Streptococcus mutans is one of the major etiological agents of dental caries (Lemos et. al., 2019, Microbiology Spectrum). Most of the virulence properties of S. mutans, such as acidogenicity, acidurance, mutacin production, competence, and biofilm formation, rely on membrane proteins. It is important to understand how membrane proteins are translocated from the site of synthesis to that of their function.
Studies in model organisms have shown that membrane protein translocation is a co-translational process, whereby nascent polypeptide chains emerging from ribosomes (Ribosome Nascent Chain) forms a complex with signal recognition particle (SRP-RNC). In order to understand the individual contributions of various machinery components, we are interested in developing a cell free (in vitro) transcription/translation/translocation system and are in the process of assembling various components. An important consideration in the in vitro system is to stall the ribosomes to produce nascent ribosome chains. That being said, the conventionally used secM stalling sequence, which is appended to the gene encoding the substrate in question to impede its translation, is lacking in S. mutans. To counter this issue, we want to take advantage of the fact that the elongation factor-P (Ef-P) facilitates translocation of proteins with consecutive prolines, and its elimination would likely impede translation of proteins with consecutive prolines. I will test this hypothesis by:
- Characterization of growth phenotype of ∆efp mutant under various environmental conditions.
I will compare the growth of S. mutans wild type and isogenic ∆efp mutant under non-stress and stress conditions, such as cold temperature, salt-stress, acid stress, and in the defined media with multiple sugars, to test whether Ef-P elimination impedes growth or not.
- Construction of an expression plasmid with putative small membrane (AtpH) fused to GFP using a poly-proline (3xPro) linker.
Resultant plasmid (pDL289 background) will be transformed in wild type and ∆efp strains, then measurement of GFP expression in both strains is expected to tell us the efficiency of poly-prolines in stalling. I expect ∆efp mutant to stall protein synthesis on encountering consecutive prolines and therefore not synthesize GFP, while wild type will have GFP expression.
I expect that on completion, this project will facilitate an essential tool to isolate ribosomes with RNCs from S. mutans for in vitro transcription/translation/translocation assay.