Abstract
Earlier studies reveal that Small protein B (SmpB), a class of well-conserved tmRNA-binding proteins, is essential for the trans-translation process, which functions as a system for translation surveillance and ribosome rescue. Here, we report a previously unrecognized mechanism by which SmpB alone positively regulates the expression of a sensor kinase, BvgS, in Aeromonas veronii. A reporter plasmid was constructed in which the promoter of bvgS was used to control the expression of the enhanced green fluorescent protein (eGFP) gene. When the reporter plasmid was co-transformed with a SmpB expression construct into E. coli, the relative fluorescence intensity increased about threefold. Transformation with a truncated form of smpB gene showed that the C-terminus had little effect, while N-terminus unexpectedly increased eGFP production. Next, a series of SmpB mutants were generated by site-directed mutagenesis. When the mutants SmpB (G11S) or SmpB (E32AG) was used in the experiment, eGFP expression dropped significantly compared with that of wild type SmpB. Further, purified SmpB was shown to bind the promoter regions of bvgS in the agarose gel retardation assay. Quantitative RT-PCR analysis showed that eGFP transcript levels increased approximately 25-fold in the presence of SmpB. Likewise, smpB knockout decreased bvgS transcripts significantly in A. veronii, and also displayed a reduced capability in salt tolerance. Collectively, the data presented here will facilitate a deeper understanding of SmpB-mediated regulatory circuits as a transcriptional factor in A. veronii.
Highlights
Trans-translation mediated by tmRNA and Small protein B (SmpB) is a vital quality control system which rescues stalled ribosomes and degrades incomplete nascent proteins for recycling in bacteria
SmpB Enhanced enhanced green fluorescent protein (eGFP) Production under the Control of bvgS Promoter To test if SmpB could regulate the expression of bvgS, we constructed a series of expression cassettes (Figure 1)
Great advances have been made in understanding the mechanism of trans-translation, which is mediated by tmRNA and SmpB for translational surveillance and ribosome rescue
Summary
Trans-translation mediated by tmRNA and SmpB is a vital quality control system which rescues stalled ribosomes and degrades incomplete nascent proteins for recycling in bacteria. This ensures the availability of ribosomes for protein synthesis and prevents the accumulation of dysfunctional proteins (Keiler, 2008). It has been reported that trans-translation is widespread in eubacteria, and required for stress tolerance, growth, and virulence (Moore and Sauer, 2007). Mutations in the trans-translation system render pathogen Francisella tularensis vulnerable to stress and become avirulent in mice (Svetlanov et al, 2012). Deletion of tmRNA or smpB genes causes a defect in the timing of DNA
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