Limited tolerance towards folded elements during secretion of the autotransporter Hbp

The Bam (Omp85) complex is involved in secretion of the autotransporter haemoglobin protease

“…The disulphide bond precluded OM translocation and targeted Hbp110C/348C for degradation by the periplasmic protease DegP. Importantly, in the absence of DegP, Hbp110C/348C accumulated in the OM, with part of the passenger exposed at the cell surface (Jong et al, 2007). Here, we have further characterized the OM association and folding of the b-domain of this mutant, which appears not to be folded into a heat-modifiable b-barrel and which is not completely inserted into the OM.…”

“…We propose Hbp110C/348C to represent a translocation intermediate that is stalled during translocation across the OM due to the incompatibility of a disulphide bond between domains of the passenger and transfer across a relatively narrow channel. Sedimentation gradient analysis and differential detergent solubilization have shown that Hbp110C/348C cofractionates with the OM mainly as pro-Hbp (Jong et al, 2007). To further confirm that Hbp110C/348C is located in the OM rather than forming aggregates in the periplasm, we performed flotation gradient analysis to show that Hbp110C/348C floats to the same position as the OM porins ( Supplementary Fig.…”

“…S1). Our previous whole-cell immunofluorescence data have also shown that Hbp110C/348C is in part surface exposed (Jong et al, 2007). To enable detection by immunofluorescence, Hbp110C/348C was expressed at a relatively high level upon induction with 200 mM IPTG.…”

“…1d). This Hbp110C/348C variant was shown to form an intramolecular disulphide bond, catalysed by DsbA, in the periplasm, indicating at least partial folding in the periplasm (Jong et al, 2007). The disulphide bond precluded OM translocation and targeted Hbp110C/348C for degradation by the periplasmic protease DegP.…”

“…1a, b). This 'classical' view of AT transport has recently been called into question as (i) crystal structures of b-domains Meng et al, 2006;Oomen et al, 2004) and molecular dynamics simulations (Khalid & Sansom, 2006) suggest that the pore formed by the bdomain only allows translocation of extended unfolded protein chains whereas various passengers can be translocated in a partially folded state (Jong et al, 2007;Skillman et al, 2005) and (ii) secretion of several ATs is significantly affected in cells depleted of BamA (or Omp85), the core component of the OM protein (OMP) assembly machinery (Jain & Goldberg, 2007;Voulhoux et al, 2003). Notably, BamA is homologous to the TpsB protein that acts as OM translocator in the 'two-partner secretion pathway', a secretion system related to the autotransporter pathway (Jacob-Dubuisson et al, 2009).…”