downstream of the initiation codon are also involved, but their role has not been elucidated.     

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.     

生殖支原体脂质相关膜蛋白激活核因子κB诱导人单核细胞表达前炎症细胞因子及凋亡

为了解生殖支原体(Mg)潜在的致病性及其脂质相关膜蛋白(LAMPs)诱导人单核细胞(THP-1)凋亡及表达前炎症细胞因子(CKs)的分子机制,用Mg提取的LAMPs刺激THP-1细胞,以ELISA法和RT-PCR方法分析CKs产生和其mRNA的表达。不同试实验组的细胞经AnnexinV联合PI染色后通过流式细胞仪检测细胞凋亡。采用EMSA方法检测LAMPs处理的THP-1细胞中核转录因子kappaB(NF-κB)的激活,并分析NF-κB抑制剂二硫代氨基甲酸吡咯烷(pyrrolidine dithiocoarbamate,PDTC)对LAMPs处理的THP-1细胞产生CKs的量和其mRNA表达及细胞凋亡的影响。LAMPs能以时间和剂量依赖方式刺激THP-1细胞产生TNF-α、IL-1β和IL-6,且能激活NF-κB诱导THP-1细胞表达CKs的mRNA及发生凋亡,PDTC能显著抑制CKs的mRNA表达水平和细胞凋亡。由于LAMPs能激活NF-κB诱导THP-1细胞表达CKs及产生细胞凋亡,因而可能是一个重要的致病因素。     

A random sequencing approach for placing markers on the physical map of Mycoplasma genitalium.

A physical map of the Mycoplasma genitalium genome has been prepared using pulsed-field gel electrophoresis. This report details recent efforts made to add markers or specific loci to this map in the absence of any mutants or system of genetic exchange. A total of 44 random clones were partially sequenced. Computer analysis was performed in an attempt to identify homologies with genes already recorded in the DNA sequence database. Clones with a large extent of homology to genes from other microorganisms have been assigned to specific loci on the M. genitalium map by hybridization to selected restriction digests. The additional data has facilitated an updated version of the physical map, and verified this random sequencing method as a useful mapping procedure as well as offering new insight into the physiological processes of this fastidious organism.     

Molecular characterization of the P1-like adhesin gene from Mycoplasma pirum.

A DNA fragment has been isolated from the genome of Mycoplasma pirum by use of a genetic probe derived from the conserved region within the genes for the major adhesins of Mycoplasma genitalium and Mycoplasma pneumoniae. A gene encoding an adhesin-like polypeptide was localized, and sequence analysis indicated a G + C content of only 28%, with A- and T-rich codons being preferentially used. A total of 91% of positions 3 were either A or T. The deduced polypeptide is 1,144 amino acids long (126 kDa) and shows 26% identity with the adhesins of M. genitalium and M. pneumoniae. Other features in common with these two membrane proteins include a similar hydropathic profile and a proline-rich C terminus. Antibodies were prepared by using as an immunogen a peptide derived from the C terminus of the M. pirum adhesin-like polypeptide and were found to recognize on immunoblots a 126-kDa polypeptide from an M. pirum cellular extract. The characterization of the adhesin-like gene is a first step toward a better understanding of the mechanisms allowing this human mycoplasma to attach to host cells.