Identification of a major facilitator protein from Escherichia coli involved in efflux of metabolites of the cysteine pathway

Abstract

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.

Recruitment of genes and enzymes conferring resistance to the nonnatural toxin bromoacetate

“…The cynS and cynT genes encode for cyanase and carbonic anhydrase, respectively (21). EamA has been shown to efflux cysteine and O-acetyl-Lserine, an intermediate in the biosynthesis of cysteine (22). Overexpression of eamA is also known to provide resistance to azaserine.…”

Characterization of NrnA homologs from Mycobacterium tuberculosis and Mycoplasma pneumoniae

“…This opens up the possibility that elevated pAp-phosphatase activity levels could be detrimental under conditions when not much pAp is expected to accumulate. Cysteine represses the sulfur assimilation pathway and hence the synthesis of pAp (Daßler et al 2000). We have two possible explanations for this phenomenon: (1) in the absence of pAp, pAp-phosphatase might act on nonphysiological substrates, which might be needed for optimal growth, and (2) a minimum amount of pAp is needed to support optimal growth.…”

The l-Cysteine/l-Cystine Shuttle System Provides Reducing Equivalents to the Periplasm in Escherichia coli

“…A Hpx Ϫ mutant strain lacking two catalases (KatE and KatG) and a peroxidase (AhpCF)(10) was kindly provided by James A. Imlay. High L-cysteine-producing plasmid pDES (supplied by Ajinomoto) is a derivative of pACYC184 containing the altered cysE gene encoding the L-cysteine feedback inhibition-insensitive mutant SAT (T167A), the wild-type ydeD gene encoding inner membrane L-cysteine transporter (5), and the altered serA gene encoding the L-serine feedback inhibition-insensitive mutant of D-3-phosphoglycerate dehydrogenase (T410stop). Each gene fragment is under the control of the constitutive promoter of the E. coli ompA gene encoding outer membrane protein A precursor (13).…”

“…coli has L-cysteine transporters in the inner membrane (YdeD, YfiK, and Bcr) (5)(6)(7), and in the outer membrane (TolC) (8). It is known that TolC associates with the inner membrane and accessories, e.g.…”