Abstract
Bacterial two-component systems (2CS) are usually involved in adaptation to external stimuli. Most 2CS consists of a histidine kinase sensor containing the Hpt domain, and a response regulator. In Pseudomonas aeruginosa PAO1, there are twelve hybrid sensors which do not have the Hpt domain. In addition, there are three genes encoding a protein with only the Hpt domain. Autophosphorylation of five of the hybrid sensors in vitro were detected in our laboratory previously. Three of them (PA2824, PA1611, PA1976) displayed an ability to transfer the phosphoryl group to HptB. In order to identify additional sensors that may phosphorylate HptB protein, cytoplasmic region of eleven hybrid sensors and the HptB protein were purified and characterized by phosphorelay assays with [γ-32P]ATP followed by SDS-PAGE and autoradiography. Five sensors showed rapid self-phosphorylation within 30 min in the time course assay. Using the reverse phosphorelay assay, a hybrid sensor RtsM, which can receive phosphoryl group from HptB was identified. The results demonstrate that these four hybrid sensors (PA2824, PA1611, PA1976, and RtsM) perform specific signal transduction through HptB to response regulators. Phosphorylated HptB can transfer the phosphoryl group to response regulator PA3346. Our laboratory also found PA3346 can dephosphorylate PA3347 on Ser56. In this study, swimming, swarming, and biofilm assays were performed to compare PA3346 complementary strains harboring PA3346 with different mutations at Asp61 and PA3347 complementary strains harboring PA3347 with different mutations at Ser56. Our results show little difference among these strains in these aspects. In order to search the downstream genes which were regulated by PA3345, PA3346 and PA3347, we performed quorum sensing assay. Secretion of signal molecular 3OC12-HSL was found to be decreased in MPA45, MJL46 and MJL47. In addition, we analyzed proteome of MPA45, MJL46 and MJL47 swarming cells by 2D gel electrophoresis and mass spectrometry to search for the genes downstream of PA3347. According to the results of the phenotype assay and proteomic analysis, we propose that PA3345□PA3346□PA3347 signal pathway plays a global regulation role in Pseudomonas aeruginosa.