Nandhakumar, M. | Search bioRxiv

bioRxiv2026

Periplasmic proteostasis enables bacterial survival during MreB cytoskeletal disruption

The bacterial actin homolog MreB is essential for various cellular processes, including cell wall biosynthesis, membrane organization, and cell polarity determination. Given its multifaceted roles, MreB is considered a potential target for antibiotic development. However, the bacterial response to MreB inhibition and the factors contributing to bacterial survival under such conditions are not well understood. In this study, RNA sequencing (RNA-seq) was used to identify genes that are differentially expressed in response to MreB inhibition by the A22 antibiotic or to deletion of the mreBCD operon. We identified 6 upregulated genes and 24 downregulated genes under both conditions. To determine whether the upregulated genes contribute to bacterial survival during MreB inhibition, we performed A22 antibiotic susceptibility assay on mutants deleted for each of the 6 upregulated genes. Our findings reveal that cells lacking DegP, a periplasmic serine protease, are highly suceptible to A22 treatment. Complementation analysis showed that wild-type DegP, but not a protease-defective mutant, mitigated the effects of A22. The morphological defects in DegP-deficient cells caused by A22 were reduced by ectopic expression of related periplasmic proteases, such as DegQ and DegS. Furthermore, elevated temperatures could alleviate the effects of A22 in a DegP-dependent manner. Overall, our study provides a comprehensive analysis of the global transcriptome-wide effects of MreB inhibition, offering new insights into bacterial response to cytoskeleton disruption. The findings highlight the critical role of DegP in bacterial survival during MreB inhibition and suggest potential avenues for developing novel combinatorial antibiotic strategies targeting MreB and DegP. HighlightsO_LIMreB disruption by A22 or mutation causes major transcriptome alterations C_LIO_LISix genes are consistently upregulated under both MreB disruption conditions C_LIO_LIDegP, a periplasmic protease, is crucial for bacterial tolerance to A22 C_LIO_LIElevated temperatures alleviate A22 toxicity in a DegP-dependent manner C_LI

microbiology↗

bioRxiv2025

KilR of E. coli Rac prophage is a dual morphogenetic inhibitor of bacterial cell shape

Bacterial cryptic prophages encode genes that reduce the viability of the host, upon induction, but also contribute to host survival during stress conditions. Rac is a cryptic prophage of Escherichia coli and it encodes a toxic protein KilR which causes morphological defects to the host. But the mechanistic basis of its action is not well understood. In this study, we provide evidence that KilR is a dual morphogenetic 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 does 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 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 is a dual morphogenetic inhibitor that affects FtsZ-mediated cell division and MreB-mediated cell elongation. Simultaneous inhibition of cell division and cell elongation are known to be crucial for bacterial survival during stress conditions like oxidative stress. Our study identifies KilR as a dual morphogenetic inhibitor, offering insights into how bacterial-phage coevolution drives the emergence of cryptic prophage elements, with specific genes enhancing bacterial fitness.

microbiology↗