Nonfunctional expression of escherichia-coli signal peptidase-i in bacillus-subtilis

Jan Maarten van Dijl, Anne de Jong, Hilde Smith, Sierd Bron, Gerard Venema

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Abstract

The Escherichia coli lep gene, encoding signal peptidase I (SPase I) was provided with Bacillus subtilis transcription/translation signals and expressed in this organism. When present on a low-copy-number plasmid, the amount of E. coli SPase I produced (per mg cell protein) in B. subtilis was half that produced in wild-type E. coli cells. The production of E. coli SPace I in B. subtilis was increased approximately fivefold by cloning the lep gene into a high-copy-number plasmid. The expression of E. coli SPase I in B. subtilis did not appear to increase the rate of processing of two hybrid secretory precursor proteins. Two observations may explain the failure of E. coli SPase I to stimulate processing of exported proteins in B. subtilis. First, the E. coli SPase I was apparently not exposed on the outside of the B. subtilis cytoplasmic membrane, indicating its incorrect insertion into the membrane. Second, in vitro processing studies, using cell-free extracts of B. subtilis producing E. coli SPase I, suggested that the enzyme was not active. A further outcome of this study was that conditions favouring processing of precursors by SPase I in cell-free extracts of E. coli did not favour processing by the corresponding enzyme in B. subtilis cell-free extracts. This suggests that significant differences exist between the two enzymes. The observation that antibodies directed against E. coli SPase I did not cross-react with B. subtilis membrane proteins supports this idea.

Original languageEnglish
Pages (from-to)2073-2083
Number of pages11
JournalJournal of general microbiology
Volume137
Issue number9
DOIs
Publication statusPublished - Sep-1991

Keywords

  • ALPHA-COMPLEMENTATION SYSTEM
  • OUTER-MEMBRANE LIPOPROTEIN
  • LEADER PEPTIDASE
  • NUCLEOTIDE-SEQUENCE
  • MOLECULAR-CLONING
  • CYTOPLASMIC MEMBRANE
  • POLYACRYLAMIDE GELS
  • HYDROPHOBIC DOMAIN
  • PLASMA-MEMBRANE
  • BETA-LACTAMASE

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