Catabolite repression of the TodS/TodT two-component system and effector-dependent transphosphorylation of TodT as the basis for toluene dioxygenase catabolic pathway control

Abstract

The TodS/TodT two-component system of Pseudomonas putida regulates the expression of the toluene dioxygenase (tod) operon for the metabolism of toluene, benzene, and ethylbenzene. The sensor kinase TodS has a complex domain arrangement containing two functional modules, each harboring a sensor and an autokinase domain separated by a receiver domain. The TodT protein is the cognate response regulator that activates transcription of the toluene dioxygenase (TOD) pathway genes at the P(todX) promoter. We report in this study that the todST operon is transcribed from a main promoter and that the +1 initiation point is located 31 nucleotides upstream from the A of the first ATG codon and is preceded by a -10/-35 canonical promoter. Expression from P(todS) is under catabolite control, and in cells growing with glucose, the level of expression from this promoter is reduced, which in turn translates to low levels of the TodS/TodT regulators and results in a decrease of transcription from the P(todX) promoter. Thus, the main underlying regulatory mechanisms of the tod structural genes are at the levels of catabolite repression control from P(todS) and transcription activation, mediated by the TodT response regulator through a regulatory cascade in which the effector enhances autophosphorylation of TodS by ATP, with subsequent transphosphorylation of TodT.

FIG. 1.

(a) Graphical overview of the tod catabolic and todST operons. The primers used to determine whether the todH and todST gene clusters form a transcriptional unit and whether todS and todT form an operon are indicated with arrows at the todH (forward) and todS (reverse) genes and at the todS (forward) and todT (reverse) genes, respectively. The promoter sequence with its −35 and −10 regions and the transcription initiation point (+1) are represented, as determined by primer extension analysis. (b) Reverse transcriptase PCR (RT-PCR) to determine transcriptional units. Experiments were carried out with primers including the todH-todS intergenic region and with RNAs isolated from wild-type P. putida DOT-T1E, without and with 1 mM toluene (lanes 1 and 2, respectively), and from P. putida DOT-T1E ΔC1C2, without and with 1 mM toluene (lanes 3 and 4, respectively). Lanes 5 and 6 are negative controls with RT enzyme but no RNA and with Taq polymerase enzyme with RNA, respectively. Lane 7 is the positive control, showing that todS and todT form an operon. Lane 8 is NEB molecular weight marker VIII.

FIG. 2.

(a) Primer extension analysis of the P_todST promoter region in P. putida DOT-T1E. Wild-type (WT) and ΔC1C2 mutant cells were used to determine the transcription initiation point (+1), in both cases in the absence of an effector (C−) or in the presence of a 1 mM concentration of the agonist toluene (T), fluorobenzene (FB), or ethylbenzene (EB). For primer extension analysis, primers were labeled at their 5′ ends with [γ-³²P]ATP and T4 polynucleotide kinase. About 10⁵ cpm of the labeled primers was hybridized to 30 μg of total RNA, and extension was carried out using avian myeloblastosis virus RT as previously described (14). Electrophoresis of cDNA products was done using a urea-polyacrylamide sequencing gel to separate the reaction products, and gels were exposed to a phosphor screen (Fuji Photo Film Co., Ltd.) for 24 to 48 h. Phosphor screens were scanned using a phosphorimaging instrument (Molecular Imager FX; Bio-Rad). (b) Primer extension analysis to determine whether both putative transcription initiation sites (S1 and S2) are active or if the lower band (S1) is a degradation product of S2. See the text for experimental procedures.

FIG. 3.

Stability assays of TodS and TodT. Pseudomonas putida DOT-T1E cells were grown on minimal medium with glucose. When cells reached a turbidity of 0.6, the culture was separated into two flasks of equal volume, to one of which toluene was added in the vapor phase. Protein production was inhibited with 30 μg/ml of tetracycline after an additional 30 min. Aliquots of 200 μl were removed at different time points after inhibition of protein production, to monitor TodS or TodT stability in the presence or absence of toluene. Ten-microliter samples were boiled for 5 min in Laemmli buffer, separated by SDS-PAGE, electroblotted to polyvinylidene difluoride (PVDF) membranes (Millipore, Madrid, Spain), and probed with polyclonal antibodies against TodS or TodT, generated in rabbits. The specific bands were detected by chemiluminescence [goat anti-rabbit IgG (H+L)-horseradish peroxidase conjugate; Caltag Labs, Burlingame, CA] and quantified with Quantity One software (Perkin Elmer).