Abstract
BackgroundMycobacterium 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.ResultsRNA-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 the WT strain, further demonstrating that PrrAB regulates energy production.ConclusionsThe 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.
Highlights
Mycobacterium smegmatis is a saprophytic bacterium frequently used as a genetic surrogate to study pathogenic Mycobacterium tuberculosis
We recently demonstrated that prrAB is not essential in M. smegmatis and that PrrAB differentially regulates triacylglycerol biosynthetic genes during ammonium limitation [19]
Phylogenetic analyses of PrrA and PrrB in mycobacteria Since prrAB orthologues are present in all mycobacterial species and prrAB is essential for viability in M. tuberculosis [15], it is reasonable to believe that PrrAB fulfills important regulatory properties in mycobacteria
Summary
Mycobacterium smegmatis is a saprophytic bacterium frequently used as a genetic surrogate to study pathogenic Mycobacterium tuberculosis. The M. tuberculosis genome harbors 11 paired TCSs, two orphaned histidine kinases, and six orphaned response regulators [13]. Of these TCSs, only MtrAB [14] and PrrAB [15] are essential for M. tuberculosis viability. Capitalizing on findings that diarylthiazole compounds inhibit M. tuberculosis growth via the PrrAB TCS, Bellale et al [17] exposed M. tuberculosis cultures to diarylthiazole and found that PrrAB modulates transcription of genes enabling metabolic adaptation to a lipid-rich environment, responsiveness to reduced oxygen tension, and production of essential ribosomal proteins and amino acid tRNA synthases
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