Cyclophilin PpiB is involved in motility and biofilm formation via its functional association with certain proteins

Abstract

PpiB belongs to the superfamily of peptidyl-prolyl cis/trans isomerases (PPIases, EC: 5.2.1.8), which catalyze the rate-limiting protein folding step at peptidyl-prolyl bonds and control several biological processes. In this study, we show that PpiB acts as a negative effector of motility and biofilm formation ability of Escherichia coli. We identify multicopy suppressors of each DppiB phenotype among putative PpiB prey proteins which upon deletion are often characterized by analogous phenotypes. Many putative preys show similar gene expression in wild-type and DppiB genetic backgrounds implying possible post-translational modifications by PpiB. We further conducted in vivo and in vitro interaction screens to determine which of them represent true preys. For DnaK, acetyl-CoA carboxylase, biotin carboxylase subunit (AccC) and phosphate acetyltransferase (Pta) we also showed a direct role of PpiB in the functional control of these proteins because it increased the measured enzyme activity of each protein and further interfered with DnaK localization and the correct folding of AccC. Taken together, these results indicate that PpiB is involved in diverse regulatory mechanisms to negatively modulate motility and biofilm formation via its functional association with certain protein substrates.

Structural and functional analysis of cyclophilin PpiB mutants supports an in vivo function not limited to prolyl isomerization activity

“…Previously, we have shown that PpiB modulates various bacterial behaviors such as swimming and swarming motility and biofilm formation ability in a negative way (Skagia et al . ). In many reported cases, elimination of the prolyl isomerase activity of a PPIase had no or little impact on protein interactions and/or chaperoning activities, indicating that PPIases can also act as chaperones or accessory components of multiprotein complexes (Göthel & Marahiel ; Chakraborty et al .…”

“…The previously described primers carry restriction sites for ligation to the pCDFDuet‐1 expression vector (Skagia et al . ). The fragments excised from amplified sequences were cloned between the corresponding sites of pCDFDuet‐1, resulting in F99A His ‐Prey S .pCDFDuet‐1.…”

“…Furthermore, the cytoplasmic cyclophilin PpiB is involved in swarming and swimming motility and biofilm formation ability (Skagia et al . ). In Bacillus subtilis , disruption of both cyclophilin PpiB and trigger factor, an FKBP, has no effect on cell viability in rich medium or under several stress conditions, but decelerates the growth in poor medium, signifying the essential role of PPIases during starvation (Göthel et al .…”

Structure-Function Analysis of the Periplasmic <b><i>Escherichia coli</i></b> Cyclophilin PpiA in Relation to Biofilm Formation

“…The swarming motility plates were prepared with 0.5% Fluka agar, 1% Bacto-Tryptone, 0.5% yeast extract, 1% NaCl, and 0.5% D-(+)-glucose. Biofilm formation and quantification assays were performed as previously described [O'Toole and Kolter, 1998;Skagia et al, 2016].…”

“…Previously, we have shown that the cytoplasmic cyclophilin PpiB has a negative effect on swimming and swarming motility and the biofilm formation ability of E. coli in a way that employs the prolyl isomerase activity of the enzyme only in the 2 latter cases [Skagia et al, 2016;Skagia et al, 2017]. Since the periplasmic cyclophilin PpiA is regulated by the Cpx 2-component system which senses also adhesion [Dorel et al, 2006;Vogt and Raivio, 2012], we hypothesized that PpiA might be involved in biofilm formation as well.…”