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A gene fusion approach to the study of pullulanase export and secretion in Eschenchia coli
Author(s) -
D'Enfert C.,
Pugsley A.P.
Publication year - 1987
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1987.tb00508.x
Subject(s) - pullulanase , biology , escherichia coli , periplasmic space , biochemistry , gene , secretion , bacterial outer membrane , microbiology and biotechnology , enzyme
Summary A series of fusions between the gene for the Klebsiella pneumoniae secreted lipoprotein pullulanase (pulA) and the genes for cytoplasmic β‐galactosidase (lacZ) or periplasmic alkaline phosphatase (phoA) were created by transposon mutagenesis using mini‐Mudlt1681 or Tn phoA , respectively. The hybrid genes were expressed in Escherichia coli K‐12 with or without the K. pneumoniae genes that promote pullulanase secretion in E. coli. We characterized seven different pulA‐lacZ gene fusions encoding hybrid polypeptides containing from 14 to c. 1060 residues of pro‐pullulanase. All but the smallest hybrid were fatty acylated and were toxic to producing cells, causing the accumulation of precursors of other exported proteins. Four different pulA‐phoA gene fusions encoded hybrids with alkallne phosphatase activity. All four hybrids were fatty acylated, but were not toxic. Although the hybrids were apparently membrane‐associated, they were not secreted into the medium either by E. coli carrying pullulanase secretion genes or by K. pneumoniae. Immunofluorescence tests indicated that the pullulanase secretion genes promoted the localization of one of these hybrids to the outer face of the E. coli outer membrane, which may have important implications for the design of live vaccine. strains and of immobilized enzymes.