Abstract
The XylR protein positively controls expression from the Pseudomonas putida TOL plasmid sigma 54-dependent "upper" pathway operon promoter (Pu) and the xylS gene promoter (Ps), in response to the presence of aromatic effectors. Two mutant XylR regulators able to stimulate transcription from Pu and Ps in the absence of effectors were isolated. These mutants exhibited single point mutations, namely Asp135-->Asn and Pro85-->Ser. Both mutations are located in the amino termini domain of XylR, which is thought to be responsible for interactions with effectors. The effector profile of XylRP85S was similar to that of wild-type XylR protein; however, XylRD135N exhibited an altered pattern of effector recognition: with m-nitrotoluene it stimulated transcription from the Pu promoter above the high basal level, whereas this nitroarene inhibited the wild-type regulator. Previous work (Delgado, A., and Ramos, J.L. (1994) J. Biol. Chem. 269, 8059-8062) showed that residue 172 was involved in effector interactions, as mutant XylRE172K also recognized m-nitrotoluene. However, double mutant XylR135N/E172K did not stimulate transcription in the absence of effector, but retained the ability to stimulate transcription with m-nitrotoluene. Transcription mediated by XylRD135N and XylRP85S from Pu::lacZ was analyzed in detail. Like the wild-type regulator, XylRD135N and XylRP85S required sigma 54 for full transcription activation, but in contrast with the wild-type regulator, XylRD135N, but not XylRP85S, stimulated transcription from Pu in the absence of the integration host factor protein. XylRD135N, also in contrast with XylR and XylRP85S, mediated transcription from a mutant Pu promoter that lacked one of the upstream regulator binding sites (delta UAS1), but not when both upstream regulator binding sites were deleted. The level of autoregulation of XylRD135N was at least 2-fold higher than that found with the wild-type XylR regulator and the mutant XylRP85S.
Highlights
The XylR protein positively controls expression from the Pseudomonas putida TOL plasmid 0-54 -d ep en d en t "upper" pathway operon promoter (Pu) and the xylS gene promoter (Ps), in response to the presence of aromatic effectors
Transcription mediated by XylRD135N and XylRP85S from Pu::lacZ was analyzed in detail
The XylR protein needs to be activated by effectors to stimulate transcription from the upper pathway operon promoter (Pu), and from the xylS gene promoter (Ps), Increased xylS mRNA levels lead to overproduction of the XylS regulator, which stimulate transcription from Pm, the meta-pathway operon promoter [2,3,4,5]
Summary
Vol 270, No 10, Issue of March 10, pp. 5144-5150, 1995 Printed in U.S.A. Single Amino Acids Changes in the Signal Receptor Domain of XylR Resulted in Mutants That Stimulate Transcription in the Absence of Effectors*. Two mutant XylR regulators able to stimulate transcription from Pu and Ps in the absence of effectors were isolated. The nonhomologous NHz-terminal domain A has been implicated in signal reception, either via a sensory protein as in the NtrBlNtrC pair, or via interaction with a chemical signal as in DmpR [7, 8] and XylR [9,10,11] This has been genetically confirmed with the latter two regulators, in which mutations in the NHz-terminal region altered effector recognition [11].1. Fine deletions in Pu [10, 16] and in Ps [17, 19], and in vivo [10] and in vitro footprinting experiments in the Pu promoter region [20], revealed that XylR recognizes a 5' motif, which appears twice, in inverted orientation These upstream activator sequences were identified around -160 (UASl) and around -130 (UAS2).
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