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Institut National de la Recherche Scientifique, INRS-Institut Armand-Frappier, 531 boul. des Prairies, Laval, Québec, Canada H7V 1B7
Correspondence
Charles M. Dozois
charles.dozois{at}iaf.inrs.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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7122. The sitABCD genes were located on the virulence plasmid pAPEC-1, and were highly similar at the nucleotide level to the chromosomally encoded sitABCD genes present in Shigella spp. A cloned copy of sitABCD conferred increased growth upon a siderophore-deficient E. coli strain grown in nutrient broth supplemented with the chelator 2,2'-dipyridyl. Ion rescue demonstrated that Sit-mediated growth promotion of this strain was due to the transport of iron. SitABCD mediated increased transport of both iron and manganese as demonstrated by uptake of 55Fe, 59Fe or 54Mn in E. coli K-12 strains deficient for the transport of iron (aroB feoB) and manganese (mntH) respectively. Isotope uptake and transport inhibition studies showed that in the iron transport deficient strain, SitABCD demonstrated a greater affinity for iron than for manganese, and SitABCD-mediated transport was higher for ferrous iron, whereas in the manganese transport deficient strain, SitABCD demonstrated greater affinity for manganese than for iron. Introduction of the APEC sitABCD genes into an E. coli K-12 mntH mutant also conferred increased resistance to the bactericidal effects of hydrogen peroxide. APEC strain 7122 derivatives lacking either a functional SitABCD or a functional MntH transport system were as resistant to hydrogen peroxide as the wild-type strain, whereas a 
sit mntH double mutant was more sensitive to hydrogen peroxide. Overall, the results demonstrate that in E. coli SitABCD represents a manganese and iron transporter that, in combination with other ion transport systems, may contribute to acquisition of iron and manganese, and resistance to oxidative stress.
The GenBank/EMBL/DDBJ accession numbers of the SitABCD-encoding DNA region of pAPEC-1 from strain 7122, and the sitA gene from E. coli CFT073 sequenced from plasmid pIJ4, are AY598030 and DQ256074, respectively.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Nataro & Kaper, 1998). The intestinal pathotypes cause diarrhoea in humans and animals, whereas pathotypes associated with urinary tract infections, neonatal meningitis and septicaemia have been collectively termed extra-intestinal pathogenic E. coli (ExPEC) (Johnson & Russo, 2002). Pathogenic E. coli strains are also classified according to the host species they infect (Gyles, 1994). Avian pathogenic E. coli (APEC) cause colibacillosis, a general term used to encompass a number of extra-intestinal infections of poultry, including respiratory infection (airsacculitis), septicaemia and cellulitis (Dho-Moulin & Fairbrother, 1999; La Ragione & Woodward, 2002). APEC strain 7122 (O78 : K80 : H9) has been used as a model strain to study molecular mechanisms of APEC pathogenicity (Brown & Curtiss, 1996; Dozois et al., 2003). 7122 has several iron transport systems, including the aerobactin and salmochelin siderophore systems, which are encoded on a large virulence plasmid pAPEC-1 and are absent from non-pathogenic E. coli K-12. In addition, strain 7122 possesses the chromosome-encoded enterobactin siderophore system and the Feo ferrous iron transporter. Siderophores mediate the acquisition of ferric (Fe3+) iron by competing with the host iron-binding proteins. Other transporters such as FeoB transport ferrous (Fe2+) iron directly from the environment (Kammler et al., 1993).
In E. coli and most other bacteria iron is a cofactor of a number of essential metabolic enzymes (Andrews et al., 2003). Importantly, iron plays a role in protection against oxidative damage, as it is a component of the sodB-encoded superoxide dismutase (FeSOD) and catalase enzymes which eliminate superoxide (O2–) and H2O2 respectively. However, excess iron levels in bacterial cells contribute to oxidative damage through the generation of free radicals (Imlay, 2003).
The global regulator Fur, upon association with its co-repressor Fe2+, represses transcription of genes encoding high-affinity iron transport systems and other proteins involved in iron metabolism, and thus tightly controls iron homeostasis of the bacterial cell (Andrews et al., 2003; McHugh et al., 2003). Iron metabolism is co-ordinately regulated with the oxidative stress response, and fur expression is positively regulated by the oxidative response regulators OxyR and SoxRS (Zheng et al., 1999). In addition to regulating genes associated with iron metabolism or transport, Fur also regulates the expression of genes required for manganese transport (Kehres et al., 2002b; Patzer & Hantke, 2001) and the manganese-dependent superoxide dismutase (MnSOD), SodA (Tardat & Touati, 1993). Hence, control of both manganese and iron transport and iron- and manganese-dependent defence against oxidative stress are coordinated.
Manganese contributes to protection against oxidative stress, is a cofactor for a number of enzymes in bacteria and other organisms (Kehres & Maguire, 2003), and can also contribute directly to the catalytic detoxification of reactive oxygen species (Horsburgh et al., 2002). In the enterobacteria, two major types of manganese transporters have been identified: a proton-dependent Nramp-related transport system typified by MntH and an ATP-binding cassette (ABC) transporter typified by SitABCD (Cellier et al., 2001; Kehres & Maguire, 2003). In addition to being regulated by iron levels and Fur, expression of these systems is also regulated by manganese levels and the regulator MntR (Kehres et al., 2002a; Patzer & Hantke, 2001). In E. coli and Salmonella enterica MntH functions as a proton-dependent divalent cation transporter that is highly selective for Mn2+ (Kehres & Maguire, 2003). ABC transporters of divalent metal cations are widely distributed phylogenetically among bacteria (Claverys, 2001). They include SitABCD from Sal. enterica and YfeABCD from Yersinia pestis. SitABCD and YfeABCD were initially identified as Fe2+ transporters (Bearden et al., 1998; Zhou et al., 1999). However, it has since been demonstrated that these transporters can also mediate the transport of Mn2+ (Bearden & Perry, 1999; Kehres et al., 2002b). A Sit homologue from Shigella flexneri 2a has been characterized (Runyen-Janecky et al., 2003), although the capacity of SitABCD homologues from either Shigella or E. coli to transport iron or manganese has not been investigated.
We have identified a SitABCD homologue in APEC strain 7122. Unlike other SitABCD and related transporters identified in the enterobacteria, which are encoded on the chromosome, the sitABCD genes in strain 7122 are encoded on the colicin-V type plasmid pAPEC-1. In this study, we characterized SitABCD by investigating its capacity to transport manganese and iron in E. coli K-12 mutants deficient in the tranport of these cations. In addition, we determined the contribution of SitABCD to the resistance of E. coli strains to hydrogen peroxide.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. In addition, clinical isolates from different sources were used. Archetypal ExPEC reference strains CFT073 (O6 : K2 : H1), CP9 (O4 : K54 : H5) and RS218 (O18 : K1 : H7) from human infections were kindly provided by Dr James R. Johnson (VA Medical Center, Minneapolis, MN, USA). Strain 536 (O6 : K15 : H31) was kindly provided by Professor Jorg Hacker (Universität Würzburg, Würzburg, Germany). Strain EB1 (O8 : K43) from a human wound infection was kindly provided by Dr Ben Otto (Vrije Universiteit, Amsterdam, The Netherlands). APEC strains MT78 (O2 : K1 : H5), MT458 (O78 : K80) and MT512 (O2 : K1 : H7) were kindly provided by Maryvonne Moulin-Schouleur (INRA, Tours, France). All strains were maintained in stock cultures at –80 °C in 25 % (v/v) glycerol following overnight culture in Luria–Bertani (LB) broth (10 g yeast extract, 5 g tryptone, and 10 g NaCl l–1). Strains and clones were routinely grown in LB broth or on LB agar plates (15 g agar l–1). E. coli strain DH5
was routinely used for plasmid cloning and recovery. Antibiotics were added as required at the following concentrations: ampicillin 100–200 µg ml–1, kanamycin 30 µg ml–1, chloramphenicol 30 µg ml–1, nalidixic acid 15 µg ml–1 and tetracycline 10 µg ml–1.
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). Restriction enzymes and DNA ligase were purchased from New England Biolabs (NEB), Invitrogen or Amersham-Pharmacia and used according to the suppliers' recommendations. Native plasmids from clinical isolates were extracted and analysed as described by Kado & Liu (1981). Recombinant plasmids, PCR products and restriction fragments were purified using plasmid mini-prep, PCR clean-up and gel extraction kits (Qiagen or Sigma) as recommended by the supplier.
PCR and DNA hybridization.
Taq DNA polymerase (NEB) was used for routine DNA amplifications (<2 kb), and Elongase (InVitrogen) was used for longer high-fidelity amplifications and cloning of genes. For amplification of products of up to 2 kb, 10 µl of a bacterial whole-cell lysate was added to a PCR reaction mixture of a final volume of 25 µl containing 6·25 pmol of each primer, 5 nmol of each dNTP and 0·5 U of Taq polymerase in 1x buffer. The PCR conditions were as follows: 94 °C for 3 min; followed by annealing for 1 min as indicated, 72 °C for 1 min and 94 °C for 1 min for 25 cycles; and a final extension at 72 °C for 10 min. For Southern blots, plasmid extracts or digested genomic DNA were separated by agarose gel electrophoresis and transferred to nylon membranes. A 663 bp DNA fragment was amplified from E. coli 7122 genomic DNA by using the sitA primer pair [CMD22, 5'-CCCTGTACCAGCGTACTGG-3'; and CMD23, 5'-CGCAGGGGGCACAACTGAT-3' with an annealing temperature (AT) of 54 °C] and was labelled by using the PCR DIG Labelling Mix (Roche). Hybridization and detection of the hybridized fragments were performed using the Digoxigenin Detection Kit as recommended by the manufacturer (Roche).
Cloning of the sitABCD and mntH genes.
A homologue of the SitABCD transporter of Sal. enterica was identified in APEC strain MT512 (O2 : K1) by DNA subtractive hybridization against the genome of the non-pathogenic avian E. coli strain EC79 (O2 : K–) (Schouler et al., 2004). Genes encoding homologues of SitABCD are also present in the genomes of uropathogenic E. coli CFT073 (Welch et al., 2002) and Sh. flexneri serotype 2a strains (Jin et al., 2002; Wei et al., 2003). Primers specific to sitA and sitD of E. coli and Sh. flexneri 2a were designed from the sequences available at GenBank. sitA-specific primers (CMD22, 5'-CCCTGTACCAGCGTACTGG-3'; and CMD23, 5'-CGCAGGGGGCACAACTGAT-3' used at an AT of 54 °C) and sitD-specific primers (CMD41, 5'-GCGTTGTGTCAGGAGTACC-3'; and CMD42, 5'-CTGTGCGCTGCTGTCGGTC-3' used at an AT of 52 °C) amplified fragments from the genomic DNA of strain 7122. The sit-specific primer pairs were used to screen a cosmid bank containing DNA inserts that hybridized with E. coli 7122 DNA that is absent from the genome of E. coli K-12 (Brown & Curtiss, 1996). A cosmid, pCA6, which was positive for PCR amplification using the sitA and sitD primer pairs was identified. A HindIII fragment containing the sit genes of 7122 was subcloned from pCA6 into pACYC184, generating pIJ28.
The sit genes from strain CFT073 were amplified from genomic DNA by PCR using primers CMD20 (5'-AAAAGCTTAAGACGACAATCGTCGGA-3'; HindIII site underlined) and CMD21 (5'-TTGAGCTCGGAATAACGCTTACCGTGTAA-3'; SacI site underlined) at an AT of 57 °C and Elongase DNA polymerase (Invitrogen) according to the manufacter's instructions. The amplification product was cloned directly into pTOPO-XL (InVitrogen), resulting in plasmid pIJ4. A segment of pIJ4 containing the sitABCD genes was obtained following digestion with HindIII and SacI. This fragment was cloned into the HindIII and SacI sites of pWSK29, resulting in plasmid pIJ5.
The mntH primer pair [CMD58, 5'-GGTTAAGCTTCCGTGCACATTCTATGTAA-3'; and CMD59, 5'-CTAAGCTTCGTAGGGCGGATAAGGCGTT-3' (HindIII sites underlined) at an AT of 64 °C] was used to amplify and clone the mntH gene from genomic DNA of strain 7122 by using the same strategy as above. Cloning of the HindIII-digested PCR product containing the mntH gene and promoter region into the HindIII site of pACYC184 generated pIJ42.
Construction of sitABCD and mntH mutant strains.
In order to construct an isogenic sitABCD : : tetAR(B) mutant of strain 7122 we used suicide-vector-based allele replacement. Plasmid pIJ5 containing the sitABCD genes from strain CFT073 was digested with PstI, resulting in removal of a section of the sitABCD operon spanning from nucleotide 149 of sitA to nucleotide 25 of sitD. A PstI fragment bearing a tetAR(B) cassette (Dozois et al., 2000) was cloned into the PstI sites of PstI-digested pIJ5, resulting in pIJ43. A BssHII fragment of pIJ43 encompassing the sitABCD : : tetAR construct was ligated to the compatible AscI sites of suicide vector pMEG-375, resulting in plasmid pIJ44. pIJ44 was used to replace the sitABCD genes with a non-functional sitABCD : : tetAR region using sacB-mediated counterselection as described by Dozois et al. (2000). A derivative of 7122, strain QT205, was confirmed as a double-crossover recombinant in which the sitABCD region had been replaced by sitABCD : : tetAR following homologous recombination.
mntH null mutants of E. coli K-12 strain 289 and APEC strains 7122 and QT205 were generated by the bacteriophage 
red recombinase-mediated mutagenesis method (Datsenko & Wanner, 2000). The mntHKO primer pair (CMD43, 5'-CTATGTTTTAGAGGCACAAGATGACGAACTATCGCGTGTAGGCTGGAGCTGCTTC-3'; and CMD44, 5'-TAGTGCCATATGCGACGCTCATTCAACTACAATCCCATATGAATATCCTCCTTAG-3' at an AT of 65 °C) was used to generate a kanamycin resistance gene cassette containing a DNA fragment from the template plasmid pKD3. The PCR product was used to delete the mntH gene as described by Datsenko & Wanner (2000). PCR amplification using the mntH primer pair (CMD58 and CMD59), which was used to clone the mntH gene, confirmed the mntH deletion mutations in strains QT99 (289 mntH), QT878 (7122 mntH) and QT1239 (7122 mntH sitABCD).
DNA sequencing and analysis of the sitABCD-encoding region.
The sitABCD operon and flanking DNA regions were sequenced from plasmid pIJ28 and from PCR-amplified fragments of strain 7122 DNA. Sequencing was achieved by generating derivatives of pIJ28 that contained the transposon Tn5seq1(Nag et al., 1988). The Tn5seq1 sequence contains SP6 and T7 primers flanking each end and facilitates bidirectional sequencing. In addition, custom primers were used to complete the sequence. DNA sequencing was done at the Genome Québec facility (McGill University, Montréal, QC, Canada). Putative ORFs were identified using the ORF Finder program and similarity searches of the DNA sequence. Predicted ORFs were obtained using BLAST programs accessed from the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov). Pairwise % identity/ % similarity of ORFs was determined using the Stretcher program available from the European Molecular Biology Open Software Suite (EMBOSS) (Rice et al., 2000).
Siderophore production, growth assays and ion-rescue experiments.
Chrome azurol S (CAS) agar plates (Schwyn & Neilands, 1987) were used to determine production of siderophores in the siderophore-negative E. coli strain 1017 containing cloned DNA from APEC strain 7122. Growth curves of this construct were obtained as described by Gong et al. (2001). The growth assay medium was nutrient broth (NB) (Difco) supplemented with 0·5 % (w/v) NaCl, thiamine (1 µg ml–1), histidine (22 µg ml–1) and 70 µM of the chelator 2,2'-dipyridyl (Sigma). To minimize possible metal contamination, the medium was prepared in polypropylene bottles that were washed twice with distilled water, and bacterial cultures were grown in disposable plastic tubes. Bacterial growth was measured every hour by spectrophotometry (OD600). For ion-rescue experiments we proceeded as for the growth assays, but the metal-restricted medium was supplemented with either 70 µM 2,2'-dipyridyl or 140 µM EDDA as chelator and 10 µM of either Mn2+, Zn2+, Fe2+ or Fe3+ as complementing ion. All experiments were performed in triplicate.
Transport of iron and manganese isotopes and isotope uptake inhibition experiments.
Isotope uptake and isotope uptake inhibition assays were performed using 54Mn, 59Fe and 55Fe isotopes purchased from Perkin Elmer. For iron uptake experiments, ferric iron (55FeCl3) was used. In addition, to determine ferrous iron uptake, the iron isotope was reduced to the ferrous state by addition of 100 mM ascorbate to the stock solution as described elsewhere (Kammler et al., 1993). Iron and manganese uptake experiments were based on a protocol modified from Silver and Kralovic (Silver & Kralovic, 1969; Kehres et al., 2002b). Briefly, the strains were grown overnight in dilute tryptone (DT) broth, comprising Bacto tryptone (4 g l–1) (Difco) supplemented with 0·25 % (w/v) NaCl, 0·4 % (w/v) glucose, thiamine (1 µg ml–1), histidine (22 µg ml–1) and aromatic amino acids (tryptophan, phenylalanine and tyrosine) (20 µg ml–1 each) with appropriate antibiotics. Cultures were adjusted to an OD600 of 0·5, centrifuged at 1600 g, and washed twice with equal volumes of room temperature DT broth (pH 7·0). In 2 ml Eppendorf tubes, 1 ml washed cells was centrifuged at 1600 g for 10 min and the bacterial pellets were suspended in DT medium containing isotope, ferric or ferrous 55Fe or ferrous 59Fe (to a final concentration of 100 nM) or 54Mn (to a final concentration of 50 nM), and samples were left to stand for 3 min at room temperature. Samples were then centrifuged at 1600 g and cells were washed twice with isotope-free DT. A 2 ml volume of scintillation cocktail was added to the cells and scintillation was measured in a Wallac Microbeta Trilux scintillation counter equipped with an Eppendorf tube adaptor plate (Perkin Elmer). Samples were analysed on the channels 5–810 for 54Mn, 5–980 for 59Fe and 5–750 for 55Fe. The scintillation cocktail Optiphase (Wallac) was purchased from Perkin Elmer. All experiments were done in triplicate. Values obtained were normalized with a positive control containing the isotope without cells and a negative control containing cells without isotope. For the isotope uptake inhibition, the samples were prepared as for the isotope uptake assays and ferrous 59Fe, ferric 55Fe or 54Mn was used. The isotopes were mixed with from 0·01 µM to 100 µM of cold competing Fe2+, Fe3+ or Mn2+ ions, and uptake was measured. All results were presented as a percentage of total isotope uptake determined in the absence of added cold ions.
Hydrogen peroxide sensitivity assay.
Sensitivity of bacterial cultures to H2O2 was determined by using an agar overlay diffusion method as described by Boyer et al. (2002). Briefly, overnight-grown cultures were used to inoculate (1/100) fresh LB medium without antibiotics, and the resulting cultures were incubated until the OD600 was 0·5. Then 100 µl of each culture was mixed with 3 ml molten top agar and poured onto an LB agar plate. A 7-mm-diameter Whatman filter disk impregnated with 10 µl 30·4 % H2O2 was placed in the centre of the plate and plates were incubated overnight at 37 °C. The inhibition zone diameters were then measured.
Statistical analyses.
Statistical analyses were performed using the Prism 4.0b software package (GraphPad Software). Statistically significant difference between two groups was established by unpaired t-test and comparison among three or more groups was done by one-way analysis of variance (ANOVA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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71227122 contains a large plasmid, pAPEC-1, that encodes virulence factors including the aerobactin and salmochelin siderophore systems and the temperature-sensitive haemagglutinin (Tsh) autotransporter (Dozois et al., 2000, 2003). Strain 7274 is a pAPEC-1 cured attenuated derivative of strain 7122 (Dozois et al., 2000). Attempts to amplify sit-specific DNA by PCR in strain 7274 were negative, suggesting that the sitABCD genes are located on plasmid pAPEC-1 of strain 7122. This finding is in contrast to E. coli CFT073 and Sh. flexneri 2a strains, which each contain one chromosomal copy of sitABCD (Jin et al., 2002; Wei et al., 2003; Welch et al., 2002). Southern blotting of plasmid DNA using a sitA-specific probe demonstrated that pAPEC-1 of strain 7122 contained the sit genes, whereas strain 7274, which had lost pAPEC-1, did not hybridize with the sitA-specific probe (Fig. 1). Further, plasmids from three other APEC strains, E. coli strain EB1 isolated from a human wound abscess, and the prototype Colicin V plasmid pColV-K30 also hybridized to the sitA probe. By contrast, archetype ExPEC strains CFT073, CP9, RS218 and 536 each contained a chromosome-encoded copy of sit genes as demonstrated by hybridization of the sitA-specific probe to total genomic DNA digested with either HindIII or SalI (Fig. 1). Further, digests from the total genomic DNA of strains MT78, MT458 and EB1 each demonstrated two DNA fragments that hybridized to the sit probe, suggesting that some E. coli strains contain a chromosomal as well as a plasmid-encoded copy of the sit genes.
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7122, Table 2). The predicted SitABCD proteins of strain 7122 (SitABCDE. coli 7122) exhibited the highest identity/similarity to SitABCD of Sh. flexneri 2a strain SA100 (Table 2). None of the genomic regions flanking sit genes from E. coli or Shigella strains identified in the DNA databases exhibited identity with the regions flanking the sitABCD genes from pAPEC-1. On pAPEC-1, a complete IS1 element is located 5' of the sit operon. The 5' and 3' regions adjacent to the sit genes are identical to a contiguous segment of E. coli plasmid p1658/97. Identity to the segment of p1658/97 in the 5' region of the sit genes includes the IS1 element and ends exactly at the end of the IS1 left repeat (position 1273 of the sit region) and the identity to p1658/97 sequence resumes 3' of the sit genes. The 3' region adjacent to sitD contains a 288 bp sequence exhibiting 93 % identity to a Tn1000-like transposase-encoding sequence, and this segment overlaps ORF1 described from p1658/97 (Fig. 2). Other ORFs 3' to the sit genes share identity at the protein level with part of a putative enolase, Eno-2, from Pseudomonas syringae, CrcB from Nitrosomonas europaea, ShiF and IucA (Table 2). ShiF is an ORF that is part of the aerobactin siderophore encoding pathogenicity islands present in Sh. flexneri 2a (Moss et al., 1999; Vokes et al., 1999) and E. coli CFT073 (Welch et al., 2002). The first gene of the aerobactin operon, iucA, is adjacent to shiF on pAPEC-1 (Fig. 2). When cosmid pCA6 or its subclone pIJ28 (sitABCD) was transferred to the E. coli siderophore-negative K-12 strain 1017, pCA6 conferred siderophore production upon this strain, whereas pIJ28 was negative for the production of siderophores. PCR analysis demonstrated that in addition to the sit genes, pCA6 also contained genes encoding aerobactin siderophore synthesis (iucABC) and uptake (iutA) (data not shown). These results are in accordance with the sequencing data, which indicate that the shiF and iucA genes are adjacent to the sit operon (Fig. 2, Table 2).
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7122 contains a potential operator sequence for the binding of the iron(II)-responsive Fur regulatory protein (Fig. 2
). In addition, a region highly similar to the binding site of the manganese-responsive regulator protein MntR was identified (Fig. 2). The potential Fur- and MntR-binding sites both overlap the predicted –10 to –35 region, suggesting that both iron and manganese levels are likely to influence regulation of sit gene expression. The predicted Fur- and MntR-binding regions were derived from the consensus sequences described by de Lorenzo et al. (1987) and Patzer & Hantke (2001) respectively, and are also conserved in PsitABCD of E. coli CFT073 and in Sh. flexneri strains (data not shown; Runyen-Janecky et al., 2003).
The sitABCDE. coli 7122 genes are most similar to those of Sh. flexneri 2a strains 301 (Jin et al., 2002) and SA100 (Runyen-Janecky et al., 2003), which are identical to each other. The 3450 bp region encompassing sitABCDE. coli 7122 contains 69 nucleotide differences with sitABCDSh. flexneri 301 and 92 differences with sitABCDE. coli CFT073, whereas sitABCDE. coli CFT073 demonstrates 62 differences with sitABCD Sh. flexneri 301. The sitA gene from the complete genome sequence of strain CFT073 contains six deletions that result in a predicted truncated SitA precursor protein of 285 amino acids, compared to the predicted 304 residue products of E. coli 7122 and Shigella strains. Because of the discrepancies between sitA sequences among strains we verified the sequence of the sitA gene of strain CFT073 from clone pIJ4 (Table 1). The DNA sequence we obtained lacked the six deletions that are present in the sequence submitted for sitA from the CFT073 genome (GenBank accession no. AE016759) and encoded a predicted 304 amino acid precursor protein, but was otherwise identical.
The characterized systems currently identified in other bacteria that are most similar to SitABCD of E. coli 7122 are SitABCD of Sal. enterica serovars Typhimurium and Typhi and YfeABCD of Y. pestis. The Sal. enterica sitA gene product (GenBank accession no. AAD41065) encodes the predicted periplasmic cation-binding protein and exhibits a percentage identity/similarity of 70/82 to SitAE. coli 7122, whereas YfeA from Y. pestis (GenBank accession no. CAC47605) demonstrates a percentage identity/similarity of 64/77 to SitAE. coli 7122.
SitABCD confers Fur-regulated repression upon an aroB feoB mutant of E. coli K-12 grown on iron-replete medium
To further investigate the role of SitABCD in the transport of iron in E. coli, we used E. coli K-12 H1771 as an indicator strain to determine fur-regulated control of iron acquisition on MacConkey-lactose agar supplemented with either 50 µM FeSO4 or 50 µM 2,2'-dipyridyl. Strain H1771 lacks both siderophore-mediated (Fe3+) and FeoB-mediated (Fe2+) transport systems and contains a Fur-regulated fhuF : : placMu53 fusion. Due to a reduced ability to obtain iron, the Fur-regulated fhuF-lacZYA fusion in this strain remains derepressed even on MacConkey-lactose plates containing added iron (Kammler et al., 1993). Introduction of pIJ28 to strain H1771 resulted in repression of fhuF-lacZYA expression and production of white colonies on MacConkey-lactose containing 50 µM Fe2+, whereas strain H1771 produced red colonies on MacConkey-lactose containing either 50 µM Fe2+ or 50 µM 2,2'-dipyridyl. These results suggest that SitABCD conferred increased iron transport ability upon strain H1771 in iron-supplemented medium and this resulted in the formation of Fur–Fe2+ complexes and repression of the fhuF-lacZYA fusion.
SitABCD promotes the growth of an E. coli K-12 enterobactin-deficient strain
The capacity of sit genes to promote the growth of the siderophore-negative strain 1017 in NB containing 75 µM of the chelator 2,2'-dipyridyl (NB-DIP) was assessed. The growth of strain 1017 that contained the sit genes (pIJ28), the aerobactin-encoding genes (pABN1), or a vector control (pACYC184) in NB-DIP was determined at hourly intervals. Strain 1017 containing pIJ28 (sitABCDE. coli 7122) grew well and at a rate similar to that of strain 1017 containing pABN1 (aerobactin system). By contrast, strain 1017 transformed with pACYC184 (vector control for plasmid pIJ28) exhibited poor growth (Fig. 3). Introduction of pIJ5 (sitABCDE. coli CFT073) complemented the growth defect of strain 1017 as effectively as did pIJ28 (data not shown). These results indicate that introduction of the SitABCD system alone effectively complements the growth defect of strain 1017 in NB-DIP.
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7122. Ferrous or ferric iron, manganese or zinc was added to NB supplemented with either 70 µM 2,2' dipyridyl or 140 µM EDDA, and growth of strain 1017(pACYC184) was determined. Following overnight culture in either NB-DIP or NB-EDDA only ferrous or ferric iron could complement the growth deficit of strain 1017(pACYC184) in a significant manner (80 %), whereas manganese complemented the growth defect only partially (40 %) and zinc did not improve growth (data not shown). The effect of addition of metals on the growth (measured at hourly intervals) of strain 1017 containing pIJ28 (sitABCDE. coli 7122) in NB-DIP is presented in Fig. 4; this clearly demonstrates the effect on growth of addition of iron compared to manganese or zinc. Further, introduction of the Sit system to strain 1017 increased growth to 80 % in NB-DIP supplemented with iron, as compared to NB without chelator, within 6 h, whereas it took strain 1017(pACYC184) over 10 h to reach a similar growth level (data not shown). These results suggest that SitABCD contributes to improved growth of strain 1017 by compensating for an iron deficit.
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7122 mediates transport of iron and manganese7122 conferred restored growth and Fur-mediated gene regulation upon iron transport deficient E. coli mutants, and since homologues of this system are characterized manganese and iron transporters (Bearden & Perry, 1999; Kehres et al., 2002b), we investigated the capacity of SitABCDE. coli 7122 to transport iron and manganese. SitABCD mediated the transport of ferrous iron and manganese under the conditions used for the assay (see Table 3). Under non-reducing conditions, in which iron is predominantly in the ferric state, the mean transport of iron by H1771 containing pIJ28 (sitABCDE. coli 7122) was not statistically different (P=0·111): a mean increase of 1597±784 c.p.m. compared to the control strain H1771(pACYC184) (Table 3). By contrast, when iron was treated with 100 mM ascorbate, to reduce it to the ferrous state, plasmid pIJ28 (sitABCDE. coli 7122) conferred a significant increase in iron uptake: a mean increase of 3008 c.p.m. compared to H1771(pACYC184) (P=0·001) (Table 3). Despite the increase in iron transport mediated by SitABCD in strain H1771, the level of iron uptake observed was considerably less than transport by the Aro+ Feo+ E. coli K-12 strain 289, which was 3·6-fold higher and 2·6-fold higher than that of H1771(pIJ28) under non-reduced and reduced conditions respectively.
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289(pACYC184), which exhibited a 1·63-fold higher mean uptake. Introduction of pIJ28 (sitABCD) to the mntH mutant strain QT99, conferred a statistically significant (P=0·01) 3·2-fold increase in uptake of 54Mn2+ compared to the vector control. Complementation of QT99 with plasmid pIJ42 (mntH) resulted in 54Mn2+ uptake 4·8-fold higher than that seen with QT99 containing pACYC184 (P=0·006) (Table 3). Hence, sitABCD cloned on vector pACYC184 effectively increased manganese transport by an mntH mutant K-12 strain, but not as effectively as reintroduction of a functional mntH allele on the same vector. Taken together, the results demonstrate that a cloned copy of the sitABCD genes from APEC strain 7122 is able to complement mutants impaired in either iron or manganese transport function, and suggest that SitABCDE. coli 7122 mediates transport of both ferrous iron and manganese under the conditions used for the assays.
SitABCD affinities for iron and manganese are dependent on the strain background
To investigate the relative affinity of SitABCD from APEC strain 7122 for iron or manganese we conducted isotope transport inhibition assays by addition of competing cold ions to iron or manganese isotopes during transport assays. Ferrous 59Fe or ferric 55Fe uptake by SitABCD was investigated in the iron transport deficient strain H1771 (aro feo), and 54Mn uptake by SitABCD was investigated in the manganese transport deficient strain QT99 (mntH) in the presence of increasing concentrations of Fe2+, Fe3+ or Mn2+ (Fig. 5). The apparent affinity or inhibition constants were determined as the concentration of cold cation required to inhibit 50 % of the isotope uptake. In strain H1771, the apparent affinity for Fe2+ was 0·9 µM and for Fe3+ it was 3·2 µM. For strain QT99 the apparent affinity for Mn2+ was 4 µM. For strain H1771, 59Fe2+ uptake was more inhibited by Fe3+ (2·5 µM) than by Mn2+ (5 µM); in the same strain 55Fe3+ uptake was more inhibited by Fe2+ (0·7 µM) and was less inhibited by Mn2+ (20 µM). In QT99 54Mn2+ uptake was better inhibited by Fe2+ (7 µM) and only poorly inhibited by Fe3+ (13 µM). Strains QT99 and H1771 differ in their native functional iron or manganese uptake systems. Strain QT99 is deficient in manganese transport, whereas H1771 is deficient in iron transport. Thus, the observed differences in SitABCD substrate specificity in these two strains are likely due to intrinsic differences in manganese and iron transport.
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289, whereas introduction of either plasmid pIJ28 (sitABCD) or pIJ42 (mntH) restored resistance to H2O2 which was similar to that of wild-type strain 289 (Fig. 6a). Analysis of variance of the means indicated that the growth inhibition zones generated by H2O2 were significantly different among the mntH mutant strain and the mutant complemented with either sitABCD or mntH (P=0·0312). By contrast, differences in growth inhibition zones were not significant among the wild-type strain and QT99 complemented with either pIJ28 (sitABCD) or pIJ42 (mntH) (P=0·0788). The wild-type pathogenic E. coli strain 7122 did not demonstrate a significant difference in resistance to H2O2 when compared to wild-type K-12 strain 289 (Fig. 6b). In addition, strains QT205 (7122 sitABCD) and QT878 (7122 mntH) were as resistant to H2O2 as wild-type APEC strain 7122. However, the sitABCD mntH of strain 7122 (QT1278) was more sensitive to H2O2 than APEC strain 7122 (Fig. 6b). These results indicate that sitABCD contributes to resistance to H2O2 in E. coli K-12 that lacks a functional MntH transporter. However, deletion of sitABCD genes from APEC strain 7122 does not cause an appreciable difference in resistance to H2O2, and the loss of both the SitABCD and MntH systems is necessary to render APEC strain 7122 sensitive to H2O2.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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7122. In addition, we demonstrated that sitABCD is also encoded by large plasmids in other pathogenic E. coli strains and that sit genes are also present on the reference colicin V plasmid pColV-K30, as well as on the genomes of certain strains (Fig. 1). Previous reports have described sit homologues that are located on the genomes of Sh. flexneri 2a strains (Jin et al., 2002; Runyen-Janecky et al., 2003; Wei et al., 2003) and ExPEC strain CFT073 (Welch et al., 2002). Similarly, operons of the related systems such as sitABCDSal. enterica (Zhou et al., 1999) and yfeABCD in Y. pestis (Bearden et al., 1998) are located on the chromosome. The presence of plasmid-located sit sequences in other E. coli strains has also been reported (Rodriguez-Siek et al., 2005; Chouler et al., 2004). The SitABCD system represents yet another metal-sequestering system that is encoded by genes located on ColV-type plasmids as well as the chromosomes of certain E. coli and Shigella strains. Other known metal transporters present on ColV-type or other conjugative plasmids in E. coli include the aerobactin and salmochelin siderophore systems encoded by the iucABCDiutA genes and iroBCDEN genes respectively (Dozois et al., 2003; Sorsa et al., 2003; Warner et al., 1981; Waters & Crosa, 1991). Thus ColV-type plasmids commonly carry genes encoding transporters important for the acquisition of metallic cations, particularly iron. In addition, both the aerobactin- and salmochelin-encoding operons are localized on the chromosomes of certain pathogenic E. coli or Shigella strains (Dobrindt et al., 2001; Moss et al., 1999; Vokes et al., 1999; Waters & Crosa, 1991), further attesting to the presence of these systems on either plasmids or pathogenicity islands.
Iron and manganese transport
In this report we have demonstrated the capacity of sitABCD from an APEC strain to function as a manganese and iron transporter. By using E. coli K-12 strains deficient in either transport of iron (ent or aroB feoB) or manganese (mntH), we investigated whether a cloned copy of sitABCD from APEC strain 7122 could compensate for mutations in these transport systems. Introduction of the sitABCD genes resulted in a significant regain in growth of the ent mutant strain 1017 in NB containing chelators (either 2,2'-dipyridyl or EDDA). The growth increase due to the presence of SitABCD was as marked as that seen after introduction of the aerobactin siderophore system (Fig. 3). Ion-rescue experiments demonstrated that only iron was able to complement the growth defect of strain 1017 (ent) to a high level (80 % of the growth yield in medium without chelator) and that in the presence of pIJ28 (sitABCD) growth was faster (6 h to reach 80 % growth yield, compared to more than 10 h in the absence of sitABCD). Addition of manganese partially restored the growth of strain 1017 (ent) (40 % maximum growth increase) whereas zinc did not contribute to any discernible growth increase. These results suggest that lack of iron and not manganese or zinc was largely responsible for the growth deficit of strain 1017. This conclusion is reinforced by the fact that aerobactin, an iron-specific siderophore, was equally able to compensate the growth of strain 1017 in NB-DIP (Fig. 3). Hence, increased growth of the E. coli K-12 ent mutant strain 1017 containing sitABCD was likely due to a greater capacity to obtain iron and suggests a role for SitABCDE. coli 7122 as an iron transporter. These findings are in accordance with previous results demonstrating that SitABCD homologues from other bacterial species may function as iron transporters (Bearden & Perry, 1999; Runyen-Janecky et al., 2003; Zhou et al., 1999).
In the aroB feoB mutant strain H1771, sitABCD conferred a significant increase in uptake of 55Fe and 59Fe under reduced conditions, suggesting it may function as a ferrous iron transporter (Table 3). However, the levels of iron uptake observed in strain H1771 containing sitABCDE. coli 7122 were significantly lower when compared to the E. coli K-12 control strain containing the functional endogenous iron transport systems, FeoB and enterobactin (Table 3). Taken together, these results suggest that, under our assay conditions, despite demonstrating a lower efficiency in iron uptake compared to the endogenous systems present in E. coli K-12, SitABCD is nevertheless able to compensate for iron transport deficiencies in E. coli K-12 and confer sufficient iron acquisition for restoration of growth under iron-limiting conditions.
Introduction of the sitABCDE. coli 7122 genes to an E. coli K-12 mntH mutant effectively restored manganese transport. In fact, when cloned copies of either sitABCDE. coli 7122 or mntH were introduced into the mntH mutant, manganese transport levels were greater than that of the wild-type parent (Table 3). This increased transport is most likely due to a plasmid copy number effect. In our studies, the first 54Mn uptake assay was conducted at pH 7·0. In subsequent 54Mn uptake assays done under acid to alkaline conditions ranging from pH 5 to pH 9, we observed that mntH mutant strain QT99 complemented with mntH (pIJ42) more effectively transported manganese under acid conditions (pH 5–6), whereas complementation with sitABCDE. coli 7122 (pIJ28) demonstrated increased manganese uptake at alkaline pH (pH 8–9) (M. Sabri & C. M. Dozois, unpublished results). These results were similar to those observed for Sal. enterica, which demonstrated that SitABCDSal. enterica mediated 54Mn uptake most effectively at alkaline pH (Kehres et al., 2002b). SitABCDSal. enterica has also been shown to be more specific for transport of manganese than iron (Kehres & Maguire, 2003). The YfeABC from Y. pestis was shown to mediate both manganese and iron uptake, although no preferential uptake of either of these metals was established (Bearden & Perry, 1999).
SitABCD and related systems are members of the Mn-Zn-Fe transport family (TC 3.A.1.15) based on bioinformatics analysis (Claverys 2001; Saier, 1999), and these tranporters have been shown to mediate uptake of iron or manganese in Y. pestis (Bearden & Perry, 1999) and manganese in Sal. enterica serovar Typhimurium (Kehres & Maguire, 2003). For SitABCDSh. flexneri no isotope uptake or isotope uptake inhibition experiments were performed, although the authors demonstrated an iron- and manganese-dependent regulation of sitABCDSh. flexneri (Runyen-Janecky et al., 2003). To determine if SitABCD exhibits a preference for uptake of iron or manganese we conducted isotope uptake and inhibition assays using ferric iron, ferrous iron or manganese. Since zinc did not demonstrate any growth rescue to strain 1017 pIJ28 (sitABCD) in NB containing chelators it was not tested for ion inhibition. Results indicated that in a strain impaired for the transport of iron, SitABCDE. coli 7122 demonstrated a higher affinity for iron than for manganese and confirmed that SitABCD displayed a higher affinity for ferrous iron than for ferric iron (Fig. 5). By contrast, when uptake and inhibition experiments were done in a strain impaired for manganese transport, SitABCDE. coli 7122 transported manganese with higher affinity than iron. The differences observed in ion transport affinities between the two strains are possibly due to the influence of other ion transport systems present in these strains which may affect the availability and/or uptake of competing cold ions. In the iron transport impaired strain H1771 (aro feo), the MntH manganese transporter is functional and may compete with the Sit system for uptake of Mn2+ ions, which could reduce the levels of cold Mn2+ ions available for the 55Fe or 59Fe uptake inhibition, thus resulting in lower apparent inhibition constants for manganese. Similarly in the manganese transport impaired strain QT99 (mntH), functional enterobactin and Feo transport systems may have influenced the apparent inhibition constants for Fe2+ and Fe3+. It is noteworthy that previous SitABCDSal. enterica transport affinity studies by Kehres & Maguire (2003) were done in strains which had functional siderophore and Feo systems, which may have led to the consequent finding that SitABCDSal. enterica exhibited a considerably lower affinity for iron than for manganese. Ion transport studies with YfeABCY. pestis used strains that contained functional MntH and Feo homologues (Bearden & Perry, 1999). It is therefore likely that some discrepancies observed in the transport specificity of Sit and related transporters that have been reported may be at least partially explained by differences in strain backgrounds as well as potential functional differences in the metal tranporters themselves.
The apparent affinities of uptake for Fe2+, Fe3+ and Mn2+ indicated that under our assay conditions SitABCDE. coli 7122 transported ferrous iron with the highest affinity (0·9 µM), followed by ferric iron (3·2 µM) and manganese (4 µM). In this regard, we believe our studies are the first to demonstrate a preference for ferrous iron uptake by a SitABCD transport system. This observation was determined by comparison of Sit-mediated 55Fe uptake under reducing (Fe2+) versus non-reducing (Fe3+) conditions (Table 3) as well as by comparison of the uptake of 59Fe2+ and 55Fe3+ isotopes and inhibition with cold Fe2+ or Fe3+ ions (Fig. 5). The role of SitABCD as both an iron and manganese transporter is further supported by the improved growth under conditions of iron deficit and H2O2 resistance phenotypes conferred upon E. coli K-12 strains that were deficient in iron or manganese transport respectively.
Protection against oxidative stress and resistance to H2O2
The ability to obtain manganese in bacteria contributes to detoxification of free radicals and protection against oxidative damage from agents such as H2O2 (Horsburgh et al., 2002; Kehres & Maguire, 2003). In our studies, increased sensitivity to H2O2 correlated with a decreased capacity to transport manganese. The E. coli K-12 mntH mutant was more sensitive to H2O2 than its wild-type parent strain, and complementation of the mntH mutant with either sitABCD or mntH restored resistance to H2O2 (Fig. 6a). Therefore sitABCD was shown to effectively compensate for the loss of MntH by restoring transport of manganese as well as resistance to H2O2 in a K-12 E. coli strain. By contrast, in APEC strain 7122, deletion of either sitABCD or mntH alone did not render it more sensitive to H2O2 (Fig. 6b). This suggested that the presence of either MntH or SitABCD sufficed for maintaining resistance to H2O2 in the absence of the other transporter. In support of this, the double sitABCD mntH mutant derivative of strain 7122 (QT1239) demonstrated a significant loss of resistance to H2O2 toxicity. These results suggest that under our assay conditions either SitABCD- or MntH-mediated manganese import systems were sufficient for maintaining a similar level of H2O2 resistance and that each of the two systems is able to compensate for the loss of function of the other transporter in the APEC strain. These findings also support a potential combined role for the SitABCD and MntH transporters for the virulence of APEC and perhaps other ExPEC.
Role of SitABCD in virulence
Inactivation of sitABCD alone in APEC strain 7122 had no appreciable effect on its sensitivity to H2O2 (Fig. 6). In line with this, following infection with the sitABCD mutant QT205, chickens developed lesions of airsacculitis, pericarditis and perihepatitis that were as severe as those observed in birds infected with the wild-type pathogenic parent strain 7122 (M. Caza, R. Curtiss III & C. M. Dozois, unpublished results). In the extra-intestinal tissues of infected chickens a combination of iron transport systems is needed for full virulence, and the APEC sit derivative QT205 retains functional siderophores and other iron and manganese transporters such as FeoB and MntH. Taken together, these results suggest that other iron and/or manganese transport systems present in APEC strain 7122 may compensate for the loss of a functional Sit transport system and provide sufficient transport for growth in iron-restricted medium and resistance to H2O2. Recently, by using signature-tagged mutagenesis (STM), the Sit tranporter was identified as a potential virulence factor in an APEC O2 strain in experimentally infected chickens (Li et al., 2005). Since STM-based studies involve co-infections with pools of mutants, in such studies it is not possible to establish if specific mutants are less able to generate lesions during infection. The potential contribution of SitABCD to the virulence of APEC strains may also differ among different APEC strains or serogroups since the pathogenic mechanisms of different strains have been shown to differ. For instance an APEC O2 strain demonstrated uptake and survival within avian phagocytes, whereas APEC O78 strain 7122 has been shown to avoid phagocytosis by avian heterophils or macrophages (Mellata et al., 2003). In E. coli, SitABCD-encoding genes are associated with clinical strains isolated from extra-intestinal infections from poultry and human urinary tract infections (Rodriguez-Siek et al., 2005; Schouler et al., 2004). Recently, transcriptome analysis of uropathogenic E. coli strain CFT073 indicated that sit genes were highly upregulated in the urine of infected mice (Snyder et al., 2004), further supporting a potential role for the Sit system during infection. Further virulence studies will be required to assess the individual and cumulative roles of SitABCD and other iron and manganese transport systems for the virulence of APEC and other pathogenic E. coli strains.
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ACKNOWLEDGEMENTS |
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Received 10 November 2005;
revised 24 November 2005;
accepted 29 November 2005.
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 K. A. Tivendale, A. H. Noormohammadi, J. L. Allen, and G. F. Browning The conserved portion of the putative virulence region contributes to virulence of avian pathogenic Escherichia coli Microbiology, February 1, 2009; 155(2): 450 - 460. [Abstract] [Full Text] [PDF]
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 W. F. Fricke, M. S. Wright, A. H. Lindell, D. M. Harkins, C. Baker-Austin, J. Ravel, and R. Stepanauskas Insights into the Environmental Resistance Gene Pool from the Genome Sequence of the Multidrug-Resistant Environmental Isolate Escherichia coli SMS-3-5 J. Bacteriol., October 15, 2008; 190(20): 6779 - 6794. [Abstract] [Full Text] [PDF]
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T. J. Johnson, Y. M. Wannemuehler, and L. K. Nolan Evolution of the iss Gene in Escherichia coli Appl. Envir. Microbiol., April 15, 2008; 74(8): 2360 - 2369. [Abstract] [Full Text] [PDF]
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G. Li, C. Ewers, C. Laturnus, I. Diehl, K. Alt, J. Dai, E.-M. Antao, K. Schnetz, and L. H. Wieler Characterization of a yjjQ mutant of avian pathogenic Escherichia coli (APEC) Microbiology, April 1, 2008; 154(4): 1082 - 1093. [Abstract] [Full Text] [PDF]
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M. Sabri, M. Caza, J. Proulx, M. H. Lymberopoulos, A. Bree, M. Moulin-Schouleur, R. Curtiss III, and C. M. Dozois Contribution of the SitABCD, MntH, and FeoB Metal Transporters to the Virulence of Avian Pathogenic Escherichia coli O78 Strain {chi}7122 Infect. Immun., February 1, 2008; 76(2): 601 - 611. [Abstract] [Full Text] [PDF]
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T. J. Johnson, S. Kariyawasam, Y. Wannemuehler, P. Mangiamele, S. J. Johnson, C. Doetkott, J. A. Skyberg, A. M. Lynne, J. R. Johnson, and L. K. Nolan The Genome Sequence of Avian Pathogenic Escherichia coli Strain O1:K1:H7 Shares Strong Similarities with Human Extraintestinal Pathogenic E. coli Genomes J. Bacteriol., April 15, 2007; 189(8): 3228 - 3236. [Abstract] [Full Text] [PDF]
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M. Moulin-Schouleur, C. Schouler, P. Tailliez, M.-R. Kao, A. Bree, P. Germon, E. Oswald, J. Mainil, M. Blanco, and J. Blanco Common Virulence Factors and Genetic Relationships between O18:K1:H7 Escherichia coli Isolates of Human and Avian Origin. J. Clin. Microbiol., October 1, 2006; 44(10): 3484 - 3492. [Abstract] [Full Text] [PDF]
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L. Runyen-Janecky, E. Dazenski, S. Hawkins, and L. Warner Role and Regulation of the Shigella flexneri Sit and MntH Systems. Infect. Immun., August 1, 2006; 74(8): 4666 - 4672. [Abstract] [Full Text] [PDF]
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), pIJ28 (
) or pABN1 (
) in NB medium containing 75 µM 2,2'-dipyridyl at 37 °C. pACYC184 is the vector for pIJ28 and serves as a negative control. pIJ28 encodes the sitABCD genes. pABN1 encodes the aerobactin gene cluster and serves as a positive control. Results represent means±SEM for three independent experiments.
