SummaryThe stringent response to amino acid starvation, whereby stable RNA synthesis is curtailed in favour of transcription of amino acid biosynthetic genes, is controlled by the alarmone ppGpp. To elucidate the extent of gene expression effected by ppGpp, we designed an experimental system based on starvation for isoleucine, which could be applied to both wild-type Escherichia coli and the multiauxotrophic relA spoT mutant (ppGpp 0 ). We used microarrays to profile the response to amino acid starvation in both strains. The wild-type response included induction of the general stress response, downregulation of genes involved in production of macromolecular structures and comprehensive restructuring of metabolic gene expression, but not induction of amino acid biosynthesis genes en masse. This restructuring of metabolism was confirmed using kinetic Biolog assays. These responses were profoundly altered in the ppGpp 0 strain. Furthermore, upon isoleucine starvation, the ppGpp 0 strain exhibited a larger cell size and continued growth, ultimately producing 50% more biomass than the wildtype, despite producing a similar amount of protein. This mutant phenotype correlated with aberrant gene expression in diverse processes, including DNA replication, cell division, and fatty acid and membrane biosynthesis. We present a model that expands and functionally integrates the ppGpp-mediated stringent response to include control of virtually all macromolecular synthesis and intermediary metabolism.

SummaryBacteria comprehensively reorganize their global gene expression when faced with starvation. The alarmone ppGpp facilitates this massive response by co-ordinating the downregulation of genes of the translation apparatus, and the induction of biosynthetic genes and the general stress response. Such a large reorientation requires the activities of multiple regulators, yet the regulatory network downstream of ppGpp remains poorly defined. Transcription profiling during isoleucine depletion, which leads to gradual starvation (over > 100 min), allowed us to identify genes that required ppGpp, Lrp and RpoS for their induction and to deduce the regulon response times. Although the Lrp and RpoS regulons required ppGpp for their activation, they were not induced simultaneously. The data suggest that metabolic genes, i.e. those of the Lrp regulon, require only a low level of ppGpp for their induction. In contrast, the RpoS regulon was induced only when high levels of ppGpp accumulated. We tested several predictions of a model that explains how bacteria allocate transcriptional resources between metabolism and stress response by discretely tuning two regulatory circuits to different levels of ppGpp. The emergent regulatory structure insures that stress survival circuits are only triggered if homeostatic metabolic networks fail to compensate for environmental deficiencies.