Monitoring intracellular levels of XylR in Pseudomonas putida with a single-chain antibody specific for aromatic-responsive enhancer-binding proteins.
We have isolated a recombinant phage antibody (Phab) that binds a distinct epitope of the subclass of the sigma(54)-dependent prokaryotic enhancer-binding proteins that respond directly to aromatic effectors, e.g., those that activate biodegradative operons of Pseudomonas spp. The DNA segments encoding the variable (V) domains of the immunoglobulins expressed by mice immunized with the C-terminal half of TouR (TouRDeltaA) of Pseudomonas stutzeri OX1 were amplified and rearranged in vitro as single-chain Fv (scFv) genes. An scFv library was thereby constructed, expressed in an M13 display system, and subjected to a panning procedure with TouR. One clone (named B7) was selected with high affinity for TouR and XylR (the regulator of the upper TOL operon of the pWW0 plasmid). The epitope recognized by this Phab was mapped to the peptide TPRAQATLLRVL, which seems to be characteristic of the group of enhancer-binding proteins to which TouR and XylR belong and which is located adjacent to the Walker B motif of the proteins. The Phab B7 was instrumental in measuring directly the intracellular levels of XylR expressed from its natural promoter in monocopy gene dosage in Pseudomonas putida under various conditions. Growth stage, the physical form of the protein produced (XylR or XylRDeltaA), and the presence or absence of aromatic inducers in the medium influenced the intracellular pool of these molecules. XylR oscillated from a minimum of approximately 30 molecules (monomers) per cell during exponential phase to approximately140 molecules per cell at stationary phase. Activation of XylR by aromatic inducers decreased the intracellular concentration of the regulator. The levels of the constitutively active variant of XylR named XylRDeltaA were higher, fluctuating between approximately 90 and approximately 570 molecules per cell, depending on the growth stage. These results are compatible with the present model of transcriptional autoregulation of XylR and suggest the existence of mechanisms controlling the stability of XylR protein in vivo.
Fraile
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Centro Nacional de Biotecnología del Consejo Superior de Investigaciones Científicas, Campus de Cantoblanco, 28049 Madrid, Spain.
Roncal
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Fernández
L A
LA
de Lorenzo
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V
D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000918:Antibody Specificity; D001426:Bacterial Proteins; D001592:Benzyl Alcohols / Q000494:pharmacology; D015153:Blotting, Western; D004268:DNA-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D018604:Epitope Mapping; D005260:Female; D015964:Gene Expression Regulation, Bacterial; D006841:Hydrocarbons, Aromatic / Q000494:pharmacology*; D007114:Immunization; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D019151:Peptide Library; D016958:Pseudomonas putida / Q000235:genetics / Q000254:growth & development / Q000276:immunology / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D001592:Benzyl Alcohols; D004268:DNA-Binding Proteins; D006841:Hydrocarbons, Aromatic; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D014157:Transcription Factors; C074322:XylR protein, Pseudomonas; C042289:3-methylbenzyl alcohol