Selectivity among anti-σ factors by Mycobacterium tuberculosis ClpX influences intracellular levels of Extracytoplasmic Function σ factors

Abstract

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 ...