KilR of Rac prophage is a dual inhibitor of bacterial cell division and elongation machineries.

Bacterial cryptic prophages not only encode genes that reduce the viability of the host upon induction but also contribute to host survival during stressful conditions. Rac is a cryptic prophage of , and it encodes a toxic protein KilR, which causes morphological defects to the host. However, the mechanistic basis of its action is not well understood. In this study, we provide evidence that KilR is a dual inhibitor that affects cell division and cytoskeletal organization. We show that KilR expression is highly toxic, as demonstrated previously, and its predicted C-terminal unstructured region plays a crucial role in its function via a length-dependent manner. Low levels of KilR expression lead to cell filamentation and disruption of Z-rings, while high levels result in rod-shaped defects and mislocalization of the MreB cytoskeletal protein. Using fluorescent fusions, we observed that KilR is diffusively localized in the cytoplasm. When MreBCD proteins are overexpressed, KilR co-localizes with them, forming membrane-associated filaments, indicating a physical association. However, overexpressed MreBCD proteins do not alleviate the KilR-associated growth defect, unlike FtsZ. Finally, we present evidence that chromosomal KilR contributes to the co-inhibition of FtsZ and MreB localization in response to oxidative stress. Our data indicate that KilR inhibits MreB-associated cytoskeletal system, in addition to its effect on FtsZ-associated cell division system. We propose that the dual inhibition activity of KilR contributes to its high level of toxicity and to its function in SOS-independent DNA damage tolerance during oxidative stress.IMPORTANCEKilR is a Rac cryptic prophage-encoded toxic protein, which contributes to host survival during oxidative stress conditions. It is known to inhibit cell division by targeting the tubulin homolog, FtsZ. In this study, we show that KilR affects FtsZ-mediated cell division and MreB-mediated cell elongation. The simultaneous inhibition of cell division and cell elongation is known to be crucial for bacterial survival during stress conditions like oxidative stress. Our study identifies KilR as a cell division and cell elongation inhibitor, offering insights into how bacterial-phage coevolution drives the emergence of cryptic prophage elements, with specific genes enhancing bacterial fitness.