RESUMO
The recombinases present in the all kingdoms in nature play a crucial role in DNA metabolism processes such as replication, repair, recombination and transcription. However, till date, the role of RecA in the deadly foodborne pathogen Listeria monocytogenes remains unknown. In this study, the authors show that L. monocytogenes expresses recA more than two-fold in vivo upon exposure to the DNA damaging agents, methyl methanesulfonate and ultraviolet radiation. The purified L. monocytogenes RecA protein show robust binding to single stranded DNA. The RecA is capable of forming displacement loop and hydrolyzes ATP, whereas the mutant LmRecAK70A fails to hydrolyze ATP, showing conserved walker A and B motifs. Interestingly, L. monocytogenes RecA and LmRecAK70A perform the DNA strand transfer activity, which is the hallmark feature of RecA protein with an oligonucleotide-based substrate. Notably, L. monocytogenes RecA readily cleaves L. monocytogenes LexA, the SOS regulon and protects the presynaptic filament from the exonuclease I activity. Altogether, this study provides the first detailed characterization of L. monocytogenes RecA and presents important insights into the process of homologous recombination in the gram-positive foodborne bacteria L. monocytogenes.
Assuntos
Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Recombinação Homóloga , Listeria monocytogenes/enzimologia , Resposta SOS em Genética , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/metabolismoRESUMO
Bacterial RecA plays an important role in the evaluation of antibiotic resistance via stress-induced DNA repair mechanism; SOS response. Accordingly, RecA became an important therapeutic target against antimicrobial resistance. Small molecule inhibitors of RecA may prevent adaptation of antibiotic resistance mutations and the emergence of antimicrobial resistance. In our study, we observed that phenolic compound p-Coumaric acid as potent RecA inhibitor. It inhibited RecA driven biochemical activities in vitro such as ssDNA binding, strand exchange, ATP hydrolysis and RecA coprotease activity of E. coli and L. monocytogenes RecA proteins. The mechanism underlying such inhibitory action of p-Coumaric acid involves its ability to interfere with the DNA binding domain of RecA protein. p-Coumaric acid also potentiates the activity of ciprofloxacin by inhibiting drastic cell survival of L. monocytogenes as well as filamentation process; the bacteria defensive mechanism in response to DNA damage. Additionally, it also blocked the ciprofloxacin induced RecA expression leading to suppression of SOS response in L. monocytogenes. These findings revealed that p-Coumaric acid is a potent RecA inhibitor, and can be used as an adjuvant to the existing antibiotics which not only enhance the shelf-life but also slow down the emergence of antibiotic resistance in bacteria.