Gallic acid inhibits Staphylococcus aureus RecA protein functions: role in countering antibiotic resistance in bacteria.

AIM: Recombinase RecA and its homologs play a key role in homologous recombination DNA repair and revive stalled replication fork DNA synthesis. RecA plays an essential role in the evolution of antibiotic-resistant strains via stress-induced DNA repair mechanisms during the SOS response. Accordingly, RecA has become an attractive target to slow down antibiotic resistance rates and prevent mutations in pathogenic bacterial species. METHODS AND RESULTS: We employed RecA conserved activities: DNA binding, displacement loop formation, strand exchange, ATP hydrolysis, and LexA cleavage, to elucidate the inhibitory role of gallic acid on S. aureus RecA functions. Gallic acid inhibition of the SOS response by western blot analysis and its antibacterial activity were measured. The gallic acid inhibited all the canonical activities of S. aureus RecA protein. CONCLUSION: The natural phenolic compound gallic acid interferes with RecA protein DNA complex formation and inhibits activities such as displacement loop formation, strand exchange reaction, ATP hydrolysis, and coprotease activity of S. aureus. Additionally, gallic acid can obstruct ciprofloxacin-induced RecA expression and eventually confer the inhibitory role of gallic acid in the SOS survival mechanism in S. aureus.

Characterization of Staphylococcus aureus RecX protein: Molecular insights into negative regulation of RecA protein and implications in HR processes.

Homologous recombination (HR) is essential for genome stability and for maintaining genetic diversity. In eubacteria, RecA protein plays a key role during DNA repair, transcription, and HR. RecA is regulated at multiple levels, but majorly by RecX protein. Moreover, studies have shown RecX is a potent inhibitor of RecA and thus acts as an antirecombinase. Staphylococcus aureus is a major food-borne pathogen that causes skin, bone joint, and bloodstream infections. To date, RecX's role in S. aureus has remained enigmatic. Here, we show that S. aureus RecX (SaRecX) is expressed during exposure to DNA-damaging agents, and purified RecX protein directly interacts physically with RecA protein. The SaRecX is competent to bind with single-stranded DNA preferentially and double-stranded DNA feebly. Significantly, SaRecX impedes the RecA-driven displacement loop and inhibits formation of the strand exchange. Notably, SaRecX also abrogates adenosine triphosphate hydrolysis and abolishes the LexA coprotease activity. These findings highlight the role of the RecX protein as an antirecombinase during HR and play a pivotal role in regulation of RecA during the DNA transactions.

Expression and Characterization of the Staphylococcus aureus RecA protein: A mapping of canonical functions.

Recombinases are responsible for homologous recombination (HR), proper genome maintenance, and accurate deoxyribonucleic acid (DNA) duplication. Moreover, HR plays a determining role in DNA transaction processes such as DNA replication, repair, recombination, and transcription. Staphylococcus aureus, an opportunistic pathogen, usually causes respiratory infections such as sinusitis, skin infections, and food poisoning. To date, the role of the RecA gene product in S. aureus remains obscure. In this study, we attempted to map the functional properties of the RecA protein. S. aureus expresses the recA gene product in vivo upon exposure to the DNA-damaging agents, ultraviolet radiation, and methyl methanesulfonate. The recombinant purified S. aureus RecA protein displayed strong single-stranded DNA affinity compared to feeble binding to double-stranded DNA. Interestingly, the RecA protein is capable of invasion and formed displacement loops and readily performed strand-exchange activities with an oligonucleotide-based substrate. Notably, the S. aureus RecA protein hydrolyzed the DNA-dependent adenosine triphosphate and cleaved LexA, showing the conserved function of coprotease. This study provides the functional characterization of the S. aureus RecA protein and sheds light on the canonical processes of homologous recombination, which are conserved in the gram-positive foodborne pathogen S. aureus.