Mapped to the genome, and 13,316,197 (S8DclpP) and 12,562,203 (S8) mapped uniquely. One thousand two hundred thirteen of the unique reads in the S8 sample aligned to the clpP gene, while none of the reads from the S8DclpP sample aligned to this gene. This finding proved that the clpP gene was not expressed in the S8DclpP mutant. The differential expression analysis of the S8DclpP and S8 strains revealed that 16 A. pleuropneumoniae genes exhibited an FDR#0.001 and an absolute value of log2Ratio absolute value 1, proving that these genes were affected by the deletion of the clpP gene (Table 2). Of these 16 genes, 4 were upregulated, including 2 genes involved in the iron acquisition system, and 12 were downregulated.DiscussionThe ClpP protease is a conserved protein that is present in many bacteria [32]. In this study, we initially determined thenucleotide level similarities in the clpP gene sequences with a BLASTN analysis. Currently, only the complete genomes of A. pleuropneumoniae serotypes 3, 5 and 7 have been sequenced. We found that the clpP gene was present in all of these genomes and that the identity of these nucleotide sequences was more than 94 . We also used PCR to confirm that the clpP gene is present in A. pleuropneumoniae serotypes 1?2 reference strains and 11 A. pleuropneumoniae isolates available in our laboratory (data not shown). These results indicated that the clpP gene is conserved in several A. pleuropneumoniae serotypes. In the present study, the clpP mutant was constructed and challenged with a variety of stressors and growth conditions to examine the physiological role of the ClpP protease in the stress tolerance, cell morphology, and biofilm formation related to A. pleuropneumoniae. To confirm that the characteristics of the S8DclpP mutant were not due to polar effects and were truly caused by the clpP gene deletion, we also constructed a complemented strain S8HB. In addition, the RNA sequencing results indicated that the expression levels of the 10 genes upstream of the clpP gene and the 10 genes downstream of the clpP gene did not change. All of these results proved that the characteristics of the S8DclpP mutant were truly caused by the deletion of the clpP gene and not by polar effects. In this study, we observed that the S8DclpP mutant exhibited a reduced growth rate at high temperatures (Figure 1C) and an impaired resistance to heat shock (Figure 2B) compared to the wild-type S8 strain and the complemented S8HB strain. Several previous studies have demonstrated growth deficits resulting from ClpP deficiency over a broad range of temperatures [7,33,34]. However, there was no significant 50-14-6 web difference between the growth curves of the S8DclpP mutant, the wild-type S8 strain and the complemented S8HB strain when these strains were cultured at a lower temperature (Figure 
1A). The observations in our studyFigure 6. Microscopy analysis of the biofilm formation. Biofilm development was monitored by confocal scanning laser microscopy (CSLM) after 12, 18, 24, and 30 h. The cells were stained with LIVE/DEAD@ BacLightTm Bacterial Viability Kit solution. The S8DclpP strain shows a reduction in biofilm formation compared to the wild-type S8 strain and the complemented S8HB strain. doi:10.1371/16960-16-0 site journal.pone.0053600.gRole of ClpP in Actinobacillus pleuropneumoniaeTable 2. The regulated genes in A. pleuropneumoniae by the deletion of the clpP gene.Gene Up-regulated genes APP7_1881 APP7_1156 APP7_1879 APP7_Log2 RatioAnnotation1.Mapped to the genome, and 13,316,197 (S8DclpP) and 12,562,203 (S8) mapped uniquely. One thousand two hundred thirteen of the unique reads in the S8 sample aligned to the clpP gene, while none of the reads from the S8DclpP sample aligned to this gene. This finding proved that the clpP gene was not expressed in the S8DclpP mutant. The differential expression analysis of the S8DclpP and S8 strains revealed that 16 A. pleuropneumoniae genes exhibited an FDR#0.001 and an absolute value of log2Ratio absolute value 1, proving that these genes were affected by the deletion of the clpP gene (Table 2). Of these 16 genes, 4 were upregulated, including 2 genes involved in the iron acquisition system, and 12 were downregulated.DiscussionThe ClpP protease is a conserved protein that is present in many bacteria [32]. In this study, we initially determined thenucleotide level similarities in the clpP gene sequences with a BLASTN analysis. Currently, only the complete genomes of A. pleuropneumoniae serotypes 3, 5 and 7 have been sequenced. We found that the clpP gene was present in all of these genomes and that the identity of these nucleotide sequences was more than 94 . We also used PCR to confirm that the clpP gene is present in A. pleuropneumoniae serotypes 1?2 reference strains and 11 A. pleuropneumoniae isolates available in our laboratory (data not shown). These results indicated that the clpP gene is conserved in several A. pleuropneumoniae serotypes. In the present study, the clpP mutant was constructed and challenged with a variety of stressors and growth conditions to examine the physiological role of the ClpP protease in the stress tolerance, cell morphology, and biofilm formation related to A. pleuropneumoniae. To confirm that the characteristics of the S8DclpP mutant were not due to polar effects and were truly 
caused by the clpP gene deletion, we also constructed a complemented strain S8HB. In addition, the RNA sequencing results indicated that the expression levels of the 10 genes upstream of the clpP gene and the 10 genes downstream of the clpP gene did not change. All of these results proved that the characteristics of the S8DclpP mutant were truly caused by the deletion of the clpP gene and not by polar effects. In this study, we observed that the S8DclpP mutant exhibited a reduced growth rate at high temperatures (Figure 1C) and an impaired resistance to heat shock (Figure 2B) compared to the wild-type S8 strain and the complemented S8HB strain. Several previous studies have demonstrated growth deficits resulting from ClpP deficiency over a broad range of temperatures [7,33,34]. However, there was no significant difference between the growth curves of the S8DclpP mutant, the wild-type S8 strain and the complemented S8HB strain when these strains were cultured at a lower temperature (Figure 1A). The observations in our studyFigure 6. Microscopy analysis of the biofilm formation. Biofilm development was monitored by confocal scanning laser microscopy (CSLM) after 12, 18, 24, and 30 h. The cells were stained with LIVE/DEAD@ BacLightTm Bacterial Viability Kit solution. The S8DclpP strain shows a reduction in biofilm formation compared to the wild-type S8 strain and the complemented S8HB strain. doi:10.1371/journal.pone.0053600.gRole of ClpP in Actinobacillus pleuropneumoniaeTable 2. The regulated genes in A. pleuropneumoniae by the deletion of the clpP gene.Gene Up-regulated genes APP7_1881 APP7_1156 APP7_1879 APP7_Log2 RatioAnnotation1.
