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CONSTRUCTION AND ANALYSIS OF IPBR/XYLS HYBRID REGULATORY PROTEINS
Citation:
Eaton, R W. AND S S. Wilkinson. CONSTRUCTION AND ANALYSIS OF IPBR/XYLS HYBRID REGULATORY PROTEINS. Presented at 1999 Annual Meeting of the American Society for Microbiology, Chicago, IL, May 30-June 3, 1999.
Description:
IpbR and XylS are related regulatory proteins (having 56% identity). IpbR responds to isopropylbenzene as well as to a variety of hydrophobic chemicals to activate expression of the isopropylbenzene catabolic pathway operon of pRE4 from ipbOP. XylS responds to substituted benzoic acids by activating transcription of the m- and p-toluate catabolic pathway operon of various TOL plasmids from xylOmPm. These two regulatory proteins can be divided into at least four functional parts: an effector (inducer)- binding region [A] at the amino-terminus, two possible helix-turn-helix DNA-binding regions (HTH-1 [C] and HTH-2 [D]) at the carboxy-terminus, and a large segment [B] separating the inducer- and DNA-binding regions. Plasmids carrying genes encoding IpbR/XylS hybrids in which these regions have been exchanged were constructed using standard recombinant DNA technology. Hybrid regulators were studied by introducing these plasmids into E. coli JM109 containing either ipbOP-lacZ (on pSSW4) or xylOmPm-lacZ (on pERD100). Following induction of cells using isopropylbenzene or m-toluate, the resulting ?-galactosidase activities in extracts were assayed. Hybrid regulator responses ranged from un-inducible to a high constitutive level. Most notably the results demonstrated that although HTH-1 of XylS is involved in the recognition of xylOm, HTH-2 of IpbR is required for recognition of ipbO. Thus the hybrid regulator XylS-ABC/IpbR-D activated expression from both ipbOP and xylOmPm in the presence of m-toulate, while IpbR-ABC/XylS-D failed to activate expression from either ipbOP or xylOmPm in the presence of isopropylbenzene or m-toluate.