Linker mutagenesis of a bacterial fatty acid transport protein

Identification of domains with functional importance

Geetha B. Kumar, Paul N Black

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

The product of the fadL gene (FadL) of Escherichia coli is a multifunctional integral outer-membrane protein required for the specific binding and transport of exogenous long-chain fatty acids [C12-C18]. FadL also serves as a receptor for the bacteriophage T2. In order to define regions of functional importance within FadL, the fadL gene has been mutagenized by the insertion of single-stranded hexameric linkers into the unique SalI restriction site that lies towards the 3′ end of the gene and into four HpaII restriction sites distributed throughout the coding region. The five insertion mutants were classified into three groups based on their specific growth rates (a) in minimal media containing the long-chain fatty acid oleate (C18:1) as a sole carbon and energy source: Oleslow, α = 0.035-0.045; Ole±, α = 0.020-0.035; and Ole-, α ≤ 0.005 (wild-type, α = 0.07-0.10). The hexameric insertion at the SalI site (fadL allele termed S1; insertion after amino acid 410) conferred an Oleslow phenotype and resulted in a reduction of long-chain fatty acid transport (36% the wild-type level). This insertion mutant, however, bound oleic acid at wild-type levels and was fully functional as a receptor for the bacteriophage T2. The modified FadL-S1 protein did not have the heat-modifiable property characteristic of wild-type FadL. Insertions in the four HpaII sites (fadL alleles termed H1, H2, H3, and H5; after amino acids 41, 81, 238, and 389, respectively) resulted in all three classes of mutants. The fadL insertion mutant H5 was defective for long-chain fatty acid transport but bound oleic acid at significant levels. Together with the S1 allele, these data suggest that the carboxyl terminus of FadL is crucial for long-chain fatty acid transport. The insertion mutants H1 and H2 were defective for both oleic acid binding and transport suggesting that the amino terminus of FadL is important for long-chain fatty acid binding and transport. The fadL linker mutant H3 was defective in oleic acid binding yet had significant levels of oleic acid transport. These studies delineated for the first time different regions of the fadL gene that encode domains of FadL implicated in the binding and transport of long-chain fatty acids.

Original languageEnglish (US)
Pages (from-to)1348-1353
Number of pages6
JournalJournal of Biological Chemistry
Volume266
Issue number2
StatePublished - Jan 15 1991

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Fatty Acid Transport Proteins
Mutagenesis
Oleic Acid
Fatty Acids
Genes
Bacteriophage T4
Bacteriophages
Alleles
Amino Acids
Protein Domains
Escherichia coli
Membrane Proteins
Carbon
Hot Temperature
Phenotype

ASJC Scopus subject areas

  • Biochemistry

Cite this

Linker mutagenesis of a bacterial fatty acid transport protein : Identification of domains with functional importance. / Kumar, Geetha B.; Black, Paul N.

In: Journal of Biological Chemistry, Vol. 266, No. 2, 15.01.1991, p. 1348-1353.

Research output: Contribution to journalArticle

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abstract = "The product of the fadL gene (FadL) of Escherichia coli is a multifunctional integral outer-membrane protein required for the specific binding and transport of exogenous long-chain fatty acids [C12-C18]. FadL also serves as a receptor for the bacteriophage T2. In order to define regions of functional importance within FadL, the fadL gene has been mutagenized by the insertion of single-stranded hexameric linkers into the unique SalI restriction site that lies towards the 3′ end of the gene and into four HpaII restriction sites distributed throughout the coding region. The five insertion mutants were classified into three groups based on their specific growth rates (a) in minimal media containing the long-chain fatty acid oleate (C18:1) as a sole carbon and energy source: Oleslow, α = 0.035-0.045; Ole±, α = 0.020-0.035; and Ole-, α ≤ 0.005 (wild-type, α = 0.07-0.10). The hexameric insertion at the SalI site (fadL allele termed S1; insertion after amino acid 410) conferred an Oleslow phenotype and resulted in a reduction of long-chain fatty acid transport (36{\%} the wild-type level). This insertion mutant, however, bound oleic acid at wild-type levels and was fully functional as a receptor for the bacteriophage T2. The modified FadL-S1 protein did not have the heat-modifiable property characteristic of wild-type FadL. Insertions in the four HpaII sites (fadL alleles termed H1, H2, H3, and H5; after amino acids 41, 81, 238, and 389, respectively) resulted in all three classes of mutants. The fadL insertion mutant H5 was defective for long-chain fatty acid transport but bound oleic acid at significant levels. Together with the S1 allele, these data suggest that the carboxyl terminus of FadL is crucial for long-chain fatty acid transport. The insertion mutants H1 and H2 were defective for both oleic acid binding and transport suggesting that the amino terminus of FadL is important for long-chain fatty acid binding and transport. The fadL linker mutant H3 was defective in oleic acid binding yet had significant levels of oleic acid transport. These studies delineated for the first time different regions of the fadL gene that encode domains of FadL implicated in the binding and transport of long-chain fatty acids.",
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