Impact of C-terminal amino acid composition on protein expression in bacteria

Abstract

The C-terminal sequence of a protein is involved in processes such as efficiency of translation termination and protein degradation. However, the general relationship between features of this C-terminal sequence and levels of protein expression remains unknown. Here, we identified C-terminal amino acid biases that are ubiquitous across the bacterial taxonomy (1582 genomes). We showed that the frequency is higher for positively charged amino acids (lysine, arginine) while hydrophobic amino acids and threonine are lower. In highly abundant proteins, the C-terminal residue is more conserved. We then studied the impact of C-terminal composition on protein levels in a library of M. pneumoniae mutants, covering all possible combinations of the two last codons. We found that charged and polar residues, in particular lysine, led to higher expression, while hydrophobic and aromatic residues led to lower expression, with a difference in protein levels up to 4-fold. Our results demonstrate that the identity of the last amino acids has a strong influence on protein expression levels and is under selective pressure in highly expressed proteins.

Protein quality control and regulated proteolysis in the genome‐reduced organism Mycoplasma pneumoniae

“…We found that hydrophobic sequences as short as a single residue can promote Lon-dependent degradation of functional proteins in M. pneumoniae. In this line, we have recently shown that protein abundances can be influenced by the identity of the last C-terminal amino acid, with hydrophobic residues associated with faster degradation rates (Weber et al, 2020). In vitro studies performed with the E. coli Lon protease has suggested that sequences rich in aromatic residues that are accessible in misfolded proteins act as Lon recognition signals (Gur & Sauer, 2008).…”

“…To model amino acid substitutions and quantitatively predict their impact on protein stability, we run the command BuildModel that predicted a ΔΔG of 3.228 kcal/mol for the F89E mutation. Disruption of activity of the Fluc variant was confirmed by using the ONE-Glo ™ Luciferase Assay System (Promega) as previously described (Weber et al, 2020). Primers and Gibson cloning strategy to generate the F89E luc variant are detailed in Reagents and Tools Table (Appendix Tables S3 and S2).…”

“…To obtain Lon and FtsH conditional mutants, M129_GP35 and M129 + pMTnTc_IndFtsH strains were transformed respectively with 3 μg of the corresponding ssDNA recombineering substrate (see above). Bacteria transformation was accomplished by electroporation as previously described (Weber et al, 2020). To allow GP35 mediated recombination, electroporated cells were cultured in 25cm 2 flasks containing 5 ml of Hayflick medium during 24 h. Then, cells were recovered and mutants selected in Hayflick agar plates containing 20 µg/ml chloramphenicol and 100 ng/ml tetracycline to induce Lon and/or FtsH expression.…”

“…All FLAG variants were expressed from the P438 promoter (Pich et al, 2006). The resulting transposon vectors were transformed into the corresponding strain (ΔIndLon or ΔIndFtsH) by electroporation as previously described (Weber et al, 2020). To allow selection of transformants in the ΔIndFtsH strain, we excised the cat resistance marker by Cre recombination as previously described (Mariscal et al, 2016).…”

FASTQINS and ANUBIS: two bioinformatic tools to explore facts and artifacts in transposon sequencing and essentiality studies

“…Wildtype M. pneumoniae strain M129 (WT) was grown in modified Hayflick medium ( 31 ) at 37°C under 5% CO 2 in tissue culture flasks. To generate M. pneumoniae mutant libraries, 2 μg of mini-transposon plasmid DNA (pMTnCat_BDPr) was electroporated as previously described ( 32 ). The resulting transformants were selected during 5 days in 5 ml of culture medium supplemented with 20 μg/ml of chloramphenicol, and then harvested in 1 ml of fresh medium.…”