PMID:16352845

Citation	De Lay, NR and Cronan, JE (2006) Gene-specific random mutagenesis of Escherichia coli in vivo: isolation of temperature-sensitive mutations in the acyl carrier protein of fatty acid synthesis.J. Bacteriol. 188:287-96
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 lambda 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.
Links	PubMed Online version:10.1128/JB.188.1.287-296.2006
Keywords	Acyl Carrier Protein; Amino Acid Substitution; Apoproteins; Escherichia coli K12; Escherichia coli Proteins; Fatty Acids; Genes, Essential; Genetic Techniques; Models, Molecular; Mutagenesis; Polymerase Chain Reaction; Recombination, Genetic; Temperature
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Materials and Methods Used

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Phenotype Annotations

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Species	Taxon ID	Strain	Gene (if known)	OMP	Phenotype	Details	Evidence	Notes
Escherichia coli		NRD53	acpP	no growth at high temperature temperature-sensitive for growth	Growth	at 42C	Plating Assay	Figure 2
Escherichia coli		NRD50	acpP	leaky growth at high temperature temperature-sensitive for growth	Growth	at 42C	Plating Assay	Figure 2
Escherichia coli		NRD51	acpP	no growth at high temperature temperature-sensitive for growth	Growth	at 37C or 42C	Plating Assay	Figure 2
Escherichia coli		NRD28	acpP (ts)	no growth at high temperature temperature-sensitive for growth	Growth	at 44C, ceased normal growth after 30 minutes, and ceased growth altogether within 150 minutes	Growth Curve	Figure 3A and 3B
Escherichia coli		NRD29	acpP (ts)	temperature-sensitive decrease in growth rate	Growth	at 44C, ceased normal growth after 30 minutes, continuing to grow at a lesser rate	Growth Curve	Figure 3A and 3B
Escherichia coli		NRD28	acpP (ts)	decreased rate of fatty acid synthesis	Metabolic Activity	one-third the rate of the permissive temperature (30C)	Biochemical Assay	Figure 3C- incorporation of [1-14C]acetate
Escherichia coli		NRD29	acpP (ts)	decreased rate of fatty acid synthesis	Metabolic Activity	one-third the rate of the permissive temperature (30C)	Biochemical Assay	Figure 3C- incorporation of [1-14C]acetate
Escherichia coli		NRD29	acpP (ts)	decreased rate of fatty acid synthesis	Metabolic Activity	one-tenth the rate of the permissive temperature (44C)	Biochemical Assay	Figure 3C- incorporation of [1-14C]acetate
Escherichia coli		NRD28	acpP (ts)	decreased rate of fatty acid synthesis	Metabolic Activity	small percentage of the rate of the permissive temperature (44C)	Biochemical Assay	Figure 3C- incorporation of [1-14C]acetate
Escherichia coli		NRD62	?acpP	essential for growth	Growth	only grew in the presence of inducer	Plating Assay	Figure 2C-acpP was supplied in trans on an inducible plasmid
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