Abstract
ABSTRACTThe alarmone (p)ppGpp plays pivotal roles in basic bacterial stress responses by increasing tolerance of various nutritional limitations and chemical insults, including antibiotics. Despite intensive studies since (p)ppGpp was discovered over 4 decades ago, (p)ppGpp binding proteins have not been systematically identified in Escherichia coli. We applied DRaCALA (differential radial capillary action of ligand assay) to identify (p)ppGpp-protein interactions. We discovered 12 new (p)ppGpp targets in E. coli that, based on their physiological functions, could be classified into four major groups, involved in (i) purine nucleotide homeostasis (YgdH), (ii) ribosome biogenesis and translation (RsgA, Era, HflX, and LepA), (iii) maturation of dehydrogenases (HypB), and (iv) metabolism of (p)ppGpp (MutT, NudG, TrmE, NadR, PhoA, and UshA). We present a comprehensive and comparative biochemical and physiological characterization of these novel (p)ppGpp targets together with a comparative analysis of relevant, known (p)ppGpp binding proteins. Via this, primary targets of (p)ppGpp in E. coli are identified. The GTP salvage biosynthesis pathway and ribosome biogenesis and translation are confirmed as targets of (p)ppGpp that are highly conserved between E. coli and Firmicutes. In addition, an alternative (p)ppGpp degradative pathway, involving NudG and MutT, was uncovered. This report thus significantly expands the known cohort of (p)ppGpp targets in E. coli.
Highlights
The alarmone (p)ppGpp plays pivotal roles in basic bacterial stress responses by increasing tolerance of various nutritional limitations and chemical insults, including antibiotics
Analysis of E. coli revealed that (p)ppGpp levels dramatically increased 10- to 100-fold during shifts to amino acid starvation [4, 14,15,16]. This response depends on the presence of RelA [(p)ppGpp synthetase I], which is active when bound to the ribosomal A-site together with cognate, uncharged tRNA [17,18,19]. (p)ppGpp reprograms RNA polymerase (RNAP) to actively transcribe genes involved in biosynthesis of amino acids, while transcription of genes encoding ribosome proteins, rRNA, and tRNA is repressed
To employ differential radial capillary action of ligand assay (DRaCALA), we used the ASKA plasmid library consisting of a complete set of E. coli K-12 genes encoding N-terminally His-tagged proteins encoded by a high-copy-number plasmid [42]
Summary
The alarmone (p)ppGpp plays pivotal roles in basic bacterial stress responses by increasing tolerance of various nutritional limitations and chemical insults, including antibiotics. Analysis of E. coli revealed that (p)ppGpp levels dramatically increased 10- to 100-fold during shifts to amino acid starvation [4, 14,15,16] This response depends on the presence of RelA [(p)ppGpp synthetase I], which is active when bound to the ribosomal A-site together with cognate, uncharged tRNA [17,18,19]. (p)ppGpp reprograms RNAP to actively transcribe genes involved in biosynthesis of amino acids, while transcription of genes encoding ribosome proteins, rRNA, and tRNA is repressed These dramatic physiological changes reduce the cell growth rate or induce dormancy [9,10,11, 13, 20, 21]. Via such transcriptional reprogramming, (p)ppGpp affects the expression of between 500 and 1,400 genes [15, 21]
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