Integration of global regulation of two aromatic-responsive sigma(54)-dependent systems: a common phenotype by different mechanisms

Abstract

Pseudomonas-derived regulators DmpR and XylR are structurally and mechanistically related sigma(54)-dependent activators that control transcription of genes involved in catabolism of aromatic compounds. The binding of distinct sets of aromatic effectors to these regulatory proteins results in release of a repressive interdomain interaction and consequently allows the activators to promote transcription from their cognate target promoters. The DmpR-controlled Po promoter region and the XylR-controlled Pu promoter region are also similar, although homology is limited to three discrete DNA signatures for binding sigma(54) RNA polymerase, the integration host factor, and the regulator. These common properties allow cross-regulation of Pu and Po by DmpR and XylR in response to appropriate aromatic effectors. In vivo, transcription of both the DmpR/Po and XylR/Pu regulatory circuits is subject to dominant global regulation, which results in repression of transcription during growth in rich media. Here, we comparatively assess the contribution of (p)ppGpp, the FtsH protease, and a component of an alternative phosphoenolpyruvate-sugar phosphotransferase system, which have been independently implicated in mediating this level of regulation. Further, by exploiting the cross-regulatory abilities of these two circuits, we identify the target component(s) that are intercepted in each case. The results show that (i) contrary to previous speculation, FtsH is not universally required for transcription of sigma(54)-dependent systems; (ii) the two factors found to impact the XylR/Pu regulatory circuit do not intercept the DmpR/Po circuit; and (iii) (p)ppGpp impacts the DmpR/Po system to a greater extent than the XylR/Pu system in both the native Pseudomonas putida and a heterologous Escherichia coli host. The data demonstrate that, despite the similarities of the specific regulatory circuits, the host global regulatory network latches onto and dominates over these specific circuits by exploiting their different properties. The mechanistic implications of how each of the host factors exerts its action are discussed.

FIG. 1.

(A) Regulatory elements of dmp and xyl systems, as described in the text. The dmpR gene is organized in a divergent configuration from its target promoter Po. The xylR gene is similarly orientated relative to Ps, the upstream activating sequences (UASs) of which overlap with the xylR promoter, while its other target promoter, Pu, is located distally on the pWW0 plasmid. Indicated PvuII and XmnI restriction sites were used as working boundaries for P_dmpR and P_xylR, respectively, in instances where the regulatory genes have to be dissected from their divergently positioned promoters. (B) Schematic representation of the chromosomal inserts in KT2440::dmpR and KT2440::xylR derivatives. (C) Schematic representation of reporter constructs (lacZ and luxAB) used in this study. White boxes and arrows, regulatory elements that originate from the dmp and xyl systems, as indicated.

FIG. 2.

Transcriptional profiles of DmpR/Po and XylR/Pu regulatory circuits in LB-grown P. putida (A, B, E, and F) and E. coli (C, D, G, and H), based on assays of luciferase (A to D) and β-galactosidase (β-gal; E to H) activities. Growth conditions are as stated in Materials and Methods. Solid symbols, log optical density at 600 nm (OD₆₀₀); open symbols, relative luciferase units (RLU) and Miller β-galactosidase units.

FIG. 3.

Luciferase transcriptional profiles of Po and Pu promoters activated by their native regulators (A and B) or nonnative regulators (C and D) in E. coli A8514 (FtsH⁺) and A8926 (FtsH⁻). Solid symbols, culture growth; open symbols, luciferase response. Note that, as has been found previously, the absolute values of reporters for different E. coli strains vary (68) and that transcriptional levels are higher in this lineage of E. coli than in the MG1655 lineage used in Fig. 2. RLU, relative luciferase units; OD₆₀₀, optical density at 600 nm.

FIG. 4.

Luciferase activity of P. putida KT2440::dmpR-Km and KT2440::xylR-Km carrying RK2-based plasmid pVI673 (Po-luxAB) or pVI674 (Pu-luxAB). Cells were grown in M9 media with 0.2% casein amino acids (CAA) and the appropriate aromatic effector and further supplemented with 10 mM glucose where indicated. Peak activities shown are averages of triplicate determinations from two independent experiments. RLU, relative luciferase activity.

FIG. 5.

Chromatographic analysis of ³²P-labeled nucleotides of the indicated E. coli and P. putida strains as described in Materials and Methods. The abilities of the strains to grow on M9 minimal medium plates supplemented with 10 mM glucose and 100 μg of thiamine/ml are indicated (bottom).

FIG. 6.

Luciferase transcriptional profiles of Po and Pu promoters activated by their native regulators (A to D) or nonnative regulators (E to H), in isogenic E. coli (A, B, E, and F) and P. putida (C, D, G, and H) (p)ppGpp⁺ and (p)ppGpp⁰ strains. E. coli (p)ppGpp⁺ MG1655Δlac and (p)ppGpp⁰ CF1693Δlac strains and P. putida (p)ppGpp⁺ KT2440-dmpR-Tel and -xylR-Tel and (p)ppGpp⁰ PP1-dmpR and -PP1-xylR strains were used. Solid symbols, culture growth; open symbols, luciferase response. RLU, relative luciferase units; OD₆₀₀, optical density at 600 nm.