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Selectivity among Anti-? Factors by Mycobacterium tuberculosis ClpX Influences Intracellular Levels of Extracytoplasmic Function ? Factors.

ABSTRACT: Extracytoplasmic function ? factors that are stress inducible are often sequestered in an inactive complex with a membrane-associated anti-? factor. Mycobacterium tuberculosis membrane-associated anti-? 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-? 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-? interactions were correlated with changes in unfoldase activities. Green fluorescent protein (GFP) chimeras or polypeptides with a length identical to that of the anti-? factor degron also demonstrate degron-dependent variation in ClpX activities. We show that degron-dependent ClpX activity leads to differences in anti-? degradation, thereby regulating the release of free ? from the ?/anti-? complex. M. tuberculosis ClpX activity thus influences changes in gene expression by modulating the cellular abundance of ECF ? factors.IMPORTANCE The ability of Mycobacterium tuberculosis to quickly adapt to changing environmental stimuli occurs by maintaining protein homeostasis. Extracytoplasmic function (ECF) ? factors play a significant role in coordinating the transcription profile to changes in environmental conditions. Release of the ? factor from the anti-? is governed by the ClpXP2P1 assembly. M. tuberculosis ECF anti-? factors have an ssrA-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 ? factors. ClpX activity thus synchronizes changes in gene expression with environmental stimuli affecting M. tuberculosis physiology.

SUBMITTER: Joshi AC

PROVIDER: S-EPMC6398269 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Project description:Bacteria respond to nutrient starvation implementing the stringent response, a stress signaling system resulting in metabolic remodeling leading to decreased growth rate and energy requirements. A well-characterized model of stringent response in Mycobacterium tuberculosis is the one induced by growth in low phosphate. The extracytoplasmic function (ECF) sigma factor SigE was previously suggested as having a key role in the activation of stringent response. In this study, we challenge this hypothesis by analyzing the temporal dynamics of the transcriptional response of a sigE mutant and its wild-type parental strain to low phosphate using RNA sequencing. We found that both strains responded to low phosphate with a typical stringent response trait, including the downregulation of genes encoding ribosomal proteins and RNA polymerase. We also observed transcriptional changes that support the occurring of an energetics imbalance, compensated by a reduced activity of the electron transport chain, decreased export of protons, and a remodeling of central metabolism. The most striking difference between the two strains was the induction in the sigE mutant of several stress-related genes, in particular, the genes encoding the ECF sigma factor SigH and the transcriptional regulator WhiB6. Since both proteins respond to redox unbalances, their induction suggests that the sigE mutant is not able to maintain redox homeostasis in response to the energetics imbalance induced by low phosphate. In conclusion, our data suggest that SigE is not directly involved in initiating stringent response but in protecting the cell from stress consequent to the low phosphate exposure and activation of stringent response. IMPORTANCE Mycobacterium tuberculosis can enter a dormant state enabling it to establish latent infections and to become tolerant to antibacterial drugs. Dormant bacteria's physiology and the mechanism(s) used by bacteria to enter dormancy during infection are still unknown due to the lack of reliable animal models. However, several in vitro models, mimicking conditions encountered during infection, can reproduce different aspects of dormancy (growth arrest, metabolic slowdown, drug tolerance). The stringent response, a stress response program enabling bacteria to cope with nutrient starvation, is one of them. In this study, we provide evidence suggesting that the sigma factor SigE is not directly involved in the activation of stringent response as previously hypothesized, but it is important to help the bacteria to handle the metabolic stress related to the adaptation to low phosphate and activation of stringent response, thus giving an important contribution to our understanding of the mechanism behind stringent response development.

Project description:Bacteria must sense alterations in their environment and respond with changes in function and/or structure in order to cope. Extracytoplasmic function sigma factors (ECF ?s) modulate transcription in response to cellular and environmental signals. The symbiotic nitrogen-fixing alphaproteobacterium Sinorhizobium meliloti carries genes for 11 ECF-like ?s (RpoE1 to -E10 and FecI). We hypothesized that some of these play a role in mediating the interaction between the bacterium and its plant symbiotic partner. The bacterium senses changes in its immediate environment as it establishes contact with the plant root, initiates invasion of the plant as the root nodule is formed, traverses several root cell layers, and enters plant cortical cells via endocytosis. We used genetics, transcriptomics, and functionality to characterize the entire S. meliloti cohort of ECF ?s. We discovered new targets for individual ?s, confirmed others by overexpressing individual ECF ?s, and identified or confirmed putative promoter motifs for nine of them. We constructed precise deletions of each ECF ? gene and its demonstrated or putative anti-? gene and also a strain in which all 11 ECF ? and anti-? genes were deleted. This all-ECF ? deletion strain showed no major defects in free-living growth, in Biolog Phenotype MicroArray assays, or in response to multiple stresses. None of the ECF ?s were required for symbiosis on the host plants Medicago sativa and Medicago truncatula: the strain deleted for all ECF ? and anti-? genes was symbiotically normal.IMPORTANCE Fixed (reduced) soil nitrogen plays a critical role in soil fertility and successful food growth. Much soil fertility relies on symbiotic nitrogen fixation: the bacterial partner infects the host plant roots and reduces atmospheric dinitrogen in exchange for host metabolic fuel, a process that involves complex interactions between the partners mediated by changes in gene expression in each partner. Here we test the roles of a family of 11 extracytoplasmic function (ECF) gene regulatory proteins (sigma factors [?s]) that interact with RNA polymerase to determine if they play a significant role in establishing a nitrogen-fixing symbiosis or in responding to various stresses, including cell envelope stress. We discovered that symbiotic nitrogen fixation occurs even when all 11 of these regulatory genes are deleted, that most ECF sigma factors control accessory functions, and that none of the ECF sigma factors are required to survive envelope stress.

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Selectivity among Anti-? Factors by Mycobacterium tuberculosis ClpX Influences Intracellular Levels of Extracytoplasmic Function ? Factors.

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