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Identification of a major facilitator protein from Escherichia coli involved in efflux of metabolites of the cysteine pathway
Author(s) -
Daßler Tobias,
Maier Thomas,
Winterhalter Christoph,
Böck August
Publication year - 2000
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2000.01924.x
Subject(s) - regulon , biology , cysteine , subcloning , biochemistry , serine , open reading frame , escherichia coli , major facilitator superfamily , efflux , amino acid , gene , gene product , peptide sequence , gene expression , mutant , enzyme
A chromosomal fragment has been identified in a gene bank from Escherichia coli , which augmented the yield of cysteine in an industrial production strain. Subcloning and genetic analysis showed that an open reading frame coding for a product of 299 amino acids (Orf299) was responsible. Orf299 was synthesized in the T7 polymerase/promoter system and exhibited the properties of an integral membrane protein. Mutational interruption of orf299 did not cause a distinct phenotype; however, transformants overexpressing orf299 had lost the ability to grow in minimal medium unless it was supplemented with a source of reduced sulphur compounds, and they excreted considerable amounts of cysteine and O‐acetyl‐ l ‐serine, especially in the presence of thiosulphate. Most of the cysteine was found to be masked in 2‐methyl‐2,4‐thiazolidinedicarboxylic acid. N‐acetyl‐ l ‐serine was also present in the medium, but it is open to question whether it represents a primary excretion product. Measurement of the induction status of the cysteine regulon by means of a cysK ′–′ lacZ gene fusion demonstrated that the regulon is not induced upon growth in the presence of a poor sulphur source and that the introduction of a constitutive cysB allele alleviates this deficiency. The results indicate that orf299 codes for an export pump for different metabolites of the cysteine pathway. Its relation to other efflux systems and the physiological role are discussed.