(p)ppGpp-dependent activation of gene expression during nutrient limitation

As rapidly growing bacteria begin to exhaust nutrients, their growth rate slows, ultimately leading to stasis or quiescence. Adaptation to nutrient limitation requires widespread metabolic remodeling that leads to lower cellular energy consumption. Examples of such changes include attenuated transcription of genes encoding ribosome components, in part mediated by the phosphorylated nucleotides guanosine tetra- and penta-phosphate, collectively (p)ppGpp. In addition, genes such as those encoding specific proteins that facilitate survival exhibit increased expression during nutrient limitation. An example is the hpf gene, encoding a broadly conserved protein responsible for protecting the ribosome from degradation under conditions limiting for ribosome synthesis. Here we show that (p)ppGpp plays a key role in the transcriptional activation of hpf as B. subtilis cells exit rapid growth. Specifically, we demonstrate that hpf transcription during nutrient limitation requires an RNAP holoenzyme containing the alternative sigma factor σH, encoded by sigH, whose expression is normally inhibited by the AbrB repressor. However, when global protein synthesis decreases, in part dependent on (p)ppGpp, AbrB levels fall, leading to increased sigH transcription and consequently hpf activation. This mechanism couples a key physiological consequence of nutrient limitation – reduced protein synthesis – with specific gene activation, thereby linking transcriptional and translational regulation. Finally, we demonstrate that (p)ppGpp is necessary for the gene expression underlying the elaboration of developmental fates including sporulation and genetic competence. Thus, the active attenuation of protein synthesis by (p)ppGpp is not only necessary for the conservation of energetic resources but also for the proper pattern of gene activation during transition to quiescence. wildtype and ppGpp0 cells were fixed in 1% formaldehyde at room temperature for 30 minutes. Fixed cells were pelleted by centrifugation at 6000 RPM for 3 min at room temperature and washed with 0.02% saline sodium citrate (SSC, Invitrogen). The cell were pelleted again and resuspended in 300 µl MAAM mix (4:1 V:V dilution of methanol to glacial acetic acid) and stored at 20oC until processing. ProBac-seq sample processing was done as described previously (33). Briefly, samples were pelleted and resuspended in PBS to remove MAAM. Following PBS wash to remove MAAM the cells were incubated with lysozyme (Readylyse, Epicenter) for 30 minutes in room temperature for cell wall hydrolysis. After an additional pelleting step and PBS wash the samples were further permeabilized by a 5 min incubation in PBS-Tween (PBS with 1% final concentration of Tween-20 detergent) and a subsequent wash in PBS to remove the detergent. Permeabilized cells were resuspended in 100 μl of probe binding buffer (5 × SSC, 30% formamide, 9 mM citric acid (pH 6.0), 0.1% Tween 20, 50 μg ml−1 heparin and 10% low molecular weight dextran sulfate purchased from Molecular Instruments as Probe Binding Buffer – Cells in Suspension). A transcriptome-wide probe-set containing approximately 30,000 mRNA binding probes designed against the B. subtilis genome (33) was added to each sample before placing the samples to incubate overnight at 50oC. After overnight incubation unbound probes were removed by a series of seven washes with probe wash buffer (5 × SSC, 30% formamide, 9 mM citric acid pH 6.0, 0.1% Tween 20 and 50 μg ml-1 heparin (Molecular Instruments)) and 2 final washes in PBS. Probed and washed cells were enumerated using a flow-cytometer and ~10,000 cells per condition were processed using the ProBac-Seq protocols as described in McNulty et al. Nature Microbiology 2023