RecA dependent and independent recombination in Pseudomonas aeruginosa and Escherichia coli

CAMILA MORO; MARIA VICTORIA BORGOGNO; MARIELA R MONTI; CARLOS E ARGARAÑA

Congreso; LIII Reunión anual de SAIB - Reunión Conjunta de Sociedades de BioCiencias; 2017

Genetic recombination participates in multiple physiological pathwaysthat are crucial for the maintenance and diversification of thegenome. The recombinase RecA is a central factor in this process,mediating the exchange between DNA molecules containing perfecthomology (homologous, HO) or very low divergence (homeologous,HE). At present multiple mechanisms are known to mediate geneticrecombination, both dependent and independent of RecA. In orderto study the recombination process in Gram-negative bacteria, weused a LacZ based system to determine both HO and HE recombination.Using this system, we determined the recombination rates inwild-type (WT) and RecA deficient (ΔrecA) strains of Pseudomonasaeruginosa and Escherichia coli. Moreover, the recombination processcreates a functional copy of the lacZ gene and thus, the recombinantclones can be detected by their β galactosidase activity.In both bacteria, HO recombination rates were approximately 60-200-fold higher than HE recombination rates. In the RecA-deficientE. coli strain, both HO and HE recombination rates were 60-foldlower than that obtained for the WT strain. The recA deletion mutantof P. aeruginosa showed that HO and HE recombination rates decreased15 and 3-fold respectively, compared with the WT strain.When the β galactosidase activity of recombined clones was determined,we found that all E. coli clones showed enzymatic activity asit was expected. In P. aeruginosa however, many clones showed noβ galactosidase activity. Furthermore, the molecular analysis of therecombined regions of these clones showed the presence of pointmutations and also deletions of as much as 800bp.These resultsindicate the existence of a significantly mutagenic recombinationmechanism, independent of RecA in P. aeruginosa, at difference ofE. coli.Keywords: P.aeruginosa, E.coli, Genetic recombination, RecA recombinase