Two distinct steps in pullulanase secretion by Escherichia coli K12

Area of Science:

• Microbiology
• Molecular Biology
• Protein Secretion

Background:

• Escherichia coli secretes extracellular lipoproteins like pullulanase.
• Efficient secretion involves multiple genetic pathways and protein translocation steps.

Purpose of the Study:

• To investigate the distinct steps and conformational changes during pullulanase secretion in E. coli.
• To characterize the secretion intermediate and its structural properties.

Main Methods:

• Uncoupling of secretion steps by manipulating export pathways (signal peptide/Sec pathway and pulC-O operon).
• Cell lysis, vesicle fractionation via isopycnic sucrose density centrifugation.
• Biochemical assays including heat inactivation, SDS denaturation, carboxymethylation, and protease susceptibility (Proteinase K, trypsin).

Main Results:

• Secretion was successfully uncoupled into cytoplasmic membrane export and subsequent cell surface translocation.
• The secretion intermediate cofractionated with intermediate-density vesicles.
• The intermediate form exhibited distinct biochemical properties compared to cytoplasmic and cell-surface forms, including resistance to denaturation and protease digestion.
• Evidence suggests the intermediate acquires higher-ordered structure, including disulfide bridges, prior to cell surface transport.

Conclusions:

• The secretion intermediate of pullulanase possesses significant higher-ordered structure, including disulfide bonds.
• This structural acquisition occurs before translocation to the cell surface.
• Findings challenge the model of secretion intermediates being threaded through membranes as unfolded polypeptides.