Abstract
Although the m-xylene-responsive sigma54 promoter Pu of Pseudomonas putida mt-2, borne by the TOL plasmid pWWO, is one of the strongest known promoters in vivo, its base-line level in the absence of its aromatic inducer is below the limit of any detection procedure. This is unusual because regulatory networks (such as the one to which Pu belongs) can hardly escape the noise caused by intrinsic fluctuations in background transcription, including that transmitted from upstream promoters. This study provides genetic evidence that the upstream-activating sequences (UAS), which serve as the binding sites for the pWW0-encoded XylR protein (the m-xylene-responsive sigma54-dependent activator of Pu), isolate expression of the upper TOL genes from any adventitious transcriptional flow originating further upstream. An in vivo test system was developed in which different segments of the Pu promoter were examined for the inhibition of incoming transcription products from an upstream promoter in P. putida and Escherichia coli. Minimal transcription filter ability was located within a 105-bp fragment encompassing the UAS of Pu. Although S1 nuclease assays showed that the UAS prevented the buildup of downstream transcripts, the mechanism seems to diverge from a typical termination system. This was shown by the fact that the UAS did not halt transcription in vitro and that the filter effect could not be relieved by the anti-termination system of lambda phage. Because the Pu promoter lies adjacent to the edge of a transposon in pWW0, the preset transcriptional filter in the UAS may isolate the upper TOL operon from undue expression after random insertion of the mobile genetic element in a new replicon.
Highlights
Bacterial promoters form part of regulatory networks through which signals are propagated faithfully from one member to the [1, 2]
This study provides genetic evidence that the upstream-activating sequences (UAS), which serve as the binding sites for the pWW0-encoded XylR protein, isolate expression of the upper TOL genes from any adventitious transcriptional flow originating further upstream
This event is assisted by the binding of the integration host factor (IHF) to the region between the UAS and the 54 RNAP attachment site (Fig. 1)
Summary
Bacterial promoters form part of regulatory networks through which signals are propagated faithfully from one member to the [1, 2]. Regulatory circuits that control the expression of metabolic programs for the biodegradation of pollutants in soil bacteria require the suppression of transcriptional noise if these organisms are to survive initial exposure [6] How cells organize their transcriptional response can be examined by analyzing the biodegradation of m-xylene by Pseudomonas putida mt-2, a function encoded by the catabolic TOL plasmid pWW0 which it carries [7]. When P. putida mt-2 is challenged with m-xylene in the medium, much of the available transcriptional machinery is reassigned to allow the bacterium to endure this general stress [10, 79] In theory this could reduce the availability of the RNAP and other transcription factors necessary for expression of the xyl genes borne by plasmid pWW0, thereby making the corresponding catabolic promoters more sensitive to cell-to-cell variations.
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