Article Figures & Data
Figures
- Fig. 1.
Construction of a frameshift mutation in thefoxA gene of S. enterica serotype Typhimurium. (A) Comparison of the nucleotide sequences of the wild-type and mutated foxA alleles, as confirmed by sequence analysis. The mutation consists of a 2-bp (AT) insertion (indicated by plus signs) and a BglII restriction enzyme recognition sequence (indicated by boldface type). The mutation was introduced into a cloned subgenic foxA fragment as described in the text by using primers FXM1 and FXM2, which have mismatching base pairs that include the BglII recognition site (B). (C and D) The mutations were confirmed by Southern hybridization analysis in which we used a subgenic foxA fragment as the probe. (C) Analysis ofBglII-digested chromosomal DNA of parental strain TML (lane 1) and the foxA derivative strain RK102 (lane 2). (D) Analysis of parental strain SL1344 (lane 1) and the foxAderivative strain BK102 (lane 2).
- Fig. 2.
Distribution of the foxA gene and the ability to utilize ferrioxamine E as a sole source of iron among subspecies of the genus Salmonella. (A) Neighbor-joining dendrogram showing the relatedness of the various subspecies, based on variation in the combined coding sequences of five housekeeping genes ofSalmonella spp. (8). The arrows indicate probable introduction by horizontal transfer of SPI1 and SPI2 and of thefoxA gene; ΔfoxA indicates possible deletion of the foxA gene in the subspecies VI lineage. Note that Reeves et al. (35) did not differentiate S. enterica subspecies IV into subspecies IV and VII; therefore, the three serotypes tested were random examples of what was originally designated subspecies IV. The hybridization data are the number of strains that exhibited positive hybridization with thefoxA-specific probe/total number of strains tested. The growth promotion data are the number of strains whose growth was promoted by ferrioxamine E (fE) or coprogen (cop) as a sole source of iron/total number of strains tested. (B) Southern blot analyses of chromosomal DNA of representative strains of each subspecies probed with a foxA-specific DNA fragment. For the SARB collection, only the first 25 of the 72 strains listed by Boyd et al. (7) were used, but all other isolates gave similar positive results with the foxA probe. For the representative serotypes of other subspecies, control hybridizations were performed by using a probe derived from the Salmonella fim operon (1) in order to validate negative foxAhybridizations.
- Fig. 3.
Growth of S. enterica serotype Typhimurium strain AR1258 (entB) (A) and its foxAderivative RK809 (B) in tryptone soy broth that was supplemented with 200 μM 2,2′-bipyridyl and ferrioxamine B (●), E (+), or G (□) (at a concentration of 1 μg/ml) or not supplemented (■). Optical densities at 620 nm (OD620) were determined at intervals during incubation with aeration by shaking at 37°C. The data are from a representative experiment; we performed at least five experiments in which similar results were obtained.
- Fig. 4.
Comparison of colonization of rabbit ileal loops byS. enterica serovar Typhimurium strain TML with colonization of rabbit ileal loops by derivative strains TML/nr and RK102 (foxA). We performed competitive colonization experiments involving wild-type strain S. enterica TML and either the nalidixic acid-resistant derivative strain TML/nr or the nalidixic acid-resistant foxA mutant strain RK102 by using a total of 10 ileal loops in two rabbits. Approximately 5-cm-long loops were inoculated with 1:1 mixtures containing 107 or 108 cells. After 28 h the proportion of nalidixic acid-resistant coliform bacteria present in the lumen and associated with the gut tissue was determined. The proportion of nalidixic acid-resistant coliform bacteria recovered was expressed as a log10 value (competitive index) and was subjected to a statistical analysis in which the Student t test was used. The asterisks indicate competitive indices significantly different (P > 0.01) from the input ratio (log101 = 0).
Tables
- Table 1.
Bacterial strains and plasmids used in this study
Strain or plasmid Relevant characteristics Source or reference S. entericaserotype Typhimurium strains ATCC 14028 Wild type ATCCa AR1258 ATCC 14028,entB 44 RK804 AR1258,tonB 18 RK809 AR1258,foxA::pMAP This study enb-7 LT2, class II ent mutant 33 WR1024 enb-7, fhuB::MudJ Laboratory stock SL1344 hisG46 17 SL1344/nr SL1344, Nalr This study BK102 SL1344/nr, foxA This study TML Wild type 12 TML/nr TML, Nalr This study RK102 TML/nr,foxA This study E. coliSM10λpir F−thi-1 thr-1 leuB6 supE44 tonA21 lacY1 recA::RP4-2-tc::Mu Kmr R. Haigh Plasmids pUC18 ColE1,bla Laboratory stock pRA17 pUC18 carrying the 287-bp foxA fragment This study pRA5 pUC18 carrying the foxA 5′ region This study pBluescriptSK ColE1, bla Laboratory stock pRA19 pBluescriptSK carrying the mutated 287-bpfoxA fragment This study pRDH10 sacRB cat oriR6K mobRP4, Tetr R. Haigh pRA21 pRDH10 carrying the 287-bp foxAfragment This study pIRS618 pSUKS1 carrying the serovar Typhimurium tonB gene 45 pMAP bla oriR6KmobRP4 44 ↵a ATCC, American Type Culture Collection.
- Table 2.
Siderophore cross-feeding of various serotype Typhimurium strainsa
Medium Concn of 2,2′-bipyridyl (μM) Strain Relevant genotype Results with the following siderophores: Ferrioxamine B Ferrioxamine E Ferrioxamine G Coprogen Enterobactin Vogel-Bonnerb 200 AR1258 entB 28 29 26 28 30 RK804 entB tonB 13 14 5 5 5 RK804(pIRS618) entB tonB+ 26 25 23 28 30 WR1024 ent fhuB 5 5 5 5 30 RK809 entB foxA 8 5 5 28 30 EWMc 400 SL1344/nr Wild type + + + + ND BK102 foxA (+) − − + ND TML/nr Wild type + + + + ND RK102 foxA (+) − − + ND ↵a Approximately 106 cells of each strain tested were spread over the surface of Vogel-Bonner or EWM agar. Sterile filter paper discs (diameter, 5 mm), each impregnated with 1 μg of a siderophore, were placed on the agar surfaces.
↵b For the Vogel-Bonner medium bioassays, the sizes (in millimeters) of growth zones around siderophore discs were recorded after 18 h of incubation at 37°C; a value of 5 mm indicates that there was no growth stimulation.
↵c In the EWM bioassays, growth was too diffuse to measure accurately as discrete zones, and so the results were recorded as follows: +, obvious growth; (+), limited growth around the disc; and −, no observable growth after 3 to 4 h of incubation at 37°C. ND, growth stimulation not determined.
