The global, ppGpp-mediated stringent response to amino acid starvation in Escherichia coli

Abstract

The 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 wild-type, 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.

Fig. 1

Growth curves and selected medium component concentrations for WT and ppGpp⁰ strains. A. and B. Growth curves and ppGpp accumulation for WT and ppGpp⁰ strains, respectively, grown at 37° C in MOPS medium with glucose plus all 20 amino acids, with a limiting amount of isoleucine. ppGpp was undetectable in the mutant strain. Time points marked in green indicate times of sampling for medium component analysis presented in C-F. Black arrows indicate sampling times for microarray analysis. Note the higher OD reached by the ppGpp⁰ strain despite being grown in identical medium. C. and D. Concentrations of carbon sources and acetate, and % O₂ saturation for WT and ppGpp⁰ strains, respectively. E. and F. Concentrations of selected amino acids. Concentrations of amino acids not shown did not change appreciably over the course of the experiments.

Fig. 2

Comparisons of microarray results for WT and ppGpp⁰ strains. Control RNA was extracted from log phase WT cells at an OD of ~0.4 grown in medium with replete isoleucine. Test RNA was harvested from WT, ΔrelA and ppGpp⁰ strains ~40 min after onset of growth arrest. All test array data were normalized to control array data before comparative analysis of strain-specific responses to isoleucine starvation. Array data presented in all figures are log₂ expression ratios (test:control). A. Upper plot: comparison of isoleucine-starved WT and ppGpp⁰ strain transcriptome profiles. Lower plot: comparison of isoleucine-starved ΔrelA and ppGpp⁰ strain transcriptome profiles. B. Upper diagram: Venn diagram comparing up regulated genes between the WT and ppGpp⁰ strain. Lower diagram: Venn diagram comparing down regulated genes between the WT and ppGpp⁰ strain. C. Heat maps of log₂ expression ratios for the WT and ppGpp⁰ strain for ribosomal protein genes, other genes involved in translation, and the general stress response. All genes shown in C differed in their expression >2-fold between the WT and ppGpp⁰ strain. Ribosomal protein and translation genes shown were all down regulated >2-fold in the WT, while all the general stress response genes shown were induced >2-fold in the WT.

Fig. 3

WT transcriptome data overlaid on selected metabolic pathways. Genes up regulated >2-fold are shown in red, while genes down regulated >2-fold are shown in green. Genes whose expression did not change >2-fold are shown in black. Where multiple gene products are required for a single conversion, the corresponding arrow is colored according to the average expression value of the corresponding genes. Amino acids are in blue font. Background colors are intended to delineate various pathways as follows: glycolysis: green (upper center), pentose phosphate pathway: dark blue (upper left), TCA cycle: yellow (lower right), threonine biosynthesis: light orange (middle left), branched chain amino acid biosynthesis: purple (lower left), serine metabolism: dark orange (middle right), acetate metabolism: light blue (middle right), glutamate biosynthesis: red (lower center), arginine degradation: light green (bottom right).

Fig. 4

ppGpp⁰ strain transcriptome data overlaid on selected metabolic pathways. Color coding is identical to that for Fig. 3.

Fig. 5

Cluster analysis of carbon sources utilized in kinetic Biolog assays for WT and ppGpp⁰ strains. White represents negligible utilization, dark blue represents maximal utilization, and bright red represents half-maximal utilization, as shown by key. Units are arbitrary. Column 1 contains data for both strains at OD ~0.3, during rapid growth before isoleucine starvation. Column 2 contains data for cells harvested at the onset of isoleucine starvation. Column 3 contains data for cells harvested 1.5 hours into growth arrest. These sampling times correspond to time points T2, T3 and T5 in Fig 6A and 6B. Clusters A, B, and C are described in the text.

Fig. 6

Growth curves and viability staining of WT and ppGpp⁰ strains. A. Growth curve of WT for viability staining. Time points where viability was tested are marked in green and labeled T1-T5. B. Growth curve of ppGpp⁰ strain for viability staining. Labels are as in (A). C. Micrographs of differentially stained WT cells harvested at time points T1-T5 as labeled in (A). Cells stained green are viable, while cells stained red have compromised membranes (non-viable). Gold scale bar = 50 μm. D. Micrographs of differentially stained ppGpp⁰ cells harvested at time points T1-T5 as labeled in (B). Staining and scale bars are identical to (C).

Fig. 7

Total protein, biomass, and RNA produced by WT (black bars) and ppGpp⁰ strains (grey bars) during isoleucine starvation. The first time point (rapid growth) corresponds to T2 in (Fig 6A and 6B). Onset of growth arrest corresponds to (T3) in (Fig 6A and 6B). Final time point (1.5 hrs into growth arrest) corresponds to (T5) in (Fig 6A and 6B). Error bars indicate standard deviations. Both strains produce a similar amount of protein, however, the ppGpp⁰ strain produces 50% more biomass and >150% more RNA than the WT.

Fig. 8

Physiological model of the ppGpp-mediated response to isoleucine starvation. Solid arrows represent positive regulation while solid lines with flat ends indicate negative regulation. Solid lines ending with both lines and arrows denote complex regulation. Dashed lines indicate functional effects (eg. Nucleotide catabolism generates intermediates which are funneled into central metabolism). Regulatory relationships are not necessarily direct.