Cloning and expression of a gene coding for the prolipoprotein signal peptidase of Escherichia coli

Abstract

An Escherichia coli mutant, Y815, has a temperature-sensitive prolipoprotein signal peptidase. IPTGinduced synthesis of the major outer membrane prolipoprotein (PLP) results in the inhibition of cell growth because of accumulation of PLP in its envelope [J. Bacterial. (1982) 152, 1163-11681. The 2000 E. coli strains of Clarke and Carbon's collection were screened for the presence of a plasmid complementing the IPTG-sensitivity of the growth of Y815. One plasmid, pLC3-13, complemented the IPTG-sensitivity.The envelope fraction prepared from Y815 transformed by pLC3-13 showed high activity of the PLP signal peptidase in vitro at high temperature. A 4 kb AccI fragment subcloned onto plasmid pHYOO1 was shown to carry the gene for the PLP signal peptidase. Cloning

The gene coding for lipoprotein signal peptidase (lspA) and that for isoleucyl‐tRNA synthetase (ileS) constitute a cotranscriptional unit in Escherichia coli

“…pYKlOT was constructed from pYKl0 by lrpP0 insertion at the XhoI site 3. RESULTS AND DISCUSSION 3.1. pHYl1 carries both the 1spA and ileS genes Plasmid pHY 11 possesses the /SPA-carrying 4.3 kb fragment derived from the E. coli chromosome [2]. Genetic complementation experiments in [4] did not support the existence of the ileS gene in this plasmid.…”

“…The IspA gene coding for the signal peptidase has been cloned [2,3] and mapped at about 0.5 min on the E. coli chromosome map near the ileS gene that codes for isoleucyl-tRNA synthetase [4].…”

Nucleotide sequence of the lspA gene, the structural gene for lipoprotein signal peptidase of Escherichia coli

“…pKD15 was constructed by insertion of the IspA gene-carrying 4.3 kb EcoRI fragment of pHYl1 [7] into the EcoRI site of pSY343, a runaway replication plasmid vector [8]. The E. cc& strain used was JE5506 [9], which was grown on L broth.…”

The major outer membrane lipoprotein and new lipoproteins share a common signal peptidase that exists in the cytoplasmic membrane of Escherichia coli

“…These in-+ To whom correspondence should be addressed Abbreviations: LP, major outer membrane lipoprotein; PAL, peptidoglycan-associated lipoprotein; NLP, new lipoprotein; pro-LP, pro-PAL, and pro-NLP, glyceridecontaining precursors of LP, PAL and NLPs, respectively; SDS, sodium dodecyl sulfate elude the major outer membrane lipoprotein (LP), the peptidoglycan-associated lipoprotein (PAL) and additional lipoproteins (NLPs) that have been found only recently. The gene (IspA) coding for signal peptidase for pro-LP has been isolated [6], and mapped on the E. coli chromosome [7]. The signal peptidase was distinct from that for the Ml3 procoat [8].…”