Backbone and sidechain 1H, 15N and 13C assignments of Tyrosine Phosphatase related to Biofilm formation A (TpbA) of Pseudomonas aeruginosa
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- Koveal, D., Jayasundera, T.B., Wood, T.K. et al. Biomol NMR Assign (2013) 7: 57. doi:10.1007/s12104-012-9376-z
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The backbone and side chain resonance assignments of the Tyrosine Phosphatase related to Biofilm formation A (TpbA) of Pseudomonas aeruginosa have been determined based on triple-resonance experiments using uniformly [13C,15N]-labeled protein. This assignment is the first step towards the determination of the 3-dimensional structure of TpbA.
Bacterial biofilms are complex communities of bacteria that are highly resistant to antibiotics and are the cause of many recalcitrant infectious diseases diagnosed in the developed world (Donlan 2002). Biofilms are formed when communities of bacterial microorganisms adhere to a suitable surface and encapsulate themselves in an extracellular matrix composed of exopolysaccharides, proteins and DNA (Karatan and Watnick 2009). The bacteria within the colony communicate with each other through quorum sensing, and once the biofilm is formed they are virtually impossible to eliminate using traditional antibiotics (Davies 2002; Ueda and Wood 2009). As a result, biofilms pose a serious medical threat, especially when they infect an immunocompromised patient or colonize medical devices that must then be surgically replaced.
The pathogenicity of the free-living bacterium Pseudomonas aeruginosa is linked to its ability to form biofilms (Ryder et al. 2007). Infection from P. aeruginosa biofilms is a major problem for patients suffering from chronic diseases, especially cystic fibrosis (CF). In most CF patients, P. aeruginosa biofilm infections typically lead to respiratory failure and death, primarily due to the insensitivity of P. aeruginosa biofilms to antibiotics (Davies 2002). Tyrosine Phosphatase related to Biofilm formation A (TpbA) is a vital regulator of P. aeruginosa biofilm formation as it negatively regulates the concentration of the small molecule 3,5-cyclic diguanylate (c-di-GMP), a positive regulator of biofilm formation (Ross et al. 1990; Lee et al. 2007; Ueda and Wood 2009).
TpbA is a cysteine-based phosphatase that contains the protein tyrosine phosphatase (PTP) signature motif C(X)5R(S/T) and is capable of dephosphorylating serines, threonines and tyrosines (Ueda and Wood 2009; Pu and Wood 2010). In response to an external stimulus from the quorum sensing molecule oxo-C12-AHL, a signaling cascade within the cell triggers the expression and translocation of TpbA from the reductive cytosolic cellular environment to the more oxidative periplasmic space, where TpbA dephosphorylates and inactivates the membrane bound diguanylate cyclase TpbB. This reduces cellular c-di-GMP levels and, in turn, biofilm formation (Pu and Wood 2010). In this way, TpbA functions to link extracellular quorum sensing signals to biofilm formation. A structural study to elucidate the molecular basis of TpbA activity is therefore critical, as it will not only enable the mechanism by which the catalytic cysteine is protected from oxidation in the periplasm to be determined, but will also provide a basis for the development of novel chemical agents that modulate TpbA activity, and, in turn, biofilm formation (Messens and Collet 2006; Ueda and Wood 2009).
Methods and experiments
TpbA (residues 29–218) was subcloned into a modified pET28a-based vector (RP1B), which encodes a Thio6His6 expression/purification tag and a TEV (tobacco etch virus protease) cleavage site (MGSDKIHHHHHHENLYFQGH). The expression plasmid was transformed into BL21 (DE3) cells (Invitrogen) and cultures were grown to an optical density (OD600) of 0.8–0.9 at 37°C with shaking at 250 rpm. Protein expression was induced by the addition of 1 mM isopropylthio-β-D-galactoside (IPTG), and the cultures were grown for an additional 18–20 h at 18°C with shaking at 250 rpm. Freshly transformed cells were grown in LB medium containing selective antibiotics. To prepare uniformly 13C/15N-labeled and 15N-labeled protein, bacterial cultures were grown in M9 minimal medium containing selective antibiotics and 4 g/l [13C]-d-glucose and/or 1 g/l 15NH4Cl (Cambridge Isotope Laboratories, Cambridge, MA) as the sole carbon and nitrogen sources, respectively.
The cells were harvested by centrifugation, resuspended in ice-cold lysis buffer (50 mM Tris pH 8.0, 500 mM NaCl, 5 mM imidazole, 0.1% Triton X-100, and EDTA-free protease inhibitor tablets (Roche)), and lysed by high-pressure cell homogenization (Avestin C3 Emulsiflex). The bacterial lysate was clarified by centrifugation at 16,000×g for 45 min at 4°C. After filtration, the supernatant was loaded onto a HisTrap HP column (GE Healthcare) equilibrated with 50 mM Tris pH 8.0, 5 mM imidazole, and 500 mM NaCl, and the His6-tagged protein was eluted with a 5–500 mM imidazole gradient. Fractions containing TpbA were pooled and digested with tobacco etch virus (TEV, in-house) protease overnight at 4°C while being dialyzed against 50 mM Tris pH 8.0, 500 mM NaCl. Cleaved protein was further purified using Ni2+-NTA immobilized metal affinity chromatography followed by size exclusion chromatography (Superdex 75 26/60; GE Healthcare) equilibrated in 10 mM Tris pH 7.8, 100 mM NaCl, 0.5 mM TCEP to a purity of >98% and a final yield of ~25 mg protein/l culture. Purified protein was concentrated to 1.1 mM. For long-term storage, the protein was flash frozen in liquid nitrogen and stored at −80°C.
All NMR measurements were performed at 298 K. The sequence-specific backbone assignment was obtained from the following experiments performed on a Bruker Avance 500 MHz spectrometer: 2D [1H,15N] HSQC, 2D [1H,13C] HSQC, 3D HNCA, 3D HNCACB, 3D CBCA(CO)NH, 3D (H)CC(CO)NH (τm = 12 ms), 3D HBHA(CO)NH, and a 3D 15N-resolved [1H,1H] NOESY spectrum (τm = 65 ms). A HNCACB spectrum was also recorded on a Bruker Avance 800 MHz spectrometer. The side chain assignment of all aliphatic residues was obtained from a 3D HC(C)H-TOCSY (τm = 12 ms) spectrum recorded on a Bruker Avance 800 MHz spectrometer, with assistance from 3D (H)CCH-TOCSY (τm = 12 ms) and 3D HC(C)H-COSY spectra recorded on a Bruker Avance 500 MHz spectrometer. The aromatic side chains were assigned using 2D [1H,1H] NOESY (τm = 65 ms) and 2D [1H,1H] TOCSY (τm = 50 ms) (800 MHz 1H Lamor frequency) acquired with a TpbA sample dissolved in 100% D2O solution after complete H/D exchange of all labile protons. The NMR spectra were processed with Topspin 1.3 (Bruker, Billerica, MA) and analyzed using the CARA software package (www.nmr.ch).
Assignment and data deposition
The authors thank Dr. David A. Critton for help at early stages of the project. This work was supported by NSF CAREER (MCB0952550) to R. P and NIH (GM089999) to T. K. W. 800 MHz NMR data were recorded at Brandeis University (NIH S10-RR017269).