The amikacin resistance gene aphA6 was first detected in the nosocomial pathogen Acinetobacter baumannii and subsequently in other genera. Analysis of 133 whole-genome sequences covering the taxonomic diversity of Acinetobacter spp. detected aphA6 in the chromosome of 2 isolates of A. guillouiae, which is an environmental species, 1 of 8 A. parvus isolates, and 5 of 34 A. baumannii isolates. The gene was also present in 29 out of 36 A. guillouiae isolates screened by PCR, indicating that it is ancestral to this species. The Pnative promoter for aphA6 in A. guillouiae and A. parvus was replaced in A. baumannii by PaphA6, which was generated by use of the insertion sequence ISAba125, which brought a -35 sequence. Study of promoter strength in Escherichia coli and A. baumannii indicated that PaphA6 was four times more potent than Pnative. There was a good correlation between aminoglycoside MICs and aphA6 transcription in A. guillouiae isolates that remained susceptible to amikacin. The marked topology differences of the phylogenetic trees of aphA6 and of the hosts strongly support its recent direct transfer within Acinetobacter spp. and also to evolutionarily remote bacterial genera. Concomitant expression of aphA6 must have occurred because, contrary to the donors, it can confer resistance to the new hosts. Mobilization and expression of aphA6 via composite transposons and the upstream IS-generating hybrid PaphA6, followed by conjugation, seems the most plausible mechanism. This is in agreement with the observation that, in the recipients, aphA6 is carried by conjugative plasmids and flanked by IS that are common in Acinetobacter spp. Our data indicate that resistance genes can also be found in susceptible environmental bacteria. Importance: We speculated that the aphA6 gene for an enzyme that confers resistance to amikacin, the most active aminoglycoside for the treatment of nosocomial infections due to Acinetobacter spp., originated in this genus before disseminating to phylogenetically distant genera pathogenic for humans. Using a combination of whole-genome sequencing of a collection of Acinetobacter spp. covering the breadth of the known taxonomic diversity of the genus, gene cloning, detailed promoter analysis, study of heterologous gene expression, and comparative analysis of the phylogenetic trees of aphA6 and of the bacterial hosts, we found that aphA6 originated in Acinetobacter guillouiae, an amikacin-susceptible environmental species. The gene conferred, upon mobilization, high-level resistance to the new hosts. This work stresses that nonpathogenic bacteria can act as reservoirs of resistance determinants, and it provides an example of the use of a genomic library to study the origin and dissemination of an antibiotic resistance gene to human pathogens.
- MeSH
- Acinetobacter účinky léků enzymologie genetika izolace a purifikace MeSH
- aminoglykosidy farmakologie MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální léková rezistence MeSH
- Escherichia coli enzymologie genetika MeSH
- fylogeneze MeSH
- kanamycinkinasa genetika MeSH
- konjugace genetická MeSH
- mikrobiální testy citlivosti MeSH
- mikrobiologie životního prostředí MeSH
- molekulární evoluce MeSH
- molekulární sekvence - údaje MeSH
- přenos genů horizontální MeSH
- promotorové oblasti (genetika) MeSH
- rozptýlené repetitivní sekvence MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- sekvenční homologie MeSH
- shluková analýza MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
We studied the in trans-silencing capacities of a transgene locus that carried the neomycin phosphotransferase II reporter gene linked to the 35S promoter in an inverted repeat (IR). This transgene locus was originally posttranscriptionally silenced but switched to a transcriptionally silenced epiallele after in vitro tissue culture. Here, we show that both epialleles were strongly methylated in the coding region and IR center. However, by genomic sequencing, we found that the 1.0 kb region around the transcription start site was heavily methylated in symmetrical and non-symmetrical contexts in transcriptionally but not in posttranscriptionally silenced epilallele. Also, the posttranscriptionally silenced epiallele could trans-silence and trans-methylate homologous transgene loci irrespective of their genomic organization. We demonstrate that this in trans-silencing was accompanied by the production of small RNA molecules. On the other hand, the transcriptionally silenced variant could neither trans-silence nor trans-methylate homologous sequences, even after being in the same genetic background for generations and meiotic cycles. Interestingly, 5-aza-2-deoxy-cytidine-induced hypomethylation could partially restore signaling from the transcriptionally silenced epiallele. These results are consistent with the hypothesis that non-transcribed highly methylated IRs are poor silencers of homologous loci at non-allelic positions even across two generations and that transcription of the inverted sequences is essential for their trans-silencing potential.
- MeSH
- alely MeSH
- epigeneze genetická MeSH
- financování organizované MeSH
- geneticky modifikované rostliny genetika metabolismus MeSH
- kanamycinkinasa genetika metabolismus MeSH
- metylace DNA MeSH
- nekódující RNA analýza MeSH
- repetitivní sekvence nukleových kyselin MeSH
- reportérové geny MeSH
- tabák genetika MeSH
- transgeny MeSH
- umlčování genů MeSH
