Rapid dephosphorylation of the TorR response regulator by the TorS unorthodox sensor in Escherichia coli

Abstract

Induction of the torCAD operon, encoding the trimethylamine N-oxide (TMAO) respiratory system, is tightly controlled by the TorS-TorR phosphorelay system in response to TMAO availability. TorS is an unorthodox sensor that contains three phosphorylation sites and transphosphorylates TorR via a four-step phosphorelay, His443-->Asp723-->His850-->Asp(TorR). In this study, we provide genetic evidence that TorS can dephosphorylate phospho-TorR when TMAO is removed. Dephosphorylation probably occurs by a reverse phosphorelay, Asp(TorR)-->His850-->Asp723, since His850 and Asp723 are both essential in this process. By using reverse transcriptase PCR, we also show that TMAO removal results in shutoff of tor operon transcription in less than 2 min. Based on our results and on analogy to other phosphorelay signal transduction systems, we propose that reverse phosphotransfer could be a rapid and efficient mechanism to inactivate response regulators.

FIG. 1

Schematic representation of the TorS-TorR phosphorelay system. TorS contains a N-terminal TMAO detector region, a primary transmitter domain (H1) with a histidine phosphorylation site (H443), a receiver domain (D1) with an aspartate phosphorylation site (D723), and a secondary transmitter domain (H2) with a histidine phosphorylation site (H850) (15). 726, location of the TMAO-constitutive mutation of TorS726; dark bars, putative transmembrane segments (16). The TorR response regulator contains a phosphoaccepting site (D53) in its receiver domain and a C-terminal DNA-binding domain. In the presence of TMAO, TorR is activated by phosphorylation via a four-step phosphorelay, as shown by the arrows overlined by a plus sign, leading to torCAD induction. The circled P corresponds to the phosphoryl group. The arrows underlined by minus signs indicate the reverse phosphorelay model for the dephosphorylation of TorR-P when TMAO is removed.

FIG. 2

Kinetic analysis of tor operon transcription followed by 1% agarose gel electrophoresis. To evaluate tor operon expression of strain LCB620 (torA-lacZ), we carried out RT-PCR with two converging oligonucleotides, one corresponding to the torC coding sequence (positions 640 to 667) and the other complementary to the beginning of lacZ (positions 64 to 41). Reverse transcription was performed with Superscript II RT (Gibco BRL) from 100 ng of total RNA prepared with the High Pure RNA isolation kit (Roche Diagnostic). PCR amplification (30 cycles) was carried out from 1/10 of the product of reverse transcription. Lane 1, 1-kb DNA ladder (Gibco BRL); lane 3, RT-PCR experiment with total RNA prepared from cells grown in the presence of TMAO just before the washing step; lanes 5, 9, and 13, RT-PCR experiments with total RNA prepared from cells grown (respectively) 2, 10, and 20 min after TMAO removal; lanes 7, 11, and 15, same experiments as for lanes 5, 9, and 13, respectively, but with TMAO added again after the washing step; lanes 2, 4, 6, 8, 10, 12, and 14, same experiments as for lanes 3, 5, 7, 9, 11, 13, and 15, respectively, but without RT; lane 16, control PCR with genomic DNA.

FIG. 3

Effect of plasmid-borne TorS derivatives on expression of the torA-lacZ chromosomal fusion of strain LCB726 pcnB. Cells were grown anaerobically on minimal medium supplemented with glucose (0.2%). No TMAO was added to the growth medium since LCB726 pcnB carries a TMAO-constitutive chromosomal mutation (torS726) (16). The construction of the pS plasmid series has been described previously (15), whereas that of the pΔS plasmid series is described in the text. The representation of each TorS allele encoded by the plasmids is shown. To avoid artifactual effects due to overproduction of the plasmid-borne TorS alleles, a pcnB mutation was transduced into strain LCB726 to lower the plasmid copy number (18). Moreover, as the plasmidic torS alleles are under the control of the leaky tac promoter, only 0.005 mM IPTG (isopropyl-β-d-thiogalactopyranoside) was used for the pS-plasmid series and no IPTG was added for the pΔS plasmid series. β-Galactosidase activities were measured on whole cells by the method of Miller (21); values represent the averages of at least three determinations with a variation of no more than 15% from the mean.

FIG. 4

Effect of plasmid-borne TorS truncated derivatives on expression of the torA-lacZ chromosomal fusion of strain LCB640 (torS⁺ pcnB). The experimental conditions were identical to those described for Fig. 3 except that 10 mM TMAO was added to the medium when indicated.