Sortase-assembled pili are virulence factors in most Gram positive pathogens, including Enterococci where they are critical factors for biofilm formation and urinary tract infection. These pili are translocated across the membrane, assembled into fibers by a pilus-polymerizing Sortase on the membrane, and are subsequently attached to the cell wall by the membrane-anchored enzyme Sortase A (SrtA). When pilus assembly goes “off pathway”, the cell must respond to pilus subunits accumulating in the membrane. In response to membrane stress of this kind, many bacteria rely on protein quality control processes that involve proteases and chaperones. In this study, we describe a critical role for the cross-kingdom conserved, membrane-anchored HtrA (high temperature requirement A) chaperone/protease in monitoring pilus biogenesis in Enterococcus faecalis. We have found that, in the absence of HtrA, “off pathway” pilus biogenesis activates a previously uncharacterized two component signal transduction system, hereafter termed MsmRS for membrane stress & morphology, which governs cell separation and morphology phenotypes. MsmRS expression is also induced upon daptomycin-mediated membrane stress. This discovery suggests that in the event of certain types of membrane stress, including aberrant pilus biogenesis, a cell separation checkpoint is triggered which we postulate may potentially provide a window for cells to respond to and recover from membrane stress events. Given the conservation of sortase-assembled pili and HtrA across Gram positive bacteria, we propose that HtrA and the MsmRS checkpoint may be a conserved mechanism by which many Gram positive pathogens monitor sortase-assembled pilus biogenesis.