The aadE*-sat4-aphA-3 Gene Cluster of Mycoplasma bovirhinis HAZ141_2 Undergoes Genomic Rearrangements Influencing the Primary Promoter Sequence

The 54 kb GC-rich prophage region of Mycoplasma bovirhinis HAZ141_2 contains three structural ‘compartments’, one of which is a highly transmittable cluster of three genes, aadE-like (aadE*), sat4, and aphA-3</i&...

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Autores principales:	Inna Lysnyansky, Ilya Borovok
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	<i>Mycoplasma</i> kanamycin neomycin nourseothricin resistance prophage Therapeutics. Pharmacology RM1-950
Acceso en línea:	https://doaj.org/article/728b35eab5864d7c911b3f2df4e73705
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spelling	oai:doaj.org-article:728b35eab5864d7c911b3f2df4e737052021-11-25T16:23:00ZThe <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 Sequence10.3390/antibiotics101113352079-6382https://doaj.org/article/728b35eab5864d7c911b3f2df4e737052021-11-01T00:00:00Zhttps://www.mdpi.com/2079-6382/10/11/1335https://doaj.org/toc/2079-6382The 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.Inna LysnyanskyIlya BorovokMDPI AGarticle<i>Mycoplasma</i>kanamycinneomycinnourseothricinresistanceprophageTherapeutics. PharmacologyRM1-950ENAntibiotics, Vol 10, Iss 1335, p 1335 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	<i>Mycoplasma</i> kanamycin neomycin nourseothricin resistance prophage Therapeutics. Pharmacology RM1-950
spellingShingle	<i>Mycoplasma</i> kanamycin neomycin nourseothricin resistance prophage Therapeutics. Pharmacology RM1-950 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
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.
format	article
author	Inna Lysnyansky Ilya Borovok
author_facet	Inna Lysnyansky Ilya Borovok
author_sort	Inna Lysnyansky
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_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_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_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
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/728b35eab5864d7c911b3f2df4e73705
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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

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