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. 2019 Feb 27;9(1):2934.
doi: 10.1038/s41598-019-39371-3.

(p)ppGpp-mediated stress response induced by defects in outer membrane biogenesis and ATP production promotes survival in Escherichia coli

Mohammad Roghanian  1 , Szabolcs Semsey  1 , Anders Løbner-Olesen  1 , Farshid Jalalvand  2
Affiliations

(p)ppGpp-mediated stress response induced by defects in outer membrane biogenesis and ATP production promotes survival in Escherichia coli

Mohammad Roghanian et al. Sci Rep. .

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.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Schematic representation of the targeted cellular processes and genes. CRISPRi-mediated transcriptional repression of 18 essential genes involved in 14 specific cellular processes was carried out in MG1655. Activation of the stringent response was monitored during the induction of the specific intracellular imbalances.
Figure 2
Figure 2
Single-cell analysis of RpoS-mCherry levels during the transcriptional repression of selected essential genes. CRISPRi-mediated shut down of expression of the indicated genes in a MG1655-derivate strain harboring the intracellular (p)ppGpp-reporter rpoS-mCherry (A) RpoS-mCherry fluorescence measurements for all investigated genes at the conclusion of the experiments at 15 h post-CRISPRi induction, or, when not applicable, just prior to the occurrence of lysis. Fluorescence was measured in >30 cells per experiment, the mean fluorescence intensities are shown. Error bars indicate standard deviations (SD). The pre-determined cutoff limit for hits we pursued is marked by the dotted line. (B) Phase contrast (left panels), the (p)ppGpp-reporter RpoS-mCherry fluorescence (middle panels) and the merged overlay images (right panels) capturing cells 15 h post-CRISPRi induction. The transcriptional repressions producing RpoS-mCherry levels exceeding the pre-determined cutoff limit are shown (valS; tRNA-charging, adk; nucleotide metabolism, lptA and lpxA; outer membrane biogenesis, and plsC; phospholipid biosynthesis). The phenotype of dnaB repression is shown as an example of the cellular process disturbances that did not yield a strong rpoS-mCherry expression for comparison. Scale bars = 10 µm.
Figure 3
Figure 3
(p)ppGpp is produced in vivo during repression of valS, adk, lptA and lpxA. Autoradiographs of the lysate of radiolabeled cells showing (p)ppGpp synthesis over time during transcriptional repression of the indicated genes in MG1655. The experimental conditions were identical for all samples and they were processed in parallel. Representative examples of three independent experiments are shown.
Figure 4
Figure 4
Cell viability during transcriptional repressions in MG1655 wt and isogenic background. Viable count of MG1655 wt (blue circles), ΔrelA (green triangles) and ΔrelA/spoT (red squares) isogenic mutants experiencing transcriptional repression of (A) lacI, (B) murC, (C) valS, (D) adk, (E) lptA, and (F) lptX. Values are given as the percentage of CFU/ml at the start of CRISPRi induction. The mean of three separate experiments is plotted, and error bars indicate the standard deviation. The endpoint means are stated in numbers.
Figure 5
Figure 5
(p)ppGpp0 cells display aberrant growth and cell division during repression of valS, adk, lptA and lpxA. Time-lapse phase contrast imaging of ΔrelA/spoT cells during transcriptional repression of the indicated genes. Scale bars = 10 µm.
Figure 6
Figure 6
The nucleoid localizes differently in (p)ppGpp0 cells during repression of valS, adk, lptA and lpxA. Fluorescence microscopy imaging of DAPI-stained ΔrelA/spoT cells 10 h post CRISPRi-mediated transcriptional repression of the indicated genes. Phase contrast (left panels), DAPI fluorescence (middle panels) and their merged overlay (right panels) are shown. Scale bars = 10 µm.
Figure 7
Figure 7
The intracellular ATP-levels are perturbed in ΔrelA and ΔrelA/spoT cells. Measurements of intracellular ATP levels in MG1655 wt (green), ΔrelA (orange) and ΔrelA/spoT (red) during different growth phases using the ratiometric fluorescent ATP-reporter QUEEN-7 µ. >100 cells were measured per sample. The mean is and SD are indicated by the black line and error bars, respectively.
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