Article Figures & Data
Figures
- FIG. 1.
Map of the S. flexneri sit and feo loci. The ORFs of the sit (A) and feo (B) loci are indicated above the representative arrows. The nucleotide sequence of the putative sitA promoter is shown. The putative Fur and MntR binding sites are boxed and underlined, respectively, and the SitA start codon is in boldface.
- FIG. 2.
The S. flexneri sitA mutants have slightly reduced growth in L broth plus EDDA. Overnight cultures of each strain were subcultured 1:1,000 into L broth containing 250 μg of EDDA/ml (A) or without EDDA (B) and grown at 37°C. The optical densities of the cultures were measured after 8 h. The data presented are the means of three experiments, and the standard deviations of the means are indicated. The presence of mutations in sitA, iucD, and feoB or the wild-type allele is indicated by − or +, respectively.
- FIG. 3.
Addition of either iron or manganese restores sitA mutant growth to parental levels in L broth containing EDDA. Overnight cultures of each strain were subcultured 1:1,000 (approximately 106 bacteria/ml) into L broth, L broth containing 125 μg of EDDA/ml (350 μM), L broth containing 125 μg of EDDA/ml and 350 μM ferric chloride (Fe), or L broth containing 125 μg of EDDA/ml and 350 μM manganese chloride (Mn). After 8 h of growth at 37°C, the number of bacteria per milliliter of cultures was determined by viable plate counts. The data presented are the means of three experiments, and the standard deviations of the means are indicated.
- FIG. 4.
S. flexneri iron acquisition mutants in Henle cell plaque assays. Confluent Henle cell monolayers were infected with 104 bacteria per 35-mm-diameter plate, and the plaques were photographed after 3 days. pEG1 and pKLS971 carry the S. flexneri sit and iuc operons, respectively.
- FIG. 5.
Induction of the sitA promoter in LB broth containing EDDA. SM100 carrying pEG2 (sitA-gfp) was grown in LB broth containing EDDA as indicated, and the fluorescence was quantitated by fluorescence-activated cell sorting after 6 h. Ten thousand bacterial cells were assayed for each experimental condition. The data presented are the means of three experiments, and the standard deviations of the means are indicated.
- FIG. 6.
Induction of the sitA promoter. SA211 (fur::Tn5) or the parent strain SA101 (fur+) carrying pEG2 (sitA-gfp) was grown in LB broth with or without 16 μg of EDDA/ml (45 μM), and the fluorescence was quantitated by fluorescence-activated cell sorting after 5 h. Where indicated, 500 μM ferric chloride (Fe) or 100 μM manganese chloride (Mn) was added to EDDA cultures. Ten thousand bacterial cells were assayed for each experimental condition. The data presented are the means of three experiments, and the standard deviations of the means are indicated.
Tables
- TABLE 1.
Bacterial strains and plasmids
Strain or plasmid Characteristics Reference or source E. coli strains DH5α endA1 hsdR17 supE44 thi-1 recA1 gyrA relA1 Δ(lacZYA-argF)U169 deoR [Φ80dlacΔ(lacZ)M15] 48 SM10 λpir pirR6K 52 HB101 F− Δ(gpt-proA)62 leu supE44 ara-14 galK2 lacY1 Δ(mcrC-mrr) rpsL20 xyl-5 mtl-1 recA13 48 1017 HB101 ent::Tn5 7 S. flexneri strains SA100 S. flexneri wild-type serotype 2a 41 SM100 SA100 Strr S. Seliger SA240 SA100 iucD::Tn5 27 SM166 SM100 sitA::cam This study SA167 SA240 (iuc::Tn5) sitA::cam This study SA190 SA100 feoB::dhfr This study SM191 SM166 (sitA::cam) feoB::dhfr This study SA192 SA190 (feoB::dhfr) iucD::Tn5 This study SM193 SA191 (sitA::cam feoB::dhfr) iucD::Tn5 This study SA514 SA100 Camr 17 SA101 SA100 derivative with deletion in virulence plasmid 6 SA211 SA101 fur::Tn5 51 Plasmids pEG2 pLR29 carrying the sitA promoter 47 pHM5 Allelic-exchange vector 46 pLAFR1 Cosmid vector 10 pLR29 pGTXN3 with RP4 mobilization region from pGP704 (35) 47 pWKS30 Low-copy-number cloning vector 54 pEG1 pLAFR1 carrying SA100 sit operon This study pSIT1 pLAFR1 carrying S. dysenteriae sit operon 42 pKL971 pLAFR1 carrying SA100 iuc operon 32 - TABLE 2.
Selected homologies of S. flexneri SitABCD to other proteins
Species (locus) % Identity at amino acid level to Shigella flexneria SitA SitB SitC SitD Salmonella enterica serovar Typhimurium (Sit) 77 77 75 64 Sinorhizobium meliloti (Sit) 70 70 68 55 Haemophilus influenzae (Yfe) 67 60 55 46 Yersinia pestis (Yfe) 64 61 59 47 Pasteurella multocida (Yfe) 62 58 53 46 Agrobacterium tumefaciens (Sit) 65 67 65 49 Synechocystis sp. (Mnt) 55 59 42 40 ↵a Homologies were calculated with the BLAST algorithm (National Center for Biotechnology Information).
- TABLE 3.
Distribution of sitA in Shigella spp. and pathogenic E. coli strains
Strain No. sitA positive/no. testeda Shigella S. flexneri 13/13 S. dysenteriae 5/5 S. boydii 6/6 S. sonnei 1/1 E. coli Enteroinvasive 6/7 Enteropathogenic 0/13 Enterohemorrhagic 0/8 Enterotoxigenic 0/1 K1 (meningitis) 0/6 K-12 0/2 ↵a The presence of sitA was determined by PCR on cultures of each isolate.
