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-bi nding proteins that respond directly to aromatic effectors, e.g., those tha t activate biodegradative operons of Pseudomonas spp. The DNA segments enco ding the variable (V) domains of the immunoglobulins expressed by mice immu nized with the C-terminal half of TouR (TouR DeltaA) of Pseudomonas stutzer i OX1 were amplified and rearranged in vitro as single-chain Fv (scFv) gene s. An scFv library was thereby constructed, expressed in an M13 display sys tem, and subjected to a panning procedure with TouR. One clone (named B7) w as selected with high affinity for TouR and XyIR (the regulator of the uppe r 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 XyIR 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 XyIR expres sed from its natural promoter in monocopy gene dosage in Pseudomonas putida under various conditions. Growth stage, the physical form of the protein p roduced (XyIR or XyIR DeltaA), and the presence or absence of aromatic indu cers in the medium influenced the intracellular pool of these molecules. Xy IR oscillated from a minimum of similar to 30 molecules (monomers) per cell during exponential phase to similar to 140 molecules per cell at stationar y phase. Activation of XyIR by aromatic inducers decreased the intracellula r concentration of the regulator. The levels of the constitutively active v ariant of XyIR named XyIR DeltaA were higher, fluctuating between similar t o 90 and similar to 570 molecules per cell, depending on the growth stage. These results are compatible with the present model of transcriptional auto regulation of XyIR and suggest the existence of mechanisms controlling the stability of XyIR protein in vivo.