The <i>aadE</i>*-<i>sat4</i>-<i>aphA-3</i> Gene Cluster of <i>Mycoplasma bovirhinis</i> HAZ141_2 Undergoes Genomic Rearrangements Influencing the Primary Promoter Sequence
The 54 kb GC-rich prophage region of <i>Mycoplasma bovirhinis</i> HAZ141_2 contains three structural ‘compartments’, one of which is a highly transmittable cluster of three genes, <i>aadE</i>-like (<i>aadE</i>*), <i>sat4</i>, and <i>aphA-3</i&...
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| Autores principales: | , |
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| Formato: | article |
| Lenguaje: | EN |
| Publicado: |
MDPI AG
2021
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| Materias: | |
| Acceso en línea: | https://doaj.org/article/728b35eab5864d7c911b3f2df4e73705 |
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| Sumario: | The 54 kb GC-rich prophage region of <i>Mycoplasma bovirhinis</i> HAZ141_2 contains three structural ‘compartments’, one of which is a highly transmittable cluster of three genes, <i>aadE</i>-like (<i>aadE</i>*), <i>sat4</i>, and <i>aphA-3</i>. In this study, we characterized recombination events and their consequences occurred within the <i>aadE*</i>-<i>sat4</i>-<i>aphA-3</i> containing region. Analysis of this region revealed direct repeats (DRs) of 155 and invert repeats (IRs) of 197 base pairs (bps) each, flanking and overlapping with the primary promoter P* located upstream of the <i>aadE</i>*. Two recombination events, including inversions via both 197 and 155-bp IRs (the latter become inverted after the initial 197-bp IRs associated inversion) and the excision of the <i>aadE</i>*-<i>sat4</i>-<i>aphA-3</i> cluster, were confirmed. Inversion via 155-IRs results in changes within the P* promoter region. Using <i>Escherichia coli</i> JM109 carrying plasmids containing derivatives of the <i>aadE</i>*-<i>sat4</i>-<i>aphA-3</i> cluster, we validated the expression of those genes from different promoters. Our results showed no difference in the minimal inhibitory concentrations (MICs) to kanamycin and neomycin and only 2-fold decrease in MIC (from 512 to 256 μg/mL) to nourseothricin between the wild type and a P* derivative promoter. However, the MICs to kanamycin and neomycin were at least 4-fold lower in the construct where <i>aphA-3</i> expressed under its P2 promoter (128 µg/mL) in comparison to the construct where <i>aphA-3</i> expressed under P1″ promoter located within the <i>sat4</i> gene (512–1024 µg/mL). PCR confirmed the excision of the <i>aadE</i>*-<i>sat4</i>-<i>aphA-3</i> cluster via 197- and 155-bp DRs, but no selection of antibiotic-sensitive <i>M. bovirhinis</i> were obtained after 100 passages in kanamycin-free medium. |
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