Regulation of RecA nucleoprotein filament stability by RecX and the effects of RecA-membrane interaction on the activities of RecA in mycobacteria

Abstract

E. coli RecA is a multifunctional protein known to be associated with the cell membrane, forming foci often located at the cell poles, which gets redistributed along the length of the cell during SOS response. Several lines of evidence suggest that RecA foci is occluded from the nucleoid during SOS response and positioned at the polar-proximal end of the cell, implicating RecA-membrane interactions. Compared to the E. coli RecA paradigm, very little is known about the in vivo properties of mycobacterial RecA proteins. Furthermore, the distribution of RecA in the cell or its interaction with the constituents of the mycobacterial cell membrane and its role in cell physiology remains to be investigated. The mycobacterial cell wall is a rigid structure that contains distinctive lipids and glycolipids, including mycolic acids, phosphatidyl inositol mannosides, phthiocerol dimycocerosates and lipoglycans. In this study, we demonstrate that mycobacterial RecA proteins specifically interact with phosphatidylinositol (PI) and cardiolipin (CL) among other acidic lipids. Interestingly, these interactions had no discernible effect on the ability of RecA proteins to bind single-strand DNA or ATP. However, PI and CL abrogated the DNA-dependent ATP activity of mycobacterial as well as E. coli RecA proteins. Further, RecA-phospholipid interaction had no significant effect on their capacity to promote strand exchange between linear double-stranded and single-stranded DNA. Furthermore, GFP-tagged RecA foci were localized at the polar ends, which are redistributed in the cell upon DNA damage and then exclusively associated with the nucleoid. The interaction of RecA protein with CL and PG, which represent two of the three major phospholipids of the M. smegmatis cell membrane, may be physiologically relevant because it provides a possible mechanism by which the membrane components may regulate RecA levels and function during the SOS response and recombinational DNA repair. Altogether, these and other findings indicate that the interaction of RecA proteins with CL and PI, the major acidic constituents of the mycobacterial membrane, may be physiologically relevant, as they provide a possible mechanism for storage of RecA in the cell and regulate recombinational DNA repair during the SOS response. This work sets the stage for future studies on the broader role of the mycobacterial cell membrane and provides a framework for further investigations into the role of cell membrane components in RecA function.