The SmpB-tmRNA Tagging System Plays Important Roles in Streptomyces coelicolor Growth and Development

Abstract

The ssrA gene encodes tmRNA that, together with a specialized tmRNA-binding protein, SmpB, forms part of a ribonucleoprotein complex, provides a template for the resumption of translation elongation, subsequent termination and recycling of stalled ribosomes. In addition, the mRNA-like domain of tmRNA encodes a peptide that tags polypeptides derived from stalled ribosomes for degradation. Streptomyces are unique bacteria that undergo a developmental cycle culminating at sporulation that is at least partly controlled at the level of translation elongation by the abundance of a rare tRNA that decodes UUA codons found in a relatively small number of open reading frames prompting us to examine the role of tmRNA in S. coelicolor. Using a temperature sensitive replicon, we found that the ssrA gene could be disrupted only in cells with an extra-copy wild type gene but not in wild type cells or cells with an extra-copy mutant tmRNA (tmRNADD) encoding a degradation-resistant tag. A cosmid-based gene replacement method that does not include a high temperature step enabled us to disrupt both the ssrA and smpB genes separately and at the same time suggesting that the tmRNA tagging system may be required for cell survival under high temperature. Indeed, mutant cells show growth and sporulation defects at high temperature and under optimal culture conditions. Interestingly, even though these defects can be completely restored by wild type genes, the ΔssrA strain was only partially corrected by tmRNADD. In addition, wildtype tmRNA can restore the hygromycin-resistance to ΔssrA cells while tmRNADD failed to do so suggesting that degradation of aberrant peptides is important for antibiotic resistance. Overall, these results suggest that the tmRNA tagging system plays important roles during Streptomyces growth and sporulation under both normal and stress conditions.

Dual Positive Feedback Regulation of Protein Degradation of an Extra-cytoplasmic Function σ Factor for Cell Differentiation in Streptomyces coelicolor

“…In this report, we show that ClpP protease is required for SigT protein degradation to remove its inhibitory effects on cell differentiation. This is consistent with the phenotypes of the ⌬clpP and ⌬ssrA mutants that display delayed morphological development and secondary metabolism (41,43). Furthermore, SigT negatively regulates the clpP1/P2 gene expression by binding to its promoter.…”

The tmRNA ribosome-rescue system

“…Several groups have developed tmRNA variants that encode various epitope tags that are resistant to proteolysis (Fig. 2 and Section III) (Barends et al, 2010; Fujihara et al, 2002; Gottesman et al, 1998; Roche and Sauer, 2001; Thibonnier et al, 2008; Yang and Glover, 2009). These tmRNA variants allow tagging activity to be assessed by immunoblot and have facilitated the identification of several ssrA-tagged proteins in a variety of bacteria (Barends et al, 2010; Collier et al, 2002; Fujihara et al, 2002; Roche and Sauer, 2001).…”

“…This ssrA(DD) peptide tag is also resistant to degradation in the cytoplasm (Keiler et al, 1996), and several studies have shown that the hydrophobic C-terminus of ssrA is critical for degradation by cytoplasmic proteases (Flynn et al, 2001; Gottesman et al, 1998; Herman et al, 1998). This recognition mode is highly conserved throughout eubacteria and tmRNA variants encoding charged C-terminal residues have been used to stabilize tagged proteins in a variety of species (Fujihara et al, 2002; Hong et al, 2007; Keiler and Shapiro, 2003b; Thibonnier et al, 2008; Wiegert and Schumann, 2001; Yang and Glover, 2009). …”

The Transfer-Messenger RNA-Small Protein B System Plays a Role in Avian Pathogenic Escherichia coli Pathogenicity

“…2 to 4% of all protein chains are tagged by the tmRNA system (66), indicating that the tmRNASmpB system plays an important and general role in bacterial pathogenesis (67). In bacteria, the tmRNA quality control system is the primary mediator of ribosome rescue and thus is critical for several aspects of bacterial fitness, including growth and development, stress responses, and pathogenesis (29,68,69). Further investigations are required to fully elucidate the mechanism by which the tmRNA-SmpB system influences the pathogenesis of APEC.…”