Figure 1. Exposure assay results of S. aureus wild-type, ΔpurN to ampicillin (10 μg/mL, (A–C)), and levofloxacin (20 μg/mL, (D–F)) in cultures at different time points. 5 h point (A,D). 9 h point (B,E). 18 h point (C,F).

Figure 1. Exposure assay results of S. aureus wild-type, ΔpurN to ampicillin (10 μg/mL, (A–C)), and levofloxacin (20 μg/mL, (D–F)) in cultures at different time points. 5 h point (A,D). 9 h point (B,E). 18 h point (C,F).

Figure 2. Drug exposure results of Newman::pRAB11, ∆purN::pRAB11, ∆purN::pRABpurN, and Newman::pRBpurN to ampicillin (A,B), vancomycin (C,D), gentamicin (E,F) and levofloxacin (G,H) at different culture times. 5-h culture (A,C,E,G); 9-h culture (B,D,F,H).

Figure 2. Drug exposure results of Newman::pRAB11, ∆purN::pRAB11, ∆purN::pRABpurN, and Newman::pRBpurN to ampicillin (A,B), vancomycin (C,D), gentamicin (E,F) and levofloxacin (G,H) at different culture times. 5-h culture (A,C,E,G); 9-h culture (B,D,F,H).

Figure 3. Comparison of the virulence of Newman::pRAB11, ∆purN::pRAB11, ∆purN::pRABpurN, and Newman::pRBpurN in S. aureus. (A) The virulence gene expression levels detected by RT-qPCR. (B) Variation of hemolysis in different strains. Hemolysis status of Newman::pRAB11 (a,e), ∆purN::pRAB11 (b,f), ∆purN::pRABpurN (c,g), and Newman::pRBpurN (d,h) cultured for 24 h (a–d) and 48 h (e–h) on blood TSA plates. The hemolysis assay of the four strains was measured in different time points cultures. (i)10 h, (j) 14 h, (k) 24 h, and (l) 48 h. (C) The biofilm formation abilities of the four S. aureus strains in 96-well plates. Comparison of OD550 and biofilm images in 96-well plate of different strains. * p < 0.05, ** p < 0.01.

Figure 3. Comparison of the virulence of Newman::pRAB11, ∆purN::pRAB11, ∆purN::pRABpurN, and Newman::pRBpurN in S. aureus. (A) The virulence gene expression levels detected by RT-qPCR. (B) Variation of hemolysis in different strains. Hemolysis status of Newman::pRAB11 (a,e), ∆purN::pRAB11 (b,f), ∆purN::pRABpurN (c,g), and Newman::pRBpurN (d,h) cultured for 24 h (a–d) and 48 h (e–h) on blood TSA plates. The hemolysis assay of the four strains was measured in different time points cultures. (i)10 h, (j) 14 h, (k) 24 h, and (l) 48 h. (C) The biofilm formation abilities of the four S. aureus strains in 96-well plates. Comparison of OD550 and biofilm images in 96-well plate of different strains. * p < 0.05, ** p < 0.01.

Figure 4. Comparative analyses of the transcriptomics of ΔpurN and wild-type, and the gltB, saeR, and saeS expression in Newman::pRAB11, ΔpurN::pRAB11, ΔpurN::pRBpurN, and Newman::pRBpurN strains. (A) DEGs and pathways involved in the comparison of ΔpurN and wild-type. The genes in the green box and red box are downregulated and upregulated genes, respectively. (B) Protein-protein interaction network of DEGs between ΔpurN and parental strain by STRING database. The line thickness of the network indicates the strength of association/binding. (C) Comparison of expression levels of the gltB, saeR, and saeS in the four S. aureus strains (* p < 0.05).

Figure 4. Comparative analyses of the transcriptomics of ΔpurN and wild-type, and the gltB, saeR, and saeS expression in Newman::pRAB11, ΔpurN::pRAB11, ΔpurN::pRBpurN, and Newman::pRBpurN strains. (A) DEGs and pathways involved in the comparison of ΔpurN and wild-type. The genes in the green box and red box are downregulated and upregulated genes, respectively. (B) Protein-protein interaction network of DEGs between ΔpurN and parental strain by STRING database. The line thickness of the network indicates the strength of association/binding. (C) Comparison of expression levels of the gltB, saeR, and saeS in the four S. aureus strains (* p < 0.05).

Figure 5. Antibiotics exposure results of Newman::pRAB11, ∆gltB::pRAB11, ΔgltB::pRBpurN and Newman::pRBpurN to ampicillin (A,B), levofloxacin (C,D), gentamicin (E,F) and vancomycin (G,H) at different culture times. 5-h culture (A,C,E,G); 9-h culture (B,D,F,H).

Figure 5. Antibiotics exposure results of Newman::pRAB11, ∆gltB::pRAB11, ΔgltB::pRBpurN and Newman::pRBpurN to ampicillin (A,B), levofloxacin (C,D), gentamicin (E,F) and vancomycin (G,H) at different culture times. 5-h culture (A,C,E,G); 9-h culture (B,D,F,H).

Figure 6. Pathways that indicate how purN is involved in persister formation and virulence in S. aureus.

Figure 6. Pathways that indicate how purN is involved in persister formation and virulence in S. aureus.

Table 1. Bacteria and plasmids used in this study.

Table 1. Bacteria and plasmids used in this study.

Strains or PlasmidRelevant Genotype and PropertySource or ReferenceS. aureus Strains    NewmanClinical isolate, ATCC 25904ATCC   ΔpurNNewman with a deletion of purNThis study   Newman::pRAB11Newman with pRAB11This study   ∆purN::pRAB11∆purN with pRAB11This study   ∆purN::pRBpurN
   ∆gltB::pRAB11
   ∆gltB::pRBpurN∆purN with pRAB11-purN
∆gltB with pRAB11
∆gltB with pRAB11-purNThis study
This study
This studyEscherichia coli strains    DC10B∆dcm in the DH10B background; Dam methylation only[33]plasmids    pMX10A pKOR1 derivate for gene knockout, CmR, AmpR[29]   pRAB11Atc inducible shuttle plasmid, CmR, AmpR[15]   pRAB11-purNOverexpression plasmid for purNThis study