Comparative transcriptomics reveals PrrAB-mediated control of metabolic, respiration, energy-generating, and dormancy pathways in Mycobacterium smegmatis

Abstract

Background Mycobacterium smegmatis is a saprophytic bacterium frequently used as a genetic surrogate to study pathogenic Mycobacterium tuberculosis. The PrrAB two-component genetic regulatory system is essential in M. tuberculosis and represents an attractive therapeutic target. In this study, transcriptomic analysis (RNA-seq) of an M. smegmatis ΔprrAB mutant was used to define the PrrAB regulon and provide insights into the essential nature of PrrAB in M. tuberculosis. Results RNA-seq differential expression analysis of M. smegmatis wild-type (WT), ΔprrAB mutant, and complementation strains revealed that during in vitro exponential growth, PrrAB regulates 167 genes (q < 0.05), 57% of which are induced in the WT background. Gene ontology and cluster of orthologous groups analyses showed that PrrAB regulates genes participating in ion homeostasis, redox balance, metabolism, and energy production. PrrAB induced transcription of dosR (devR), a response regulator gene that promotes latent infection in M. tuberculosis and 21 of the 25 M. smegmatis DosRS regulon homologues. Compared to the WT and complementation strains, the ΔprrAB mutant exhibited an exaggerated delayed growth phenotype upon exposure to potassium cyanide and respiratory inhibition. Gene expression profiling correlated with these growth deficiency results, revealing that PrrAB induces transcription of the high-affinity cytochrome bd oxidase genes under both aerobic and hypoxic conditions. ATP synthesis was ~64% lower in the ΔprrAB mutant relative to WT strain, further demonstrating that PrrAB regulates energy production. Conclusions The M. smegmatis PrrAB twocomponent system regulates respiratory and oxidative phosphorylation pathways, potentially to provide tolerance against the dynamic environmental conditions experienced in its natural ecological niche. PrrAB positively regulates ATP levels during exponential growth, presumably through transcriptional activation of both terminal respiratory branches (cytochrome c bc1 -aa3 and cytochrome bd oxidases), despite transcriptional repression of ATP synthase genes. Additionally, PrrAB positively regulates expression of the dormancy-associated dosR response regulator genes in an oxygen-independent manner, which may serve to fine-tune sensory perception of environmental stimuli associated with metabolic repression. Background 3 Two-component systems (TCSs) participate in signal transduction pathways and are ubiquitously found in bacteria, archaea, some lower eukaryotes and plants [1-4]. TCSs recognize specific environmental stimuli [5] and integrate an adaptive response, frequently by modulating transcription [6]. A prototypical TCS consists of a membrane-bound histidine kinase sensor and a cytoplasmic DNAbinding response regulator. In pathogenic bacteria, TCSs act as virulence factors that regulate diverse survival mechanisms, such as antibiotic resistance [7], phosphate limitation [8], low oxygen tension [9], and evasion of immune responses [10]. Though mammalian proteins bearing histidine kinase sequence m...