Highlights d Crystal structure of apo B. subtilis Rel d Cryo-EM structure of the Reldstalled ribosome complex d Rel is regulated by the TGS domain in absence of the ribosome d Rel homodimerization masks the tRNA interaction interface

When bacteria experience growth-limiting environmental conditions, the synthesis of the hyperphosphorylated guanosine derivatives (p)ppGpp is induced by enzymes of the RelA/ SpoT homology (RSH)-type protein family. High levels of (p)ppGpp induce a process called "stringent response", a major cellular reprogramming during which ribosomal RNA (rRNA) and transfer RNA (tRNA) synthesis is downregulated, stress-related genes upregulated, messenger RNA (mRNA) stability and translation altered, and allocation of scarce resources optimized. The (p)ppGpp-mediated stringent response is thus often regarded as an all-ornothing paradigm induced by stress. Over the past decades, several binding partners of (p)ppGpp have been uncovered displaying dissociation constants from below one micromolar to more than one millimolar and thus coincide with the accepted intracellular concentrations of (p)ppGpp under non-stringent (basal levels) and stringent conditions. This suggests that the ability of (p)ppGpp to modulate target proteins or processes would be better characterized as an unceasing continuum over a concentration range instead of being an abrupt switch of biochemical processes under specific conditions. We analyzed the reported binding affinities of (p)ppGpp targets and depicted a scheme for prioritization of modulation by (p)ppGpp. In this ranking, many enzymes of e.g., nucleotide metabolism are among the first targets to be affected by rising (p)ppGpp while more fundamental processes such as DNA replication are among the last. This preference should be part of (p)ppGpp's "magic" in the adaptation of microorganisms while still maintaining their potential for outgrowth once a stressful condition is overcome.

ParB-like CTPases mediate the segregation of bacterial chromosomes and low-copy number plasmids. They act as DNA-sliding clamps that are loaded at parS motifs in the centromere of target DNA molecules and spread laterally to form large nucleoprotein complexes serving as docking points for the DNA segregation machinery. Here, we solve crystal structures of ParB in the pre-and post-hydrolysis state and illuminate the catalytic mechanism of nucleotide hydrolysis. Moreover, we identify conformational changes that underlie the CTP-and parS-dependent closure of ParB clamps. The study of CTPase-deficient ParB variants reveals that CTP hydrolysis serves to limit the sliding time of ParB clamps and thus drives the establishment of a well-defined ParB diffusion gradient across the centromere whose dynamics are critical for DNA segregation. These findings clarify the role of the ParB CTPase cycle in partition complex assembly and function and thus advance our understanding of this prototypic CTP-dependent molecular switch.

Bacterial flagella differ in their number and spatial arrangement. In many species, the MinD-type ATPase FlhG (also YlxH/FleN) is central to the numerical control of bacterial flagella, and its deletion in polarly flagellated bacteria typically leads to hyperflagellation. The molecular mechanism underlying this numerical control, however, remains enigmatic. Using the model species Shewanella putrefaciens, we show that FlhG links assembly of the flagellar C ring with the action of the master transcriptional regulator FlrA (named FleQ in other species). While FlrA and the flagellar C-ring protein FliM have an overlapping binding site on FlhG, their binding depends on the ATP-dependent dimerization state of FlhG. FliM interacts with FlhG independent of nucleotide binding, while FlrA exclusively interacts with the ATP-dependent FlhG dimer and stimulates FlhG ATPase activity. Our in vivo analysis of FlhG partner switching between FliM and FlrA reveals its mechanism in the numerical restriction of flagella, in which the transcriptional activity of FlrA is down-regulated through a negative feedback loop. Our study demonstrates another level of regulatory complexity underlying the spationumerical regulation of flagellar biogenesis and implies that flagellar assembly transcriptionally regulates the production of more initial building blocks.