Abstract
Two-component regulatory systems (TCSSs) are key regulatory elements responsible for the adaptation of bacteria to environmental stresses. A classical TCSS is typically comprised of a sensory histidine kinase and a corresponding response regulator. Here, we used homologous recombination to construct a Mycobacterium smegmatis mutant defective in the synthesis of cytosolic histidine kinase PdtaS (Msmeg_1918). The resulting ΔpdtaS mutant strain was tested in the Phenotype Microarray screening system, which allowed us to identify aminoglycoside antibiotic sensitivity, tetracyclines antibiotic resistance as well as membrane transport and respiration, as the main processes affected by removal of pdtaS. The antibiotic sensitivity profiles were confirmed by survival assessment and complementation studies. To gain insight into the molecular mechanisms responsible for the observed phenotype, we compared ribosomal RNA and protein profiles of the mutant and wild-type strains. We carried out Northern blotting and qRT-PCR to compare rRNA levels and analyzed ribosome sedimentation patterns of the wild-type and mutant strains on sucrose gradients. Isolated ribosomes were further used to estimate relative abundance of individual proteins in the ribosomal subunits using label free mass spectrometry analysis. Additionally, the ΔpdtaS mutant revealed lower activity of the respiratory chain as measured by the rate of TTC (triphenyltetrazolium chloride) reduction, while at the same time showing only insignificant changes in the uptake of aminoglycosides. We postulate that deficiency of PdtaS affects the oxidative respiration rates and ribosomal composition causing relevant changes to intrinsic resistance or susceptibility to antibiotics targeting ribosomes, which are commonly used to treat mycobacterial infections.
Highlights
The genus Mycobacterium possesses pathogens known to cause serious diseases in mammals, including tuberculosis (Mycobacterium tuberculosis) and leprosy (Mycobacterium leprae) in humans as well as saprophytes or non-pathogenic soil bacteria, e.g., Mycobacterium smegmatis
The resulting strain was subjected to a second crossover event screening to generate double crossovers and the pKD10 plasmid was later swapped efficiently with an empty pMV306 plasmid, which was lost by culturing bacteria without antibiotic pressure
To investigate the possible pathways that the PdtaS/R system could potentially control in the mycobacterial cell, we have generated a directed and unmarked mutant strain lacking PdtaS in the saprophytic cousin of M. tuberculosis, M. smegmatis
Summary
The genus Mycobacterium possesses pathogens known to cause serious diseases in mammals, including tuberculosis (Mycobacterium tuberculosis) and leprosy (Mycobacterium leprae) in humans as well as saprophytes or non-pathogenic soil bacteria, e.g., Mycobacterium smegmatis. This is the case for Escherichia coli’s CheY, which, upon phosphorylation by its partner sensory kinase, CheA, acts as a flagellar motor switch, binding to the flagella, activating its movement and allowing bacteria to travel to more favorable, nutrient rich niches (Dyer et al, 2009) Another extraordinary group of response regulators includes the ANTAR (AmiR and NasR Transcriptional Antitermination Regulator) domain containing proteins. These regulators are believed to form specific antiterminator structures with the newly transcribed RNA, binding stem loop structures or sequences present either in the 5 UTR regions of regulated transcripts or within the polycistronic RNA species, preventing premature RNA polymerase disengagement (Shu and Zhulin, 2002; Boudes et al, 2012) One of these putative antiterminator proteins, PdtaR (Rv1626), exists in M. tuberculosis and is known to be phosphorylated by its cognate sensory kinase PdtaS (Rv3220c) (Morth et al, 2005). The molecular mechanism of PdtaS-dependent sensitization of mycobacteria to aminoglycosides is discussed in this paper
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