Spx, the central regulator of the heat and oxidative stress response in B. subtilis, can repress transcription of translation‐related genes

Abstract

Spx is a Bacillus subtilis transcription factor that interacts with the alpha subunits of RNA polymerase. It can activate the thiol stress response regulon and interfere with the activation of many developmental processes. Here, we show that Spx is a central player orchestrating the heat shock response by up-regulating relevant stress response genes as revealed by comparative transcriptomic experiments. Moreover, these experiments revealed the potential of Spx to inhibit transcription of translation-related genes. By in vivo and in vitro experiments, we confirmed that Spx can inhibit transcription from rRNA. This inhibition depended mostly on UP elements and the alpha subunits of RNA polymerase. However, the concurrent up-regulation activity of stress genes by Spx, but not the inhibition of translation related genes, was essential for mediating stress response and antibiotic tolerance under the applied stress conditions. The observed inhibitory activity might be compensated in vivo by additional stress response processes interfering with translation. Nevertheless, the impact of Spx on limiting translation becomes apparent under conditions with high cellular Spx levels. Interestingly, we observed a subpopulation of stationary phase cells that contains raised Spx levels, which may contribute to growth inhibition and a persister-like behaviour of this subpopulation during outgrowth.

The alarmone (p)ppGpp is part of the heat shock response ofBacillus subtilis

“…We experimentally established that 55°C was an appropriate temperature to examine the impact of severe heat on B. subtilis cells growing on agar plates. In addition to exposure to these various heat conditions, we also examined other potentially proteotoxic stresses, such as salt and oxidative stress [5,14,41].…”

“…S5, see S1 Text for a detailed analysis, Dataset S1, Dataset S2, Dataset S3). Since down-regulation of “stable” rRNA is a hallmark of the SR, we introduced a previously established chromosomal rrnJ p1- lacZ fusion into the assessed strains, thereby allowing us to follow the activity of this rRNA promoter using the lacZ reporter [14]. Only small changes between wildtype and (p)ppGpp 0 strains (45 genes significantly regulated, Fig.…”

“…3), but also at 48 °C, in the same RNA-seq experiment that was used to investigate the thermoresistance (37/53 °C) and thermotolerance (48°/53 °C) conditions (Fig.4 & Fig. 1A) [5, 14]. Thermotolerant cells (48/53 °C) exhibited a pronounced up-regulation of the heat-specific stress response (median 3.4-fold up-regulated) or general stress response (median 3.0-fold up-regulated) as well as comprehensive down-regulation of translation-related genes (median 2.6-fold down-regulated, Fig.…”

“…During exposure to heat and oxidative stress, we and others previously observed in B. subtilis a pronounced down-regulation of rRNA and r-protein genes that resembled the pattern of the SR [13,14,39,40]. Thus, we hypothesized that the alarmone (p)ppGpp and the SR-like response could be part of the heat shock response of B. subtilis .…”

“…thioredoxin [5,12,13]. Interestingly, Spx can also mediate the inhibition of cell growth by the concurrent transcriptional down-regulation of many translation-related genes [14].…”