Pendorf, Hamburg, Germany) at order JI-101 various time points. The experiments were carried out in triplicate.Scanning electron microscopyFor scanning electron microscopy, A. pleuropneumoniae strains S8, S8DclpP and S8HB were grown to similar optical densities at the OD600 value of approximately 0.5, 2.0 and 2.7, respectively. Then washed 3 times with phosphate-buffered saline (PBS; in mM: 137 NaCl, 2.7 KCl, 1.8 KH2PO4 and 10 Na2HPO4, pH 7.4). Cultures were then fixed with 2.5 glutaraldehyde and 1 OsO4, dehydrated in a graded ethanol series and embedded in isoamyl acetate. The cells were dried using a critical point drying method, mounted on aluminum stubs and shadowed with gold. A scanning electron microscope (S-3400N, Hitachi, Japan) at 5 kV was used to visualize cells.Differential expression analysisThe raw sequence reads were filtered by the Illumina pipeline. All of the low-quality reads, reads with adaptor contamination, and reads with a copy number of one were excluded from the analysis, while the clean 18325633 reads were mapped to the A. pleuropneumoniae serotype 7 strain S8 reference sequences (Genbank accession No. ALYN00000000.1). To identify the genes affected by the deletion of the clpP gene, the libraries were initially compared. To complete this procedure, the number of reads for each coding region was determined, the number of total reads was normalized between the libraries and the ratio of S8 reads to S8DclpP reads was purchase Gracillin calculated. The differentially expressed genes were detected as previously described [28], with the false discovery rate (FDR) being set below 0.01 [29]. An FDR#0.001 and a log2Ratio absolute value 1 was set as the threshold for significant differences in gene expression.Polystyrene microtiter plate biofilm assayThe microtiter plate biofilm assay is a static assay that is particularly useful for examining biofilm formation [27]. The wellsRole of ClpP in Actinobacillus pleuropneumoniaeRole of ClpP in Actinobacillus pleuropneumoniaeFigure 2. Impaired stress tolerance of the A. pleuropneumoniae S8DclpP mutant. Overnight cultures were inoculated into fresh medium and grown to an OD600 value of approximately 0.6. Cells were then treated with (A) 1 mM H2O2 for 30 min, * p,0.01, (B) 52uC heat shock for 20 min, *p,0.01, or (C) 0.3 M KCl for 1 hour, * p,0.05. * denotes P values (t test) for comparison to S8DclpP. doi:10.1371/journal.pone.0053600.gThe Blast2GO program [30] was used to obtain GO annotations for molecular functions, biological processes and cellular component ontologies (http://www.geneontology.org).The Kyoto Encyclopedia of Genes and Genomes pathway database [31] (http://www.genome.jp/kegg) was used to make pathway assignments. The BlastN program (http://blast.ncbi.nlm.Figure 3. The growth curves of the A. pleuropneumoniae in iron-restricted and iron supplemented conditions. Overnight cultures of the S8 ( ), S8DclpP (#) and S8HB (n) strains were diluted into fresh medium and then incubated in (A) BHI containing 30 mM of the iron chelator EDDHA or (B) BHI containing 30 mM of the iron chelator EDDHA and 10 mM FeSO4. Growth was monitored by OD600 at various time points. Points indicate the mean values, and error bars indicate standard deviations. doi:10.1371/journal.pone.0053600.gRole of ClpP in Actinobacillus pleuropneumoniaenih.gov/) was also used to compare these sequences to the A. pleuropneumoniae serotype 7 strain AP76 reference sequences (Genbank accession No. CP001091.1) and obtain a historical annotation.42uC.Pendorf, Hamburg, Germany) at various time points. The experiments were carried out in triplicate.Scanning electron microscopyFor scanning electron microscopy, A. pleuropneumoniae strains S8, S8DclpP and S8HB were grown to similar optical densities at the OD600 value of approximately 0.5, 2.0 and 2.7, respectively. Then washed 3 times with phosphate-buffered saline (PBS; in mM: 137 NaCl, 2.7 KCl, 1.8 KH2PO4 and 10 Na2HPO4, pH 7.4). Cultures were then fixed with 2.5 glutaraldehyde and 1 OsO4, dehydrated in a graded ethanol series and embedded in isoamyl acetate. The cells were dried using a critical point drying method, mounted on aluminum stubs and shadowed with gold. A scanning electron microscope (S-3400N, Hitachi, Japan) at 5 kV was used to visualize cells.Differential expression analysisThe raw sequence reads were filtered by the Illumina pipeline. All of the low-quality reads, reads with adaptor contamination, and reads with a copy number of one were excluded from the analysis, while the clean 18325633 reads were mapped to the A. pleuropneumoniae serotype 7 strain S8 reference sequences (Genbank accession No. ALYN00000000.1). To identify the genes affected by the deletion of the clpP gene, the libraries were initially compared. To complete this procedure, the number of reads for each coding region was determined, the number of total reads was normalized between the libraries and the ratio of S8 reads to S8DclpP reads was calculated. The differentially expressed genes were detected as previously described [28], with the false discovery rate (FDR) being set below 0.01 [29]. An FDR#0.001 and a log2Ratio absolute value 1 was set as the threshold for significant differences in gene expression.Polystyrene microtiter plate biofilm assayThe microtiter plate biofilm assay is a static assay that is particularly useful for examining biofilm formation [27]. The wellsRole of ClpP in Actinobacillus pleuropneumoniaeRole of ClpP in Actinobacillus pleuropneumoniaeFigure 2. Impaired stress tolerance of the A. pleuropneumoniae S8DclpP mutant. Overnight cultures were inoculated into fresh medium and grown to an OD600 value of approximately 0.6. Cells were then treated with (A) 1 mM H2O2 for 30 min, * p,0.01, (B) 52uC heat shock for 20 min, *p,0.01, or (C) 0.3 M KCl for 1 hour, * p,0.05. * denotes P values (t test) for comparison to S8DclpP. doi:10.1371/journal.pone.0053600.gThe Blast2GO program [30] was used to obtain GO annotations for molecular functions, biological processes and cellular component ontologies (http://www.geneontology.org).The Kyoto Encyclopedia of Genes and Genomes pathway database [31] (http://www.genome.jp/kegg) was used to make pathway assignments. The BlastN program (http://blast.ncbi.nlm.Figure 3. The growth curves of the A. pleuropneumoniae in iron-restricted and iron supplemented conditions. Overnight cultures of the S8 ( ), S8DclpP (#) and S8HB (n) strains were diluted into fresh medium and then incubated in (A) BHI containing 30 mM of the iron chelator EDDHA or (B) BHI containing 30 mM of the iron chelator EDDHA and 10 mM FeSO4. Growth was monitored by OD600 at various time points. Points indicate the mean values, and error bars indicate standard deviations. doi:10.1371/journal.pone.0053600.gRole of ClpP in Actinobacillus pleuropneumoniaenih.gov/) was also used to compare these sequences to the A. pleuropneumoniae serotype 7 strain AP76 reference sequences (Genbank accession No. CP001091.1) and obtain a historical annotation.42uC.
