Abstract
Cellular growth requires a high level of coordination to ensure that all processes run in concert. The role of the nucleotide alarmone (p)ppGpp has been extensively studied in response to external stresses, such as amino acid starvation, in Escherichia coli, but much less is known about the involvement of (p)ppGpp in response to perturbations in intracellular processes. We therefore employed CRISPRi to transcriptionally repress essential genes involved in 14 vital processes and investigated whether a (p)ppGpp-mediated response would be induced. We show that (p)ppGpp is produced and required for a pertinent stress response during interference with outer membrane biogenesis and ADP synthesis specifically. When these processes were perturbed via the transcriptional repression of essential genes, wild type E. coli MG1655 ceased growing and entered a semi-dormant state, whereas isogenic (p)ppGpp0 cells continued to grow uncontrollably to the point of lysis. Furthermore, in vivo measurements revealed that the ATP levels were intrinsically offset in (p)ppGpp0 cells, further indicating a role for the alarmone in cellular energy homeostasis. In summary, our investigation suggests that (p)ppGpp acts as a coordinator of cell growth in response to imbalances in outer membrane biogenesis and adenosine ribonucleotide synthesis, elucidating novel roles for (p)ppGpp in bacterial physiology.
Highlights
Bacteria, as all other life forms, encounter a variety of stresses in nature that must be efficiently sensed and countered in order to ensure surviva[1]
In order to screen for novel roles for (p)ppGpp in cellular physiology, we employed Clustered regularly interspaced short palindromic repeats interference (CRISPRi) to disrupt 14 essential cellular processes (cell division, cytoskeleton biogenesis, DNA replication, essential GTPase activity, lipoprotein incorporation in the outer membrane, lipopolysaccharides (LPS) biosynthesis and outer membrane biogenesis, nucleotide metabolism, outer membrane protein assembly, peptidoglycan synthesis, phospholipid biosynthesis, extracellular secretion, transcription, translation and tRNA aminoacylation) by repressing 18 essential genes in E. coli MG1655 (Fig. 1, Supplementary Table S1), collectively referred to as intracellular imbalances
The best studied model for stringent response (SR) activation, the effector alarmone (p)ppGpp is synthesized and transiently halts macromolecular biosynthesis to greatly decelerate cellular growth rates in order to allow the amino acid pools to recover to growth-permissive levels, ensuring that nutrients are present in sufficient quantities for coordinated growth
Summary
As all other life forms, encounter a variety of stresses in nature that must be efficiently sensed and countered in order to ensure surviva[1]. In order to screen for novel roles for (p)ppGpp in cellular physiology, we employed CRISPRi to disrupt 14 essential cellular processes (cell division, cytoskeleton biogenesis, DNA replication, essential GTPase activity, lipoprotein incorporation in the outer membrane, lipopolysaccharides (LPS) biosynthesis and outer membrane biogenesis, nucleotide metabolism, outer membrane protein assembly, peptidoglycan synthesis, phospholipid biosynthesis, extracellular secretion, transcription, translation and tRNA aminoacylation) by repressing 18 essential genes in E. coli MG1655 (Fig. 1, Supplementary Table S1), collectively referred to as intracellular imbalances. Our results implicate (p)ppGpp in regulation of cell growth during imbalances in outer membrane biogenesis and adenosine ribonucleotide synthesis in E. coli, indicating hitherto overlooked roles in bacterial physiology
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