Crystal structure of a putative type I restriction-modification S subunit from Mycoplasma genitalium

The crystal structure of the eubacteria Mycoplasma genitalium ORF MG438 polypeptide, determined by multiple anomalous dispersion and refined at 2.3 A resolution, reveals the organization of S subunits from the Type I restriction and modification system. The structure consists of two globular domains, with about 150 residues each, separated by a pair of 40 residue long antiparallel alpha-helices. The globular domains correspond to the variable target recognition domains (TRDs), as previously defined for S subunits on sequence analysis, while the two helices correspond to the central (CR1) and C-terminal (CR2) conserved regions, respectively. The structure of the MG438 subunit presents an overall cyclic topology with an intramolecular 2-fold axis that superimposes the N and the C-half parts, each half containing a globular domain and a conserved helix. TRDs are found to be structurally related with the small domain of the Type II N6-adenine DNA MTase TaqI. These relationships together with the structural peculiarities of MG438, in particular the presence of the intramolecular quasi-symmetry, allow the proposal of a model for S subunits recognition of their DNA targets in agreement with previous experimental results. In the crystal, two subunits of MG438 related by a crystallographic 2-fold axis present a large contact area mainly involving the symmetric interactions of a cluster of exposed hydrophobic residues. Comparison with the recently reported structure of an S subunit from the archaea Methanococcus jannaschii highlights the structural features preserved despite a sequence identity below 20%, but also reveals important differences in the globular domains and in their disposition with respect to the conserved regions.     

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.     

In vitro replication of a DNA fragment containing the vicinity of the origin of E. coli DNA replication.

Summary The restriction nuclease cleavage pattern of E. coli DNA synthesized in vitro in the cellophane membrane system (Schaller et al., 1972) is similar to the one obtained after labelling E. coli in vivo. This is shown for exponentially growing cells and for cells synchronized by amino acid starvation followed by thymine starvation. In synchronized cells a piece of some 180 kilobase pairs is labelled containing oriC and neighbouring regions at 82 min on the genetic map of E. coli. A pulse label in vitro is incorporated into the same piece of DNA, but the center of this region, i.e. the EcoR1 fragment of 8.6 kbp length which contains the oriC region (Marsh and Worcel, 1977; v. Meyenburg et al., 1977; Yasuda and Hirota, 1977) is missing.     

Efficient gene transfer of high copy number mibilizable plasmids containing a marineRhodobacter origin of replication

The replication region of pRD31, a 3.1-kb endogenous plasmid from a marineRhodobacter species, has recently been localized and sequenced. We report here incorporation of this replicon into a narrow-host-range mobilizable pBR325 derivative. This has allowed us to establish an efficient conjugative gene transfer system for a hydrogen-producing marine species ofRhodobacter that is unable to grow aerobically. Efficient transfer was obtained (1.2×10^–3 transconjugants per recipient cell), and hybrid plasmids replicated with a high copy number (>10) and good stability. Southern hybridization analysis indicated that the new vectors, pRDP203 and pRDP203s, were maintained in marineRhodobacter sp. NKPB0021 as autonomous replicons without detrimental structural rearrangements, confirming their suitability for use as shuttle vectors.     

Treatment of Mycoplasma genitalium. Observations from a Swedish STD Clinic

Objectives

To evaluate therapy for Mycoplasma genitalium infection with doxycycline or azithromycin 1 g compared to five days of azithromycin (total dose 1.5 g).

Methods

A retrospective case study was performed among patients attending the STD-clinic in Falun, Sweden 1998–2005. All patients with a positive PCR test for M. genitalium were routinely offered a test of cure (toc). Response to doxycycline for 9 days, azithromycin 1 g single dose and extended azithromycin (500 mg on day 1 followed by 250 mg o.d. for 4 days) was determined. In patients with treatment failure after azithromycin, macrolide resistance was monitored before and after treatment. Furthermore, the rate of macrolide resistance was monitored for positive specimens available from 2006–2011.

Results

The eradication rate after doxycycline was 43% (48% for women and 38% for men), for azithromycin 1 g 91% (96% for women and 88% for men) and for extended azithromycin 99% (100% for women and 93% for men). Macrolide resistance developed in 7/7 examined (100%) of those testing positive after azithromycin 1 g, but in none of those treated with extended azithromycin. Macrolide resistance before treatment increased from 0% in 2006 and 2007 to 18% in 2011.

Conclusions

These findings confirm the results from other studies showing that doxycycline is inefficient in eradicating M. genitalium. Although azithromycin 1 g was not significantly less efficient than extended dosage, it was associated with selection of macrolide resistant M. genitalium strains and should not be used as first line therapy for M. genitalium. Monitoring of M. genitalium macrolide resistance should be encouraged.     

Analysis of the autonomous replication behavior in human cells of the dihydrofolate reductase putative chromosomal origin of replication.

Chinese hamster genomic DNA sequences from the region downstream of the dihydrofolate reductase (DHFR) gene reported to contain a chromosomal origin of bidirectional DNA replication (OBR-1) were tested for their ability to support autonomous DNA replication in human cells. A 13.3 kilobase fragment containing OBR-1 and surrounding sequences supported replication in short-term and long-term replication assays, while a 4.5 kb fragment containing OBR-1 did not support substantial replication in either assay. These results are consistent with our previous observations that large fragments of human DNA support replication, while smaller fragments are less efficient. The replication activities of plasmids containing OBR-1 were no greater than those of randomly chosen human fragments of similar size. Furthermore, two-dimensional gel analysis of plasmids containing OBR-1 indicated that initiation does not preferentially occur within the OBR-1 region. These results suggest that in the context of autonomous replication, the DHFR sequences tested do not contain genetic information specifying site-specific replication initiation. Possible implications of these results for chromosomal replication are discussed.     

Localizing the replication origin region on the physical map of the Mycoplasma capricolum genome.

Four lines of evidence argue that the replication origin of the Mycoplasma capricolum genome lies within the 46-kb BamHI fragment bordered by two BamHI sites of the total of nine BamHI sites that have been located on the physical map (M. Miyata, L. Wang, and T. Fukumura, FEMS Microbiol. Lett. 79:329-334, 1991). First, this fragment lost its labeling in preference to other fragments when log-phase cultures were incubated in the presence of chloramphenicol for various times to inhibit the initiation of new rounds of replication and then further incubated with radioactive dTMP to allow DNA elongation to continue. Second, the relative frequencies of various restriction fragments of the genome DNA from exponentially growing cells decreased with increasing distance from the putative origin. Third, preferential labeling occurred when radioactive dTMP was added to cultures of a DNA elongation-defective, temperature-sensitive mutant with a simultaneous temperature downshift. Fourth, the M. capricolum homolog of the dnaA gene, which is located near the replication origin in many other bacteria, was found in the 46-kb fragment.     

An essential gene for replication of the mini-F plasmid from origin I

Summary We constructed a series of defective mini-F plasmids, which have deletion(s) in the replication origin I and/or origin II, and their derivatives, which do not produce F3 protein, by insertion of the XhoI fragment of Tn5 into the XhoI site at 41.0 F (kilobases on the coordinate map of F-plasmid). Using these mutant mini-F plasmids, we found that F3 protein is essential for the replication of mini-F from origin I, but not from origin II.     

Functional analysis of the Mycoplasma genitalium MG312 protein reveals a specific requirement of the MG312 N-terminal domain for gliding motility

The human pathogen Mycoplasma genitalium is known to mediate cell adhesion to target cells by the attachment organelle, a complex structure also implicated in gliding motility. The gliding mechanism of M. genitalium cells is completely unknown, but recent studies have begun to elucidate the components of the gliding machinery. We report the study of MG312, a cytadherence-related protein containing in the N terminus a box enriched in aromatic and glycine residues (EAGR), which is also exclusively found in MG200 and MG386 gliding motility proteins. Characterization of an MG_312 deletion mutant obtained by homologous recombination has revealed that the MG312 protein is required for the assembly of the M. genitalium terminal organelle. This finding is consistent with the intermediate-cytadherence phenotype and the complete absence of gliding motility exhibited by this mutant. Reintroduction of several MG_312 deletion derivatives into the MG_312 null mutant allowed us to identify two separate functional domains: an N-terminal domain implicated in gliding motility and a C-terminal domain involved in cytadherence and terminal organelle assembly functions. In addition, our results also provide evidence that the EAGR box has a specific contribution to mycoplasma cell motion. Finally, the presence of a conserved ATP binding site known as a Walker A box in the MG312 N-terminal region suggests that this structural protein could also play an active function in the gliding mechanism.     

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.     

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.     

Transformation of Penicillium chrysogenum by a vector containing a mitochondrial origin of replication

Summary In order to establish a transformation system for P. chrysogenum autonomously replicating vectors were constructed using mitochondrial DNA sequences from the fungus. A physical map of the mt DNA of a production strain was established using ten different restriction enzymes. Unexpectedly, the mt DNA of this strain proved to be significantly smaller than that of a second strain from a culture collection (27 kb versus 49 kb). Various fragments representing about 71% of the 27 kb mt DNA were cloned and, at first, preselected for replicating activity in an intermediate host (Saccharomyces cerevisiae). Two of these fragments also promoted autonomous replication in P. chrysogenum, which was confirmed by isolation of bulk DNA and transfer into E. coli. For selection of transformants in P. chrysogenum the prokaryotic kanamycin resistance gene was used which increased about twofold the resistance against G418. Present address: Institut für Biotechnologie, Fachgebiet Mikrobiologie, Techn. Universität Berlin, Seestr. 13, D-1000 Berlin 65