Genitalium Genome Research Articles

BigDNA: Primer Design Software for Overlap-Based Assembly of Phage Genomes and Larger DNAs.

Gibson assembly and assembly-in-yeast are strategies to create long synthetic DNAs from diverse fragments, for example, when engineering bacteriophage genomes. Design for these methods requires terminal sequence overlaps in the fragments, determining the order of assembly. Design to rebuild a genomic fragment that is too long for a single PCR presents a puzzle since some candidate joint regions cannot yield satisfactory primers for the overlap. No existing overlap assembly design software is open-source, and none explicitly supports rebuilding. We describe here bigDNA software that solves the rebuilding puzzle by recursive backtracking, with options to remove or introduce genes; it also tests for mispriming on the template DNA. BigDNA was tested with 3082 prophages and other genomic islands (GIs), from 20 to 100 kb, and the synthetic Mycoplasma genitalium genome. Rebuilding assembly design succeeded for all but ∼1% of GIs. BigDNA will speed and standardize assembly design.

Alternative conformation of the C-domain of the P140 protein from Mycoplasma genitalium.

The human pathogen Mycoplasma genitalium is responsible for urethritis in men, and for cervicitis and pelvic inflammatory disease in women. The adherence of M.genitalium to host target epithelial cells is mediated through an adhesion complex called Nap, which is essential for infectivity. Nap is a transmembrane dimer of heterodimers of the immunodominant proteins P110 and P140. The M.genitalium genome contains multiple copies of portions that share homology with the extracellular regions of P140 and P110 encoded by the genes mg191 and mg192, respectively. Homologous recombination between the genes and the copies allows the generation of a large diversity of P140 and P110 variants to overcome surveillance by the host immune system. Interestingly, the C-terminal domain (C-domain) of the extracellular region of P140, which is essential for thefunction of Nap by acting as a flexible stalk anchoring the protein to the mycoplasma membrane, presents a low degree of sequence variability. In the present work, the X-ray crystal structures of two crystal forms of a construct of the P140 C-domain are reported. In both crystal forms, the construct forms a compact octamer with D4 point-group symmetry. The structure of the C-domain determined in this work presents significant differences with respect to the structure of the C-domain found recently in intact P140. The structural plasticity of the C-domain appears to be a possible mechanism that may help in the functioning of the mycoplasma adhesion complex.

Subtractive proteomics and systems biology analysis revealed novel drug targets in Mycoplasma genitalium strain G37

Mycoplasma genitalium is one of the sexually transmitted pathogens that cause significant morbidity in the host. The development of effective therapeutic procedures is urgently needed to counter the multi-drug resistant events imposed by this pathogen. In the current version of M. genitalium G37 genome, 512 open reading frames have been identified. The function of 91 proteins encoded by M. genitalium genes was found to be hypothetical and these proteins were termed hypothetical proteins (HPs). This study aims to carry out functional characterization of HPs by a systems biology approach. Functional assignments of 61 HPs were made with high confidence. They belong to different functional groups, such as DNA-binding proteins, helicases and transporters. Approximately 26% of HPs were identified as transporters, suggesting that M. genitalium is likely to rely on the exogenous nutrient supply for survival. A group of 20 proteins was predicted to be virulence factors, indicating the pathogenic characteristics of M. genitalium. Among the coding proteins, six proteins were pathogen-specific and could serve as potential drug targets by subtractive proteomics analysis. Network analysis of the HPs suggested that several critical proteins were involved in SOS response and stringent response in M. genitalium. These findings provided a better picture of M. genitalium genome and novel clues for studying the potential infection mechanism in this bacterium.

Loop-mediated isothermal amplification (LAMP) for the rapid detection of Mycoplasma genitalium

Mycoplasma genitalium is a sexually transmissible, pathogenic bacterium and a significant cause of nongonococcal urethritis in both men and women. Due to the difficulty of the culture of M. genitalium from clinical samples, the laboratory diagnosis of M. genitalium infection is almost exclusively carried out using nucleic acid amplification tests. Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification technology, utilising a set of 4 primers specific to 6 distinct regions of the target DNA sequence, in order to amplify target DNA in a highly specific and rapid manner. A LAMP assay was designed to the pdhD gene of M. genitalium, and the limit of detection of the assay was determined as 10fg of M. genitalium genomic DNA, equating to ~16 copies of the M. genitalium genome, which was equally sensitive as a gold standard 16S rRNA polymerase chain reaction assay.

Venter's Build-a-Bug Workshop

News reports on May 20, 2010, heralded a new era in scientific research, as well as a new way of thinking about the nature of life. Craig Venter and his team had created a cell controlled by an entirely synthetic genome. So, were the sensational headlines warranted? And just how much of an advance is the latest report from the Venter Institute? Craig Venter (Chairman and President of the J. Craig Venter Institute, Rockville, Md) always seems to be doing something scientifically brilliant and yet controversial. In the 1990s, he was sequencing the human genome through his then company, Celera, with the aim of owning the information and charging researchers for access. Then, in 2007, he sequenced the first complete diploid human genome of one individual—himself. An impressive, if some might say self-centered, feat. His new article,1 reporting the first man-made cell, continues the brilliant-yet-controversial theme, with some claiming that Venter is now “playing God.” The motivation behind Venter's latest work comes from his longstanding interest in deciphering the minimal genetic instructions required for cellular life. This interest was born back in the 1990s, and as a first step toward his goal, Venter chose to study a bacterium thought to have one of the smallest genomes of any replicating cell— Mycoplasma genitalium . Through extensive mutational analysis of the microbe, Venter and his team predicted that of its 480 protein-coding genes, only 300 or so were essential.2 Armed with this knowledge, Venter envisioned that he might be able to build his own minimal microbial genome from scratch. To show that such a synthetic genome is fully …

Tandem repeat coupled with endonuclease cleavage (TREC): a seamless modification tool for genome engineering in yeast

The complete synthetic Mycoplasma genitalium genome (∼583 kb) has been assembled and cloned as a circular plasmid in the yeast Saccharomyces cerevisiae. Attempts to engineer the cloned genome by standard genetic methods involving the URA3/5-fluoroorotic acid (5-FOA) counter-selection have shown a high background of 5-FOA resistant clones derived from spontaneous deletions of the bacterial genome maintained in yeast. Here, we report a method that can seamlessly modify the bacterial genome in yeast with high efficiency. This method requires two sequential homologous recombination events. First, the target region is replaced with a mutagenesis cassette that consists of a knock-out CORE (an18-bp I-SceI recognition site, the SCEI gene under the control of the GAL1 promoter, and the URA3 marker) and a DNA fragment homologous to the sequence upstream of the target site. The replacement generates tandem repeat sequences flanking the CORE. Second, galactose induces the expression of I-SceI, which generates a double-strand break (DSB) at the recognition site. This DSB promotes intra-molecular homologous recombination between the repeat sequences, and leads to an excision of the CORE. As a result, a seamless modification is generated. This method can be adapted for a variety of genomic modifications and may provide an important tool to modify and design natural or synthetic genomes propagated in yeast.

The dawn of synthetic genomics.

The first month of 2008 was unusually quiet in terms of microbial genome sequencing. Still, even the relatively short list of newly released genomes includes several interesting environmental microorganisms, such as the anoxygenic phototroph Chloroflexus aurantiacus, the toxic bloom-forming cyanobacterium Microcystis aeruginosa, and the methylotroph Methylobacterium extorquens (Table 1). However, arguably the biggest news was the announcement by J. Craig Venter and colleagues that they ‘have synthesized a 582 970 bp Mycoplasma genitalium genome’ (Gibson et al., 2008). The authors used chemically synthesized oligonucleotides ∼50 nucleotides in length to assemble ‘cassettes’ 5–7 kb in length, then to join them by in vitro recombination to produce intermediate assemblies, gradually increasing in size. Finally, four 144 kb pieces were cloned in Escherichia coli as bacterial artificial chromosomes, transferred into yeast and assembled into a full-length genome. The genome of the resulting strain, named M. genitalium JCVI-1.0, was virtually identical to the genome of the original strain M. genitalium G37. It was not immediately clear whether this technically very challenging and truly monumental work had any purpose beyond just serving as a proof of principle. However, J. C. Venter and colleagues have a record of overcoming enormous technical challenges and launching entirely new areas of biotechnology. It might be simply too early right now to ask them to explain the future of this work. In any case, the era of synthetic biology has officially begun and who knows what kind of molecules people will be synthesizing 10 or 20 years from now.

Short tandem repeat sequences in the Mycoplasma genitalium genome and their use in a multilocus genotyping system

BackgroundSeveral methods have been reported for strain typing of Mycoplasma genitalium. The value of these methods has never been comparatively assessed. The aims of this study were: 1) to identify new potential genetic markers based on an analysis of short tandem repeat (STR) sequences in the published M. genitalium genome sequence; 2) to apply previously and newly identified markers to a panel of clinical strains in order to determine the optimal combination for an efficient multi-locus genotyping system; 3) to further confirm sexual transmission of M. genitalium using the newly developed system.ResultsWe performed a comprehensive analysis of STRs in the genome of the M. genitalium type strain G37 and identified 18 loci containing STRs. In addition to one previously studied locus, MG309, we chose two others, MG307 and MG338, for further study. Based on an analysis of 74 unrelated patient specimens from New Orleans and Scandinavia, the discriminatory indices (DIs) for these three markers were 0.9153, 0.7381 and 0.8730, respectively. Two other previously described markers, including single nucleotide polymorphisms (SNPs) in the rRNA genes (rRNA-SNPs) and SNPs in the MG191 gene (MG191-SNPs) were found to have DIs of 0.5820 and 0.9392, respectively. A combination of MG309-STRs and MG191-SNPs yielded almost perfect discrimination (DI = 0.9894). An additional finding was that the rRNA-SNPs distribution pattern differed significantly between Scandinavia and New Orleans. Finally we applied multi-locus typing to further confirm sexual transmission using specimens from 74 unrelated patients and 31 concurrently infected couples. Analysis of multi-locus genotype profiles using the five variable loci described above revealed 27 of the couples had concordant genotype profiles compared to only four examples of concordance among the 74 unrelated randomly selected patients.ConclusionWe propose that a combination of the MG309-STRs and MG191-SNPs is efficient for general epidemiological studies and addition of MG307-STRs and MG338-STRs is potentially useful for sexual network studies of M. genitalium infection. The multi-locus typing analysis of 74 unrelated M. genitalium-infected individuals and 31 infected couples adds to the evidence that M. genitalium is sexually transmitted.

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.

Transcriptional heat shock response in the smallest known self-replicating cell, Mycoplasma genitalium.

Mycoplasma genitalium is a human bacterial pathogen linked to urethritis and other sexually transmitted diseases as well as respiratory and joint pathologies. Though its complete genome sequence is available, little is understood about the regulation of gene expression in this smallest known, self-replicating cell, as its genome lacks orthologues for most of the conventional bacterial regulators. Still, the transcriptional repressor HrcA (heat regulation at CIRCE [controlling inverted repeat of chaperone expression]) is predicted in the M. genitalium genome as well as three copies of its corresponding regulatory sequence CIRCE. We investigated the transcriptional response of M. genitalium to elevated temperatures and detected the differential induction of four hsp genes. Three of the up-regulated genes, which encode DnaK, ClpB, and Lon, possess CIRCE within their promoter regions, suggesting that the HrcA-CIRCE regulatory mechanism is functional. Additionally, one of three DnaJ-encoding genes was up-regulated, even though no known regulatory sequences were found in the promoter region. Transcript levels returned to control values after 1 h of incubation at 37 degrees C, reinforcing the transient nature of the heat shock transcriptional response. Interestingly, neither of the groESL operon genes, which encode the GroEL chaperone and its cochaperone GroES, responded to heat shock. These data suggest that M. genitalium selectively regulates a limited number of genes in response to heat shock.

Genome sequencing in microfabricated high-density picolitre reactors

The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.

Transcriptional starts for cytadherence-related operons of Mycoplasma genitalium

One mechanism of mycoplasma cytadherence possessed by several mycoplasmas, including Mycoplasma genitalium, necessitates coordination of multiple adhesins and adherence-associated proteins. The genes encoding these adherence-related proteins are located in three different regions of the M. genitalium genome and exhibit an operon-like organization with surrounding genes. To understand whether genes encoding adherence-related proteins in M. genitalium are regulated as operons, we performed transcriptional and reverse transcription-polymerase chain reaction (RT-PCR) analyses on the loci mg191 (encoding major cytadhesin P140 localized at the specialized tip organelle) and mg218 (encoding high molecular mass cytadherence-related protein MG218 required for tip-mediated adherence). Primer extension suggested that transcription of mg191 was under the control of two transcriptional starts, one immediately upstream of mg191 (Prm MG191) and the other upstream of mg190 (Prm MG190). In contrast, mg218 appeared to be transcribed by a single transcriptional start, located upstream of mg217. RT-PCR indicated that transcription was continuous from mg190 to mg192 and mg217 to mg219, suggesting that these loci constitute true operons. Additional data revealed heretofore undetected similarities between adherence-related operons of M. genitalium and Mycoplasma pneumoniae.

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.

Enhanced genome annotation using structural profiles in the program 3D-PSSM

A method (three-dimensional position-specific scoring matrix, 3D-PSSM) to recognise remote protein sequence homologues is described. The method combines the power of multiple sequence profiles with knowledge of protein structure to provide enhanced recognition and thus functional assignment of newly sequenced genomes. The method uses structural alignments of homologous proteins of similar three-dimensional structure in the structural classification of proteins (SCOP) database to obtain a structural equivalence of residues. These equivalences are used to extend multiply aligned sequences obtained by standard sequence searches. The resulting large superfamily-based multiple alignment is converted into a PSSM. Combined with secondary structure matching and solvation potentials, 3D-PSSM can recognise structural and functional relationships beyond state-of-the-art sequence methods. In a cross-validated benchmark on 136 homologous relationships unambiguously undetectable by position-specific iterated basic local alignment search tool (PSI-Blast), 3D-PSSM can confidently assign 18 %. The method was applied to the remaining unassigned regions of the Mycoplasma genitalium genome and an additional 13 regions were assigned with 95 % confidence. 3D-PSSM is available to the community as a web server http://www.bmm.icnet.uk/servers/3dpssm

Three views of microbial genomes

We describe here GenomeAtlases as a method for visualising three different aspects of complete microbial chromosomes: repeats, DNA structural characteristics, and base composition. We have applied this method to all publicly available genomes, and find a general strand preference of global repeats. The atlas for the Mycoplasma genitalium genome is presented as an example, and results from all three views are consistent with known characteristics of the genome.

Gene identification in bacterial and organellar genomes using GeneScan

The performance of the GeneScan algorithm for gene identification has been improved by incorporation of a directed iterative scanning procedure. Application is made here to the cases of bacterial and organnellar genomes. The sensitivity of gene identification was 100% in Plasmodium falciparum plastid-like genome (35 kb) and in 98% in the Mycoplasma genitalium genome (∼580 kb) and the Haemophilus influenzae Rd genome (∼1.8 Mb). Sensitivity was found to improve in both the Open Reading Frames (ORFs) which have been identified as genes (by homology or by other methods) and those that are classified as hypothetical. False positive assignments (at the nucleotide level) were 0.25% in H. influenzae genome and 0.3% in M. genitalium. There were no false positive assignments in the plastid-like genome. The agreement between the GeneScan predictions and GeneMark predictions of putative ORFs was 97% in M. genitalium genome and 86% in H. influenzae genome. In terms of an exact match between predicted genes/ORFs and the annotation in the databank, GeneScan performance was evaluated to be between 72% and 90% in different genomes. We predict five putative ORFs that were not annotated earlier in the GenBank files for both M. genitalium and H. influenzae genomes. Our preliminary analysis of the newly sequenced G+C rich genome of Mycobacterium tuberculosis H 37R v also shows comparable sensitivity (99%).

Functional insights from structural predictions: analysis of the Escherichia coli genome.

Fold assignments for proteins from the Escherichia coli genome are carried out using BASIC, a profile-profile alignment algorithm, recently tested on fold recognition benchmarks and on the Mycoplasma genitalium genome and PSI BLAST, the newest generation of the de facto standard in homology search algorithms. The fold assignments are followed by automated modeling and the resulting three-dimensional models are analyzed for possible function prediction. Close to 30% of the proteins encoded in the E. coli genome can be recognized as homologous to a protein family with known structure. Most of these homologies (23% of the entire genome) can be recognized both by PSI BLAST and BASIC algorithms, but the latter recognizes an additional 260 homologies. Previous estimates suggested that only 10-15% of E. coli proteins can be characterized this way. This dramatic increase in the number of recognized homologies between E. coli proteins and structurally characterized protein families is partly due to the rapid increase of the database of known protein structures, but mostly it is due to the significant improvement in prediction algorithms. Knowing protein structure adds a new dimension to our understanding of its function and the predictions presented here can be used to predict function for uncharacterized proteins. Several examples, analyzed in more detail in this paper, include the DPS protein protecting DNA from oxidative damage (predicted to be homologous to ferritin with iron ion acting as a reducing agent) and the ahpC/tsa family of proteins, which provides resistance to various oxidating agents (predicted to be homologous to glutathione peroxidase).

The minimal cell genome: "on being the right size".

The minimal cell genome: "on being the right size".

Characterization of repetitive DNA in the Mycoplasma genitalium genome: possible role in the generation of antigenic variation.

We have characterized a family of repetitive DNA elements with homology to the MgPa cellular adhesion operon of Mycoplasma genitalium, a bacterium that has the smallest known genome of any free-living organism. One element, 2272 bp in length and flanked by DNA with no homology to MgPa, was completely sequenced. At least four others were partially sequenced. The complete element is a composite of six regions. Five of these regions show sequence similarity with nonadjacent segments of genes of the MgPa operon. The sixth region, located near the center of the element, is an A+T-rich sequence that has only been found in this repeat family. Open reading frames are present within the five individual regions showing sequence homology to MgPa and the adjacent open reading frame 3 (ORF3) gene. However, termination codons are found between adjacent regions of homology to the MgPa operon and in the A+T-rich sequence. Thus, these repetitive elements do not appear to be directly expressible protein coding sequences. The sequence of one region from five different repetitive elements was compared with the homologous region of the MgPa gene from the type strain G37 and four newly isolated M. genitalium strains. Recombination between repetitive elements of strain G37 and the MgPa operon can explain the majority of polymorphisms within our partial sequences of the MgPa genes of the new isolates. Therefore, we propose that the repetitive elements of M. genitalium provide a reservoir of sequence that contributes to antigenic variation in proteins of the MgPa cellular adhesion operon.

The Minimal Gene Complement of Mycoplasma genitalium

The complete nucleotide sequence (580,070 base pairs) of the Mycoplasma genitalium genome, the smallest known genome of any free-living organism, has been determined by whole-genome random sequencing and assembly. A total of only 470 predicted coding regions were identified that include genes required for DNA replication, transcription and translation, DNA repair, cellular transport, and energy metabolism. Comparison of this genome to that of Haemophilus influenzae suggests that differences in genome content are reflected as profound differences in physiology and metabolic capacity between these two organisms.