Escherichia coli signal peptides direct inefficient secretion of an outer membrane protein (OmpA) and periplasmic proteins (maltose-binding protein, ribose-binding protein, and alkaline phosphatase) in Bacillus subtilis

Abstract

Signal peptides of gram-positive exoproteins generally carry a higher net positive charge at their amino termini (N regions)

Expression of Escherichia coli SecB in Bacillus subtilis facilitates secretion of the SecB-dependent maltose-binding protein of E. coli

“…Genes encoding E. coli envelope proteins were placed under transcriptional control of the Bacillus amyloliquefaciens alkaline protease promoter (aprp) (30) provided by the shuttle vector pBE1020 (6). The pBE1020 derivatives pBE1064, pBE1065, and pBE1146, carrying operon fusions .F(aprp-rbsB,,-malE)(Hyb), 'F(aprpmalE), and 1D(aprp-ompA), respectively, have been described previously (6).…”

“…Genes encoding E. coli envelope proteins were placed under transcriptional control of the Bacillus amyloliquefaciens alkaline protease promoter (aprp) (30) provided by the shuttle vector pBE1020 (6). The pBE1020 derivatives pBE1064, pBE1065, and pBE1146, carrying operon fusions .F(aprp-rbsB,,-malE)(Hyb), 'F(aprpmalE), and 1D(aprp-ompA), respectively, have been described previously (6). secB was obtained by PCR amplification (DNA Thermal Cycler and Gene Amp Kit; Perkin-Elmer Co., Norwalk, Conn.) of MC4100 chromosomal DNA with PCR primers that introduced an NdeI restriction site overlapping the translation initiation site and an XbaI site immediately 3' to the termination codon.…”

“…Cultures of BE2000 harboring the appropriate plasmids were grown at 30'C to late logarithmic phase in modified S7 media and pulse-chase radiolabeled with L-[35S]methionine (DuPont Co. NEN Research Products, Boston, Mass.) as previously described (6). At various times after initiation of chase, samples were processed for immunoprecipitation (23) with polyclonal rabbit antisera specific for MBP, RBP, OmpA, or SecB (kind gift of P. J. Bassford, Jr.).…”

“…However, several secretory proteins of E. coli, including the maltosebinding protein (MBP) and OmpA, were shown to be only inefficiently secreted in B. subtilis (6). In E. coli, efficient secretion of these proteins requires the molecular chaperone SecB (7,18,19).…”

SecA proteins of Bacillus subtilis and Escherichia coli possess homologous amino-terminal ATP-binding domains regulating integration into the plasma membrane

“…Although we also undertook experiments to determine whether secA mutant strains containing div or various div-secA chimeras promoted better translocation of the gram-positive preprotein, levansucrase (34), than the secA control strain, we were unable to perform these experiments because of toxicity of the levansucrase-producing plasmids in certain of our strains. It has been noted previously that gram-positive and gram-negative signal peptides exhibit statistical differences in both overall length and charge distribution (59), and there are limits on their interchangeability depending on the specific protein context (9,31). Clearly, additional genetic and biochemical studies will be required to characterize the various interactions that Div and SecA have with the components of the secretion pathway and to determine the basis of the observed species specificity.…”