Abstract
Certain bacterial two-component sensor kinases possess a histidine-containing phosphotransfer (Hpt) domain to carry out a multistep phosphotransferring reaction to a cognate response regulator. Pseudomonas aeruginosa PAO1 contains three genes that encode proteins with an Hpt domain but lack a kinase domain. To identify the sensor kinase coupled to these Hpt proteins, a phosphorelay profiling assay was performed. Among the 12 recombinant orphan sensor kinases tested, 4 of these sensors (PA1611, PA1976, PA2824, and RetS) transferred the phosphoryl group to HptB (PA3345). The in vivo interaction between HptB and each of the sensors was also confirmed using the bacterial two-hybrid assay. Interestingly, the phosphoryl groups from these sensors all appeared to be transferred via HptB to PA3346, a novel phosphatase consisting of an N-terminal receiver domain and a eukaryotic type Ser/Thr phosphatase domain, and resulted in a significant increase of its phosphatase activity. The subsequent reverse transcription-PCR analysis revealed an operon structure of hptB-PA3346-PA3347, suggesting a coordinate expression of the three genes to carry out a signal transduction. The possibility was supported by the analysis showing PA3347 is able to be phosphorylated on Ser-56, and this phosphoryl group could be removed by PA3346 protein. Finally, analysis of PA3346 and PA3347 gene knock-out mutants revealed that these genes are associated with bacterial swarming activity and biofilm formation. Together, these results disclose a novel multistep phosphorelay system that is essential for P. aeruginosa to respond to a wide spectrum of environmental signals.
Highlights
An intermediate group of sensors are known as the hybrid sensors
The hybrid-type sensors, which contain a kinase and a receiver domain but lack an histidine-containing phosphotransfer (Hpt) domain, are believed to require another protein to provide the Hpt domain for their signal transduction (Fig. 1) [4, 5]. An example of such a system has been demonstrated in E. coli, in which the hybrid sensor RcsC is dependent on YojN, an Hpt domain-containing protein, to signal and activate the response regulator RcsB [5, 6]
The phosphorylated sensor proteins were incubated with an Hpt protein, and the transfer of the phosphoryl group was examined by autoradiography (Fig. 2, A–E)
Summary
EndA1 hsdR17 supE44 thi-1 recA1 gyrA96 relA1 lacFЈ proAB lacIqZ⌬M15Tn10͔ MRFЈ K, ⌬(mcrA) 183⌬͓(mcr CB-hsdSMR-mrr) 173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac (FЈ proAB lacIq Z ⌬M15 Tn5(Kmr))͔. PE1397 pE2798 pE3604 pE3714 pE4843 pE5364 pTRG-1611 pTRG-1976 pTRG-2824 pTRG-RetS pBT-HptB pMMB46. His tag protein expression vector, Kmr His tag protein expression vector, Apr Apr; broad-host-range expression vector Tcr; oriTϩ sacBϩ, gene replacement vector GST tag protein expression vector, Apr Cmr, p15A origin of replication, lac-UV5 , cI open reading frame Tcr, ColE1 origin of replication, lac-UV5 promoter, RNAP␣ open reading frame Kmr; a hptA containing fragment cloned into pET30a Kmr; a hptB containing fragment cloned into pET30b Kmr; a hptC containing fragment cloned into pET30b Kmr; a PA1611HD containing fragment cloned into pET30a Apr, a fragment containing residues 456–859 of PA1243 coding region cloned into pET100 Apr, a fragment containing residues 172–541 of PA1396 coding region cloned into pET100 Apr, a fragment containing residues 460–882 of PA1976 coding region cloned into pET100 Apr, a fragment containing residues 160–565 of PA1992 coding region cloned into pET100 Apr, a fragment containing residues 310–700 of PA2177 coding region cloned into pET100 Apr, a fragment containing residues 589–993 of PA 2583 coding region cloned into pET100 Apr, a fragment containing residues 248–787 of PA2824 coding region cloned into pET100 Apr, a fragment containing residues 749–1160 of PA3271 coding region cloned into pET100 Apr, a fragment containing residues 383–920 of PA3462 coding region cloned into pET100 Apr, a fragment containing residues 386–796 of PA3974 coding region cloned into pET100 Apr, a fragment containing residues 390–943 of RetS coding region cloned into pET100 Kmr; a fragment containing the receiver domain of regulator PA3346 from residue 1 to 161 cloned into pET30a Kmr; the fragment containing the receiver domain of regulator PA0034 from residues 1 to 115 cloned into pET30a Apr, a fragment containing entire PA1397 coding region cloned into pET100 Apr, a fragment containing entire PA2798 coding region cloned into pET100 Apr, a fragment containing entire PA3604 coding region cloned into pET100 Apr, a fragment containing entire PA3714 coding region cloned into pET100 Apr, a fragment containing entire PA4843 coding region cloned into pET100 Apr, a fragment containing entire PA5364 coding region cloned into pET100 Apr, a fragment containing residues 202–652 of PA1611 coding region cloned into pTRG Apr, a fragment containing residues 468–882 of PA1976 coding region cloned into pTRG Apr, a fragment containing residues 248–787 of PA2824 coding region cloned into pTRG Apr, a fragment containing residues 392–859 of RetS coding region cloned into pTRG Apr, a fragment containing entire HptB coding region cloned into pBT Apr, a fragment containing entire PA3346 coding region fused with a N-terminal His tag cloned into pMMB66 Apr, a fragment containing entire PA3347 coding region fused with a N-terminal His tag cloned into pMMB66 Apr, a fragment containing entire hptB coding region cloned into pMMB66 Apr, a fragment containing entire PA3347 coding region fused with a N-terminal GST tag cloned into pGEX-5X-1 Apr, a fragment containing entire PA3346 coding region cloned into pET100
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