Culture of Geobacillus sp. E263 and infection of GVE2
The deep-sea thermophile Geobacillus sp. E263 (China General Microbiological Culture Collection Center accession no. CGMCC1.7046) was cultured at 60°C with shaking in TTM medium (0.2% NaCl, 0.4% yeast extract, 0.8% tryptone; pH 7.0). The host strain cultures in the mid-exponential phase were infected with its thermophilic bacteriophage GVE2 at a multiplicity of infection (MOI) of 5 and cultured at 60°C.
Protein recombinant expressions in E. coli and antibody preparations
The AST, GroEL and MreB genes of Geobacillus sp. E263 and the vp371 gene of GVE2 were cloned into pGEX-4 T-2 vector (Novagen, Germany) and expressed in E. coli BL21 (DE3) as glutathione S-transferase (GST)-tagged fusion proteins. The recombinant plasmids were confirmed by DNA sequencing. To obtain the recombinant proteins, the recombinant bacteria were induced using isopropyl-β-D- thiogalactoside (IPTG) when the optical density of bacteria was 0.6 at 600 nm. After further incubation for 12 h at 16°C, the induced cells were harvested by centrifugation at 6,000×g for 10 min. The recombinant proteins were purified by affinity chromatography using Glutathione Sepharose resins under native conditions according to the recommended protocol (Qiagen, USA).
The purified recombinant fusion proteins were used as antigens to immunize mice according to a standard procedure . The immunoglobulin G (IgG) fractions of the antiserum were purified with protein A-Sepharose (Bio-Rad) and stored at −80°C until use. As determined by enzyme-linked immunosorbent assay, the antisera dilutions were 1:10,000. The specificity of antibodies was confirmed using Western blotting with the recombinant proteins, virus-infected bacteria and host cells.
The GVE2-infected Geobacillus sp. E263 was collected by centrifugation at 7,000× g for 10 min. The precipitate was re-suspended in 0.1 M Tris–HCl (pH 7.5). After sonication for 5 min, the suspension was centrifuged at 12,000×g for 15 min. The appropriate immunoprecipitation antibody was added to the supernatant and incubated for 2 h at 4°C. Protein A Sepharose slurry (Bio-Rad) was subsequently added, followed by incubation for 2 h at 4°C. Nonspecific binding proteins were removed by five successive rinses with phosphate buffered saline (PBS). The Protein A Sepharose was finally eluted with glycine solution (0.1 M; pH 1.8). The eluant was collected and analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Mass spectrometry (MS) analysis
The protein bands of the SDS-PAGE were excised, trypsinyzed and analyzed using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) MS. A 1.5-μL aliquot was spotted onto a MALDI-TOF sample plate with an equal volume of matrix, a saturated solution of α-cyano-4-hydroxycinnamic acid (Sigma, USA) in 0.1% trifluoroacetic acid and 50% acrylonitrile. The samples were analyzed using a Bruker AutoFlex MALDI-TOF mass spectrometer (Bruker Daltonics, USA). All peptide mass finger printings were externally calibrated using standard peptide mixtures and internally calibrated using the masses of trypsin autolysis products to reach a typical mass measurement accuracy of 100 ppm. All acquired sample spectra were processed using Bruker Flexcontrol 2.4 operation software (Bruker Daltonics) in a default mode with an MS tolerance of 0.2 Da and a tandem MS tolerance of 0.6 Da. Protein identification was performed using Mascot software (version 2.1; Matrix Science, London, UK) and GPS Explorer software (version 3.6; Applied Biosystems, USA) against the NCBInr database and the ORF database of Geobacillus kaustophilus HTA426 in a local database that was generated using a shotgun approach. To eliminate protein redundancy in the database under different names and accession numbers, the single protein member belonging to the species G. kaustophilus HTA426 or otherwise had the highest protein score (top rank) was singled out from the multi-protein family.
Northern blot analysis
Total RNAs were respectively isolated from thermophilic Geobacillus sp. E263 before and after GVE2 infection using Trizol reagent (Invitrogen, USA), followed by incubation with RNase-free DNase I (TakaRa, Japan) for 30 min at 37°C. After electrophoresis on a 1.2% agarose gel in 1× Tris-borate-ethylenediaminetetraacetic acid buffer, the RNAs were transferred to a nylon membrane (Amersham Biosciences, USA). The blots were probed with digoxigenin (DIG)-labeled vp371, GroEL, or AST, respectively. Bacterial 16S rRNA gene was used as a control. In vitro RNA labeling, hybridization, and signal detection were conducted according to the manufacturer's instructions for the DIG High Prime DNA Labeling and Detection Starter Kit II (Roche, Germany).
Protein samples separated by SDS-PAGE were transferred to a nitrocellulose membrane (Bio-Rad) in electroblotting buffer (25 mM Tris, 190 mM glycine, 20% methanol; pH 8.5) for 70 min. The resulting membrane was immersed in blocking buffer (0.1% skim milk, PBS; pH 7.2) at 4°C overnight, followed by incubation with a polyclonal mouse anti-GST-AST IgG, anti-GST-GroEL IgG or anti-GST-VP371 for 3 h, respectively. The membrane was then incubated in alkaline phosphate-conjugated goat anti-mouse IgG (Sigma) for 1 h and detected using NBT and BCIP solutions (BBI, Canada).
Glutathione S-transferase (GST) pull-down assay
The purified GST, GST-MreB, GST-AST and GST-VP371 proteins were incubated with glutathione beads for 2 h at 4°C. The overnight cultures of Geobacillus sp. E263 and Δast mutant were collected by centrifugation at 7000×g for 30 min and resuspended with GST binding buffer [200 mM NaCl, 20 mM Tris–HCl, 1 mM EDTA (ethylene diamine tetraacetic acid), 1 mM PMSF (phenylmethanesulfonyl fluoride), pH 7.6]. The suspension was sonicated for 15 min and centrifuged at 10000×g for 15 min. Subsequently the supernatant was incubated with GST, GST-MreB, GST-AST or GST-VP371 coupled glutathione beads for 5 h at 4°C with gentle rotation. Non-specific binding proteins were removed by five washes using GST binding buffer. Then the proteins bound were eluted with elution buffer (10 mM glutathione, 50 mM Tris–HCl, pH 8.0), and detected by Western blot.
Bacterial two-hybrid assay
To characterize the interactions between AST and GroEL of Geobacillus sp. E263 and the VP371 of GVE2, bacterial two hybrid assay was conducted, using the BacterioMatch two-hybrid system (Stratagene, USA). This system uses a reporter gene cassette that is incorporated into an F’ episome and contains the ampicillin (carbenicillin resistance) and β-galactosidase genes. The reporter strain (kanamycin resistance) harbors lacIq on the F’ episome to repress bait and target synthesis. If the bait (on the pBT vector, which has chloramphenicol-resistance) and target (on the pTRG vector, which has tetracycline resistance) fusion proteins interact with each other, transcription of the reporter genes are activated and represent carbenicillin resistance. Screening for protein–protein interactions involves assaying for growth on LB agar with chloramphenicol, tetracycline, carbenicillin and kanamycin (LB-CTCK). The AST gene was amplified using primers 5′-GTGCGGCCGC ATGAAGCTGGCAA AACGG-3′ (NotI in italics) and 5′-GTGGATCC TTAGGCCCGCGCCTCCAT-3′ (BamHI in italics) and cloned into the pBT (Stratagene, USA) to construct the pBT-AST plasmid. The GroEL gene was cloned into the pTRG (Stratagene) using primers 5′-AT GCGGCCGC ATGGCAAAACAAATCAAG-3′ (Not I in italics) and 5′-ATCTCGAG T TACATCATGCCGCCCAT-3′ (XhoI in italics), yielding the pTRG-GroEL plasmid. To construct the recombinant pBT-vp371, the vp371 gene was cloned into the pBT with primers 5′-GTGCGGCCGC ATGCCGAAGGAATTACGTG AAC-3′ (NotI in italics) and 5′-GTGGATCC TTAAGCAAGTTGTACTTCACCG-3′ (BamHI in italics). For the pTRG-vp371 construct, the vp371 gene was cloned into the pTRG with primers 5′-ATGCGGCCGC ATGCCGAAGGAATTACGTGAAC-3′ (NotI in italics) and 5′-ATCTCGAG TTAAGCAAGTTGTACTTCACCG-3′ (XhoI in italics). All of the recombinant plasmids were confirmed using DNA sequencing.
The constructs of pBT and pTRG were co-transformed into the competent cells of the BacterioMatch® Two-Hybrid System Reporter Strain (Stratagene). The resulting bacterial cells were subsequently plated on LB medium containing tetracycline, chloramphenicol, and kanamycin or the LB-CTCK medium. The plates were incubated for 24–36 h at 30°C and then the colonies were examined.
The antibodies against AST, GroEL, and VP371 were respectively labeled using an Alexa Fluor®532 Protein Labeling Kit, 350 Protein Labeling Kit, and 488 Protein Labeling Kit according to the manufacturer’s instructions (Invitrogen). As controls, the antibodies against GST and MreB were labeled with Alexa Fluor® 488 Protein Labeling Kit, respectively. Briefly, the antibody solution was added to1 M bicarbonate (pH 8.3) and then mixed with the reactive dye. After incubation at room temperature for 1 h, the mixture was loaded onto the purification resin. PBS (pH 7.4) was subsequently added and the labeled antibody was collected.
Overnight cultures of Geobacillus sp. E263 were diluted in TTM medium containing 0.01 M MgCl2 and grown at 60°C. When the OD600 reached 0.3–0.6, the bacteria were infected with GVE2 at an MOI of 5. For imaging, the GVE2-infected and virus-free Geobacillus sp. E263 were immobilized on slides (Sigma) covered with a thin 1% agarose film. The labeled antibodies against AST, GroEL, VP371, GST, and/or GroEL were added to the cultures that were permeabilized by 0.1% Triton X-100. The mixtures were incubated overnight at 4°C. The samples were examined under a Leica TCS SP5 confocal microscope (Germany). The digital images were acquired and analyzed using LAS AF version 2.0.0 software. Images of fluorescent samples were deconvolved within LAS AF and assembled using Adobe Photoshop version 7. Image manipulation was kept to a minimum.
Isothermal titration calorimetry
All proteins were purified and dialyzed into PBS (pH7.4) overnight at 4°C. Protein concentration was determined using ultraviolet absorbance at 280 nm on a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The titration experiments were conducted on a VP-ITC isothermal titration calorimeter (ITC) from MicroCal™, Inc. (Northampton, MA, USA) at 25°C. A 250-μL syringe was used for the ITC injections at a stirring speed of 307 rpm. The injections (10 μL each) were administered every 120 s. The AST, VP371, and GST concentrations in the syringe were 30–50 μM, whereas the GroEL, AST, and GST concentrations in the cell were 6–10 μM. All samples were degassed for 10–30 min prior to use, and all experiments were done at least in triplicate. To calculate the thermodynamic changes of the interactions between GroEL and the other two proteins, the interactions were measured at 35°C, 50°C, and 60°C. The results were analyzed using Origin 7(MicroCal™ LLC ITC) and fitted to a “three sets of sites” model. In this way, the thermodynamic association constant (Ka) and enthalpy change (ΔH) can be calculated directly. The Gibbs free energy change (ΔG) was calculated using the equation ΔG =−RTlnKa, where R was the molar gas constant and T was the absolute temperature at which the experiment was conducted. The entropy change of the interaction was calculated according to the equation TΔS = ΔH − ΔG.