Article Figures & Data
Figures
- FIG. 1.
Schematic diagram of the region comprising the iro locus and flanking sequences carried on plasmid pAPEC-1 of APEC strain χ7122. Numbers within arrows correspond to designated ORFs within the sequence (accession no. AF449498). Black arrows indicate the iro genes. Nucleotide identities between segments of the χ7122 iro coding region and those of UPEC strain CFT073 (accession no. AE014075) (60) and Klebsiella pneumoniae plasmid pLVKP (accession no. AY378100) (9) are illustrated within boxed segments, with numbers representing the percent nucleotide identity between segments. Additional sequences of CFT073 and pLVKP that are absent from the pAPEC-1 region are illustrated with diagonal lines and described in text boxes. Plasmid constructs described in the text are illustrated above pAPEC-1 containing the iro locus region. Primers used to generate clones by PCR are indicated with the symbol > for sense primers or < for antisense primers. Corresponding restriction endonuclease sites used to generate the plasmids are indicated. See Materials and Methods for details.
- FIG. 2.
Scheme showing molecular structures of each the catecholate siderophores that were analyzed. Enzymes predicted to play a role in processing of salmochelins, enterobactin, or DHBS derivatives based on biochemical studies of Lin et al. (36) and Zhu et al. (62) are indicated.
- FIG. 3.
Bacterial numbers present in the lungs, spleens, and livers of chickens infected with wild-type APEC strain χ7122, isogenic mutant derivatives, and iro plasmid-complemented strains. Data points represent bacterial counts from tissues isolated from different chickens (n = 5 to 11) 48 h postinfection. Horizontal bars represent the median bacterial CFU. Statistical differences compared with the wild-type strain are noted: * and **, P < 0.05 and P < 0.01, respectively, using the two-tailed Mann-Whitney test.
- FIG. 4.
Mean distribution of siderophores produced by wild-type APEC χ7122 in iron-poor M63 medium (A) and in air sacs (B) and pericardia (C) of infected chickens. The error values are standard errors of the means. Bold values indicate significant differences for the same molecule compared to levels present in iron-poor M63 medium. The underlined value indicates a significant difference between the air sacs and the pericardial membrane. (D) Relative total percent distribution of siderophores belonging to each siderophore group. *, significant difference for the same group of siderophores between in vivo and in vitro conditions (P < 0.05) using the unpaired t test.
- FIG. 5.
Mean concentrations of enterobactin and DHBS molecules and salmochelins detected in culture supernatants. The amount of each molecule was normalized with the internal standard, and quantities were summed up according to the related siderophore group: salmochelins or enterobactin and nonglucosylated DHBS derivatives (Enterobactin/DHBS). Error bars represent the standard errors of the means. ¤, significant difference between the total enterobactin and DHBS molecules produced by strain χ7304 compared to χ7122; ¤¤, significant difference between the total enterobactin and DHBS molecules and total salmochelins produced by strain χ7304(pYA3661) compared to χ7122. *, significant difference between the total enterobactin and DHBS molecules produced compared to χ7304(pYA3661); **, significant difference between the total enterobactin and DHBS molecules and salmochelins produced compared to χ7304(pYA3661) (P < 0.05).
Tables
- TABLE 1.
Bacterial strains and plasmids used for this study
Bacterial strain or plasmid Genotype or phenotypea Source or reference Strains χ7122 Avian pathogenic; O78:K80:H9; gyrA Nalr 44 χ7304 ΔiroBCDEN::nptII ΔiucABCD iutA::xylE Kmr Nalr 13 DH5α F− (Δ80d lacZΔM15) Δ(lacZYA-argF)U169 endA1 recA1 hsdR17(rK− mK+) deoR thi-1 supE44 gyrA96 relA1 Bethesda Research Laboratories Plasmids pACYC184 p15A replicon; Cmr Tcr 6 pBC SK (−) ColE1 origin; Cmr Stratagene pYA3661 pACYC184::iroBCDEN; Cmr 13 pIJ33 pACYC184::iroN; Cmr This study pIJ34 pACYC184::iroBCDE; Cmr This study pIJ37 pACYC184::iroBDEN; Cmr This study pIJ53 pACYC184::iroB; Cmr This study pIJ121 pACYC184::iroBC; Cmr This study pIJ135 pACYC184::iroBCN; Cmr This study pIJ136 pACYC184::iroBCEN; Cmr This study pIJ137 pACYC184::iroBCDN; Cmr This study ↵a Cm, chloramphenicol; Km, kanamycin; Nal, nalidixic acid.
- TABLE 2.
Distribution of iro genes among APEC and avian fecal commensal isolates according to lethality class
Lethality classa No. (%) of E. coli isolates APEC Avian fecal commensal Total iro positiveb Total iro positiveb LC1 222 203 (91)c 1 1 (100) LC2 38 24 (63) 12 6 (50) LC3 38 17 (45) 19 3 (16) Total 298 244 (82)d 32 10 (31) ↵a Lethality classes were defined by lethality in 1-day-old chicks as follows: LC1, 50% lethal dose (LD50) of <108 CFU; LC2, LD50 of ≥108 CFU; LC3, not lethal at ≥108 CFU (14).
↵b Positive PCR amplification of the iroB and iroN genes.
↵c iro sequences were significantly associated with APEC isolates from LC1 as compared to APEC isolates from LC2 or LC3 (P < 0.0001) using Fisher's exact test.
↵d iro sequences were significantly associated with APEC isolates relative to environmental isolates (P < 0.0001) using Fisher's exact test.
- TABLE 3.
Score-based evaluation of gross lesions in organs of infected chickens
E. coli strain Mean lesion score ± SEMa Air sacsb Liver and heartc χ7122 2.7 ± 0.2 3.2 ± 0.3 χ7304 (Δiro ΔiucABC iutA) 1.5 ± 0.4* 0.8 ± 0.3** χ7304(pYA3661 iroBCDEN) 2.8 ± 0.3 3.2 ± 0.2 χ7304(pIJ34 iroBCDE) 1.6 ± 0.4* 1.3 ± 0.5* χ7304(pIJ37 iroBDEN) 1.4 ± 0.3** 1.0 ± 0.2** χ7304(pIJ135 iroBCN) 1.4 ± 0.3** 0.8 ± 0.1** χ7304(pIJ136 iroBCEN) 1.9 ± 0.3 1.4 ± 0.1** χ7304(pIJ137 iroBCDN) 2.7 ± 0.3 2.4 ± 0.4 ↵a Lesion scores are presented as the mean (± standard error of the mean [SEM]) of values attributed to gross lesions of airsacculitis and combined lesions of perihepatitis/and perihepatitis as described in reference 33.
↵b Mean lesion scores for airsacculitis in both caudal thoracic air sacs.
↵c Combined lesion scoring values for pericarditis and perihepatitis. Statistical differences compared with the wild-type strain using the two-tailed Mann-Whitney test are noted: * and **, P < 0.05 and P < 0.001, respectively.
- TABLE 4.
Mean concentrations of salmochelins detected in culture supernatants
Strain (genotype) Concn (μM) of salmochelina Monoglucosylated molecules Diglucosylated molecules Triglucosylated molecules MGE Linear MGE S1 SX DGE (S4) Linear DGE (S2) S5 TGE Linear TGE χ7122 1.7 ± 0.28 1.1 ± 0.11 5.1 ± 0.12 9.8 ± 0.33 0.31 ± 0.03 1.1 ± 0.02 0.45 ± 0.02 ND ND χ7304b ND ND ND ND ND ND ND ND ND χ7304(pYA3661 iroBCDEN)b 0.22 ± 0.02 0.14 ± 0.03 9.2 ± 0.94 31.0 ± 4.4 0.06 ± 0.004 2.9 ± 0.07 1.7 ± 0.18 ND ND χ7304(pIJ34 iroBCDE)c 0.09 ± 0.01 0.07 ± 0.01 8.6 ± 1.1 26.1 ± 3.9 0.04 ± 0.005 8.8 ± 0.98 1.5 ± 0.23 ND 0.06 ± 0.01 χ7304(pIJ137 iroBCDN)c 0.17 ± 0.001 0.22 ± 0.01 9.9 ± 0.55 20.4 ± 1.1 0.42 ± 0.02 22.7 ± 1.1 1.5 ± 0.05 0.04 ± 0.001 0.19 ± 0.01 χ7304(pIJ136 iroBCEN)c 0.09 ± 0.01 0.06 ± 0.01 4.0 ± 0.33 19.6 ± 1.8 0.23 ± 0.02 5.6 ± 0.94 1.7 ± 0.09 ND 0.04 ± 0.004 χ7304(pIJ53 iroB)c ND ND 0.04 ± 0.001 ND ND ND ND ND ND χ7304(pIJ121 iroBC)c 0.09 ± 0.01 ND 1.3 ± 0.02 3.2 ± 0.26 0.52 ± 0.02 5.4 ± 0.12 0.43 ± 0.02 0.25 ± 0.01 0.13 ± 0.01 χ7304(pIJ135 iroBCN)c 0.13 ± 0.01 0.04 ± 0.002 1.1 ± 0.04 3.5 ± 0.40 0.46 ± 0.01 5.1 ± 0.03 0.46 ± 0.04 0.29 ± 0.01 0.15 ± 0.01 χ7304(pIJ37 iroBDEN)c ND ND 1.3 ± 0.1 31.7 ± 3.5 ND 0.71 ± 0.07 0.05 ± 0.005 ND ND ↵a Values represent means ±standard errors of the means of at least three independently grown cultures. Bold values indicate significant differences (P < 0.05) using a two-tailed unpaired t test. ND, molecule not detected in samples.
↵b Statistical differences were determined by using χ7122 as the reference strain.
↵c Statistical differences were determined by using χ7304(pYA3661 iroBCDEN) as the reference.
- TABLE 5.
Mean concentrations of enterobactin-related molecules detected in culture supernatants
Strain (genotype) Concn (μM) of enterobactin-related moleculea: Enterobactin (DHBS)3 (DHBS)2 DHBS χ7122 12.1 ± 3.4 25.3 ± 4.6 33.1 ± 4.7 11.6 ± 1.2 χ7304b 62.9 ± 7.2 62.7 ± 6.2 86.6 ± 9.3 25.9 ± 3.9 χ7304(pYA3661 iroBCDEN)b 1.5 ± 0.16 1.9 ± 0.25 9.6 ± 1.01 27.1 ± 3.7 χ7304(pIJ34 iroBCDE)c 0.9 ± 0.07 0.9 ± 0.11 10.9 ± 1.9 20.8 ± 3.1 χ7304(pIJ137 iroBCDN)c 3.8 ± 0.14 5.1 ± 0.23 22.2 ± 0.9 13.9 ± 0.8 χ7304(pIJ136 iroBCEN)c 0.6 ± 0.06 0.8 ± 0.12 8.6 ± 0.57 15.0 ± 1.3 χ7304(pIJ53 iroB)c 1.6 ± 0.35 0.6 ± 0.10 0.5 ± 0.01 ND χ7304(pIJ121 iroBC)c 3.7 ± 0.20 2.9 ± 0.09 10.7 ± 0.4 1.6 ± 0.10 χ7304(pIJ135 iroBCN)c 4.6 ± 0.36 3.9 ± 0.29 11.8 ± 0.5 2.6 ± 0.15 χ7304(pIJ37 iroBDEN)c 2.7 ± 0.25 3.7 ± 0.35 15.3 ± 1.5 8.2 ± 1.12 ↵a Values represent means ± the standard errors of the means of at least three independently grown cultures. Bold values indicate significant differences (P < 0.05) using a two-tailed unpaired t test. ND, molecule not detected in samples.
↵b Statistical differences were determined by using χ7122 as the reference strain.
↵c Statistical differences were determined by using χ7304(pYA3661 iroBCDEN) as the reference strain.
