Abstract
Consistent with a role in catalyzing rate-limiting step of protein folding, removal of genes encoding cytoplasmic protein folding catalysts belonging to the family of peptidyl-prolyl cis/trans isomerases (PPIs) in Escherichia coli confers conditional lethality. To address the molecular basis of the essentiality of PPIs, a multicopy suppressor approach revealed that overexpression of genes encoding chaperones (DnaK/J and GroL/S), transcriptional factors (DksA and SrrA), replication proteins Hda/DiaA, asparatokinase MetL, Cmk and acid resistance regulator (AriR) overcome some defects of Δ6ppi strains. Interestingly, viability of Δ6ppi bacteria requires the presence of transcriptional factors DksA, SrrA, Cmk or Hda. DksA, MetL and Cmk are for the first time shown to exhibit PPIase activity in chymotrypsin-coupled and RNase T1 refolding assays and their overexpression also restores growth of a Δ(dnaK/J/tig) strain, revealing their mechanism of suppression. Mutagenesis of DksA identified that D74, F82 and L84 amino acid residues are critical for its PPIase activity and their replacement abrogated multicopy suppression ability. Mutational studies revealed that DksA-mediated suppression of either Δ6ppi or ΔdnaK/J is abolished if GroL/S and RpoE are limiting, or in the absence of either major porin regulatory sensory kinase EnvZ or RNase H, transporter TatC or LepA GTPase or Pi-signaling regulator PhoU.
Highlights
To be functionally active, all newly synthesized polypeptides must rapidly fold into their native three-dimensional structure
Individually all six cytoplasmic peptidyl-prolyl cis/trans isomerases (PPIs) are non-essential for bacterial viability, collectively the removal of all such enzymes confers synthetic lethality, which is manifested by the inability of such ∆6ppi bacteria to grow on rich medium and the temperature sensitivity (Ts) on rich as well as minimal medium [1]
We further characterized various growth defects of ∆6ppi bacteria and show that they exhibit highly pleiotropic phenotypes. These include the inability to grow under fast-growing conditions of rich medium, and the sensitivity to: (i) DNA-damaging agents such as nalidixic acid indicating that the genome integrity of ∆6ppi bacteria is compromised; (ii) cell envelope-destabilizing agents; and (iii) the exposure to ethanol and the inability to grow on minimal medium, when glycerol was used as the sole carbon source
Summary
All newly synthesized polypeptides must rapidly fold into their native three-dimensional structure. E.g., Escherichia coli, as well as in other organisms, these factors are molecular chaperones and folding catalysts, whose requirements vary, depending on the individual functional or folding requirement of client protein(s). For the peptide bonds preceding proline, steric hindrance is comparable between two isomers. They are nearly isoenergetic (about 0.5 kcal/mol of free energy difference) and both conformations are thermodynamically possible [4,5]. The E. coli cytoplasm contains six PPIs, which cover all three families Their in vivo physiological requirement and specific substrates that require the PPIase activity have not been fully addressed
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