HipA-triggered growth arrest and β-lactam tolerance in Escherichia coli are mediated by RelA-dependent ppGpp synthesis

Abstract

Persistence is a phenomenon whereby a subpopulation of bacterial cells enters a transient growth-arrested state that confers antibiotic tolerance. While entrance into persistence has been linked to the activities of toxin proteins, the molecular mechanisms by which toxins induce growth arrest and the persistent state remain unclear. Here, we show that overexpression of the protein kinase HipA in Escherichia coli triggers growth arrest by activating synthesis of the alarmone guanosine tetraphosphate (ppGpp) by the enzyme RelA, a signal typically associated with amino acid starvation. We further demonstrate that chemically suppressing ppGpp synthesis with chloramphenicol relieves inhibition of DNA replication initiation and RNA synthesis in HipA-arrested cells and restores vulnerability to β-lactam antibiotics. HipA-arrested cells maintain glucose uptake and oxygen consumption and accumulate amino acids as a consequence of translational inhibition. We harness the active metabolism of HipA-arrested cells to provide a bacteriophage-resistant platform for the production of biotechnologically relevant compounds, which may represent an innovative solution to the costly problem of phage contamination in industrial fermentations.

Fig 1

HipA expression activates ppGpp synthesis by the enzyme RelA, which is required for complete growth arrest and the persistent state. (A) Growth curves of WT and ΔrelA E. coli MG1655 (solid black and red, respectively) bearing the plasmid pHipA. At the indicated time, 50 ng/ml anhydrous tetracycline (aTc) was added to the cultures, inducing growth arrest after ∼45 min (dotted lines). (B) Specific growth rates of WT and ΔrelA strains measured 1 h after addition of various amounts of aTc. Error bars represent averages of at least 32 data point pairs. (C) Representative chromatogram of nucleotide extract of WT and ΔrelA plate-grown cultures after 90 min of HipA induction. mAu, milliabsorbance units. (D) ppGpp concentration measured in cells during exponential growth, cells starved for isoleucine by the addition of valine (+Val), and cells experiencing HipA-mediated growth arrest (+aTc). Error bars represent the standard deviations of the results of at least three measurements. (E) Timing of ppGpp synthesis in response to HipA expression and growth arrest. A hipA-gfpmut2 operon enabled HipA expression to be indirectly monitored with GFP fluorescence.

Fig 2

Growth arrest by HipA induction causes intracellular accumulation of amino acids, indicating that HipA does not trigger ppGpp synthesis by causing amino acid depletion. Light gray and dark gray bars represent log₂ amino acid concentration ratios of HipA-arrested cells to growing cells, as measured in cells 1 and 2 h after HipA induction by addition of aTc. Black bars indicate ratios (HipA-arrested cells/growing cells) of flux through indicated amino acid pools determined using kinetic flux profiling. A value of 0 indicates no change. For concentration measurements, error bars represent the standard deviations of the results of three biological replicates. Error bars in flux bars represent uncertainty in flux determination, as described in Materials and Methods. Asterisks indicate amino acid fluxes for which values could not be determined.

Fig 3

RelA-dependent synthesis of ppGpp is responsible for the inhibition of macromolecular synthesis observed in HipA-arrested cells. (A and B) Chloramphenicol relieves the inhibition of DNA replication initiation (A) and RNA synthesis (B) imposed by ppGpp. DNA/OD and RNA/OD values were normalized to the value at 1 h. Data represent the results of biological duplicates. (C) Deactivation of ppGpp synthesis by chloramphenicol resensitizes HipA-arrested cells to β-lactam antibiotics. Carbenicillin (100 μg/ml) was added to all cultures at the time indicated. Error bars represent standard deviations of the results of biological triplicates. (D) HipA expression does not enable ΔrelA cells to resist ampicillin (added to 100 μg/ml). Shown are the results of biological triplicates.

Fig 4

HipA-arrested cells maintain consume glucose and oxygen. Data represent glucose and oxygen consumption by a growing culture (top) and a HipA-arrested culture (bottom). At the indicated time (vertical dashed line), HipA expression was induced by the addition of aTc. Examples shown are representative of the results of three biological replicates.

Fig 5

HipA-arrested cells can maintain a heterologous pathway that enables production of mevalonate and are unaffected by infection by phage. (A) Production of mevalonate by HipA-arrested cells. A plasmid-encoded mevalonate pathway was induced with IPTG (at 0.17 days), and cells were subjected to growth arrest by HipA induction 5 h later (0.36 days). At the times indicated with arrows, fresh medium was added to the culture to replenish the supply of glucose for further production. (B) Production of mevalonate in HipA-arrested cells is unaffected by phage infection. Data represent mevalonate titers of phage-infected cultures expressed as ratios to titers obtained from uninfected cultures.