The REC domain mediated dimerization is critical for FleQ from Pseudomonas aeruginosa to function as a c-di-GMP receptor and flagella gene regulator

Journal of Structural Biology xxx (2015) xxx–xxx

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The REC domain mediated dimerization is critical for FleQ from Pseudomonas aeruginosa to function as a c-di-GMP receptor and flagella gene regulator Tiantian Su a,1, Shiheng Liu a,1, Kang Wang a, Kaikai Chi a, Deyu Zhu a, Tiandi Wei a, Yan Huang a, Liming Guo b, Wei Hu a, Sujuan Xu a, Zong Lin c, Lichuan Gu a,⇑ a

State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Jinan 250100, Shandong, China Rizhao Center for Diseases Prevention and Control, Rizhao Health Bureau, Rizhao 276826, Shandong, China c Department of Biotechnology and Biomedicine, Yangtze Delta Region Institute of Tsinghua University, Jiaxing, Zhejiang 314006, China b

a r t i c l e

i n f o

Article history: Received 23 May 2015 Received in revised form 27 August 2015 Accepted 7 September 2015 Available online xxxx Keywords: Flagella Biofilm Transcription factor Structure–function Cyclic di-GMP Pseudomonas aeruginosa

a b s t r a c t FleQ is an AAA+ ATPase enhancer-binding protein that regulates both flagella and biofilm formation in the opportunistic pathogen Pseudomonas aeruginosa. FleQ belongs to the NtrC subfamily of response regulators, but lacks the corresponding aspartic acid for phosphorylation in the REC domain (FleQR, also named FleQ domain). Here, we show that the atypical REC domain of FleQ is essential for the function of FleQ. Crystal structure of FleQR at 2.3 Å reveals that the structure of FleQR is significantly different from the REC domain of NtrC1 which regulates gene expression in a phosphorylation dependent manner. FleQR forms a novel active dimer (transverse dimer), and mediates the dimerization of full-length FleQ in an unusual manner. Point mutations that affect the dimerization of FleQ lead to loss of function of the protein. Moreover, a c-di-GMP binding site deviating from the previous reported one is identified through structure analysis and point mutations. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction Besides the critical function in motility, flagella have been demonstrated as an important virulence and colonization factor for numerous pathogenic bacteria including Pseudomonas aeruginosa, Campylobacter jejuni, Proteus mirabilis, Vibrio cholerae and Helicobacter pylori (Moens and Vanderleyden, 1996; Ottemann and Miller, 1997). Formation and assembly of the polar flagellum in P. aeruginosa require more than 40 genes, which are organized in a four-tiered hierarchy of transcriptional regulation (Dasgupta et al., 2003). FleQ is the master regulator, which in concert with r54, activates transcription of the class II genes, including regulator proteins FleSR and FleN, and more than 20 flagella proteins (Arora et al., 1997; Jyot et al., 2002). In addition to flagella genes, FleQ also regulates expression of biofilm-related genes, including genes for Pel and Psl exopolysaccharide (EPS) production in response to cyclic diguanosine monophosphate (c-di-GMP) (Baraquet and Harwood, 2013; Hickman and Harwood, 2008; Baraquet et al., 2012). FleQ binds ⇑ Corresponding author. 1

E-mail address: [email protected] (L. Gu). The first two authors should be regarded as joint first authors.

to the two FleQ binding sites in pelA promoter region, and functions not only as a repressor in the absence of c-di-GMP but also as an activator in the presence of c-di-GMP (Baraquet et al., 2012). Baraquet et al. reported that c-di-GMP binds to the Walker A motif of FleQ by competing with ATP and inhibits FleQ ATPase activity, thereby depressing the ability of FleQ to activate gene expression (Baraquet and Harwood, 2013). Notably, the effect of c-di-GMP on flagella gene expression is much weaker than that on EPS gene expression, indicating that FleQ regulates flagella genes and EPS gene expression through different mechanisms (Hickman and Harwood, 2008). Sequence analysis shows FleQ belongs to the NtrC enhancer binding protein (EBP) family. It consists of an N-terminal REC domain (also named FleQ domain), a conserved central AAA+/ ATPase domain, and a helix-turn-helix DNA binding domain (Arora et al., 1997). The AAA+/ATPase domain has ATP hydrolysis activity and can remodel the r54-RNA polymerase closed complex through direct interaction with r54. However, unlike typical regulators, the REC domain of FleQ lacks the aspartic acid residue which acts as the phosphate acceptor in NtrC and carries a serine residue instead. No gene encoding a corresponding sensor kinase has been identified upstream of the fleQ gene (Arora et al., 1997). Although how the REC domain of FleQ plays a role in the function of the full

http://dx.doi.org/10.1016/j.jsb.2015.09.002 1047-8477/Ó 2015 Elsevier Inc. All rights reserved.

Please cite this article in press as: Su, T., et al. The REC domain mediated dimerization is critical for FleQ from Pseudomonas aeruginosa to function as a c-diGMP receptor and flagella gene regulator. J. Struct. Biol. (2015), http://dx.doi.org/10.1016/j.jsb.2015.09.002

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T. Su et al. / Journal of Structural Biology xxx (2015) xxx–xxx

length protein remains obscure, the lack of the potential phosphorylation site has implied an unknown distinctive mechanism. The lack of a phosphorylation site in the REC domain also occurs in other homologs of FleQ. Transcriptional regulator FlbD from Caulobacter crescentus, for example, also lacks the aspartic acid which serves as an acceptor for phosphate group in its REC domain. Accordingly, no sensor kinase has been identified to perform phosphorylation on FlbD (Ramakrishnan and Newton, 1990). The activity of FlbD is regulated by FliX, a conserved trans-acting factor which has no demonstrated histidine kinase activity, through direct physical interaction (Dutton et al., 2005; Muir and Gober, 2002; Muir and Gober, 2004; Muir et al., 2001). Similar phenomenon is also observed in FlrA, the master regulator of flagella biosynthesis from V. cholerae (Prouty et al., 2001). The wide spread of the lack of the phosphorylation site in REC domains in bacteria suggests that this family of proteins adopt a distinctive mechanism of extensive importance to regulate downstream gene expression. Recently, Yang et al. have obtained the native and Se-Met labelled crystals of the FleQ REC domain from Stenotrophomonas maltophilia, which diffracted to resolutions of 2.08 and 2.58 Å, respectively (Yang et al., 2014). FleQ from S. maltophilia also lacks the phosphorylation site (with a Ser59 instead), and it shares a sequence identity of 44% to FleQ from P. aeruginosa PAO1. The molecular mechanism for FleQ to perform its function in cells has been studied for many years. Recently it has been reported that the activity of FleQ is negatively regulated by its anti-activator FleN through direct protein–protein interaction (Dasgupta and Ramphal, 2001). FleN is a putative ATPase, but contains a deviant Walker A motif. The expression of FleN has been known to be under the control of FleQ and FleQ interacts with FleN both in the presence and absence of ATP/c-di-GMP. It was also known that the DNA binding specificity of FleQ was not affected by the interaction with FleN (Dasgupta and Ramphal, 2001; Hickman and Harwood, 2008) and that disruption of FleN led to upregulation of FleQ-dependent promoters (Dasgupta et al., 2000). Consequently, in wild-type strain, FleN inhibits FleQ activity and the cell assembles a polar flagellum, while a fleN mutant strain is multiflagellate and has reduced motility (Dasgupta et al., 2000). Lately, Jain and Kazmierczak determined that the aflagellate phenotype of P. aeruginosa PA103 is caused by a conservative amino acid mutation (G240V) of FleQ, which lies in the Walker B motif and might involve in ATP hydrolysis (Jain and Kazmierczak, 2014). Since FleQ plays a vital role in regulating gene expression related to flagella and biofilm formation, both of which are crucial virulence factors in P. aeruginosa, uncovering its molecular mechanism may help us better understand how P. aeruginosa thrives in different environments and successfully establishes infection in human hosts. In this paper, we solved the crystal structure of FleQ REC domain (designated as FleQR) at 2.3 Å. Our result shows that FleQR forms an unusual active dimer, and the FleQR mediated dimerization is essential for FleQ to function in vivo. The c-diGMP binding site was further clarified close to Walker A motif through our biochemical experiments. 2. Materials and methods 2.1. Gene knockout and complementation The unmarked fleQ deletion mutant strain DfleQ was constructed by using previously described sac B-based allelic exchange strategy (Hoang et al., 1998). A 1.4 kb DNA fragment was obtained by overlapping two 700 bp DNA fragments which are located upstream and downstream of the fleQ gene respectively. The fragment was ligated into pEX18Gm plasmid and transferred into PAO1 from Escherichia coli DH5a through conjunction by the aid of pRK2013 plasmid. Merodiploid strains were selected from

VBMM (VB medium containing 0.3% citrate) plates supplemented with 100 lg mL
1 gentamicin. The DfleQ strains were obtained from LB plates containing 20% sucrose, followed by PCR verification. For complementation, wild-type fleQ and fleQ mutant genes were subcloned into pUCP18 plasmids between EcoRI and HindIII restriction sites. The recombinant plasmids were electroporated into DfleQ strains and expressed without induction. 2.2. Motility assay Flagella-mediated swimming motility was performed on LB plates with 0.3% agar as described by O’Toole et al. (1999). Single colonies were poked into the plates using toothpicks, and the plates were incubated at 37 °C for about 6 h. Migration of the cells from the point of inoculation represents their motility ability. Three independent experiments were done for each strain. Swarming motility was assayed on 0.5% soft agar plates.

2.3. RNA isolation and real-time quantitative PCR WT PAO1 and mutant strains were grown overnight in 5 mL LB medium and subcultured into 30 mL LB medium. Bacterial cells were harvested for total RNA isolation when OD600 reached 0.4– 0.6. Total RNA was isolated using MiniBEST Universal RNA Extraction Kit (TaKaRa). The quality of purified RNA was analyzed by electrophoresis and quantified by Nanodrop 1000 Spectrophotometer (NanoDrop Technologies). The cDNA was synthesized using PrimeScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time). Primers for RT-PCR listed in Table S1 were designed using the program PrimerExpress (Applied Biosystems). The RT-PCR reactions were performed on the CFX96 Real-Time PCR Detection System (Bio-Rad). The cycling parameters were: initial denaturation at 95 °C for 10 min, and 40 cycles of 95 °C for 10 s, 60 °C for 15 s and 72 °C for 20 s. The dissociation curve analysis revealed single amplicons of an appropriate melting temperature. The relative transcript abundance was calculated according to the 2
DDCt method (Livak and Schmittgen, 2001). The housekeeping gene rpoC (Wang et al., 2014) was used as the internal control. Three individual replicates were performed with three independent cultures grown on different days. Standard errors were calculated from these independent replicates.

2.4. Protein expression and purification Sequences encoding the REC domain (the first 139 amino acids) of FleQ were amplified from P. aeruginosa PAO1 genomic DNA and inserted into PGL01, a modified vector based on pET15b (Invitrogen) with a PreScission protease (PPase) cleavage site to remove the His tag. FleQ was overexpressed in E. coli BL21 (DE3). The cells were cultured in LB medium supplemented with 100 lg mL
1 ampicillin at 37 °C until OD600 reached 0.8, followed by overnight induction with 0.12 mM isopropyl 1-thio-b-D-galactopyranoside at 22 °C. For protein purification, bacterial cells were harvested and resuspended in lysis buffer (25 mM Tris pH 8.0, 200 mM NaCl), and lysed by sonication. The soluble fraction containing FleQ was loaded onto a nickel affinity column (Chelating Sepharose Fast Flow, GE Healthcare), and treated with PPase to remove the His tag. The protein was further purified by ion exchange (Source 15Q HR 16/10, GE Healthcare) and size-exclusion chromatography (Superdex 200 10/300 GL, GE Healthcare). Selenomethioninelabeled FleQ was expressed from BL21 (DE3) in M9 medium supplemented with selenomethionnine. The induction condition and purification procedure was the same with native protein.

Please cite this article in press as: Su, T., et al. The REC domain mediated dimerization is critical for FleQ from Pseudomonas aeruginosa to function as a c-diGMP receptor and flagella gene regulator. J. Struct. Biol. (2015), http://dx.doi.org/10.1016/j.jsb.2015.09.002

T. Su et al. / Journal of Structural Biology xxx (2015) xxx–xxx

2.5. Crystallization, data collection and structure determination The crystallization conditions were primarily screened with Hampton Research crystal screen kits by sitting drop vapor diffusion at 20 °C. Native crystals were obtained under the condition with reservoir solutions containing 0.2 M sodium acetate, 0.1 M Tris pH 8.5, and 34% w/v polyethylene glycol 4000. Selenomethionine-labeled crystals were obtained under the condition containing 0.2 M sodium chloride, 0.1 M Tris pH 8.5, and 28% w/v polyethylene glycol 3350. Optimization was performed using hanging drop method. For data collection, crystals were soaked in a solvent identical to the reservoir solution with 15% v/v glycerol as a cryoprotectant and then flash frozen in liquid nitrogen. All diffraction data were collected at Shanghai Synchrotron Radiation facility (SSRF) on beam line BL17U. Diffraction data sets were processed using the HKL2000 software suite (Otwinowski and Minor, 1997). The structure of FleQ REC domain was solved by single-wavelength anomalous dispersion (SAD) phasing. The atomic models were initially built using PHENIX (Adams et al., 2002) and refined using COOT (Emsley and Cowtan, 2004). One monomer from the resulting structure was subsequently selected as the search model for molecular replacement with the high resolution native data set using PHASER (McCoy et al., 2007; Winn et al., 2011). The structure of FleQ REC domain was finally determined at 2.3 Å. All of the structure representations were generated using the PyMOL program (http://www.pymol.org).

The release of Pi was monitored by immediately reading the absorbance at 360 mm as a function of time. A standard curve using KH2PO4 as the source for inorganic phosphate was performed. Each experiment was repeated at least twice, and control without substrate was subtracted. 2.10. Isothermal titration calorimetry The binding affinities of WT FleQ and mutant proteins with cdi-GMP were measured by an ITC-200 microcalorimeter (GE Healthcare) at 25 °C. All protein samples (30 lM) and c-di-GMP (600 lM) were prepared in buffer containing 10 mM Tris–HCl (pH 8.0) and 100 mM NaCl. The solutions were centrifuged to remove any possible precipitate before use. Twenty injections were measured with a time interval of 120 s for each sample. Except for the first injection, each titration contains 2 lL of ligand solution. The blank buffer was titrated as a contrast. To evaluate the effect of ATPcS on c-di-GMP binding, 1 mM ATPcS and 1 mM MgCl2 was added to reaction buffer. The titration data were analyzed with Origin software using a single-site binding model. 2.11. Accession code Coordinate and structure factor files have been deposited in the Protein Data Bank (http://www.rcsb.org/pdb) under ID code 4WXM.

2.6. Oligomeric state analysis by native gel PAGE

3. Results

Purified proteins (8 lg) were mixed with CAB buffer (20 mM Tris–HCl pH 8.0, 100 mM NaCl, 1 mM MgCl2, 1 mM DTT and 4% glycerol) and loaded on a 7% native gel PAGE, followed by electrophoresis for 2 h at 120 V in 0.5  TBE (46 mM Tris base, 46 mM boric acid, 1 mM EDTA) at 4 °C, and stained with Coomassie brilliant blue G-250. NativeMark Unstained (Invitrogen) served as the standard.

3.1. The REC domain is essential for the in vivo function of FleQ

2.7. Circular dichroism spectroscopy assay WT FleQ and all the mutant proteins were analyzed at 25 °C on a J-810 spectropolarimeter (Jasco). All the proteins were prepared in buffer containing 25 mM Tris (pH 8.0) and 200 mM NaCl. CD spectra of the proteins at a final concentration of 14 lM were collected from 250 to 200 nm at a scan speed of 200 nm/min with a path length of 0.1 cm. 2.8. Biofilm formation assay Biofilm formation assay was carried out using a modification of previously reported protocol (O’Toole and Kolter, 1998b). Borosilicate glass tubes inoculated with 1:100 dilutions from overnight LB cultures were incubated standing for 10 h at 30 °C. After incubation, cells were washed off, and the tubes were stained with 0.1% crystal violet solution for 30 min and washed thoroughly with water. 2.9. ATPase activity ATPase activity was assayed as previously described (Baraquet and Harwood, 2013) using the EnzChek Phosphate Assay Kit (Invitrogen). WT FleQ or mutant protein (1 lM) was added to the reaction mixtures and incubated for 10 min at room temperature. The reactions were started by the addition of 1 mM ATP. Where necessary, a final concentration of 10 lM or 50 lM c-di-GMP was preincubated with the protein for 30 min on ice before the assay.

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Sequence analysis has shown that the REC domain of FleQ (FleQR, also named FleQ domain) lacks a phosphorylation site which is critical for many receiver domain containing proteins (Arora et al., 1997). We thus performed experiments to test if FleQR plays a role in the function of FleQ in vivo. An unmarked fleQ deletion mutant was made by using previously described sacB-based allelic exchange strategy (Choi and Schweizer, 2005; Hoang et al., 1998; Schweizer and Hoang, 1995; Windgassen et al., 2000). The mutant strain (DfleQ) was completely non-motile in motility assays (Fig. 1A). Motility of the DfleQ strain was restored by full length fleQ gene provided by pUCP18 plasmid, while a fleQ truncation (fragment 139-end) with REC domain deletion could not complement the mutation (Fig. 1A). Since FleQ is the master regulator that activates flagella gene expression (Arora et al., 1997; Dasgupta et al., 2003; Jyot et al., 2002), we guess that the truncation of REC domain may disrupt FleQ function through affecting the transcription of flagella genes. Thus real-time quantitative PCR was performed to evaluate the transcription levels of flagella genes in mutant strains in comparison with WT PAO1. Four genes (fleN, fleR, flhA, fliE) related to flagella formation were analyzed. All these genes showed obviously decreased transcription in DfleQ strain (Fig. 1B–E). The strain complemented with full length fleQ gene had transcription levels that were comparable to WT PAO1 (Fig. 1B–E), while the fleQ REC truncation strain had transcription levels similar to that of DfleQ strain (Fig. 1B–E), indicating that the REC domain of FleQ is essential for transcription activation of flagella genes. Moreover, FleQ has been known to play a significant role in regulating EPS formation. We also performed real-time quantitative PCR to evaluate the transcription levels of pelA and pslA in fleQ REC truncation strain. We found that the DfleQ strain had 40 fold increased level of pelA transcript and 2.6 fold increased level of pslA transcript compared with WT PAO1 (Fig. 1F and G). The fleQ REC truncation strain had comparable transcript levels with DfleQ

Please cite this article in press as: Su, T., et al. The REC domain mediated dimerization is critical for FleQ from Pseudomonas aeruginosa to function as a c-diGMP receptor and flagella gene regulator. J. Struct. Biol. (2015), http://dx.doi.org/10.1016/j.jsb.2015.09.002

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T. Su et al. / Journal of Structural Biology xxx (2015) xxx–xxx

Fig. 1. REC domain regulates the activity of FleQ in vivo. (A) The swimming motility of WT PAO1 strain, fleQ deletion strain (DfleQ) and complement strains (DfleQ + fleQ, DfleQ + fleQ 1–139 and DfleQ + fleQ 139-end) was measured on 0.3% soft agar plates. The experiment has been repeated for three times. (B–G) The transcriptional fold changes of fleN, fleR, flhA, fliE, pelA and pslA genes are presented for fleQ deletion strain.

Please cite this article in press as: Su, T., et al. The REC domain mediated dimerization is critical for FleQ from Pseudomonas aeruginosa to function as a c-diGMP receptor and flagella gene regulator. J. Struct. Biol. (2015), http://dx.doi.org/10.1016/j.jsb.2015.09.002

T. Su et al. / Journal of Structural Biology xxx (2015) xxx–xxx

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Fig. 2. Overall structure of FleQ REC domain. (A) Cartoon representation of FleQR in the asymmetric unit. The monomers are shown in different colors. (B) Structure of the FleQR monomer with the secondary structure elements labeled. (C) The topology view of FleQR generated by Pro-origami (Stivala et al., 2011).

strain (Fig. 1F and G). From these results, we infer that the REC domain is also essential for EPS gene regulation.

3.2. Overall structure of FleQ REC domain Since crystal structure of FleQ could be helpful in uncovering the molecular mechanism by which FleQ regulates flagella biosynthesis and biofilm formation, we have made extensive efforts to determine the three-dimensional structure of FleQ by X-ray crystallography. However, perhaps due to the structural heterogeneity of full-length FleQ, we failed to get qualified crystals. Fortunately, the crystal of FleQ REC domain (residues 1–139) diffracted well. The structure of FleQR was solved by single-wavelength anomalous dispersion (SAD) phasing at 2.3 Å. The final model of FleQR contains five peptide chains in the asymmetric unit (Fig. 2A). Each peptide chain has 129 amino acids (1–129) visible in the density. The last 10 residues at the C-terminal are invisible due to the poor electron densities in this area. The monomer of FleQR is made up of five bstrands and five a-helices, forming a classic REC domain (b/a)5 fold (Fig. 2B). The five parallel b-strands comprise the b-sheet core with the b2b1b3b4b5 topology and are surrounded by three a-helices (a2, a3 and a4) on one side and two a-helices (a1 and a5) on the other side (Fig. 2B and C). Data collection and structure refinement statistics are summarized in Table 1. A similarity search against the Protein Data Bank (PDB) was performed using the Dali server (Holm and Rosenstrom, 2010). The topology of FleQR is similar to the structures of many other determined response regulator proteins. A large part of the homologues belong to the NtrC family, which is consistent with the BLAST results by NCBI database. These includes FleQ homologues NtrC4 from Aquifex aeolicus (PDB code 3DZD; Z-scores 13.6) (Batchelor et al., 2008) and NtrC1 from A. aeolicus (PDB code 1NY5; Z-scores

13.4) (Lee et al., 2003). Superimposition of FleQR to NtrC1 receiver domain shows that the largest structural deviations occur in the b4–a4 loop and in the a4 helix, which notably, is crucial for active dimerization in NtrC1. The a4 helix in FleQR is drastically shortened to only one helical turn (Fig. 3A). The receiver domain of StyR (PDB code 1YIO; Z score 15.2), which regulates styrene catabolism in Pseudomonas fluorescens ST is most similar to FleQR. StyR also possesses a shortened a4 helix (Fig. 3A), and adopts an activelike structure in the unphosphorylated state (Milani et al., 2005). All the homologous proteins of FleQ mentioned above regulate gene expression through phosphorylation, as is the situation in most two-component response regulators. Compared to NtrC1 and StyR, FleQ lacks the conserved phosphate receiver aspartic acid which is commonly situated at the C-terminal of b3 strand, and carries a serine instead (Fig. 3B). Besides, the conformation of D12 is significantly different from the corresponding residue that makes a part of the phosphorylation pocket in its homologous structures. This indicates that FleQ may function in a unique way distinctly different from other phosphorylated regulators.

3.3. Unique dimerization of the REC domain As predicated by PISA (Krissinel and Henrick, 2007), the FleQR protein could potentially form two different dimers in the crystal structure, dimer AB and dimer BC (Fig. 4A). Molecule A interacts with B through a novel dimerization interface that has not been observed in other response regulators. The interface spans about 570 Å2, which accounts for about 8% of the total FleQR surface. Residues buried in the interface mainly come from a1, loop b5– a5 and the amino-terminal of a5. Surface potential analysis shows that dimer AB is mainly formed by hydrophobic interaction in which F26 of each monomer is deeply buried in its partner mole-

Please cite this article in press as: Su, T., et al. The REC domain mediated dimerization is critical for FleQ from Pseudomonas aeruginosa to function as a c-diGMP receptor and flagella gene regulator. J. Struct. Biol. (2015), http://dx.doi.org/10.1016/j.jsb.2015.09.002

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T. Su et al. / Journal of Structural Biology xxx (2015) xxx–xxx

Table 1 Data collection and refinement statistics. Data collection

Native

SAD

Space group

C2

P1

Unit-cell parameters a, b, c (Å) a, b, c (°) Wavelength (Å) Resolution (Å) Measured reflections Unique reflections Completeness (%) Redundancy I/r(I) Rmerge (%)a Wilson B-factors (Å2)

144.40, 58.45, 92.21 90.00, 118.35, 90.00 0.97917 50.00–2.30 (2.38–2.30) 110,445 30,312 100 (99.6) 3.6 (3.7) 33.0 (4.1) 6.2 (39.7) 48.6

62.63, 68.44, 78.23 70.65, 88.88, 68.97 0.97924 50.00–3.10 (3.21–3.10) 79,688 20,578 99.0 (98.6) 3.9 (3.9) 20.9 (3.9) 9.2 (52.5)

Refinement Protein molecules in ASUb Residues Water molecules Total no. of atoms Rworking (Rfree) RMSD bond length (Å) RMSD bond angles (°) Mean B-factors (Å2) Ramachandran plot (%) Most favored Additional allowed Disallowed

5 510 30 5131 0.2104/0.2710 0.009 1.274 70.2 93.3 6.7 0.0

a Rmerge = RhklRi|Ii(hkl)
hI(hkl)i|/RhklRiIi(hkl), where hI(hkl)i is the mean intensity of multiply recorded reflections. b ASU means asymmetric units.

cule thus playing a critical role in dimerization (Figs. 4B and 5A). The alternative dimer BC is mediated mainly through a5 (Fig. 4C), which was in agreement with the situation in NtrC1 (Doucleff et al., 2005). However, the a4 helix and b4–a4 loop of FleQ show significant conformational difference compared to NtrC1 and no longer contribute to dimer formation. This results in a smaller interface (690 Å2) relative to NtrC1 (870.8 Å2). According to the above-mentioned structural information, we guess FleQR would form AB dimer in solution. This idea is confirmed by gel-filtration analysis which shows that FleQR exists as a dimer in solution while FleQR F26N (FleQR with the point mutation F26N) forms a monomer (Fig. 5B). From the structure we can easily see that the AB dimer is significantly different from the widespread classic BC dimer. In BC dimer, the two long helixes a5 of the two monomers are parallel and extend directly to the AAA domains. In AB dimer, however, the two long helixes a5 of

the two monomers contact with each other head to head and would be perpendicular to the linker leading to the AAA domains. Consequently we name the classic BC dimer ‘‘lengthwise dimer”, the unique AB dimer ‘‘transverse dimer”. The distinctive AB ‘‘transverse dimer” is very likely to represent a distinctive mechanism to coordinate biological function. However the question is: is the full-length FleQ activity really dependent on the AB ‘‘transverse dimer”? To answer this question we first need to clarify if full-length FleQ form dimer or higher-order oligomer. Indeed, the dimeric nature of FleQ in solution was supported by the result of native gel PAGE analysis (Fig. 6A). 3.4. Dimerization mediated by Helix a1 is crucial for the function of FleQ For further verifying the biologically relevant dimer interface, a variety of site directed substitutions were introduced into the fleQ gene. Plasmids carrying wild-type or mutated fleQ genes were complemented into the DfleQ strain, and the resulting strains were subjected to biochemical assays to assess the influence of mutated residues on the function of FleQ. The mutation sites were determined according to the result of a structure based sequence analysis. A blast search against NCBI nr database was performed and proteins that share sequence identities between 40% and 60% with FleQR were selected for conservative analysis. All chosen proteins belong to the FleQ superfamily so that they may function with a similar mechanism. The sequence alignment was carried out by T-Coffee (Notredame et al., 2000) and prepared by ESPript (Gouet et al., 1999) (Fig. 4D). Sixteen conserved residues were chosen for mutagenesis, and all of the selected residues were either mapped to the two supposed dimer interfaces or to other potential charged areas that could mediate signal transduction. Five substitutions (R18E, F26A, L27N, P108G and Y111A) occur at residues that are buried in the dimer AB interface (Fig. 4B). Six other substitutions (D116R, H119A, R120E, V123N, E126R and M127N) occur at residues that are parts of dimer BC interface (Fig. 4C). Only five substitutions (E4K, D12K, D15K, E37K and E61K) are not close to either interface. Besides, almost all these residues are located within the conserved motifs predicted by MEME suit (Fig. 4E). The results showed that five substituted strains (L27N, P108G, Y111A, H119A, and D12K) were observed with obviously defective motility when compared to WT PAO1 (data not shown), however, only FleQ Y111A and H119A were soluble during purification. Subsequently, we generated hydrophilic mutations Y111Q and H119N. Also, we constructed F26N for the reason that F26 located in the

Fig. 3. Comparison of FleQR with NtrC1 and StyR. (A) Superposition of the FleQR structure to NtrC1 and StyR. FleQR is shown in green. NtrC1 and StyR are shown in blue and yellow, respectively. The a4 Helixes that share great differences are circled in red. (B) Close-up view of the phosphorylation pocket. Residues related to phosphorylation are labeled and shown in stick modes. FleQ lacks the corresponding D51 and carries a serine instead.

Please cite this article in press as: Su, T., et al. The REC domain mediated dimerization is critical for FleQ from Pseudomonas aeruginosa to function as a c-diGMP receptor and flagella gene regulator. J. Struct. Biol. (2015), http://dx.doi.org/10.1016/j.jsb.2015.09.002

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Fig. 4. Two potential dimerization interfaces and conservative analysis. (A) Molecules from the asymmetric unit are shown in cartoon and labeled as a, b, c, d and e. The two potential dimer interfaces are boxed. (B) Stereo view of the dimer ab interface. Residues in the interface are shown in stick modes. (C) Stereo view of the dimer bc interface. The interface is mainly formed by Helix a5, and the related residues are shown in stick modes. (D) Multiple sequence alignment of the regulatory domains of FleQ from different species sharing identities from 40% to 60%. The alignment was carried out by T-Coffee, and the results were generated using ESPript. Only parts of the alignment results are shown, and the residues conserved in all six sequences are highlighted in red. The residues F26, Y111, and H119 are marked with black triangles. Pa: Pseudomonas aeruginosa PAO1, To: Thalassolituus oleivorans MIL-1, Oa: Oleispira antarctica RB-8, Bm: Bermanella marisrubri, Mgp: Marine gamma proteobacterium HTCC2143, Hg: Hahella ganghwensis. (E) Conserved motifs predicted by MEME suite (Bailey and Elkan, 1994).

Fig. 5. F26N disrupts dimerization of FleQ REC domain. (A) The hydrophobic binding pocket of Phe26. Chain A is shown in cartoon, and chain B is shown with electrostatic potential surface. (B) Oligomeric analysis of FleQ REC domain by gel filtration. FleQR forms dimers in solution, and FleQR F26N exits as monomers. Rib (ribonuclease A from bovine pancreas, 13.7 KD) and Alb (albumin from chicken egg white, 44.3 KD) are used as protein markers.

center of dimer AB interface and might play a vital role according to structural analysis. Complemental strain bearing substitution F26N showed nonmotile behavior, as the DfleQ strain did. Substitutions Y111Q and H119N led to much smaller swimming zones compared to fleQ complementary strain on soft agar plates (Fig. 7A). The swarming motility of these strains was also reduced, but the reduction was more modest than the reduction in swimming motility (Fig. 7B). The secondary structures of substituted FleQ proteins were in good agreement with native protein when examined by circular dichroism spectroscopy (Fig. 6B). That means the phenotypes observed

are not due to misfolding of the mutant proteins. The impacts of these mutants on FleQ function were also observed in biofilm formation assays. Previous studies have proposed that flagella are essential for the initial biofilm formation, probably because flagella propelled cell motility is needed to bring bacteria cells to a certain surface, to which the bacteria will eventually attach as well as in the process of initial adhesion (O’Toole and Kolter, 1998a; O’Toole and Kolter, 1998b). Our observations showed that F26N, Y111Q and H119N substitutions decreased biofilm formation as they did in motility (Fig. 7C).

Please cite this article in press as: Su, T., et al. The REC domain mediated dimerization is critical for FleQ from Pseudomonas aeruginosa to function as a c-diGMP receptor and flagella gene regulator. J. Struct. Biol. (2015), http://dx.doi.org/10.1016/j.jsb.2015.09.002

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Fig. 6. Oligomeric state of FleQ and mutants. (A) Oligomeric state measured by native gel PAGE. NativeMarker Unstained (Invitrogen) served as the standard. (B) Circular dichroism spectroscopy results. The aggregational properties of mutant proteins are in good agreement with native FleQ. (C) Gel filtration analysis results of FleQ and mutant proteins at different concentrations are shown in different colors.

3.5. Point mutations that disrupt dimerization of REC domains effectively affect flagella and EPS gene transcription RT-PCR experiments were performed to evaluate if these mutations disrupt FleQ function through affecting the transcription levels of flagella genes in these mutant strains. As shown in Fig. 1B–E, the four selected genes did exhibit decreased transcription levels, especially for fleN and fleR. Notably, the transcription levels of fleN and fleR in the F26N substituted strain were lower than those in Y111Q and H119N. Next we performed another assay to test if the ATPase activity of FleQ is affected by these mutations. As shown in Fig. 7D, F26N mutant showed the greatest decrease. H119N also exhibited decreased activity, but not as much as F26N. No obvious decrease was observed for Y111Q. These results taken together suggest that ATPase activity could be necessary but not sufficient for FleQ function. Baraquet et al. found that FleQ mainly formed active dimers in solution, accompanied by some tetramers and hexamers (Baraquet and Harwood, 2013). Dimerization of FleQ has been also confirmed by our data, which showed that the purified FleQ existed as dimers when examined by native gel PAGE (Fig. 6A). In this respect, FleQ is different from typical EBPs which exist as inactive dimers and will form higher-order oligomers (mostly hexamers) when activated (Bush and Dixon, 2012). Thus we wondered if the substitutions

which lead to dysfunctional FleQ affect FleQ oligomeric state in solution. Subsequently, the substitutions were subjected to gel filtration analysis. FleQF26N forms dimers at a relatively higher concentration (10.0 lM), which is consistent with the WT FleQ, but tends to form monomers when protein concentration is low (3.9 and 1.7 lM) (Fig. 6C). In contrast, the wild-type FleQ forms dimers regardless of protein concentration (Fig. 6C). It is worth noting that FleQR F26N exists as monomer in solution (Fig. 5B). Since the physiological protein concentration is quite low in vivo, it is reasonable to speculate that substitution F26N also tends to form monomer in cells. FleQ Y111Q shows a behavior similar to wild-type FleQ (Fig. 6C). Y111 is located on the edge of dimer AB interface, so Y111Q may weaken but not disrupt the interaction between the two monomers and result in an unstable dimer without proper function. H119 is located on the BC interface, and H119N reduces the electronic repulsion between H119 and R120 and may result in a strengthened BC interface. Indeed, partial aggregation was observed in FleQ H119N, indicating that the strengthened BC interface and a stable AB interface lead to the polymerization of FleQ (Fig. 6C). The oligomeric state of F26N, Y111Q and H119N substitution proteins were also assessed by running a native gel PAGE (Fig. 6A). The results were consistent with that of gel filtration analysis. Notably, the mutants tended to form more tetramer than

Please cite this article in press as: Su, T., et al. The REC domain mediated dimerization is critical for FleQ from Pseudomonas aeruginosa to function as a c-diGMP receptor and flagella gene regulator. J. Struct. Biol. (2015), http://dx.doi.org/10.1016/j.jsb.2015.09.002

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Fig. 7. The in vivo functional studies of FleQ mutants. (A) Swimming motility assay of fleQ mutant strains. (B) Swarming motility was tested on 0.5% agar plates. (C) Biofilm formation was measured on borosilicate glass tubes at 30 °C for 10 h, and stained with 0.1% crystal violet. (D) ATPase activity of 1 lM FleQ, FleQ F26N, FleQ Y111Q, FleQ H119N and FleQ 139-end was assayed with 1 mM ATP. Data presented are averages of two replicates.

the WT FleQ. This is probably because all these mutants have relatively stronger BC dimerization than AB dimer, which allow FleQ to form oligomers in solution as many REC domain containing proteins do (Batchelor et al., 2008; De et al., 2009; Doucleff et al., 2005). As mentioned above, our results indicated that the dimer AB interface is most likely the biologically relevant dimer interface. This structure has not been observed previously in response regulators. Structural analysis revealed that this interface is mainly formed through hydrophobic interactions. The benzyl side chain of the residue F26 from one monomer is tightly embedded into the hydrophobic groove of the adjacent monomer formed by a1helix, b5–a5 loop and the N-terminal of a5 (Fig. 5A). Thus the hydrophobic interactions mediated by the two F26 from each monomer are much stronger than the hydrophobic interactions between other surface residues. This may explain why F26N can disrupt FleQ dimers and caused such a great damage to FleQ function. By contrast, Y111 is located at the N-terminal of a5 and the benzyl side chains of two residues from both monomers are parallel to each other, generating a relative weaker hydrophobic interaction (Fig. 4B). This is consistent with our gel filtration results (Fig. 6C). Structural analysis shows that the hydrogen-bond between R18 and D19 also contributes to the dimerization, but is not as important. We also evaluated EPS gene (pelA and pslA) transcription levels of mutant strains F26N, Y111Q and H119N. The RT-PCR results showed that all mutants have higher transcription levels compared with fleQ complementary strain (Fig. 1F and G), indicating that dimerization might also be important for FleQ to regulate EPS gene transcription. 3.6. Further clarification of the c-di-GMP binding site FleQ has been determined as a novel c-di-GMP receptor with no conserved c-di-GMP binding sites (Hickman and Harwood, 2008). It was also reported that c-di-GMP binds to the Walker A motif (Lys180), and depresses the ability of FleQ to activate flagella gene expression by inhibiting its ATPase activity (Baraquet and Harwood, 2013). However, c-di-GMP is much bigger than ATP and usually tends to exist as dimer. As a result, it is unlikely that these two kinds of molecules bind to the same site. We guess

c-di-GMP binding site might be close to ATP binding site, thus binding of c-di-GMP would interfere with the ATPase activity. Since c-di-GMP binding nearly always involves arginine residues, we inferred the c-di-GMP binding site should contain arginine residues close to the ATP binding site. To identify the possible c-di-GMP binding site, we made a structural model of FleQresidues134–397 (Fig. 8A). Through structural analysis of FleQR and the model of FleQresidues134–397, we found a cleft at the junction of REC domain and AAA+ domain, which is surrounded by four arginines (R138, R144, R185 and R334). We hypothesized that these four arginines might participate in binding c-di-GMP. To verify this hypothesis, we constructed mutant strains with substitutions R138A, R144A, R185A or R334A, and tested the motility and biofilm formation of these strains using methods mentioned above. The experiment results showed that all these substitutions had almost no obvious effect on the bacteria motility (Fig. 8B). In contrast, R144A and R185A resulted in a significantly decreased biofilm formation (Fig. 8B). This is consistent with previously reported transcriptome data that c-di-GMP had more modest influence on flagella gene expression than on EPS gene expression (Hickman and Harwood, 2008). This indicated that substitutions R144A and R185A may have influenced the binding of c-di-GMP. To further test this hypothesis, we performed isothermal titration calorimetry (ITC) with both WT FleQ and mutant proteins. We found that WT FleQ bound c-di-GMP with a Kd of 1.6 lM (Fig. 8C). However, FleQ R144A and FleQ R185A could not bind cdi-GMP with a heat release similar to buffer control (Fig. 8D–F). All these data indicated that R144 and R185 contribute substantially to c-di-GMP binding in FleQ. As previously reported (Baraquet and Harwood, 2013), K180 is located in the Walker A motif (spatially close to R144 and R185) and probably involved in c-di-GMP binding too. Our result is also in agreement with the recently published work on FlrA, the master regulator of flagella biosynthesis in V. cholerae. FlrA is a FleQ orthologue with the sequence identity of 53%, which is reported to be able to bind c-di-GMP (Srivastava et al., 2013). R135 and R176 were identified to be necessary for c-di-GMP binding (Srivastava et al., 2013). Sequence alignment showed that R135 and R176 of FlrA are exactly the equivalent counterparts of R144 and R185 in FleQ. Further sequence alignment research showed that these two arginines are highly

Please cite this article in press as: Su, T., et al. The REC domain mediated dimerization is critical for FleQ from Pseudomonas aeruginosa to function as a c-diGMP receptor and flagella gene regulator. J. Struct. Biol. (2015), http://dx.doi.org/10.1016/j.jsb.2015.09.002

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Fig. 8. R144A and R185A cannot bind c-di-GMP. (A) The structural model of FleQresidues134–397 dimer. R144 and R185 are shown in stick mode. (B) Swimming motility and biofilm formation of arginine mutant strains. Results of motility and biofilm formation are representatives of three independent experiments. (C–F) Measurement of c-diGMP binding to FleQ by ITC. 20 injections of c-di-GMP solution were titrated into WT FleQ (C), FleQ R144A (D), FleQ R185A (E) and buffer (F). The integrated heat data of WT FleQ gives following parameters: n = 2.13 ± 0.0469, K = 6.08E5 ± 1.74E5 M
1, and DH =
6769 ± 226.5 cal/mol. FleQ R144A and FleQ R185A have a heat release similar to buffer control.

conserved in FleQ homologues from different bacteria. This implies that the same mechanism could be wide spread in bacteria kingdom. Since dimerization is crucial for the function of FleQ, we further examined whether it is essential for c-di-GMP binding. Our ITC results showed that FleQ F26N bound c-di-GMP with approximately 6 folds decrease in binding affinity, and that binding of cdi-GMP to FleQ Y111Q and FleQ H119N decreased by 2.6 and 1.6 folds respectively compared to wild type FleQ (Fig. 9A–C). This indicates that dimerization of FleQ is also important for c-di-GMP binding.

4. Discussion FleQ is an unusual EBP with abnormal features. It belongs to the NtrC two-component subfamily but its REC domain lacks the aspartic acid which serves as a phosphorylation site in other members of NtrC family. Accordingly, the corresponding sensor kinase gene is also missing from the genome. In FleQ REC domain, a serine residue (S59) takes the place of the potential phosphorylated aspartic acid. Substitution of Ser59 with alanine or glycine did

not affect FleQ function (data not shown). This indicates that Ser59 does not serve as phosphorylation site as the aspartic acid does in other members of NtrC family, and FleQ may perform its function in cells with a novel mechanism independent of phosphorylation. From our results, we have identified that the function of FleQ is dependent on its REC domain, and the truncated FleQ (fragment 139-end) cannot activate flagella gene transcription, which has been verified by motility assay and RT-PCR experiments (Fig. 1A–E). Also, REC domain is essential for FleQ to regulate EPS gene expression (Fig. 1F and G). Consistently, truncated FleQ exhibited an obviously decreased ATPase activity compared with fulllength FleQ (Fig. 7D). FleQ is able to bind to a variety of gene promoters, whereas there is no evident consensus on FleQ binding sites. FleQ regulates flagella gene expression dependent on r54-RNA polymerase complex but regulates EPS gene expression in concert with r70 (Baraquet et al., 2012; Dasgupta et al., 2003), indicating different regulatory mechanisms. Surprisingly, as an important transcriptional regulator, FleQ has relative weak DNA binding affinities as previously reported (Hickman and Harwood, 2008). This could be due to the lack of a glycine in the C-terminal DNA binding domain that plays an important role in DNA binding (Jyot et al., 2002). But

Please cite this article in press as: Su, T., et al. The REC domain mediated dimerization is critical for FleQ from Pseudomonas aeruginosa to function as a c-diGMP receptor and flagella gene regulator. J. Struct. Biol. (2015), http://dx.doi.org/10.1016/j.jsb.2015.09.002

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Fig. 9. C-di-GMP binding of FleQ mutants and its effect on ATPase activity. (A–C) C-di-GMP binding affinities of FleQ F26N (A), FleQ Y111Q (B), and FleQ H119N (C) analyzed by ITC. The Kd values are 9.5, 4.1 and 2.6 lM respectively. (D) Binding of c-di-GMP to FleQ measured by ITC at the presence of 1 mM ATPcS and 1 mM MgCl2. The Kd value is 5.5 lM. (E) C-di-GMP inhibits the ATPase activity of FleQ.

why FleQ binds DNA weakly is not clear, although it could have some unknown advantages. Typical EBPs form inactive dimers but assemble into higher oligomers (mostly hexamers) upon activation. However, FleQ forms mainly active dimers, accompanied by very few tetramers and hexamers in solution (Baraquet and Harwood, 2013). This may indicate that FleQ retains the ability to form higher oligomers but the FleQ dimer is more stable, which turns out to be suitable for transcription activation. Structural analysis of FleQR suggests two potential dimer interfaces, dimer AB and dimer BC. Dimer BC is similar to the inactive dimer of NtrC superfamily, which is termed as a ‘‘lengthwise dimer”. While dimer AB is determined to be a novel dimer which hasn’t been reported in other regulators. In contrast to dimer BC, it is termed as a ‘‘transverse dimer”. The ‘‘transverse dimer” interface is mainly formed through hydrophobic interactions by residues from a1, loop b5–a5 and the amino terminal of a5, among which the F26 seemly plays a vital role. Disruption of this interface resulted in a FleQ that tends to form

monomer with significantly reduced ATPase activity (Figs. 6C and 7D). The motility and biofilm formation in F26N mutant strain also greatly decreased compared with WT PAO1 (Fig. 7A–C). Moreover, the transcription levels of the flagella genes (especially for fleN and fleR) in the F26N substituted strain were lower than that in WT PAO1 (Fig. 1B–E). From above all, we termed dimer AB of FleQ as the active dimer interface. The dimeric nature of FleQ was verified by the native gel PAGE (Fig. 6A). FleQ is able to bind c-di-GMP, but it does not contain known conserved c-di-GMP binding sites. Baraquet et al. reported that cdi-GMP binds to the Walker A motif by competing with ATP, and inhibits FleQ ATPase activity (Baraquet and Harwood, 2013). This is consistent with our results (Fig. 9E). However, our data has indicated that R144 and R185 are important for c-di-GMP binding. Then why does c-di-GMP still inhibit FleQ ATPase activity? Analysis of the structural model showed that R144 and R185 are close to Walker A motif. Thus binding of c-di-GMP would interfere with the ATPase activity. In turn, the binding of ATP or ADP may influence

Please cite this article in press as: Su, T., et al. The REC domain mediated dimerization is critical for FleQ from Pseudomonas aeruginosa to function as a c-diGMP receptor and flagella gene regulator. J. Struct. Biol. (2015), http://dx.doi.org/10.1016/j.jsb.2015.09.002

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University (PCSIRT13028). This work was also supported by Zhejiang Natural Science Foundation Grant LR12C05001. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jsb.2015.09.002. References

Fig. 10. Model for the molecular mechanism of FleQ regulation on flagella genes. FleQ forms active dimer and undergoes conformational changes upon regulation of c-di-GMP or other signal molecules.

the binding of c-di-GMP, which has been confirmed by ITC data. When 1 mM ATPcS was added to the reaction buffer, binding of c-di-GMP to FleQ was obviously decreased (Fig. 9D). All these data indicated that R144 and R185 are important for c-di-GMP binding and the binding of c-di-GMP and ATP can interfere with each other. As previously reported, K180 is located in the Walker A motif (spatially close to R144 and R185) and probably involved in c-di-GMP binding too. Based on our findings and previous knowledge of FleQ, we propose a simple model for FleQ regulation on flagella genes (Fig. 10). Normally, FleQ forms active dimers in vivo and binds to flagella promoters. The active form of FleQ uses ATP hydrolysis to drive the formation of FleQ-r54-RNA polymerase open complex and activates transcription. Upon regulation from unknown signals, such as binding of c-di-GMP or interaction with FleN, FleQ probably undergoes conformational changes, which may lead to disruption of the active dimer interface. Subsequently, this may result in decreased ATPase activity and DNA binding affinity, and the transcription will be shut off. Notably, our hypothesis is based on the structures of FleQ REC domain, homologous proteins and related biochemical data. Thus, this model may not be complete, and what structural changes that happen in the output domains are not clear until the structures of full-length FleQ and the related protein complexes are determined. Homologues of FleQ have been identified in many bacteria, including other Pseudomonas species, C. crescentus (Ramakrishnan and Newton, 1990), Vibrio (Kim and McCarter, 2000; Klose and Mekalanos, 1998) and Legionella species (McCarter, 2006). These homologues regulate flagella gene expression, and it is possible that they can also regulate biofilm formation like FleQ does. Structural and biochemical characterization of FleQ may help understand the functions of its homologues, and adds to understanding of the regulation of flagella and biofilm formation in these bacteria. Conflict of interest The authors declare they have no conflicts of interest. Acknowledgments We thank the research group of Prof. Luyan Zulie Ma at Institute of Microbiology, Chinese Academy of Sciences for proving the strains and plasmids required for gene knockout as well as valuable advices. We also thank the staff at beamline BL17U1 at the Shanghai Synchrotron Radiation facility for assistance with data collection. This work was supported by National Nature Science Foundation of China Grant No. 31470732, New Century Excellent Talents in University (NCET81280336), the Shandong Provincial Funds for Distinguished Young Scientists (JQ 201307) and Program for Changjiang Scholars and Innovative Research Team in

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Please cite this article in press as: Su, T., et al. The REC domain mediated dimerization is critical for FleQ from Pseudomonas aeruginosa to function as a c-diGMP receptor and flagella gene regulator. J. Struct. Biol. (2015), http://dx.doi.org/10.1016/j.jsb.2015.09.002