Highlights d USP28 forms an active dimer d USP25 adopts an auto-inhibited tetrameric state d Cancer-associated mutations lead to active USP25 d Neither substrate nor ubiquitin chains disrupt the USP25 tetramer

Worldwide dissemination of antibiotic resistance in bacteria is facilitated by plasmids that encode postsegregational killing (PSK) systems. These produce a stable toxin (T) and a labile antitoxin (A) conditioning cell survival to plasmid maintenance, because only this ensures neutralization of toxicity. Shortage of antibiotic alternatives and the link of TA pairs to PSK have stimulated the opinion that premature toxin activation could be used to kill these recalcitrant organisms in the clinic. However, validation of TA pairs as therapeutic targets requires unambiguous understanding of their mode of action, consequences for cell viability, and function in plasmids. Conflicting with widespread notions concerning these issues, we had proposed that the TA pair kis-kid (killing suppressor-killing determinant) might function as a plasmid rescue system and not as a PSK system, but this remained to be validated. Here, we aimed to clarify unsettled mechanistic aspects of Kid activation, and of the effects of this for kis-kid-bearing plasmids and their host cells. We confirm that activation of Kid occurs in cells that are about to lose the toxin-encoding plasmid, and we show that this provokes highly selective restriction of protein outputs that inhibits cell division temporarily, avoiding plasmid loss, and stimulates DNA replication, promoting plasmid rescue. Kis and Kid are conserved in plasmids encoding multiple antibiotic resistance genes, including extended spectrum β-lactamases, for which therapeutic options are scarce, and our findings advise against the activation of this TA pair to fight pathogens carrying these extrachromosomal DNAs.PemK | mRNA interferase | parD | plasmid stability | RNase

Extracytoplasmic function σ factors that are stress inducible are often sequestered in an inactive complex with a membrane-associated anti-σ factor.Mycobacterium tuberculosismembrane-associated anti-σ factors have a small, stable RNA gene A (ssrA)-like degron for targeted proteolysis. Interaction between the unfoldase, ClpX, and a substrate with an accessible degron initiates energy-dependent proteolysis. Four anti-σ factors with a mutation in the degron provided a set of natural substrates to evaluate the influence of the degron on degradation strength in ClpX-substrate processivity. We note that a point mutation in the degron (X-Ala-Ala) leads to an order-of-magnitude difference in the dwell time of the substrate on ClpX. Differences in ClpX/anti-σ interactions were correlated with changes in unfoldase activities. Green fluorescent protein (GFP) chimeras or polypeptides with a length identical to that of the anti-σ factor degron also demonstrate degron-dependent variation in ClpX activities. We show that degron-dependent ClpX activity leads to differences in anti-σ degradation, thereby regulating the release of free σ from the σ/anti-σ complex.M. tuberculosisClpX activity thus influences changes in gene expression by modulating the cellular abundance of ECF σ factors.IMPORTANCEThe ability ofMycobacterium tuberculosisto quickly adapt to changing environmental stimuli occurs by maintaining protein homeostasis. Extracytoplasmic function (ECF) σ factors play a significant role in coordinating the transcription profile to changes in environmental conditions. Release of the σ factor from the anti-σ is governed by the ClpXP2P1 assembly.M. tuberculosisECF anti-σ factors have anssrA-like degron for targeted degradation. A point mutation in the degron leads to differences in ClpX-mediated proteolysis and affects the cellular abundance of ECF σ factors. ClpX activity thus synchronizes changes in gene expression with environmental stimuli affectingM. tuberculosisphysiology.

Extracytoplasmic Function σ factors that are stress inducible are often sequestered in 10 an inactive complex with a membrane-associated anti-σ factor. M. tuberculosis membrane-11 associated anti-σ factors have a small stable RNA gene A-like degron for targeted proteolysis. 12 Interaction between the unfoldase, ClpX, and the substrate with an accessible degron initiates 13 energy-dependent proteolysis. Four anti-σ factors with a mutation in the degron provided a set 14 of natural substrates to evaluate the influence of the degron on degradation strength in ClpX-15 substrate processivity. We note that a point mutation in the degron (XXX-Ala-Ala) leads to an 16 order of magnitude difference in the dwell time of the substrate on ClpX. Differences in 17 ClpX/anti-σ interactions were correlated with change in unfoldase activity. GFP chimeras or 18 polypeptides of identical length with the anti-σ degron also demonstrate degron-dependent 19 variation in ClpX activity. We show that degron-dependent ClpX activity leads to differences in 20 anti-σ factor degradation thereby regulating the release of free σ from the σ/anti-σ complex. M. 21 2 tuberculosis ClpX activity thus influences changes in gene expression by modulating the cellular 22 abundance of ECF σ factors. 23 Importance: The ability of Mycobacterium tuberculosis to quickly adapt to the changing 24 environmental stimuli occurs by maintaining protein homeostasis. Extra-cytoplasmic function 25 (ECF) σ factors play a significant role in coordinating the transcription profile to changes in 26 environmental conditions. Release of the σ factor from the anti-σ is governed by the ClpXP2P1 27 assembly. M. tuberculosis ECF anti-σ factors have a ssrA-like degron for targeted degradation. 28 A point mutation in the degron leads to differences in ClpX mediated proteolysis and affects the 29 cellular abundance of ECF σ-factors. ClpX activity thus synchronizes changes in gene 30 expression with environmental stimuli affecting M. tuberculosis physiology. 31 Introduction 34 Mycobacterium tuberculosis encounters diverse host microenvironments including 35 acidification of phagosomes, nitrogen intermediates, reactive oxygen species, nutrient starvation, 36 DNA damage, phosphate deprivation and hypoxia (1). Extracytoplasmic Function (ECF) σ 37 factors are non-essential and stress inducible and they contribute significantly to bacterial 38 survival alongside one-and two-component systems (2). M. tuberculosis has ten ECF σ factors-39 of which four are localized in an inactive complex with membrane associated anti-σ factors (3,40 4). The membrane associated anti-σ factors (RsdA, RsmA, RskA and RslA in M. tuberculosis) 41 share a common structural organization comprising of an extra-cytoplasmic domain that is a 42 receptor for environmental stress connected to the cytoplasmic anti-σ domain by a single trans-43 3 membrane helix ( Figure 1). The stress-induced release of an ECF σ factor from the σ/anti-σ 44 factor complex governs the intra-cellular levels of these transcription initiation ...