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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| Format: | Article |
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MDPI AG
2021-11-01
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| Series: | Antibiotics |
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| Online Access: | https://www.mdpi.com/2079-6382/10/11/1335 |
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| author | Inna Lysnyansky Ilya Borovok |
| author_facet | Inna Lysnyansky Ilya Borovok |
| author_sort | Inna Lysnyansky |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-10T05:46:11Z |
| format | Article |
| id | doaj.art-728b35eab5864d7c911b3f2df4e73705 |
| institution | Directory Open Access Journal |
| issn | 2079-6382 |
| language | English |
| last_indexed | 2024-03-10T05:46:11Z |
| publishDate | 2021-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Antibiotics |
| spelling | doaj.art-728b35eab5864d7c911b3f2df4e737052023-11-22T22:10:05ZengMDPI AGAntibiotics2079-63822021-11-011011133510.3390/antibiotics10111335The <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 SequenceInna Lysnyansky0Ilya Borovok1Mycoplasma Unit, Division of Avian Diseases, Kimron Veterinary Institute, Beit Dagan 50250, IsraelThe Shmunis School of Biomedicine and Cancer Research, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel Aviv 69978, IsraelThe 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.https://www.mdpi.com/2079-6382/10/11/1335<i>Mycoplasma</i>kanamycinneomycinnourseothricinresistanceprophage |
| spellingShingle | Inna Lysnyansky Ilya Borovok 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 Antibiotics <i>Mycoplasma</i> kanamycin neomycin nourseothricin resistance prophage |
| title | 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | 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 |
| topic | <i>Mycoplasma</i> kanamycin neomycin nourseothricin resistance prophage |
| url | https://www.mdpi.com/2079-6382/10/11/1335 |
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