Rate and molecular structure of DNA direct repeats rearrangements in Pseudomonas aeruginosa

MORO CAMILA; MARIELA R. MONTI; PEZZA ROBERTO; CARLOS E. ARGARAÑA

Congreso; LVIII Reunión Anual de SAIB; 2022

Genetic rearrangement of DNA repeated sequences has been proven important for the adaptation of different bacterial species, due to its capacity to alter the chromosomal structure. Long direct repeats can be deleted or expanded by different pathways depending on the level of homology and presence of different factors involved in DNA recombination, replication and repair. Homologous recombination is catalyzed by the RecA recombinase, which searches for homology between two DNA sequences and then promotes the exchange between DNA strands. It is also known that components of the Mismatch Repair System (MRS) have an inhibitory function over the recombination of divergent sequences. We used a genetic reporter system previously generated in our laboratory, that consists of a plasmid containing two direct repeats (1268 bp) separated by a spacer, to determine the in vivo recombination (deletion of one repeat and the spacer) rate for several mutant strains. We also investigated the influence of sequence homology between repeats using two versions of the reporter system; one containing repeats with perfect homology (HO) and another called homeologous (HE) which contains repeats with 5% of divergence. Previous results from our laboratory indicate the coexistence of both RecA dependent and independent recombination mechanisms in this species, both resulting in the deletion of one of the repeats. We found that in the WT strain, HO recombination (HOR) rate (1.23x10-4) was 60 fold higher than the HER rate. In the ΔrecA strain the HOR rate was 4 fold higher than the HER rate. More importantly, the HOR rate represents 1% of the rate found in the wt WT strain, whereas the HER represents 14%. This differs from our results in E. scherichia coli, where HER rate in the ΔrecA strain represents 5% of the rate of the wt WT strain. We also found that the HER rates in strains lacking factors involved in the Mismatch Repair System ( ∆mutS and ∆mutL) were 6 and 2 fold higher than in the WT strain. Interestingly, deletion of the MutS protein had a much higher effect on a ∆recA background; with an HER rate 30 fold higher than the one found in the RecA deficient strain. The homeologous repeat system contains 64 polymorphisms that can be used as molecular markers to identify the approximate recombination sites in recombined clones. These polymorphisms are not distributed evenly in the repeats, creating regions with different densities. We obtained the DNA sequences for multiple clones (46 - 59) of each strain and determined the approximate recombination sites. We found that the distribution of the recombination sites varied in the different mutants. In the absence of MutS, recombination occurs in a region with high mismatch density in 30% of the clones, in contrast with only 7% observed in the WT strain and 5% in the ΔrecA strain. The double mutant strain ∆mutS/∆recA however, showed 11% of recombination sites in this area. Different to the observed in E. coli, our results indicate that there is a significant percentage of HER that occurs independently of the RecA recombinase in P. aeruginosa. Also we found that the MMR system has an inhibitory effect in the deletion of direct repeats, both dependent and independent of RecA, moreover, the effect of mutS seems to be stronger on the independent pathway. Additionally, in the absence of MutS, RecA seems to prefer not a region with a perfect homology but regions that contain a certain percentage of mismatches.