To construct the recombinant pBT-vp371, the vp371 gene was cloned into the pBT with primers 5′-GTGCGGCCGCATGCCGAAGGAATTACGTG

AAC-3′ (NotI in italics) and 5′-GTGGATCCTTAAGCAAGTTGTACTTCACCG-3′ (BamHI in italics). For the pTRG-vp371 construct, the vp371 gene was cloned into the pTRG with primers 5′-ATGCGGCCGCATGCCGAAGGAATTACGTGAAC-3′ (NotI in italics) and 5′-ATCTCGAGTTAAGCAAGTTGTACTTCACCG-3′ (XhoI in italics). All of the recombinant plasmids were confirmed using DNA sequencing. The constructs Selleck MK 1775 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. Antibody labeling 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 find more solution was added to1 M bicarbonate (pH 8.3) and then mixed with the reactive dye. After incubation at room temperature for 1 h, RAD001 datasheet the mixture was loaded onto the purification resin. PBS (pH 7.4) was subsequently added and the labeled antibody was collected. Immunofluorescence microscopy 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% Astemizole 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.