The general protein‐export pathway is directly required for extracellular pullulanase secretion in Escherichia coli k12

Abstract

Pullulanase is an extracellular, cell surface-anchored lipoprotein produced by Gram-negative bacteria belonging to the genus Klebsiella. Its correct localization in recombinant Escherichia coli requires the products of 14 genes that are linked to the enzyme structural gene in the Klebsiella chromosome. In addition, we show here that six sec genes (secA, secB, secD, secE, secF and secY) are all required for processing of the prepullulanase signal peptide to occur. This implies that pullulanase crosses the cytoplasmic membrane via the general export pathway of which the sec gene products are essential components. Removal or drastic alteration of the prepullulanase signal peptide cause the enzyme to remain cytoplasmic. We propose that pullulanase secretion occurs in two steps, the first of which is common to all signal peptide-bearing precursors of exported and secreted proteins, whereas the second is specifically involved in translocating pullulanase to the cell surface.

XpsD, an Outer Membrane Protein Required for Protein Secretion by Xanthomonas campestris pv. campestris, Forms a Multimer

“…Mutations in the xpsD gene caused the accumulation of extracellular enzymes in the periplasm (1). These enzymes are probably exported from cytoplasm in the first step via a Sec-like pathway (7). Homologues of XpsD are widespread in Gram-negative bacteria.…”

Neisseria gonorrhoeae prepilin export studied in Escherichia coli

“…Cells grown in L broth were converted into spheroplasts as described previously (33). More than 98% of the cells were converted into spheroplasts (as judged by light microscopy) within 15 min, at which time different concentrations of trypsin or proteinase K were added and the mixtures were incubated at 30°C.…”

“…Immunoblots of gels loaded with increasing amounts of PhoA hybrid proteins and developed with antiPhoA serum and 35S-labelled protein A were quantified by counting the radioactivity in immunoreactive bands excised from the nitrocellulose sheets. For pulse-chase labelling and immunoprecipitation, cells were grown in low-potassium minimal medium and labelled at 37°C with [35S]methionine (200 ,u.Ci/ml) essentially as described previously (33). Labelling was arrested by adding cold methionine (0.1%), and incubation was continued (chase).…”

“…Immunoblotting and SDS-polyacrylamide gel electrophoresis (PAGE) were performed as previously described (33,45). Immunoblots of gels loaded with increasing amounts of PhoA hybrid proteins and developed with antiPhoA serum and 35S-labelled protein A were quantified by counting the radioactivity in immunoreactive bands excised from the nitrocellulose sheets.…”

“…Samples were collected and immediately mixed with an equal volume of 2% SDS and heated to 100°C for 5 min. Immunoprecipitation with antiPhoA serum was then carried out, and samples were analyzed by SDS-PAGE and autoradiography as described previously (33).…”