A mutation (lspA, prolipoprotein signal peptidase) rendering the prolipoprotein signal peptidase temperature-sensitive in Escherichia coli has been analyzed. The mutation was mapped in the dnaJ-rpsT-ileS-dapB region by interrupted mating with various Hfr strains and P1 phage transduction. lambda transducing phage lambda ddapB2 that carries the rpsT-ileS-dapB region was shown to complement the lspA mutation. Plasmid pLC3-13 which had been isolated from Clarke and Carbon's collection as a plasmid carrying the lspA locus was shown to carry the dnaJ and rpsT loci. Complementation analysis with plasmids carrying various DNA fragments derived from pLC3-13 showed that the lspA locus is between the rpsT and ileS loci. The wildtype allele was dominant over the lspA allele.
The nucleotide sequence of the tspA gene coding for lipoprotein signal peptidase of Escherichia coti was determined and the amino acid sequence of the peptidase was deduced from it. The molecular mass and amino acid composition of the predicted lipoprotein signai peptidase were consistent with those of the signal peptidase purified from ceils harboring the tspA gene-carrying plasmid. The peptidase most probably has no cleavable signal peptide. The tspA gene was preceded by the iteS gene coding for isoleucyltRNA synthetase and the tandem termination codons of the iteS gene overlapped with the initiation codon of the tspA gene.Lipoprotein signat peptidase DNA sequence Lipoprotein
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
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