Table 1.
Strains, plasmids and cell line used in this study.
Table 2.
Primers designed and used in this study.
Figure 1.
Detection of iucA (A, B), iucC (D, E) and iutA (C, F) gene expression by RT-PCR.
(A). Detection of iucA gene transcription in E058 and E058ΔiucA by RT-PCR. Templates: lanes 1, 3, 5: cDNA derived from total RNA of E058. Lane 7: total RNA from E058 without activation of RT. Lanes 8, 10, 12: cDNA derived from total RNA of E058ΔiucA. Lane 14: total RNA from E058ΔiucA without activation of RT. Lanes 2, 4, 6: pAPEC-O2-ColV-like DNA from E058. Lanes 9, 11, 13: pAPEC-O2-ColV-like DNA from E058ΔiucA. Primers: Lanes 1, 2, 8, 9: GF/GR. Lanes 3, 4, 10, 11: iucA-F/iucA-R. Lanes 5, 6, 7, 12, 13, 14: BF/BR (Table 2). (B). Detection of iucA gene transcription in E058 and E058ΔiucAΔiutA by RT-PCR. Templates: lanes 1, 3, 5: cDNA derived from total RNA of E058. Lane 7: total RNA from E058 without activation of RT. Lanes 8, 10, 12: cDNA derived from total RNA of E058ΔiucAΔiutA. Lane 14: total RNA from E058ΔiucAΔiutA without activation of RT. Lanes 2, 4, 6: pAPEC-O2-ColV-like DNA from E058. Lanes 9, 11, 13: pAPEC-O2-ColV-like DNA from E058ΔiucAΔiutA. Primers: Lanes 1, 2, 8, 9: GF/GR. Lanes 3, 4, 10, 11: iucA-F/iucA-R. Lanes 5, 6, 7, 12, 13, 14: BF/BR (Table 2). (C). Detection of iutA gene transcription in E058 and E058ΔiucAΔiutA by RT-PCR. Templates: lanes 1, 3, 5: cDNA derived from total RNA of E058. Lane 7: total RNA from E058 without activation of RT. Lanes 8, 10, 12: cDNA derived from total RNA of E058ΔiucAΔiutA. Lane 14: total RNA from E058ΔiucAΔiutA without activation of RT. Lanes 2, 4, 6: pAPEC-O2-ColV-like DNA from E058. Lanes 9, 11, 13: pAPEC-O2-ColV-like DNA from E058ΔiucAΔiutA. Primers: Lanes 1, 2, 8, 9: DF/DR. Lanes 3, 4, 10, 11: iutA-F/iutA-R. Lanes 5, 6, 7, 12, 13, 14: O2F/O2R (Table 2). (D). Detection of iucC gene transcription in E058 and E058ΔiucC by RT-PCR. Templates: lanes 1, 3, 5: cDNA derived from total RNA of E058. Lane 7: total RNA from E058 without activation of RT. Lanes 8, 10, 12: cDNA derived from total RNA of E058ΔiucC. Lane 14: total RNA from E058ΔiucC without activation of RT. Lanes 2, 4, 6: pAPEC-O2-ColV-like DNA from E058. Lanes 9, 11, 13: pAPEC-O2-ColV-like DNA from E058ΔiucC. Primers: Lanes 1, 2, 8, 9: BF/BR. Lanes 3, 4, 10, 11: iucC-F/iucC-R. Lanes 5, 6, 7, 12, 13, 14: DF/DR (Table 2). (E). Detection of iucC gene transcription in E058 and E058ΔiucCΔiutA by RT-PCR. Templates: lanes 1, 3, 5: cDNA derived from total RNA of E058. Lane 7: total RNA from E058 without activation of RT. Lanes 8, 10, 12: cDNA derived from total RNA of E058ΔiucCΔiutA. Lane 14: total RNA from E058ΔiucCΔiutA without activation of RT. Lanes 2, 4, 6: pAPEC-O2-ColV-like DNA from E058. Lanes 9, 11, 13: pAPEC-O2-ColV-like DNA from E058ΔiucCΔiutA. Primers: Lanes 1, 2, 8, 9: BF/BR. Lanes 3, 4, 10, 11: iucC-F/iucC-R. Lanes 5, 6, 7, 12, 13, 14: DF/DR (Table 2). (F). Detection of iutA gene transcription in E058 and E058ΔiucCΔiutA by RT-PCR. Templates: lanes 1, 3, 5: cDNA derived from total RNA of E058. Lane 7: total RNA from E058 without activation of RT. Lanes 8, 10, 12: cDNA derived from total RNA of E058ΔiucCΔiutA. Lane 14: total RNA from E058ΔiucCΔiutA without activation of RT. Lanes 2, 4, 6: pAPEC-O2-ColV-like DNA from E058. Lanes 9, 11, 13: pAPEC-O2-ColV-like DNA from E058ΔiucCΔiutA. Primers: Lanes 1, 2, 8, 9: DF/DR. Lanes 3, 4, 10, 11: iutA-F/iutA-R. Lanes 5, 6, 7, 12, 13, 14: O2F/O2R (Table 2). A 200 bp marker (Takara) was used as the molecular size standard (M).
Figure 2.
Growth curves of E058 wild-type strain and its mutants.
(A) The E058(□), E058ΔiutA(▪), E058ΔiucA(▴), E058ΔiucC(×), E058ΔiucAΔiutA (*) and E058ΔiucCΔiutA (•) strains were grown in LB broth at 37°C, respectively, and their growth curves were determined by measuring viable counts (CFU ml−1). (B) Growth curves of the E058 and its mutants in LB containing 200 µM 2,2′-dipyridyl (DIP). The data represent averages of three independent assays.
Figure 3.
Bactericidal activity of SPF chicken serum against the wild type strain and mutants.
Strains E058 (black bar), E058ΔiutA (white bar), E058ΔiucA (gray bar), E058ΔiucAΔiutA (gray dots), E058ΔiucC (gray oblique lines), E058ΔiucCΔiutA (gray grids). HI represents the group of heat-inactivated 25% SPF chicken serum used as controls for each strain tested. Data represent averages of three independent assays.
Figure 4.
Competition assays between wild-type E058 and mutants E058ΔiutA, E058ΔiucA, E058ΔiucC, E058ΔiucAΔiutA or E058ΔiucCΔiutA inoculated simultaneously.
Negative competitive index (CI) values indicate a decreased capacity of the mutants to compete for growth with the wild-type strain. Horizontal bars indicate the mean log10CI values. Each data point represents a sample from an individual chicken. Statistically significant differences in CI values between E058 and its mutants are indicated with asterisks (* P<0.05, ** P<0.01, *** P<0.001).
Figure 5.
Colonization and persistence of wild-type strain E058 and its mutants E058ΔiutA, E058ΔiucA, E058ΔiucC, E058ΔiucAΔiutA and E058ΔiucCΔiutA.
Data are presented as log10 CFU.g−1 of bacteria from tissues, and horizontal bars represent mean values. Each data point represents a sample from an individual chicken. Statistically significant differences are indicated with asterisks (** P<0.01, *** P<0.001), as determined by the Mann Whitney U test.