Abstract
The autogenous regulation of ribosomal protein (r-protein) synthesis plays a key role in maintaining the stoichiometry of ribosomal components in bacteria. In this work, taking the rpsO gene as a classic example, we addressed for the first time the in vivo regulation of r-protein synthesis in the mycobacteria M. smegmatis (Msm) and M. tuberculosis (Mtb). We used a strategy based on chromosomally integrated reporters under the control of the rpsO regulatory regions and the ectopic expression of Msm S15 to measure its impact on the reporter expression. Because the use of E. coli as a host appeared inefficient, a fluorescent reporter system was developed by inserting Msm or Mtb rpsO-egfp fusions into the Msm chromosome and expressing Msm S15 or E. coli S15 in trans from a novel replicative shuttle vector, pAMYC. The results of the eGFP expression measurements in Msm cells provided evidence that the rpsO gene in Msm and Mtb was feedback-regulated at the translation level. The mutagenic analysis showed that the folding of Msm rpsO 5′UTR in a pseudoknot appeared crucial for repression by both Msm S15 and E. coli S15, thus indicating a striking resemblance of the rpsO feedback control in mycobacteria and in E. coli.
Highlights
Bacterial ribosomes have been the targets of a majority of reported clinical antibiotics to date; ribosome biogenesis and its regulation are central to the development of new antimicrobials
The ability of r-proteins synthesized in excess over rRNA to inhibit the expression of their own mRNAs has already been shown for most E. coli r-protein operons [2,3,5,6,7,8]
In this article, taking the rpsO gene as a classic example, we address the in vivo regulation of r-protein synthesis in M. smegmatis (Msm) and M. tuberculosis (Mtb)
Summary
A comparison of the Msm rpsO transcript levels for cells bearing pS15Msm v.2 and pS15Msm v.3 revealed a seven-fold increase, indicating that the transcription and overall expression of the GC-rich Msm rpsO coding sequence become more efficient with the E. coli 50 UTR, despite the presence of the same E. coli rpsO promoter (Figure 2D). Msm rpsO’-‘lacZ reporter under the control of the E. coli rpsO promoter (Figure 2B) were transformed with the pS15Msm v.3 or with an empty vector, and the β-galactosidase levels were measured in transformants. 2021, 22, x FOR PEER REVIEW only had a marginal impact on the expression of Eco rpsO’-‘lacZ (Figure 3B), indicating that despite a high homology level (Figure 3C), Msm S15 is not capable of recognizing the E. coli rpsO operator, whereas E. coli S15 has the ability to bind the heterological rpsO 50 UTR we compared a set of amino acid residues.
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