Construction of a tyrP-lac operon fusion strain and its use in the isolation and analysis of mutants derepressed for tyrP expression

Kasian, P A; Pittard, J

doi:10.1128/jb.160.1.175-183.1984

Cited by 21 publications

(3 citation statements)

References 43 publications

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“…The TyrR protein controls the expression of at least eight transcription units which constitute the TyrR regulon (19,20). Although the regulation is primarily by repression, expression of two of these transcription units, mtr and tyrP, is activated by the TyrR protein (12,16,22,23,25). The DNA-binding sites for the TyrR protein have been extensively characterized (reviewed by Pittard and Davidson, [20]) and are related to the palindrome TGTAAAN6TIT ACA.…”

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confidence: 99%

“…Furthermore, Cui and Somerville (9) have recently reported that amino acid substitutions at position 3 resulted in the complete loss of TyrR-mediated activation of mtr by tyrosine and that substitutions at position 2 resulted in a partial loss of this activation. In this paper we have considerably extended the analysis of the amino-terminal end of the protein to include residues 4, 5, 6, 7, 8, 9, 10, and 16 and have, in addition, studied the effects of small internal deletions further into the gene. The 2.2-kb ScaI-BamHI fragment carrying the wild-type tyrR gene was purified from plasmid pMU1065 (27) and cloned into the EcoRV-BamHI sites of M13tgl30 (17).…”

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Mutations in the tyrR gene of Escherichia coli which affect TyrR-mediated activation but not TyrR-mediated repression

1993

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Site-directed mutagenesis has been used to further characterize amino acid residues necessary for the activation of gene expression by the TyrR protein. Amino acid substitutions have been made at positions 2, 4, 5, 6, 7, 8, 9, 10, and 16. TyrR mutants with amino acid substitutions V-5-->P (VP5), VF5, CS7, CR7, DR9, RI10, RS10, and ER16 show no or very little activation of expression of either mtr or tyrP. In each case, however, the ability to repress aroF is unaltered. Amino acid substitutions at positions 4, 6, and 8 have no effect on activation. Small internal deletions of residues 10 to 19, 20 to 29, or 30 to 39 also destroy phenylalanine- or tyrosine-mediated activation of mtr and tyrP. In these mutants repression of aroF is also unaltered. In activation-defective tyrR mutants, expression of mtr is repressed in the presence of tyrosine. This tyrosine-mediated repression is trpR dependent and implies an interaction between TrpR and TyrR proteins in the presence of tyrosine.

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Mutations in the tyrR gene of Escherichia coli which affect TyrR-mediated activation but not TyrR-mediated repression

1993

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“…In Escherichia coli it has been demonstrated that two distinct transport systems exist for the accumulation of tyrosine (6,7): the general aromatic system which also transports phenylalanine and tryptophan and the tyrosinespecific system. Mutations which inactivate the general aromatic system have been located in a single gene aroP at min 2 on the chromosome (6), and mutations affecting the tyrosine-specific system have been located in a gene tyrP at min 42 (17). The control of the expression of both these genes is affected by the tyrR protein combined with phenylalanine, tyrosine, or tryptophan in the case of aroP and tyrosine or phenylalanine in the case of tyrP (31).…”

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Cloning of the tyrP gene and further characterization of the tyrosine-specific transport system in Escherichia coli K-12

Wookey¹,

Pittard²,

Forrest³

et al. 1984

J Bacteriol

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The tyrP gene which codes for a component of the tyrosine-specific transport system of Escherichia coli has been cloned on a 2.8-kilobase insert into plasmid pBR322. Transposon mutagenesis, using TnlOOO, indicates that the tyrP+ gene is at least 1.1 kilobase in length. Labeling of the tyrP protein in maxicells with [35S]methionine indicates an apparent molecular weight of ca. 24,500. Sedimentation analysis reveals that the tyrP protein is associated with the cell membrane apd is not free in the cytoplasm or periplasm. Strains with many copies of the tyrP+ gene show an enhanced uptake of tyrosine, but the expression of the system is still modulated by tyrosine and phenylalanine in the presence of the tyrR+ regulator protein. Accumulated

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Screening of an Escherichia coli promoter library for a phenylalanine biosensor

Mahr

Boeselager

Wiechert

et al. 2016

Appl Microbiol Biotechnol

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In recent years, the application of transcription factor-based biosensors for the engineering of microbial production strains opened up new opportunities for industrial biotechnology. However, the design of synthetic regulatory circuits depends on the selection of suitable transcription factor-promoter pairs to convert the concentration of effector molecules into a measureable output. Here, we present an efficient strategy to screen promoter libraries for appropriate parts for biosensor design. To this end, we pooled the strains of the Alon library containing about 2000 different Escherichia coli promoter-gfpmut2 fusions, and enriched galactose- and L-phenylalanine-responsive promoters by toggled rounds of positive and negative selection using fluorescence-activated cell sorting (FACS). For both effectors, responsive promoters were isolated and verified by cultivation in microtiter plates. The promoter of mtr, encoding an L-tryptophan-specific transporter, was identified as suitable part for the construction of an L-phenylalanine biosensor. In the following, we performed a comparative analysis of different biosensor constructs based on the mtr promoter. The obtained data revealed a strong influence of the biosensor architecture on the performance characteristics. For proof-of-principle, the mtr sensor was applied in a FACS high-throughput screening of an E. coli MG1655 mutant library for the isolation of L-phenylalanine producers. These results emphasize the developed screening approach as a convenient strategy for the identification of effector-responsive promoters for the design of novel biosensors.

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Construction of a tyrP-lac operon fusion strain and its use in the isolation and analysis of mutants derepressed for tyrP expression

Cited by 21 publications

References 43 publications

Mutations in the tyrR gene of Escherichia coli which affect TyrR-mediated activation but not TyrR-mediated repression

Mutations in the tyrR gene of Escherichia coli which affect TyrR-mediated activation but not TyrR-mediated repression

Cloning of the tyrP gene and further characterization of the tyrosine-specific transport system in Escherichia coli K-12

Screening of an Escherichia coli promoter library for a phenylalanine biosensor

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