The *-- Gene Cluster of HAZ141_2 Undergoes Genomic Rearrangements Influencing the Primary Promoter Sequence.

The 54 kb GC-rich prophage region of HAZ141_2 contains three structural 'compartments', one of which is a highly transmittable cluster of three genes, -like (), , and . In this study, we characterized recombination events and their consequences occurred within the -- 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 *. 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 -- cluster, were confirmed. Inversion via 155-IRs results in changes within the P promoter region. Using JM109 carrying plasmids containing derivatives of the -- 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 expressed under its P2 promoter (128 µg/mL) in comparison to the construct where expressed under P1″ promoter located within the gene (512-1024 µg/mL). PCR confirmed the excision of the *-- cluster via 197- and 155-bp DRs, but no selection of antibiotic-sensitive were obtained after 100 passages in kanamycin-free medium.