mRNA Degradation Rates Are Coupled to Metabolic Status in Mycobacterium smegmatis

Abstract

The success of Mycobacterium tuberculosis as a human pathogen is due in part to its ability to survive stress conditions, such as hypoxia or nutrient deprivation, by entering nongrowing states. In these low-metabolism states, M. tuberculosis can tolerate antibiotics and develop genetically encoded antibiotic resistance, making its metabolic adaptation to stress crucial for survival. Numerous bacteria, including M. tuberculosis, have been shown to reduce their rates of mRNA degradation under growth limitation and stress. While the existence of this response appears to be conserved across species, the underlying bacterial mRNA stabilization mechanisms remain unknown. To better understand the biology of nongrowing mycobacteria, we sought to identify the mechanistic basis of mRNA stabilization in the nonpathogenic model Mycobacterium smegmatis. We found that mRNA half-life was responsive to energy stress, with carbon starvation and hypoxia causing global mRNA stabilization. This global stabilization was rapidly reversed when hypoxia-adapted cultures were reexposed to oxygen, even in the absence of new transcription. The stringent response and RNase levels did not explain mRNA stabilization, nor did transcript abundance. This led us to hypothesize that metabolic changes during growth cessation impact the activities of degradation proteins, increasing mRNA stability. Indeed, bedaquiline and isoniazid, two drugs with opposing effects on cellular energy status, had opposite effects on mRNA half-lives in growth-arrested cells. Taken together, our results indicate that mRNA stability in mycobacteria is not directly regulated by growth status but rather is dependent on the status of energy metabolism. IMPORTANCE The logistics of tuberculosis therapy are difficult, requiring multiple drugs for many months. Mycobacterium tuberculosis survives in part by entering nongrowing states in which it is metabolically less active and thus less susceptible to antibiotics. Basic knowledge on how M. tuberculosis survives during these low-metabolism states is incomplete, and we hypothesize that optimized energy resource management is important. Here, we report that slowed mRNA turnover is a common feature of mycobacteria under energy stress but is not dependent on the mechanisms that have generally been postulated in the literature. Finally, we found that mRNA stability and growth status can be decoupled by a drug that causes growth arrest but increases metabolic activity, indicating that mRNA stability responds to metabolic status rather than to growth rate per se. Our findings suggest a need to reorient studies of global mRNA stabilization to identify novel mechanisms that are presumably responsible.

Environmental Triggers of lrgA Expression in Streptococcus mutans

“…One is that our use of a fluorescent reporter provides higher temporal resolution for detecting and probing the burst of lrgAB activity that occurs at stationary phase, which transcriptional studies may have missed. It is possible that metabolic changes in response to oxygen concentration have affected RNA stability in prior studies (Vargas-Blanco et al, 2019). Further, the low-oxygen conditions in and Ahn et al (2012) were less well defined than in the present study.…”

The impact of leadered and leaderless gene structures on translation efficiency, transcript stability, and predicted transcription rates inMycobacterium smegmatis

“…sigA transcripts in both M. tuberculosis and M. smegmatis were reported to have relatively short half-lives [15, 61], and we hypothesized that this property was conferred in part by the 5’ UTR. We therefore sought to determine the impacts of the M. smegmatis sigA 5’ UTR on expression and mRNA stability.…”

“…RNA extraction, measurement of mRNA abundance, and mRNA stability analysis from M. smegmatis cultures were conducted in biological triplicates as described in [15]. Briefly, mRNA abundance was measured by quantitative PCR (qPCR) using iTaq SYBR green (Bio-Rad) on an Applied Biosystems 7500 with 400 pg of cDNA and 0.25 µM each primer in 10 µL reactions.…”

“…For mRNA stability analysis, 5 mL M. smegmatis cultures were treated with rifampicin at a final concentration of 150 µg/mL to halt transcription, and snap-frozen in liquid nitrogen after 0, 1, 2, or 4 min. Abundance over time was determined for sigA and yfp using qPCR and used to estimate mRNA half-lives as described above and in [15]. As is typical for bacteria, plotting log2 abundance over time produced a biphasic decay curve consistent with a period of faster exponential decay, followed by a period of much slower exponential decay.…”

“…Importantly, it has been shown in Escherichia coli and other bacteria that transcription and translation are physically coupled, and thus 5’ UTR-mediated modulation of translation could have repercussions on transcription rate as well [10-14]. Translation blocks in M. smegmatis have been shown to decrease transcription as well [15], suggesting that transcription-translation coupling occurs in mycobacteria, although the extent and consequences are unknown. 5’ UTRs can also regulate gene expression by altering mRNA turnover rates.…”