The Role of RecX in Mycobacteria

Manglani, Sapna Lakhi (2010). The Role of RecX in Mycobacteria. MPhil thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.000100c3

Abstract

Tuberculosis is a major global public health problem which causes millions of deaths every year. It is caused by the bacterium Mycobacterium tuberculosis, which survives and replicates within host macrophages, in which it has to overcome the effects of various DNA damaging agents in order to survive.

In bacteria, the RecA protein regulates DNA-damage repair. RecA also plays a central role in regulation of the SOS response and homologous recombination. In Mycobacterium smegmatis and Mycobacterium tuberculosis, downstream of, overlapping and co-transcribed with recA is the recX gene. Using a recA-recX deletion mutant of M. smegmatis, it has been shown that the over expression of recA is toxic in the absence of recX, suggesting that RecX plays a regulatory role on RecA.

This project is aimed at investigating the hypothesis that RecX plays a regulatory role in RecA function in Mycobacterium tuberculosis. Various in vivo and in vitro studies were carried out to test this hypothesis and characterise the nature of RecA regulation by RecX.

To investigate their mode of interaction, the purified RecX protein was used in DNA mobility shift assays to look for interactions with the M. smegmatis and M. tuberculosis recA upstream regions. No interaction was observed, suggesting that RecX does not regulate recA expression at the transcriptional level. A positive interaction was found for RecA and RecX in a yeast two-hybrid study, demonstrating a post-translational mode of interaction and regulation.

The in vitro studies included LexA cleavage and ATP hydrolysis studies. When activated RecA is incubated with LexA, it results in the RecA-mediated auto catalytic cleavage of LexA. When RecX was added with RecA, a clear inhibition in LexA cleavage was observed. All reactions promoted by RecA in vitro are dependent on its binding to single-stranded DNA in the presence of ATP to produce an active nucleoprotein filament. It was found in ATPase assays, that RecX inhibits RecA ATP hydrolysis activity.

To investigate the function of RecX in vivo, an assay was carried out to determine whether recX promotes survival when expression of recA is induced by DNA damage. A recA-recX deletion mutant of M smegmatis complemented with M. smegmatis recA+recX showed a higher percentage survival following DNA damage than when complemented with recA alone. Using the same recA-recX deletion strain of M. smegmatis in a homologous recombination assay, it was found that there is an increase in the frequency of double crossover events in recA+recX strains when compared to ΔrecX strains. Further in vivo studies demonstrated that recX enhances the recovery of RecA levels following pulse DNA damage.