Ed [11]. The bound dye was solubilized in 100 mL of 70 ethanol, and the optical density of the ethanol-dye solution was measured using a microplate reader (Bio-Tek Instruments, Winooski, VT, USA).Confocal laser scanning microscopy Stress resistance assaysStress resistance assays were conducted using A. pleuropneumoniae wild-type S8 strain, the S8DclpP mutant and the complemented S8HB strain. These strains were grown in BHI at 37uC. At an OD600 value of approximately 0.6, cells from 1 ml of broth cultures were centrifuged at 5,000 g for 5 min. For the heat-shock assay, cells were resuspended in BHI and placed in a 52uC water bath for 20 min. For the oxidative tolerance assay, the cells were resuspended in 1 ml of BHI supplemented with 1 mM hydrogen peroxide for 30 min. For the osmotic tolerance assay, the cells were resuspended in 1 ml of BHI supplemented with 0.3 M potassium chloride for 1 h. The control samples of each strain were resuspended in 1 ml of BHI without any treatment. Then, the cultures 18325633 from each stress resistance assay were serially diluted in BHI, and spread on BHI plates for CFU counting. Stress resistance was calculated as [(stressed sample CFU ml21)/(control sample CFU ml21)]6100. The experiments were carried out in triplicate. The same biofilm assay protocol was used as mentioned above. After 16 h of incubation, the wells were washed with water to remove non-adherent bacteria. Cells were then stained with LIVE/DEAD @ BacLight Tm Bacterial Viability Kit solution (Molecular Probes, Eugene, Oregon, 24272870 USA), incubated for 20 min at room temperature in the dark, and washed with water. The plate was examined with a confocal microscope (TCS SP5, Leica Microsystems, Hamburg, Germany). SYTO 9 nucleic acid stain was excited at 488 nm and detected using 520 nm filters. Propidium iodide was excited at 488 nm and detected using 572 nm filters.RNA isolation, cDNA library construction and sequencingA. pleuropneumoniae strains S8 and S8DclpP were grown to early log phase (OD600 nm = 0.5) in BHI. The cells were collected at 4uC, and the RiboPure-Bacteria kit (Ambion) was used according to the manufacturer’s instructions to isolate RNA. Both of the samples were quantified and examined for protein and reagent contamination with a Nanodrop ND-1000 purchase I-BRD9 spectrophotometer (NanoDrop, Wilmington, DE, USA). The RNA samples exhibiting a 23S/16S rRNA band intensity of 2:1, a spectroscopic A260/ A280 nm ratio of 1.8?.0, and an A260/A230 nm ratio greater than 1.5 were selected for analysis. A total of 20 mg of RNA was equally pooled from the S8 and S8DclpP strains for cDNA library preparation. Illumina sequencing was performed at the Beijing Genomics Institute (BGI)-Shenzhen in Shenzhen, China (http://www. genomics.cn/index) according to the manufacturer’s instructions (Illumina, San Diego, CA). The cDNA libraries were prepared according to Illumina’s protocols and sequenced using the Illumina HiSeq 2000.Iron utilization assaysThe iron utilization assay has been described previously [25,26]. Briefly, A. pleuropneumoniae strains S8, S8DclpP and S8HB were grown in 5 ml of BHI for about 20 h and then diluted into fresh BHI medium containing 30 mM of the iron chelator ethylenediamine dihydroxyphenylacetic acid (EDDHA) to similar optical densities at an OD600 value of approximately 0.2. In the iron supplementation culture, 10 mM FeSO4 was added to the iron chelated I-CBP112 culture after the addition of EDDHA. OD600 was determined using an Eppendorf Biophotometer (Ep.Ed [11]. The bound dye was solubilized in 100 mL of 70 ethanol, and the optical density of the ethanol-dye solution was measured using a microplate reader (Bio-Tek Instruments, Winooski, VT, USA).Confocal laser scanning microscopy Stress resistance assaysStress resistance assays were conducted using A. pleuropneumoniae wild-type S8 strain, the S8DclpP mutant and the complemented S8HB strain. These strains were grown in BHI at 37uC. At an OD600 value of approximately 0.6, cells from 1 ml of broth cultures were centrifuged at 5,000 g for 5 min. For the heat-shock assay, cells were resuspended in BHI and placed in a 52uC water bath for 20 min. For the oxidative tolerance assay, the cells were resuspended in 1 ml of BHI supplemented with 1 mM hydrogen peroxide for 30 min. For the osmotic tolerance assay, the cells were resuspended in 1 ml of BHI supplemented with 0.3 M potassium chloride for 1 h. The control samples of each strain were resuspended in 1 ml of BHI without any treatment. Then, the cultures 18325633 from each stress resistance assay were serially diluted in BHI, and spread on BHI plates for CFU counting. Stress resistance was calculated as [(stressed sample CFU ml21)/(control sample CFU ml21)]6100. The experiments were carried out in triplicate. The same biofilm assay protocol was used as mentioned above. After 16 h of incubation, the wells were washed with water to remove non-adherent bacteria. Cells were then stained with LIVE/DEAD @ BacLight Tm Bacterial Viability Kit solution (Molecular Probes, Eugene, Oregon, 24272870 USA), incubated for 20 min at room temperature in the dark, and washed with water. The plate was examined with a confocal microscope (TCS SP5, Leica Microsystems, Hamburg, Germany). SYTO 9 nucleic acid stain was excited at 488 nm and detected using 520 nm filters. Propidium iodide was excited at 488 nm and detected using 572 nm filters.RNA isolation, cDNA library construction and sequencingA. pleuropneumoniae strains S8 and S8DclpP were grown to early log phase (OD600 nm = 0.5) in BHI. The cells were collected at 4uC, and the RiboPure-Bacteria kit (Ambion) was used according to the manufacturer’s instructions to isolate RNA. Both of the samples were quantified and examined for protein and reagent contamination with a Nanodrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA). The RNA samples exhibiting a 23S/16S rRNA band intensity of 2:1, a spectroscopic A260/ A280 nm ratio of 1.8?.0, and an A260/A230 nm ratio greater than 1.5 were selected for analysis. A total of 20 mg of RNA was equally pooled from the S8 and S8DclpP strains for cDNA library preparation. Illumina sequencing was performed at the Beijing Genomics Institute (BGI)-Shenzhen in Shenzhen, China (http://www. genomics.cn/index) according to the manufacturer’s instructions (Illumina, San Diego, CA). The cDNA libraries were prepared according to Illumina’s protocols and sequenced using the Illumina HiSeq 2000.Iron utilization assaysThe iron utilization assay has been described previously [25,26]. Briefly, A. pleuropneumoniae strains S8, S8DclpP and S8HB were grown in 5 ml of BHI for about 20 h and then diluted into fresh BHI medium containing 30 mM of the iron chelator ethylenediamine dihydroxyphenylacetic acid (EDDHA) to similar optical densities at an OD600 value of approximately 0.2. In the iron supplementation culture, 10 mM FeSO4 was added to the iron chelated culture after the addition of EDDHA. OD600 was determined using an Eppendorf Biophotometer (Ep.
