Increased expression of the bifunctional protein PrlF suppresses overproduction lethality associated with exported β-galactosidase hybrid proteins in Escherichia coli

D. R. Kiino, G. J. Phillips, T. J. Silhavy

Research output: Contribution to journalArticle

14 Scopus citations

Abstract

We have cloned and determined the nucleotide sequence of the prlF gene. An open reading frame predicting a 111-amino-acid protein (M(r) 12,351) with an acidic carboxy terminus was identified. The DNA sequence preceding this open reading frame revealed a putative promoter and a ribosome-binding site. The nucleotide sequence of the prlF1 mutation revealed a 7-base-pair duplication resulting in a slightly smaller predicted gene product of M(r) 12,009 that lacked the acidic carboxy terminus. Maxicell analysis of prlF and prlF1 subclones identified peptides of sizes similar to those predicted by the nucleotide sequences. The prlF sequence was shown to be expressed in vivo by both maxicell analysis and construction of a prlF-lacZ fusion. Two kanamycin resistance insertions within the prlF open reading frame were introduced into the chromosome, replacing the wild-type gene. In contrast to the prlF1 mutation, these insertions had no detectable effect on cell growth or on the β-galactosidase activity or maltose sensitivity (two sensitive indicators of hybrid protein export) conferred by the lamB-lacZ42-1 gene fusion. Overproduction of the wild-type prlF gene product from a plasmid carrying an active hybrid promoter, however, conferred a prlF1 phenotype. In addition, both the prlF1 mutation and both kanamycin resistance insertions increased the β-galactosidase activity of a prlF-lacZ fusion. These results suggest that prlF is autoregulated and that overproduction of the prlF gene product increases the export efficiency of β-galactosidase hybrid proteins from the cytoplasm.

Original languageEnglish (US)
Pages (from-to)185-192
Number of pages8
JournalJournal of bacteriology
Volume172
Issue number1
DOIs
StatePublished - 1990

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology

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