EF-P Posttranslational Modification Has Variable Impact on Polyproline Translation in Bacillus subtilis.

Area of Science:

• Molecular Biology
• Microbiology
• Protein Biochemistry

Background:

• Elongation factor P (EF-P) is crucial for bacterial protein synthesis, particularly for translating polyproline sequences.
• EF-P requires posttranslational modification (PTM) for its function, but the pathways are diverse across species.
• In Bacillus subtilis, EF-P is modified with a 5-aminopentanol moiety at Lys32.

Purpose of the Study:

• To identify genes involved in the 5-aminopentanolylation of EF-P in Bacillus subtilis.
• To elucidate the novel EF-P posttranslational modification pathway in B. subtilis.
• To investigate the functional consequences of EF-P modification on translation and cellular phenotypes.

Main Methods:

• Forward genetic screen to identify genes essential for EF-P modification.
• Tandem mass spectrometry to analyze PTMs in mutant strains.
• Structural analysis of EF-P modifications.
• Phenotypic characterization of PTM mutants under various growth conditions.
• In vivo polyproline reporter assays.

Main Results:

• Identified genes ynbB, gsaB, and ymfI as required for 5-aminopentanolylation, and yaaO, yfkA, and ywlG as influencing modification levels.
• Evidence suggests 5-aminopentanol is assembled directly on EF-P via a novel pathway.
• EF-P PTM mutants exhibit varied phenotypes, differing from the efp mutant and wild-type depending on growth conditions.
• Phenotypic differences correlate with variations in polyproline translation defects and EF-P context dependence.

Conclusions:

• Established a novel EF-P posttranslational modification pathway in Bacillus subtilis.
• Demonstrated that EF-P modifications can be directly assembled on the protein.
• Highlighted a unique relationship between EF-P modification state, polyproline translation efficiency, and context dependence.