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In this study, we demonstrated that both the expression of most ribosomal protein genes and the amount of ribosomes were decreased in the Δaa3 mutant of Mycobacterium smegmatis, in which the major terminal oxidase (aa3 cytochrome c oxidase) of the respiratory electron transport chain (ETC) is inactivated, compared to those in the wild-type strain. Deletion of the rel gene encoding the major (p)ppGpp synthetase in the background of the Δaa3 mutant restored the reduced expression of ribosomal protein genes, suggesting that inhibition of the respiratory ETC leads to the Rel-dependent stringent response (SR) in this bacterium. Both a decrease in the expression of ribosomal protein genes by overexpression of rel and the increased expression of rel in the Δaa3 mutant relative to the wild-type strain support the Rel-dependent induction of SR in the Δaa3 mutant. We also demonstrated that the expression of ribosomal protein genes was decreased in M. smegmatis exposed to respiration-inhibitory conditions, such as KCN and bedaquiline treatment, null mutation of the cytochrome bcc1 complex, and hypoxia. The MprBA-SigE-SigB regulatory pathway was implicated in both the increased expression of rel and the decreased expression of ribosomal protein genes in the Δaa3 mutant of M. smegmatis.

The respiratory electron transport chain (ETC) of Mycobacterium smegmatis is terminated with two terminal oxidases, the aa3 cytochrome c oxidase and the cytochrome bd quinol oxidase. The bd quinol oxidase with a higher binding affinity for O2 than the aa3 oxidase is known to play an important role in aerobic respiration under oxygen-limiting conditions. Using relevant crp1 (MSMEG_6189) and crp2 (MSMEG_0539) mutant strains of M. smegmatis, we demonstrated that Crp1 plays a predominant role in induction of the cydAB operon under ETC-inhibitory conditions. Two Crp-binding sequences were identified upstream of the cydA gene, both of which are necessary for induction of cydAB expression under ETC-inhibitory conditions. The intracellular level of cAMP in M. smegmatis was found to be increased under ETC-inhibitory conditions. The crp2 gene was found to be negatively regulated by Crp1 and Crp2, which appears to lead to significantly low cellular abundance of Crp2 relative to Crp1 in M. smegmatis. Our RNA sequencing analyses suggest that in addition to the SigF partner switching system, Crp1 is involved in induction of gene expression in M. smegmatis exposed to ETC-inhibitory conditions.

The partner switching system (PSS) of the SigF regulatory pathway in Mycobacterium smegmatis has been previously demonstrated to include the anti-sigma factor RsbW (MSMEG_1803) and two anti-sigma factor antagonists RsfA and RsfB. In this study, we further characterized two additional RsbW homologs and revealed the distinct roles of three RsbW homologs [RsbW1 (MSMEG_1803), RsbW2 (MSMEG_6129), and RsbW3 (MSMEG_1787)] in the SigF PSS. RsbW1 and RsbW2 serve as the anti-sigma factor of SigF and the protein kinase phosphorylating RsfB, respectively, while RsbW3 functions as an anti-SigF antagonist through its protein interaction with RsbW1. Using relevant mutant strains, RsfB was demonstrated to be the major anti-SigF antagonist in M. smegmatis. The phosphorylation state of Ser-63 was shown to determine the functionality of RsfB as an anti-SigF antagonist. RsbW2 was demonstrated to be the only protein kinase that phosphorylates RsfB in M. smegmatis. Phosphorylation of Ser-63 inactivates RsfB to render it unable to interact with RsbW1. Our comparative RNA sequencing analysis of the wild-type strain of M. smegmatis and its isogenic Δaa3 mutant strain lacking the aa3 cytochrome c oxidase of the respiratory electron transport chain revealed that expression of the SigF regulon is strongly induced under respiration-inhibitory conditions in an RsfB-dependent way.

The BipA (BPI-inducible protein A) protein is highly conserved in a large variety of bacteria and belongs to the translational GTPases, based on sequential and structural similarities. Despite its conservation in bacteria, bipA is not essential for cell growth under normal growth conditions. However, at 20°C, deletion of bipA causes not only severe growth defects but also several phenotypic changes such as capsule production, motility, and ribosome assembly, indicating that it has global regulatory properties. Our recent studies revealed that BipA is a novel ribosome-associating GTPase, whose expression is cold-shock-inducible and involved in the incorporation of the ribosomal protein (r-protein) L6. However, the precise mechanism of BipA in 50S ribosomal subunit assembly is not completely understood. In this study, to demonstrate the role of BipA in the 50S ribosomal subunit and possibly to find an interplaying partner(s), a genomic library was constructed and suppressor screening was conducted. Through screening, we found a suppressor gene, rplT, encoding r-protein L20, which is assembled at the early stage of ribosome assembly and negatively regulates its own expression at the translational level. We demonstrated that the exogenous expression of rplT restored the growth of bipA-deleted strain at low temperature by partially recovering the defects in ribosomal RNA processing and ribosome assembly. Our findings suggest that the function of BipA is pivotal for 50S ribosomal subunit biogenesis at a low temperature and imply that BipA and L20 may exert coordinated actions for proper ribosome assembly under cold-shock conditions.