Affiliation:
1. Departments of Microbiology
2. Biochemistry, University of Illinois, Urbana, Illinois 61801
Abstract
ABSTRACT
Acyl carrier proteins (ACPs) are very small acidic proteins that play a key role in fatty acid and complex lipid synthesis. Moreover, recent data indicate that the acyl carrier protein of
Escherichia coli
has a large protein interaction network that extends beyond lipid synthesis. Despite extensive efforts over many years, no temperature-sensitive mutants with mutations in the structural gene (
acpP
) that encodes ACP have been isolated. We report the isolation of three such mutants by a new approach that utilizes error-prone PCR mutagenesis, overlap extension PCR, and phage λ Red-mediated homologous recombination and that should be generally applicable. These mutants plus other experiments demonstrate that ACP function is essential for the growth of
E. coli
. Each of the mutants was efficiently modified with the phosphopantetheinyl moiety essential for the function of ACP in lipid synthesis, and thus lack of function at the nonpermissive temperature cannot be attributed to a lack of prosthetic group attachment. All of the mutant proteins were largely stable at the nonpermissive temperature except the A68T/N73D mutant protein. Fatty acid synthesis in strains that carried the D38V or A68T/N73D mutations was inhibited upon a shift to the nonpermissive temperature and in the latter case declined to a small percentage of the rate of the wild-type strain.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
28 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献