N-regulated genes APP7_0616 APP7_2064 APP7_1497 APP7_0617 APP7_0418 APP7_0419 APP7_1517 APP7_1695 APP7_1286 APP7_1284 APP7_0747 APP7_1152 22.30679 22.12199 21.78015 21.5762 21.44626 21.33961 21.26523 21.25314 21.1338 21.0894 21.05714 21.04195 pyridoxal biosynthesis lyase PdxS tRNA 2-thiouridine synthesizing protein A hypothetical protein glutamine amidotransferase subunit PdxT RNA polymerase sigma-70 factor putative sigma-E factor negative regulatory protein hypothetical protein hypothetical protein maltose/maltodextrin import ATP-binding protein MalK maltose operon periplasmic protein putative methylation subunit, type III restriction-modification system hexosaminidasedoi:10.1371/journal.pone.0053600.tsuggest a more restricted role for ClpP in A. pleuropneumoniae, independent of cold stress. Iron is an essential factor for the growth of A. pleuropneumoniae, and low iron availability in the host represents a major stress for the pathogen. A. pleuropneumoniae has evolved a highly sophisticated system for iron acquisition that includes transferrin receptor complexes TbpA/TbpB, TonB-ExbB-ExbD, Afu, ABC transporter and so on [26]. In the current study, 25331948 the S8DclpP mutant was shown to exhibit a faster growth compared to the wild-type S8 strain and the complemented S8HB strain. In contrast, this result was not reported in studies on the role of ClpP in other bacteria. Based on this result, we hypothesize that the ClpP protease might regulate the expression of some of the genes involved in iron uptake, thus regulating virulence. Therefore, we also transcriptionally profiled the effects of the deletion of the clpP gene in A. pleuropneumoniae. The results of this analysis showed that 2 genes encoding a putative periplasmic iron/siderophore binding protein and a Fe(III) dicitrate ABC transporter were upregulated. This finding JSH-23 biological activity indicated that the inactivation of the ClpP protease affects the expression of these two iron acquisition proteins. Bacterial biofilm MedChemExpress IPI549 formation is a complex, multifactorial process requiring genes involved in adherence, metabolism, quorum sensing, and the stress response [35]. It has been shown that several genes involved in these factors mentioned above affect the biofilm formation by A. pleuropneumoniae, including the genes coding for polysaccharide PGA [36]; ArcA, which is a regulator involved in anaerobic metabolism [37]; and LuxS, which is a regulator involved in quorum sensing [38]. This study is the first to show the role of stress protein in A. pleuropneumoniae biofilm formation. We found that the clpP mutation slowed down biofilm formation. In addition, the similar growth rates of the S8 and S8DclpP strains at 37uC (Figure 1B) suggest that it is unlikely that the reduced biofilm formation observed in the clpP mutant is due to the inherently different growth rates between strains. The results of the RNA sequencing analysis indicated that a gene encoding hexosaminidase was downregulated in the S8DclpP mutant. It has been shown that hexosaminidase removes the terminal residues from glycoproteins and exposes b-linked glucosamine, thus mediating biofilm formation in A.pleuropneumoniae [36,39]. Our findings indicate that the ClpP protease regulates the expression of the gene encoding hexosaminidase, thus contributing to the attenuation of biofilm formation. In addition, we also found that the expression of some genes encoding hypothetical proteins was upregulated or downregulated. These changes might also affect biofilm f.N-regulated genes APP7_0616 APP7_2064 APP7_1497 APP7_0617 APP7_0418 APP7_0419 APP7_1517 APP7_1695 APP7_1286 APP7_1284 APP7_0747 APP7_1152 22.30679 22.12199 21.78015 21.5762 21.44626 21.33961 21.26523 21.25314 21.1338 21.0894 21.05714 21.04195 pyridoxal biosynthesis lyase PdxS tRNA 2-thiouridine synthesizing protein A hypothetical protein glutamine amidotransferase subunit PdxT RNA polymerase sigma-70 factor putative sigma-E factor negative regulatory protein hypothetical protein hypothetical protein maltose/maltodextrin import ATP-binding protein MalK maltose operon periplasmic protein putative methylation subunit, type III restriction-modification system hexosaminidasedoi:10.1371/journal.pone.0053600.tsuggest a more restricted role for ClpP in A. pleuropneumoniae, independent of cold stress. Iron is an essential factor for the growth of A. pleuropneumoniae, and low iron availability in the host represents a major stress for the pathogen. A. pleuropneumoniae has evolved a highly sophisticated system for iron acquisition that includes transferrin receptor complexes TbpA/TbpB, TonB-ExbB-ExbD, Afu, ABC transporter and so on [26]. In the current study, 25331948 the S8DclpP mutant was shown to exhibit a faster growth compared to the wild-type S8 strain and the complemented S8HB strain. In contrast, this result was not reported in studies on the role of ClpP in other bacteria. Based on this result, we hypothesize that the ClpP protease might regulate the expression of some of the genes involved in iron uptake, thus regulating virulence. Therefore, we also transcriptionally profiled the effects of the deletion of the clpP gene in A. pleuropneumoniae. The results of this analysis showed that 2 genes encoding a putative periplasmic iron/siderophore binding protein and a Fe(III) dicitrate ABC transporter were upregulated. This finding indicated that the inactivation of the ClpP protease affects the expression of these two iron acquisition proteins. Bacterial biofilm formation is a complex, multifactorial process requiring genes involved in adherence, metabolism, quorum sensing, and the stress response [35]. It has been shown that several genes involved in these factors mentioned above affect the biofilm formation by A. pleuropneumoniae, including the genes coding for polysaccharide PGA [36]; ArcA, which is a regulator involved in anaerobic metabolism [37]; and LuxS, which is a regulator involved in quorum sensing [38]. This study is the first to show the role of stress protein in A. pleuropneumoniae biofilm formation. We found that the clpP mutation slowed down biofilm formation. In addition, the similar growth rates of the S8 and S8DclpP strains at 37uC (Figure 1B) suggest that it is unlikely that the reduced biofilm formation observed in the clpP mutant is due to the inherently different growth rates between strains. The results of the RNA sequencing analysis indicated that a gene encoding hexosaminidase was downregulated in the S8DclpP mutant. It has been shown that hexosaminidase removes the terminal residues from glycoproteins and exposes b-linked glucosamine, thus mediating biofilm formation in A.pleuropneumoniae [36,39]. Our findings indicate that the ClpP protease regulates the expression of the gene encoding hexosaminidase, thus contributing to the attenuation of biofilm formation. In addition, we also found that the expression of some genes encoding hypothetical proteins was upregulated or downregulated. These changes might also affect biofilm f.
