Abstract
Secretion of the Escherichia coli toxin hemolysin A (HlyA) is catalyzed by the membrane protein complex HlyB-HlyD-TolC and requires a secretion sequence located within the last 60 amino acids of HlyA. The Hly translocator complex exports a variety of passenger proteins when fused N-terminal to this secretion sequence. However, not all fusions are secreted efficiently. Here, we demonstrate that the maltose binding protein (MalE) lacking its natural export signal and fused to the HlyA secretion signal is poorly secreted by the Hly system. We anticipated that folding kinetics might be limiting secretion, and we therefore introduced the "folding" mutation Y283D. Indeed this mutant fusion protein was secreted at a much higher level. This level was further enhanced by the introduction of a second MalE folding mutation (V8G or A276G). Secretion did not require the molecular chaperone SecB. Folding analysis revealed that all mutations reduced the refolding rate of the substrate, whereas the unfolding rate was unaffected. Thus, the efficiency of secretion by the Hly system is dictated by the folding rate of the substrate. Moreover, we demonstrate that fusion proteins defective in export can be engineered for secretion while still retaining function.
Highlights
Introduction of a Second Slow FoldingMutation in maltose binding protein (MalE)(Y283D)-HlyAc Further Enhances Secretion—Above, it was shown that slow folding variants of MalE-HlyAc fusion protein are secreted at a higher level than the wild-type fusion protein
Mutations That Affect Folding of MalE Facilitate Secretion via Hly System—The Hly type 1 secretion systems (T1SS) is able to export a large variety of proteins that are C-terminally fused to a fragment containing the hemolysin A (HlyA) secretion sequence [12, 22,23,24, 37]
We introduced the previously described “slow folding” suppressor mutations V8G, A276G, and Y283D in MalE and investigated the secretion of these MalE variants when fused to the HlyAc fragment (Fig. 2A)
Summary
Bacterial Strains and Plasmids—The E. coli strain WM2429 used in this study for expression and secretion analysis is a BW25113 derivative [31] that lacks the araBAD genes and is designed for arabinose-dependent induction systems. The pBADHisB plasmid was used as the vector for the fusion proteins expressed from an arabinose promoter, whereas pK184 was used as the vector for the production of HlyB and HlyD from a lac promoter (isopropyl 1-thio--Dgalactopyranoside-inducible) Cells harboring both plasmids were grown in the presence of ampicillin (100 g/ml) and kanamycin (30 g/ml). Cells were grown in LB medium containing isopropyl 1-thio--D-galactopyranoside at a final concentration of 1.5 mM for the production of the inner membrane proteins HlyB and HlyD. Cells were grown with agitation at 30 °C to an A600 of 0.8, and production of the HlyAc fusion proteins was induced by the addition of arabinose to a final concentration of 10 mM. Fobs is the fluorescence observed at a certain time point, F0 is the fluorescence observed at time 0, F∞ is the fluorescence at infinite time, and k is the rate constant
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