Difference between revisions of "PMID:16352845"

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'''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
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!align=left  |Abstract
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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.
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[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16352845 PubMed]
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Online version:[http://dx.doi.org/10.1128/JB.188.1.287-296.2006 10.1128/JB.188.1.287-296.2006]
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!align=left  |Keywords
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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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==Main Points of the Paper ==
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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
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==Notes==
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==References==
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<references/>

Revision as of 14:49, 16 March 2011

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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Main Points of the Paper

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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
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Notes

References

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