Genetically probing the regions of ribose-binding protein involved in permease interaction.
The ribose-binding protein (RBP) of Escherichia coli, located in the periplasm, binds to ribose and mediates transport and chemotaxis. The regions on the tertiary structure of RBP that interact with the membrane permease, an ABC transporter, were genetically probed by screening a mutation using the chimeric receptor Trz. Trz is a hybrid protein between the periplasmic domain of chemoreceptor Trg and the cytoplasmic portion of osmosensor EnvZ, which provides a system for monitoring the chemotactic interaction of RBP on MacConkey agar plates when coupled with a reporter lacZ fused to an ompC gene. The expression of ompC can be increased by an interaction of ribose-bound RBP with Trz. A transport defect, either in the binding protein or in the membrane permease, causes a signalling-constitutive Lac+ phenotype of Trz even in the absence of ribose. This appears to be due to the presence of a small amount of ribose, which is normally taken up by the high-affinity transport system. By taking advantage of this, we have designed a system for genetic screening that permits a selection for mutations in the binding protein, causing specific defects in permease interaction but not in tactic interaction. Mutant RBPs that were isolated were unable to perform normal ribose uptake and to utilize ribose as a carbon source, while other functions such as taxis and sugar-binding properties were not substantially affected. The mutational changes were repeatedly found in several residues of RBP, concentrating on three surface regions and comprising two domains of the tertiary structure. We suggest that the two regions, including residues 52 and 166, are specifically involved in the permease interaction while the third region, including residues 72, 134, and others, recognizes both the permease and the chemosensory receptor.
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Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Republic of Korea.
Park
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Park
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D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000596:Amino Acids / Q000502:physiology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002633:Chemotaxis; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D007700:Kinetics; D008565:Membrane Proteins; D008958:Models, Molecular; D009097:Multienzyme Complexes; D009154:Mutation; D033902:Periplasmic Binding Proteins; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012266:Ribose / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
D018528:ATP-Binding Cassette Transporters; D000596:Amino Acids; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D033902:Periplasmic Binding Proteins; C028003:RbsB protein, E coli; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C041476:Trg protein, E coli; D012266:Ribose; C057843:envZ protein, E coli