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 two-component 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.

Elucidating the role of c-di-AMP in Mycobacterium smegmatis: phenotypic characterization and functional analysis

“…As described earlier (30), M. smegmatis cells were grown till early exponential phase (OD 600 ~0.6), washed twice with PBS and dissolved in RNA later solution with TRIzol (Invitrogen) and placed on ice. Subsequent steps of RNA extraction, DNA synthesis, library preparation, and alignment were carried out at Clevergene, Bangalore, India.…”

PrrA modulates Mycobacterium tuberculosis response to multiple environmental cues and is critically regulated by serine/threonine protein kinases

“…Interestingly, prrAB is not essential in the non- pathogenic bacterium Mycobacterium smegmatis , and a study with a M. smegmatis Δ prrAB mutant reported possible roles of PrrAB in dormancy pathways [55, 56]. M. smegmatis has two dosR orthologs, and expression of dosR2 , the ortholog located in a different genomic location from the dosR1 and dosS orthologs, was decreased in the M. smegmatis Δ prrAB mutant in rich media, even in the absence of NO or hypoxia signals [55]. Unlike in Mtb, expression of several genes in the DosR regulon were additionally reported as differentially expressed in the absence of NO or hypoxia signals, with the Δ prrAB mutant affecting expression of a small subset of those genes [55].…”

“…M. smegmatis has two dosR orthologs, and expression of dosR2 , the ortholog located in a different genomic location from the dosR1 and dosS orthologs, was decreased in the M. smegmatis Δ prrAB mutant in rich media, even in the absence of NO or hypoxia signals [55]. Unlike in Mtb, expression of several genes in the DosR regulon were additionally reported as differentially expressed in the absence of NO or hypoxia signals, with the Δ prrAB mutant affecting expression of a small subset of those genes [55]. These differences likely reflect fundamental differences in the role of PrrA in Mtb versus M. smegmatis , given the non-essentiality of PrrA in the latter.…”

“…Early studies had indicated that prrAB transcript levels increased under nitrogen-limiting conditions [35], while a report utilizing a Mtb mutant with decreased prrA expression (transposon mutant in prrA promoter) suggested its importance early in macrophage infection [36]. Interestingly, prrAB is not essential in the non- pathogenic bacterium Mycobacterium smegmatis , and a study with a M. smegmatis Δ prrAB mutant reported possible roles of PrrAB in dormancy pathways [55, 56]. M. smegmatis has two dosR orthologs, and expression of dosR2 , the ortholog located in a different genomic location from the dosR1 and dosS orthologs, was decreased in the M. smegmatis Δ prrAB mutant in rich media, even in the absence of NO or hypoxia signals [55].…”

The Two-Component Locus MSMEG_0244/0246 Together With MSMEG_0243 Affects Biofilm Assembly in M. smegmatis Correlating With Changes in Phosphatidylinositol Mannosides Acylation

“…The mutant strain exhibited clumping in ammonium-limited medium and significantly reduced growth during ammonium and hypoxic stress and was shown to influence triacylglycerol species during ammonium stress in M.sm ( Maarsingh and Haydel, 2018 ). Transcriptome analysis of a msmeg_5662/msmeg_5663 knockout mutant provided first evidence that the two-component system regulates respiratory and oxidative phosphorylation pathways and positively regulates expression of the dormancy-associated DosR response regulator genes in an oxygen independent manner ( Maarsingh et al, 2019 ).…”