RESUMO
Mycoplasma pulmonis is a natural rodent pathogen, considered a privileged model for studying respiratory mycoplasmosis. The complete genome of this bacterium, which belongs to the class Mollicutes, has recently been sequenced, but studying the role of specific genes requires improved genetic tools. In silico comparative analysis of sequenced mollicute genomes indicated the lack of conservation of gene order in the region containing the predicted origin of replication (oriC) and the existence, in most of the mollicute genomes examined, of putative DnaA boxes lying upstream and downstream from the dnaA gene. The predicted M. pulmonis oriC region was shown to be functional after cloning it into an artificial plasmid and after transformation of the mycoplasma, which was obtained with a frequency of 3 x 10(-6) transformants/CFU/ micro g of plasmid DNA. However, after a few in vitro passages, this plasmid integrated into the chromosomal oriC region. Reduction of this oriC region by subcloning experiments to the region either upstream or downstream from dnaA resulted in plasmids that failed to replicate in M. pulmonis, except when these two intergenic regions were cloned with the tetM determinant as a spacer in between them. An internal fragment of the M. pulmonis hemolysin A gene (hlyA) was cloned into this oriC plasmid, and the resulting construct was used to transform M. pulmonis. Targeted integration of this genetic element into the chromosomal hlyA by a single crossing over, which results in the disruption of the gene, could be documented. These mycoplasmal oriC plasmids may therefore become valuable tools for investigating the roles of specific genes, including those potentially implicated in pathogenesis.
Assuntos
Cromossomos Bacterianos , Replicação do DNA , Mycoplasma/genética , Plasmídeos/genética , Origem de Replicação , Proteínas de Bactérias/genética , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Proteínas de Escherichia coli/genética , Deleção de Genes , Vetores Genéticos , Proteínas Hemolisinas/genética , Mycoplasma/crescimento & desenvolvimento , Recombinação Genética , Transformação BacterianaRESUMO
M. hominis and U. urealyticum are the better-known mycoplasma species pathogenic to the human genitourinary tract, causing mainly urethritis, bacterial vaginosis and pregnancy complications. In HIV-infected patients, the prevalence and role of these species is still not well known. The aim of this work was to determinate the prevalence of these species in this group of male patients (HIV group), in comparison to a group of men with clinical symptoms of urethritis (STD group). M. hominis was isolated from 7.5 per cent patients (8/106) and U. urealyticum from 18.9 per cent patients (20/106) from the HIV group, being among these 62.5 per cent and 85 per cent in significant concentrations, respectively. In the STD group these rates were 0.9 per cent (1/110) for M. hominis and 13.6 per cent (15/110) for U. urealyticum, being 100 per cent and 93.3 per cent in significant concentrations, respectively. We could demonstrate infection rates by these mycoplasma species in the HIV group as high as the one found in the STD one, what may indicate the occurrence of opportunistic infections in our population. This fact is discussed here because in immunosuppressed patients, specially M. hominis has been reported causing severe infections, even systemically.
