Many facets of ribosome biogenesis and function, including ribosomal RNA (rRNA) transcription, 70S assembly and protein translation, are negatively impacted upon induction of a nutrient stress-sensing signalling pathway termed the stringent response. This stress response is mediated by the alarmones guanosine tetra- and penta-phosphate ((p)ppGpp), the accumulation of which leads to a massive cellular response that slows growth and aids survival. The 70S bacterial ribosome is an intricate structure, with assembly both complex and highly modular. Presiding over the assembly process is a group of P-loop GTPases within the TRAFAC (Translation Factor Association) superclass that are crucial for correct positioning of both early and late stage ribosomal proteins (r-proteins) onto the rRNA. Often described as ‘molecular switches’, members of this GTPase superfamily readily bind and hydrolyse GTP to GDP in a cyclic manner that alters the propensity of the GTPase to carry out a function. TRAFAC GTPases are considered to act as checkpoints to ribosome assembly, involved in binding to immature sections in the GTP-bound state, preventing further r-protein association until maturation is complete. Here we review our current understanding of the impact of the stringent response and (p)ppGpp production on ribosome maturation in prokaryotic cells, focusing on the inhibition of (p)ppGpp on GTPase-mediated subunit assembly, but also touching upon the inhibition of rRNA transcription and protein translation.

The stringent response is a bacterial signaling network that utilizes the nucleotides pppGpp and ppGpp to reprogram cells in order to survive nutritional stresses. However, much about how these important nucleotides control cellular reprogramming is unknown.

The cytokine interleukin-1β (IL-1β) has pivotal roles in antimicrobial immunity, but also incites inflammatory disease. Bioactive IL-1β is released following proteolytic maturation of the pro-IL-1β precursor by caspase-1. UBE2L3, a ubiquitin conjugating enzyme, promotes pro-IL-1β ubiquitylation and proteasomal disposal. However, actions of UBE2L3 in vivo and its ubiquitin ligase partners in this process are unknown. Here we report that deletion ofUbe2l3in mice reduces pro-IL-1β turnover in macrophages, leading to excessive mature IL-1β production, neutrophilic inflammation and disease following inflammasome activation. An unbiased RNAi screen identified TRIP12 and AREL1 E3 ligases of the Homologous to E6 C-terminus (HECT) family in adding destabilising K27-, K29- and K33- poly-ubiquitin chains on pro-IL-1β. We show that precursor abundance determines mature IL-1β production, and UBE2L3, TRIP12 and AREL1 limit inflammation by shrinking the cellular pool of pro-IL-1β. Our study uncovers fundamental processes governing IL-1β homeostasis and provides molecular insights that could be exploited to mitigate its adverse actions in disease.

Enterobacter cloacae is a Gram-negative nosocomial pathogen of the ESKAPE ( Enterococcus, Staphylococcus, Klebsiella, Acinetobacter, Pseudomonas , and Enterobacter spp.) priority group with increasing multi-drug resistance via the acquisition of resistance plasmids. However, E. cloacae can also display forms of antibiotic refractoriness, such as heteroresistance and tolerance. Here, we report that E. cloacae displays transient heteroresistance to aminoglycosides, which is accompanied with the formation of small colony variants (SCVs) with increased minimum inhibitor concentration (MIC) of gentamicin and other aminoglycosides used in the clinic, but not other antibiotic classes. To explore the underlying mechanisms, we performed RNA sequencing of heteroresistant bacteria, which revealed global gene expression changes and a signature of the CpxRA cell envelope stress response. Deletion of the cpxRA two-component system abrogated aminoglycoside heteroresistance and SCV formation, pointing to its indispensable role in these processes. The introduction of a constitutively active allele of cpxA led to high aminoglycoside MICs , consistent with cell envelope stress response driving these behaviors in E. cloacae . Cell envelope stress can be caused by environmental cues, including heavy metals. Indeed, bacterial exposure to copper increased gentamicin MIC in the wild-type but not in the Δ cpxRA mutant. Moreover, copper exposure also elevated the gentamicin MICs of clinical isolates from bloodstream infections, suggesting that CpxRA- and copper-dependent aminoglycoside resistance is broadly conserved in E. cloacae strains. Altogether, we establish that E. cloacae relies on transcriptional reprogramming via the envelope stress response pathway for transient resistance to a major class of frontline antibiotic. IMPORTANCE: Enterobacter cloacae is a bacterium that belongs to the WHO high-priority group and an increasing threat worldwide due its multi-drug resistance. E. cloacae can also display heteroresistance, which has been linked to treatment failure. We report that E. cloacae shows heteroresistance to aminoglycoside antibiotics. These are important frontline microbicidal drugs used against Gram-negative bacterial infections; therefore, understanding how resistance develops among sensitive strains is important. We show that aminoglycoside resistance is driven by the activation of the cell envelope stress response and transcriptional reprogramming via the CpxRA two-component system. Furthermore, heterologous activation of envelope stress via copper, typically a heavy metal with antimicrobial actions, also increased aminoglycoside MICs of the E. cloacae type strain and clinical strains isolated from bloodstream infections. Our study suggests aminoglycoside recalcitrance in E. cloacae could be broadly conserved and cautions against the undesirable effects of copper.

The cytokine interleukin-1β (IL-1β) has pivotal roles in antimicrobial immunity, but also incites inflammatory pathology. Bioactive IL-1β is released following proteolytic maturation of the pro-IL-1β precursor by caspase-1 inflammasomes. UBE2L3/UBCH7, a conserved ubiquitin conjugating enzyme, promotes pro-IL-1β ubiquitylation and proteasomal disposal. However, UBE2L3 actions in vivo and ubiquitin ligases involved in this process are unknown. Here we report that deletion of Ube2l3 in mice markedly reduces pro-IL-1β turnover in macrophages, leading to excessive mature IL-1β production, neutrophilic inflammation and disease symptoms following inflammasome activation. A family-wide siRNA screen identified two ubiquitin ligases, TRIP12 and AREL1, which we show add K27-, K29- and K33- poly-ubiquitin chains on lysine residues in the ‘pro’ domain and destabilise pro-IL-1β. Mutation of ubiquitylation sites increased pro-IL-1β stability, but did not affect proteolysis by caspase-1. The extent of mature IL-1β production is therefore determined by precursor abundance, and UBE2L3, TRIP12 and AREL1 limit inflammation by shrinking the cellular pool of pro-IL-1β. Our study has uncovered fundamental processes governing IL-1β homeostasis and provided molecular insights that could be exploited to mitigate its adverse actions in disease.

During nutrient limitation, bacteria produce the alarmones (p)ppGpp as effectors of the stress signalling network termed the stringent response. Screening for (p)ppGpp-binding targets within Staphylococcus aureus identified four ribosome-associated GTPases (RA-GTPases), RsgA, RbgA, Era and HflX, each of which are cofactors in ribosome assembly, where they cycle between the ON (GTP-bound) and OFF (GDP-bound) states. Entry into the OFF-state from the ON-state occurs upon hydrolysis of GTP, with GTPase activity increasing substantially upon ribosome association. When bound to (p)ppGpp, GTPase activity is inhibited, reducing 70S ribosome assembly. Here, we sought to determine how (p)ppGpp impacts RA-GTPase-ribosome interactions by examining the affinity and kinetics of binding between RA-GTPases and ribosomes in various nucleotide-bound states. We show that RA-GTPases preferentially bind to 5′-diphosphate-containing nucleotides GDP and ppGpp over GTP, which is likely exploited as a regulatory mechanism within the cell. Binding to (p)ppGpp reduces stable association of RA-GTPases to ribosomal subunits compared to the GTP-bound state both in vitro and within bacterial cells by inducing the OFF-state conformation. We propose that in this conformation, the G2/switch I loop adopts a conformation incompatible with ribosome association. Altogether, we highlight (p)ppGpp-mediated inhibition of RA-GTPases as a major mechanism of stringent response-mediated growth control.