Crystallization and preliminary X-ray crystallographic analysis of a putative nonribosomal peptide synthase AmbB from Pseudomonas aeruginosa

A truncated form of AmbB from Pseudomonas aeruginosa that contains a phosphopantetheine binding (PB) domain and a condensation domain has been crystallized and diffraction data were collected to 2.45 Å resolution.

Abstract

AmbB is a putative nonribosomal peptide synthase from Pseudomonas aeruginosa, which is involved in the production of IQS, a potent cell–cell communication signal molecule that integrates the quorum-sensing mechanism and stress response. It consists of 1249 amino acids and contains an AMP-binding domain, a phosphopantetheine-binding (PB) domain and a condensation (C) domain. In this report, a truncated form of AmbB that contains the PB domain and the condensation domain was overexpressed with an N-terminal GST tag in Escherichia coli and purified as a monomer using affinity and size-exclusion chromatography. The recombinant AmbBc (comprising residues 727–1249 of full-length AmbB) was crystallized using the hanging-drop vapour-diffusion method and a full data set was collected to 2.45 Å resolution using a synchrotron-radiation source. The crystals belonged to space group P6₁22 or P6₅22, with unit-cell parameters a = b = 87.81, c = 286.8 Å, α = 90, β = 90, γ = 120°, and contained one molecule per asymmetric unit.

1. Introduction  

Bacteria are highly social organisms and are capable of cell-to-cell communication via quorum sensing (QS), which mainly coordinates various bacterial community activities including virulence and biofilm formation (Fuqua & Greenberg, 2002 ▶; Federle & Bassler, 2003 ▶). Psedomonas aeruginosa, which is responsible for chronic pneumonia in cystic fibrosis patients, has evolved a complicated regulatory QS network to regulate bacterial virulence. Three QS systems (las, rhl and pqs) that function in a hierarchical manner have been identified in P. aeruginosa (Venturi, 2006 ▶; Williams & Cámara, 2009 ▶). Among them, the las system controls the expression of the rhl and pqs genes, which is at the top of the regulatory networks (Pesci et al., 1999 ▶; de Kievit et al., 2002 ▶; Gilbert et al., 2009 ▶).

However, the central role of the las system in controlling the rhl and pqs QS systems is dependent on growth and environmental conditions. For example, phosphate-depletion stress has recently been found to favour las-independent activation of the rhl and pqs QS systems (Jensen et al., 2006 ▶; Dekimpe & Déziel, 2009 ▶; Smith et al., 2006 ▶). More importantly, clinical isolates frequently contain loss-of-function mutations in the central las system (Smith et al., 2006 ▶; Tingpej et al., 2007 ▶; Wilder et al., 2009 ▶; Karatuna & Yagci, 2010 ▶; Hoffman et al., 2009 ▶). Investigation of the mechanism that may functionally substitute for las led to the identification of a new QS signal, 2-(2-hydroxyphenyl)-thiazole-4-carbaldehyde (IQS), that integrates QS and stress response (Lee et al., 2013 ▶).

l-2-Amino-4-methoxy-trans-3-butenoic acid (AMB; methoxy­vinylglycine) is a secondary metabolite which is thought to be a potent antibiotic (Sahm et al., 1973 ▶; Scannel et al., 1972 ▶; Tisdale, 1980 ▶; Berkowitz et al., 2006 ▶). A five-gene cluster ambABCDE has been identified as being involved in AMB biosynthesis and exportation using biochemical assays combined with site-directed mutagenesis in strain PAO of P. aeruginosa (Lee et al., 2010 ▶). However, a recent study revealed that the ambBCDE gene cluster is involved in the production of IQS rather than AMB biosynthesis (Lee et al., 2013 ▶), as deletion of either ambB, ambD, ambC or ambE led to reduced production of IQS, while a null mutation of ambA had no effect on IQS production (Lee et al., 2013 ▶). IQS modulates AMB production through the pqs and rhl QS systems and is essential for full bacterial virulence (Lee et al., 2013 ▶).

AmbC and AmbD are putative dioxygenases with over 42% identity to each other, while AmbB and AmbE are putative non­ribosomal peptide synthases (NRPS) that share ∼24% sequence identity but have different domain architectures (Lee et al., 2010 ▶). AmbB contains an AMP-binding domain, a phosphopantetheine-binding (PB) domain and a condensation (C) domain (Fig. 1 ▶). The condensation domain is responsible for amide-bond formation, which is the central chemical step in nonribosomal peptide synthesis including IQS synthesis (Stachelhaus et al., 1998 ▶).

Figure 1.

Conserved domains of AmbB predicted by the SMART program (Letunic et al., 2011 ▶).

As a first step towards the structural elucidation of the molecular mechanism underlying IQS production, we overexpressed, purified and crystallized a truncated form of AmbB containing the PB domain and the C domain. A 2.45 Å resolution X-ray diffraction data set has been collected.

2. Materials and methods  

2.1. Gene cloning and expression  

The ambB gene (PA2305) encoding a truncated AmbB (UniProt entry Q9I1H0) containing the PB and C domains (AmbBc; amino acids 727–1249 containing a V733A mutation) was amplified from P. aeruginosa PAO1 genomic DNA by PCR using the forward primer 5′-CGGGATCCACCGGCGCCGAGCC-3′ and the reverse primer 5′-CCGCTCGAGTTAGGAAGCGTTGCAGCCC-3′, and inserted into BamHI and XhoI restriction-endonuclease sites in pGEX-6p-1 vector (GE Healthcare), with a GST tag at the N-terminus. AmbBc was expressed as a GST-fusion protein in Escherichia coli strain BL21 (DE3) CodonPlus. The cells were grown in LB medium at 310 K supplemented with ampicillin (0.1 mg ml⁻¹) and chloramphenicol (0.05 mg ml⁻¹). Isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM when the optical density at 600 nm reached ∼0.6 and the cells were incubated overnight at 291 K. The cells were harvested by centrifugation for 30 min at 3220g and stored at 193 K for protein purification.

2.2. Protein purification  

The frozen cells were thawed at room temperature and resuspended in lysis buffer (20 mM Tris–HCl pH 8.0, 500 mM NaCl, 2 mM DTT) containing 2 mM benzamidine, 100 µM PMSF, 1 mg ml⁻¹ hen egg-white lysozyme. The resuspended cells were incubated on ice for 30 min and then broken using a Soniprep (Sanyo). The lysed cells were centrifuged at 40 555g at 277 K for 30 min.

The clarified cell lysate was mixed with Glutathione Sepharose beads (GE Healthcare) pre-equilibrated with lysis buffer for 1 h. The unbound proteins were then washed away with lysis buffer after loading onto the column. The GST-AmbBc was eluted with lysis buffer containing 20 mM reduced glutathione. The eluted GST-AmbBc was mixed with PreScission protease at a ratio of 50:1(w:w) and incubated at 277 K overnight to cleave off the GST tag. After desalting using a HiPrep Desalting column (GE Healthcare) with desalting buffer (20 mM Tris–HCl pH 8.0, 500 mM NaCl, 2 mM DTT), the protein sample was passed through a Glutathione Sepharose column to remove the cleaved GST fragment and other contaminants. The flowthrough fractions were concentrated and applied onto a Superdex 200 column (HiLoad 26/60 Prep Grade, GE Healthcare) pre-equilibrated with a buffer consisting of 20 mM Tris–HCl pH 8.0, 150 mM NaCl, 2 mM DTT. The fractions containing AmbBc were pooled and concentrated to a final concentration of 11 mg ml⁻¹ and stored at 193 K.

2.3. Crystallization  

Initial crystallization trials were carried out by the hanging-drop vapour-diffusion method in 96-well crystallization plates at both 277 and 289 K. A total of 960 conditions were screened using commercially available crystallization screening kits (Qiagen) and the Phoenix crystallization robot (Art Robbins Instruments). A typical drop consisting of 200 nl protein solution mixed with 200 nl precipitant solution was equilibrated against 60 µl reservoir solution. Initial hits were obtained after 7 d in three conditions [0.1 M ammonium sulfate, 0.1 M HEPES pH 7.5, 10% PEG 4000 from The MbClass II Suite at 277 K; 0.1 M HEPES pH 7.5, 10% PEG 8000, 10% ethylene glycol (EG) of The PEGs II Suite at 277 K; and 0.85 M HEPES pH 7.5, 8.5% PEG 8000, 15% glycerol from The Cryos Suite at 289 K]. Subsequent optimizations were performed manually by mixing 2 µl protein solution with 2 µl precipitant solution and equilibrating against 500 µl reservoir solution using the hanging-drop vapour-diffusion technique in 24-well crystallization plates at 277 K. Several methods including additive screening and microseeding were used for optimization but without obvious effects on crystal morphology (Fig. 2 ▶ a). Finally, MEGA-9 from the detergent screening kit (Hampton Research) was found to change the crystal form (Fig. 2 ▶ b). The rod-shaped crystals (30 × 40 × 300 µm) used for data collection (Fig. 2 ▶ c) were obtained from a condition consisting of 0.1 M Tris pH 7.5, 11% PEG 6000, 2.5% EG/MPD, 25 mM MEGA-9.

Figure 2.

(a) Clusters of needle-like crystals after screening. (b) The rod-like crystals obtained using the detergent MEGA-9. (c) Optimized AmbBc crystals used for data collection.

2.4. Data collection and processing  

Crystals were harvested with cryoloops (CrystalCap HT) and transferred from the mother liquor into cryoprotectant solution consisting of the reservoir solution supplemented with 20%(v/v) glycerol. All crystals were flash-cooled in liquid nitrogen and exposed to X-rays at 100 K. X-ray data sets were collected on beamline BL17U at the Shanghai Synchrotron Radiation Facility (SSRF) in China. A total of 400 diffraction images with 0.5° oscillation per image and an exposure time of 1 s per image were collected. The crystal diffracted to better than 2.5 Å resolution (Fig. 3 ▶). Diffraction images were indexed, integrated and scaled using HKL-2000 (Otwinowski & Minor, 1997 ▶). Data-collection and processing statistics are summarized in Table 1 ▶.

Figure 3.

A representative X-ray diffraction pattern of an AmbBc crystal. The outer circle indicates a resolution of 2.5 Å.

Table 1. Data-collection and processing statistics for AmbBc crystals.

Values in parentheses are for the outermost resolution shell.

Beamline, synchrotron	BL17U, SSRF, Shanghai, China
Wavelength (Å)	0.9793
Detector	ADSC Q315
Data-collection temperature (K)	100
Crystal-to-detector distance (mm)	320
Rotation range per image (°)	0.5
Total rotation range (°)	100
Exposure time per image (s)	1
Resolution range (Å)	59.0–2.45
Space group	P6122 or P6522
Unit-cell parameters (Å, °)	a = 87.81, b = 87.81, c = 286.79, α = 90, β = 90, γ = 120
Total No. of measured intensities	273037
Unique reflections	24816
Mosaicity	0.50
Completeness (%)	99.6 (99.8)
Multiplicity	11.0 (11.5)
R merge † (%)	11.6 (93.0)
R meas ‡ (%)	12.7 (96.0)
〈I/σ(I)〉	14.7 (3.2)
Matthews coefficient (Å3 Da−1)	2.74
Solvent content (%)	55.0

^† R _merge = , where I_i(hkl) is the intensity of an individual reflection and 〈I(hkl)〉 is the average intensity of that reflection.

^‡ R _meas = , where N(hkl) is the multiplicity, l_i(hkl) is the intensity of the ith measurement of reflection hkl and 〈I(hkl)〉 is the average value over multiple measurements.

3. Results and discussion  

We have expressed and purified AmbBc using GST-affinity and size-exclusion chromatography (SEC). The SEC elution profile showed a single peak (Fig. 4 ▶ a) at an apparent molecular weight (MW) of 57 kDa, which is close to the calculated MW of 55 799.70 Da, suggesting that AmbBc may exist as a monomer in solution. SDS–PAGE with Coomassie staining showed that purified AmbB is at least 95% pure (Fig. 4 ▶ b). About 8 mg pure AmbBc could be obtained from 2 l cell culture. The identity of the purified AmbBc has been confirmed by mass spectrometric analysis.

Figure 4.

Purification of AmbBc. (a) Superdex-200 gel-filtration column chromatogram showing a peak at an apparent molecular mass of 57 kDa; (b) SDS–PAGE gel of purified AmbBc showing a final purity of ∼95%. The left lane contains molecular-mass marker (labelled in kDa).

Initially, clusters of conjoined thin needle-like crystals of AmbBc were observed. However, all attempts to obtain larger and single crystals failed until we used the detergent screening kit to identify MEGA-9, which changed the crystal morphology from a needle-like to a rod-like shape (Fig. 2 ▶ b). A complete data set to 2.45 Å resolution (Fig. 3 ▶, Table 1 ▶) was collected from a single crystal using a synchrotron-radiation source. The crystals belonged to space group P6₁22 or P6₅22, with unit-cell parameters a = b = 87.81, c = 286.79 Å, α = 90, β = 90, γ = 120°. There is one molecule per asymmetric unit, giving a solvent content of 55.0% and a Matthews coefficient of 2.74 ų Da⁻¹ (Matthews, 1968 ▶).

As AmbB has no structural homologue available in the Protein Data Bank, heavy-atom soaks were performed with Heavy Atom Screen M1 (Hampton Research). Crystals were soaked in heavy-atom solutions at concentrations of 0.1–0.5 mM overnight and of 1 mM for 2 and 4 h. In addition, we expressed and purified selenomethionine-substituted AmbBc and set up crystallization using the same condition as that used for the native protein. Further crystallization and structural determination of AmbBc using the single-wavelength anomalous dispersion method is under way.