A Mutant Isoform of ObgE Causes Cell Death by Interfering with Cell Division

Abstract

Cell division is a vital part of the cell cycle that is fundamental to all life. Despite decades of intense investigation, this process is still incompletely understood. Previously, the essential GTPase ObgE, which plays a role in a myriad of basic cellular processes (such as initiation of DNA replication, chromosome segregation, and ribosome assembly), was proposed to act as a cell cycle checkpoint in Escherichia coli by licensing chromosome segregation. We here describe the effect of a mutant isoform of ObgE (ObgE∗) that causes cell death by irreversible arrest of the cell cycle at the stage of cell division. Notably, chromosome segregation is allowed to proceed normally in the presence of ObgE∗, after which cell division is blocked. Under conditions of rapid growth, ongoing cell cycles are completed before cell cycle arrest by ObgE∗ becomes effective. However, cell division defects caused by ObgE∗ then elicit lysis through formation of membrane blebs at aberrant division sites. Based on our results, and because ObgE was previously implicated in cell cycle regulation, we hypothesize that the mutation in ObgE∗ disrupts the normal role of ObgE in cell division. We discuss how ObgE∗ could reveal more about the intricate role of wild-type ObgE in division and cell cycle control. Moreover, since Obg is widely conserved and essential for viability, also in eukaryotes, our findings might be applicable to other organisms as well.

Image-Based Dynamic Phenotyping Reveals Genetic Determinants of Filamentation-Mediated β-Lactam Tolerance

“…For showing the physiology of filamented cells, an overnight culture of the fkpB was diluted and grown to 10 6 CFU/ml and then treated with cephalexin (200 µg/ml) for 2 h. Subsequently, a 1 ml aliquot was washed with PBS buffer to remove the antibiotic and then stained with DAPI (4' ,6-diamidino-2-phenylindole) from Sigma-Aldrich. DAPI staining was performed as described before (Dewachter et al, 2017). To show recovery of filamented cells on agar pads cell cultures were treated with appropriate amount of cephalexin for 2 h and then the antibiotic was washed away.…”

GTP Binding Is Necessary for the Activation of a Toxic Mutant Isoform of the Essential GTPase ObgE

“…ObgE* contains a K268I amino acid substitution, which is located in the G domain of the protein [19,21]. This mutant protein causes a severe loss of viability in E. coli, thereby providing an easy way to assess one aspect of ObgE activity [19,21]. Our results indicate that, besides the mutated G domain, also the N-terminal domain of ObgE* is necessary to confer toxicity.…”

“…In order to do so, we induced ObgE*-Venus expression on plate and scored colony development. The fluorescent fusion protein ObgE*-Venus retains full toxicity [19] and was used to enable a counter selection against non-fluorescent mutants in which ObgE* expression is abolished. Yellow fluorescent colonies correspond to mutants that express full-length ObgE* but are still able to grow.…”

“…It plays a role in a variety of important cellular processes, such as ribosome assembly, the stringent response, morphological development, and antibiotic tolerance [2,6,[13][14][15]. Its essential function, however, is thought to stem from its involvement in cell cycle progression, where it has been shown to influence the initiation of DNA replication, chromosome segregation, and cell division [2,[16][17][18][19][20]. None of these roles of Obg have been characterized mechanistically, leaving the exact molecular functions of Obg enigmatic.…”

“…More specifically, we investigate the influence of different protein domains and nucleotide binding on the toxic effect of a mutant isoform of ObgE, called ObgE*. ObgE* contains a K268I amino acid substitution, which is located in the G domain of the protein [19,21]. This mutant protein causes a severe loss of viability in E. coli, thereby providing an easy way to assess one aspect of ObgE activity [19,21].…”