Abstract
The toxin components of toxin-antitoxin modules, found in bacterial plasmids, phages, and chromosomes, typically target a single macromolecule to interfere with an essential cellular process. An apparent exception is the chromosomally encoded toxin component of the E. coli CbtA/CbeA toxin-antitoxin module, which can inhibit both cell division and cell elongation. A small protein of only 124 amino acids, CbtA, was previously proposed to interact with both FtsZ, a tubulin homolog that is essential for cell division, and MreB, an actin homolog that is essential for cell elongation. However, whether or not the toxic effects of CbtA are due to direct interactions with these predicted targets is not known. Here, we genetically separate the effects of CbtA on cell elongation and cell division, showing that CbtA interacts directly and independently with FtsZ and MreB. Using complementary genetic approaches, we identify the functionally relevant target surfaces on FtsZ and MreB, revealing that in both cases, CbtA binds to surfaces involved in essential cytoskeletal filament architecture. We show further that each interaction contributes independently to CbtA-mediated toxicity and that disruption of both interactions is required to alleviate the observed toxicity. Although several other protein modulators are known to target FtsZ, the CbtA-interacting surface we identify represents a novel inhibitory target. Our findings establish CbtA as a dual function toxin that inhibits both cell division and cell elongation via direct and independent interactions with FtsZ and MreB.
Highlights
IntroductionIn E. coli, as in most other bacteria, cell shape is defined by the peptidoglycan sacculus [1], which is built by the coordinated efforts of two major protein complexes, the cell elongation complex and the cell division complex (reviewed in [2,3,4])
Encoded toxin-antitoxin systems, which consist of a small toxin protein that is co-produced with a neutralizing antitoxin, are a potential avenue for the identification of novel antibiotic targets
Our study is focused on the CbtA toxin of E. coli, which was known to inhibit both bacterial cell division and bacterial cell elongation
Summary
In E. coli, as in most other bacteria, cell shape is defined by the peptidoglycan sacculus [1], which is built by the coordinated efforts of two major protein complexes, the cell elongation complex and the cell division complex (reviewed in [2,3,4]). The cell elongation complex directs the insertion of new cell wall material into the E. coli lateral sidewall, causing a newly divided rod cell to increase in length (while maintaining a constant width). Once the elongated cell has approximately doubled its mass, the division complex (or divisome) builds a new septal wall at mid-cell, forming two equivalently sized rod-shaped daughter cells [2,5,6]. In vivo fluorescence imaging studies have shown that MreB forms dynamic filament patches that move circumferentially along the long axis of the cell, directing the lateral incorporation of cell wall material [17,18,19]
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