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Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky, Lexington, KY 40536-0298, USA
Correspondence
Stanislav Forman
standa{at}uky.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Present address: Interdisciplinary Program in Biomedical Sciences, UNC-Chapel Hill, CB#7100, Chapel Hill, NC 27599-7100, USA.
The list of the oligonucleotides used in this study and alignments of aerobactin receptor and biosynthesis proteins are available as supplementary data with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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), Yersinia pestis contains a plethora of iron/haem transport systems (Perry, 2004; Perry & Fetherston, 2004). There are two haem transport systems, designated Hmu and Has. The Y. pestis Has system resembles the well-characterized Has haemophore transport system of Serratia marcescens (Debarieux & Wandersman, 2004). However, the Y. pestis Has system does not appear to be functional (Rossi et al., 2001). The Hmu system is virtually identical to the Hem haem transport system that was originally identified and characterized in Yersinia enterocolitica (Stojiljkovic & Hantke, 1992, 1994), and is the haem transport system in Y. pestis responsible for utilization of haem and haemoproteins (Hornung et al., 1996; Thompson et al., 1999). There are five Y. pestis systems that have been shown to transport iron: Ybt, Yfe, Yfu, Feo and Yiu. The yersiniabactin (Ybt) system, which has been extensively studied, synthesizes and transports the siderophore yersiniabactin. Feo appears to transport ferrous iron in a number of organisms, including Y. pestis (Perry et al., 2007). The Yfe, Yfu and Yiu systems are all ABC transporters that were originally identified by their ability to enhance the growth of an Escherichia coli enterobactin-biosynthetic mutant under iron-deficient conditions and were subsequently shown to function in Y. pestis as well (Bearden & Perry, 1999; Bearden et al., 1998; Gong et al., 2001; Hantke, 2004; Kirillina et al., 2006; Perry, 2004; Perry & Fetherston, 2004).
Here we examine the functionality of two additional systems, one homologous to the aerobactin system of E. coli and another to the ferric hydroxamate uptake (Fhu) system of E. coli. The E. coli aerobactin biosynthetic operon consists of four genes, iucABCD, with the receptor encoded by a separate gene, iutA (Payne & Mey, 2004). In Y. pestis, iucA has undergone a frameshift, generating two separate open reading frames [iucA1 (y3380) and iucA2 (y3381) in strain KIM10+] (Deng et al., 2002). The remaining genes are intact and the proteins encoded share 67–74 % similarity with their E. coli counterparts (Fig. 1). Our results indicate that the Y. pestis aerobactin biosynthetic gene products are unable to produce aerobactin or complement an E. coli iucB : : cam mutant. The Fhu system of Y. pestis consists of three genes, fhuCDB, which closely resemble the corresponding genes in E. coli (Braun et al., 2004). Y. pestis lacks a gene homologous to fhuA, the E. coli receptor for ferrichrome, but does contain a gene (y2556) that is 85 % identical to the ferrichrome receptor of Y. enterocolitica (FcuA) (Koebnik et al., 1993). The Y. pestis aerobactin receptor (IutA) as well as the putative FcuA ferrichrome receptor (y2556) and the Fhu ABC transporter are functional, since we show here that Y. pestis can utilize exogenous aerobactin and ferrichrome.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. Unless indicated otherwise, E. coli strains were grown in liquid or solidified Luria broth (LB). Y. pestis strains were grown on tryptose blood agar (TBA) plates. Retention of the pgm locus was confirmed for Y. pestis by culture on Congo red plates at 26–30 °C (Jackson & Burrows, 1956).
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) resulted in the identification of clone 1985 (renamed pAero1985; Table 1
). Based upon restriction enzyme analysis and DNA sequencing, pAero1985 contains a 9035 bp insert that encompasses the putative aerobactin biosynthetic genes (iucA1-A2-B-C-D) and the outer-membrane receptor (iutA) (data not shown). The ends of the insert in subclone pAeroYp1 (Table 1) were sequenced (Northwestern University Biotechnology Laboratory) using primers HCW and SCCW, which bind to sequences in the pBR322 vector.
In Y. pestis strains KIM10+, CO92, 91001, Angola, Antiqua and Nepal516, iucA has undergone a frameshift mutation (Deng et al., 2002; Parkhill et al., 2001; Song et al., 2004). The frameshift mutation in Y. pestis iucA1/iucA2 is the result of a single base deletion in the L179 codon. To correct this, primers IucA(G)01 and IucA(G)02 in combination with outside primers IucA(SalI) and IucA(BsrGI) with pAeroYp1 as a template were used to generate a PCR fragment with a corrected iucA gene. We used a two-step PCR cloning procedure (Forman et al., 2006) to construct and introduce the repaired SalI–BsrGI fragment into pAeroYp1, generating pAeroYp4 (iucA-D-iutA+).
Comparison of the Y. pestis IucA amino acid sequence with that of Yersinia pseudotuberculosis IucA, which does not have a frameshift in any of the three genomes sequenced, showed a substitution of the first base in the codon (TAT
CAT), which results in a Y225H change in IucA2 of Y. pestis. To reverse this change, a two-step PCR cloning procedure, with pAeroYp1 as the primary target DNA and the primer set IucA(Tyr)01/IucA(Tyr)02 and the outside primers IucA(SalI)/IucA(BsrGI), was used to generate a PCR fragment with an altered iucA-H225Y. The resulting SalI–BsrGI product was ligated into pAeroYp4 to generate pAeroYp5. This plasmid encodes the entire iucA-D-iutA locus with an iucA-H225Y similar to the Y. pseudotuberculosis iucA gene. All final iuc locus constructs were sequenced by Elim Biopharmaceuticals to confirm that the sequences were correct.
The 139 bp iuc promoter region, between divergent ORFs iucA and y3378, was amplified by PCR from pAeroYp1 using primers iucP1and iucP2. The PCR products were digested with AscI and cloned into the AscI site of pNEB193. The AscI fragment of one clone (pNEBiucP) with the correct nucleotide sequence was inserted into the AscI site of pEU730. The iucP1 and M13(–40) primers were used to identify clones in which lacZ transcription was under the control of the iuc promoter. One clone with the proper orientation was named pEUiucP. To obtain a reporter plasmid containing the iuc promoter region inserted in the opposite orientation, pNEBiucP was digested with Asp718 and PacI. The promoter fragment was gel-purified and cloned into the same sites in pEU730, resulting in plasmid pEUiucP-Op.
Primers Fhu-1 and Fhu-2 were used to generate a 302 bp DIG-labelled probe that hybridized to clone 1310 (renamed pFhuYp1310) in the KIM6+ genomic library (Perry et al., 1990). This clone contains an approximately 8.3 kb insert that includes the Y. pestis fhuCDB operon. An approximately 5.4 kb subclone yielded pFhuYp1 (Table 1).
Construction of 
iutA, fhuCD and shiF : : cam mutations.
To generate a deletion within the fhu operon of Y. pestis, pFHUYp1 was digested with EcoRV to remove a 467 bp fragment that encompasses the 3' end of fhuC and the 5' end of fhuD, creating pFhuYp2 (see Fig. 1b). An approximately 2.7 kb fragment containing the deletion was subcloned from pFhuYp2 into pKNG101, yielding pKNGFhuCD (Table 1), and was introduced into the chromosome of KIM6 by allelic exchange. Strains in which the deleted genes had replaced the wild-type copies were identified by PCR using primers Fhu-1 and Fhu-2. One mutant was selected and named KIM6-2114 (fhuDC2114).
To construct a deletion within the iutA gene of Y. pestis, an approximately 3.0 kb ScaI–EcoRV fragment containing a ClaI deletion, which removes 930 bp from the iutA gene (see Fig. 1a), was subcloned from pAeroYp3 into the suicide vector pKNG101, resulting in pKNGIutA (Table 1). This plasmid was electroporated into Y. pestis KIM6 and transformants were selected on TBA plates containing streptomycin (50 µg ml–1). As previously described (Bearden & Perry, 1999), cells grown overnight in Heart Infusion broth (Difco) without antibiotic selection were used to select sucrose-resistant isolates that had completed the allelic exchange. Strains in which the wild-type gene was replaced by the deleted version were identified by PCR using primers Iut-1 and Iut-2. One isolate was selected and named KIM6-2113 (iutA2113).
The sequence for the shiF gene from E. coli CFT073 (Welch et al., 2002) was used to generate primers for mutagenesis of the shiF gene in pColV-K30. The primers Shi-cat1 and Shi-cat2 (Supplementary Table S1) were used to amplify the chloramphenicol cassette from pKD3 (Datsenko & Wanner, 2000). The PCR product was electroporated into E. coli strain 1017 (pColV-K30, pKD46) and the cells were plated on LB plates containing 15 µg chloramphenicol ml–1. The allelic exchange, which deletes 930 bp from the shiF homologue in pColV-K30, was verified by PCR with primers ShiF-1 and ShiF-2 (Supplementary Table S1). The resulting plasmid was named pColV-K30shiF : : cam (Table 1).
β-Galactosidase assays.
Reporter plasmids were electroporated into Y. pestis KIM6 and KIM6-2030 (fur : : kan-9) strains to analyse transcriptional regulation of the iucA and y3378 promoters. Y. pestis strains containing the reporter plasmid were acclimated to iron-deficient or iron-surplus conditions [
six generations in the defined deferrated medium PMH2 without added iron or with 10 µM FeCl3 (Gong et al., 2001)] and harvested during exponential growth at 3 °C. β-Galactosidase activities from whole-cell lysates were measured spectrophotometrically with a Genesys 5 spectrophotometer (Spectronic Instruments) following cleavage of o-nitrophenyl β-D-galactopyranoside (ONPG) and the results are expressed in Miller units (Miller, 1992). Data presented are the means and standard deviations derived from two independent experiments.
Protein expression analysis.
In vitro transcription/translation assays were used to examine protein expression from the various cloned aerobactin loci. Purified and concentrated template DNA at approximately 2 µg per reaction was incubated with T7 S30 E. coli extract (Promega) and Trans35S-label [50 µCi (1.85 MBq) of [35S] -methionine (70 %) and [35S]-cysteine (15 %) per reaction; MP Biomedicals] for 3 h at 37 °C, following the manufacturer's specifications. Polypeptides in boiled and unboiled samples were separated by SDS-PAGE using 12 % acrylamide. Gels were dried and newly synthesized polypeptides visualized by exposure to Blue Lite autoradiography film (ISC BioExpress). 14C-methylated molecular mass markers (range 14 000–220 000; Sigma) were used to estimate the molecular masses of the polypeptides.
Siderophore bioassays.
pColV-K30 was isolated from E. coli LG1315 and electroporated into E. coli strain 1017. Cells containing the plasmid were selected on LB agar plates containing 500 µM ethylenediamine-di(o-hydroxyphenyl-acetic acid). E. coli 1017 cells containing pColV-K30 were grown overnight at 3 °C in nutrient broth (NB) containing 50 µM 2,2'-dipyridyl (DIP). All other E. coli strains were grown in NB with 25 µM DIP. The cells from these cultures were pelleted and the supernatants filtered through a 0.22 µm filter. Assuming 1 ml of culture at an OD620 of 1.0 is 7.5x108 cells, approximately 105 cells ml–1 were incorporated into NB agar plates containing either 50 µM DIP for plates with LG1522 or 75 µM DIP for plates containing 1017(pAeroYp1). Filtered supernatants to be tested for siderophore activity were added to wells in the plates, which were incubated overnight at 37 °C.
Y. pestis strains were grown through two transfers at 37 °C in deferrated PMH2 (Gong et al., 2001). Two hundred microlitres of a KIM6-2046.1 culture with or without pBGL2 at an OD620 of 0.2 were incorporated into PMH2 agar plates containing 75 µM DIP. Filtered supernatants from iron-deficient cultures were placed into wells and the plates were incubated at 37 °C overnight.
The ability of E. coli RK4375 derivatives to use ferrichrome was tested in a plate assay. RK4375 cultures were grown overnight at 37 °C in LB medium containing 100 µM DIP and incorporated at a concentration of 105 cells ml–1 into LB plates supplemented with 500 µM DIP. A 25 µl aliquot of a 1 mM solution of ferrichrome (Sigma) was added to wells in the agar and the plates were incubated overnight at 37 °C. To test for ferrichrome use by Y. pestis, strains were grown through two transfers in deferrated PMH2 at 37 °C and 0.04 OD620 units of the cultures were included in solidified PMH2 containing 0.5 mM Na2CO3, 100 µM MnCl2, 4 mM CaCl2 and 50 µM DIP. One millimolar ferrichrome (25 µl aliquot) was added to wells and the plates were incubated at 37 °C overnight. To visualize growth of Y. pestis around the wells, the plates were overlaid with TBA containing 0.05 % aesculin and 0.05 % ferric citrate.
Growth curves in the presence of ferrichrome.
KIM6 and KIM6-2114 (fhuCD) cells were grown on TBA slants at 30 °C and used to inoculate deferrated PMH2 to an OD620 of 0.1. After overnight growth at 37 °C, the cultures were diluted to an OD620 of 0.1 into deferrated PMH2 containing or lacking 10 µM ferrichrome and incubated at 37 °C. Optical density readings using a Genesys 5 spectrophotometer (Spectronic Instruments) were taken at hourly intervals. PMH2 is a defined medium (Gong et al., 2001). Chelex 100 (Bio-Rad) was used to remove contaminating iron as previously described (Staggs & Perry, 1991).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) revealed the presence of an operon with genes homologous to E. coli iucABCD and iutA. The gene order in the Y. pestis aerobactin operon is the same as in E. coli. In all of the Y. pestis strains sequenced to date (CO92, KIM10+, 91001, Antiqua, Nepal516 and Angola) (Chain et al., 2006; Deng et al., 2002; Parkhill et al., 2001; Song et al., 2004), iucA contains a mutation which splits the gene into two ORFs [Fig. 1; iucA1 (y3380) and iucA2 (y3381) in KIM] with the corresponding proteins IucA1 being 62 % similar to residues 1–184 and IucA2 72 % similar to residues 219–575 of E. coli IucA, respectively (Fig. 1). The predicted amino acid sequences of the remaining Y. pestis aerobactin biosynthetic genes have from 67 to 74 % similarity to their E. coli counterparts. The putative Y. pestis aerobactin receptor, IutA, is 73 % similar and 67 % identical to E. coli IutA (Supplementary Fig. S1, available with the online version of this paper). In contrast, none of the three Y. pseudotuberculosis genome sequences analysed has the iucA frameshift mutation and they show 98–100 % identity with the Y. pestis Iuc/Iut amino acid sequences (Supplementary Figs S2–S5).
A potential Fur-binding site (FBS) is located 44 bp upstream of the putative start of iucA1. This FBS matches the E. coli FBS consensus sequence (Braun & Hantke, 1991) in 16 out of 19 bases. The FBS starts 2 bp upstream of a putative –10 region and overlaps the putative –35 element. This element may also regulate a divergent promoter for an ORF, Y3378, with similarity to major facilitator superfamily efflux proteins.
Sequence analysis of the fhuCDB operon
In a number of enteric organisms, aerobactin is transported across the inner membrane by the Fhu system (Payne & Mey, 2004). The Y. pestis genomes contain genes homologous to the E. coli fhuCDB operon (Fig. 1). Unlike E. coli, the Y. pestis operon does not contain the gene encoding the ferrichrome receptor, FhuA. In another region of the Y. pestis chromosome, a putative ferrichrome receptor (y2556) is encoded which is more closely related to FcuA, the ferrichrome receptor of Y. enterocolitica (85 % identity and 88 % similarity) (Koebnik et al., 1993).
Regulation of iuc genes and iutA
Generally, transcription of genes encoding iron uptake systems is repressed by iron-surplus conditions. To test whether the 139 bp region upstream of iucA is an iron- and Fur-regulated promoter, we constructed an iucA : : lacZ transcriptional fusion in a single-copy vector, pEUIucP. Cells of KIM6(pEUiucP) and KIM6-2030(pEUiucP) (fur : : kan-9) were grown at 37 °C in iron-deficient PMH medium (Staggs & Perry, 1991) in the presence or absence of added iron and assayed for β-galactosidase activity. The expression of iucA : : lacZ in KIM6 grown with surplus iron was repressed 23-fold compared to that of the iron-deficient culture. In the absence of Fur, the iucA : : lacZ promoter was no longer repressed by surplus iron in the medium (Fig. 2). Thus, the iucA promoter requires a functional Fur protein for repression by iron. To determine whether this region serves as a divergent promoter for y3378, an ORF with similarity to ORFs upstream of iucA in other bacteria encoding the aerobactin system (termed shiF in Shigella), the 139 bp between y3378 and iucA was cloned in the opposite orientation (pEUiucP-Op) as well. β-Galactosidase activity from this reporter exhibited a 10-fold repression by iron in a Fur+ strain. However, iron-deficient expression levels were 8-fold lower for this orientation than for the iucA promoter orientation. Although weaker than the activity for the iuc locus, this region appears to serve as an iron- and Fur-regulated promoter for expression of y3378 as well (Fig. 2). Similar shiF-like genes lie upstream of the aerobactin locus in a number of bacteria, suggesting a possible role in the aerobactin system. Annotations of shiF-like genes indicate it is a member of the COG0477 permeases of the major facilitator superfamily. However, we found no experimental studies on the function of these proteins. COG0477 has no recognized conserved domain and is defined simply as proteins with 12 to 14 transmembrane domains arranged as two groups of six or seven. Analysis of Y3378 with TMHMM (Sonnhammer et al., 1998) predicts only 11 transmembrane domains with no division into two groups. While it is possible that Y3378 serves as an aerobactin exporter, an insertion in a similar ORF in pColV-K30 (pColV-K30shiF : : cam; Table 1) still allowed aerobactin secretion in E. coli (data not shown). This indicates either that ShiF is not essential for the functioning of the aerobactin system, or that another system encoded within the E. coli genome can also function to export aerobactin.
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). The insert of pAero1985 includes 373 bp of DNA upstream of the putative start codon for iucA1, containing the putative promoter elements and the iucA2, iucB, iucC, iucD and iutA genes. A subclone of the Y. pestis aerobactin region, pAeroYp1 (Table 1), was introduced into the E. coli strain 1017. E. coli 1017 is a derivative of HB101 with a transposon insertion within the enterobactin biosynthetic operon and is thus unable to produce enterobactin (Daskaleros et al., 1991). Supernatants isolated from E. coli 1017(pAeroYp1) cells grown under iron-deficient conditions were unable to feed LG1522, an E. coli strain carrying a pColV-K30 plasmid with a mutation in the aerobactin biosynthetic genes (Carbonetti & Williams, 1984; Valvano et al., 1986). However, LG1522 was fed by supernatants isolated from an E. coli 1017 strain carrying wild-type ColV-K30. In addition, supernatants from KIM6, which encodes the iucA1-D-iutA locus but lacks the ybt locus which encodes the yersiniabactin iron transporter, were unable to restore growth of KIM6-2046.1 (irp2-2046 : : kan), a strain unable to synthesize the Ybt siderophore, on PMH2 plates containing 75 µM DIP (data not shown). These results suggest that Y. pestis genes iucA1, iucA2, iucB, iucC and iucD do not produce enzymes capable of aerobactin synthesis. To determine if the Y. pestis genes downstream of the iucA1/iucA2 frameshift are functional, pAeroYp1 was electroporated into E. coli strain CFT073-ENT/IUC. CFT073-ENT/IUC is a uropathogenic strain of E. coli with kanamycin and chloramphenicol gene cassettes disrupting entF and iucB, respectively. Thus, this strain does not synthesize either enterobactin or aerobactin, although it can use both of these siderophores (Torres et al., 2001). Supernatants isolated from CFT073-ENT/IUC(pAeroYp1) grown under iron-deficient conditions were unable to feed LG1522 (data not shown). This suggests that the Y. pestis iucBCD genes could not replace their E. coli counterparts in CFT073-ENT/IUC or were not expressed, and therefore did not restore aerobactin synthesis.
In vitro transcription/translation analysis using pAeroYp1 showed a polypeptide with a molecular mass similar to that predicted for the IutA OM receptor (Fig. 3). However, no polypeptides with masses similar to that predicted for IucB, IucC or IucD were apparent (Fig. 3). This suggests that the downstream iucB-D genes may not be translated due to the frameshift mutation in iucA1/iucA2.
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iutA2113) was unable to use aerobactin as an iron source (Fig. 4a). However, KIM6-2113 cells carrying pAeroYp1, which encodes the Y. pestis aerobactin locus (iucA1-A2-B-C-D-iutA), were able to use aerobactin as an iron source (data not shown). Thus, Y. pestis does produce a functional aerobactin receptor (IutA). These results are in contrast to an earlier study which indicated that Y. pestis could not use aerobactin as an iron source (Perry & Brubaker, 1979). While that study used a different Y. pestis biotype (strain Kuma+), one other likely explanation is that the concentration of conalbumin used to inhibit growth was too high to be overcome by exogenous aerobactin under the growth conditions tested. Supernatants from E. coli 1017(pColV-K30) also stimulated the growth of E. coli 1017(pAeroYp1) on NB plates containing 75 µM DIP, while supernatants from E. coli 1017(pBR322) did not (data not shown).
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). This is similar to the Y. pseudotuberculosis iucA-D-iutA loci in strains IP32953 (Chain et al., 2004) and PB1 except for a Y225H change in the Y. pestis locus. Consequently, we altered this residue to Y225 in pAeroYp5. In vitro transcription/translation products from pAeroYp5 yielded polypeptides with molecular masses similar to those predicted for IutA, IucA, IucB, IucC and IucD (Fig. 3). For reasons that are unclear, the polypeptide probably corresponding to IucB was only apparent in unboiled samples. Nevertheless, these results indicate that pAeroYp5 expresses the Y. pestis Iuc proteins, providing further evidence that the iucA1/iucA2 frameshift prevents translation of iucB, iucC and iucD, but not iutA. Iron-deficient culture supernatants from E. coli 1017 carrying pBR322, pAeroYp4 or pAeroYp5 all failed to stimulate growth of E. coli LG1522(pBR322) under iron-chelated conditions, indicating that aerobactin was not produced by any of these recombinant strains. In contrast, E. coli 1017(pColV-K30) supernatants did stimulate growth (data not shown).
Supernatants from Y. pestis KIM6 cultures carrying pBR322, pAeroYp4 or pAeroYp5 also failed to stimulate the growth of E. coli LG1522(pBR322) under iron-chelated conditions. Thus, correcting the frameshift mutation and the IucA-Y225H alteration failed to lead to aerobactin synthesis by Y. pestis (data not shown). However, Y. pestis KIM6 carrying either pKLS711 or pColV-K30shiF : : cam, which both encode functional aerobactin loci (Table 1), did produce aerobactin. Supernatants from iron-deficient cultures of these strains supported the growth of Y. pestis KIM6(pBR322) on PMH2 plates containing 75 µM DIP, while supernatants from Y. pestis strains carrying pBR322 (negative control), pAeroYp4 and pAeroYp5 did not stimulate growth of the indicator strain (Fig. 4b).
We assessed the ability of iron-starved cells of Y. pestis KIM6(pBR322) (Ybt–), KIM6(pKLS711) and KIM6(pColV-K30shiF : : cam) to grow on solidified PMH2 containing 75 µM DIP at 37 °C. Under these iron-chelated conditions Ybt+ but not Ybt– cells of Y. pestis are capable of growth. Only KIM6(pKLS711) cells grew well on these plates. KIM6(pColV-K30shiF : : cam) cells were able to grow on PMH2 plates containing 50 µM DIP (data not shown). The ability of KIM69(pKLS711) to grow at a higher DIP concentration than KIM6(pColV-K30shiF : : cam) is probably due to a higher plasmid copy number for pKLS711 compared to that of pColV- K30. Overall these results indicate that, at least under these in vitro conditions, aerobactin can replace the yersiniabactin iron transport system in supplying iron to Y. pestis cells.
The Y. pseudotuberculosis iuc locus does not produce aerobactin
We also tested supernatants from Y. pseudotuberculosis PB1-2046.1/0 cultured in deferrated PMH2. PB1-2046.1/0 is a derivative of the sequenced PB1 strain that has a kan insertion in irp2, making it unable to synthesize the yersiniabactin siderophore (Perry et al., 1999). The genome sequence of strain PB1 shows an iucA-D-iutA locus with no obvious defects and is identical to the same locus in strain IP32953 (Chain et al., 2004). Despite this, the PB1-2046.1/0 supernatants did not support the growth of E. coli LG1522, indicating that Y. pseudotuberculosis is unable to synthesize aerobactin. This correlates with a survey that failed to demonstrate aerobactin production in five different Y. pseudotuberculosis strains (Stuart et al., 1986).
The reason for this lack of aerobactin production by Y. pseudotuberculosis and the Y. pestis recombinant iucA-H225Y-iucB-D-iutA locus is unclear. However, conference of aerobactin production in Y. pestis by iuc genes from Shigella flexneri or pColV-K30 suggests the defect lies within the Yersinia iuc genes. A comparison of predicted amino acid sequences of IucA, IucB, IucC and IucD between the Yersinia and pColV-K30 identified a number of residue differences in all four proteins (Supplementary Figs S2–S5, available with the online version of this paper). No study of amino acid residues critical for the enzymic activity of the Iuc proteins has been published. Consequently, any one or multiple non-conserved residues in the Yersinia Iuc proteins could be responsible for the inability to synthesize a functional aerobactin molecule.
The Fhu ABC transporter of Y. pestis is functional
The ability of Y. pestis to utilize exogenous aerobactin suggests that the FhuCDB transporter is functional. To clearly demonstrate this, a clone containing the Y. pestis fhu operon was identified from the Y. pestis KIM10+ genomic library and the region encompassing fhuCDB was subcloned, generating pFhuYp1. This recombinant plasmid was introduced into E. coli strain RK4375, which contains a Tn10 insertion within fhuC and is thus unable to grow in the presence of ferrichrome (Kadner et al., 1980). The cloned Y. pestis fhuCDB genes were able to complement the defect in RK4375 and restored its ability to use ferrichrome as an iron source (data not shown). A fhuCD mutation was introduced into Y. pestis KIM6 by allelic exchange. The growth of KIM6-2114 (fhuCD2114) in liquid medium was similar to that of the parental strain under iron-deficient conditions in PMH2. However, growth of the mutant was significantly reduced relative to KIM6 when ferrichrome was present (Fig. 5). By plate assays, KIM6 used both ferrichrome and aerobactin as an iron source while KIM6-2113 (iutA2113) was able to use ferrichrome but not aerobactin, and KIM6-2114 (fhu) could use neither (Fig. 4a). When KIM6-2114 was complemented with pFhuYp1, the ability to use aerobactin as an iron source was restored (data not shown).
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fhuCD mutation abrogates the ability of Y. pestis to use either aerobactin or ferrichrome as iron sources, while a iutA mutant is defective in aerobactin but not ferrichrome utilization. Although Y. pestis has sequences homologous to iucABCD, these gene products do not appear to synthesize aerobactin and are unable to complement defects in the aerobactin biosynthetic pathway in E. coli. Although a frameshift mutation in iucA, which is conserved across different biotypes of Y. pestis, was thought to be the likely cause of this defect, repair of the frameshift failed to enable aerobactin biosynthesis. In addition, Y. pseudotuberculosis, which encodes an aerobactin locus with no obvious defects, is also unable to synthesize aerobactin (Stuart et al., 1986; this study). One likely cause of this defect may be amino acid variations in the four Yersinia aerobactin biosynthetic enzymes compared to known functional enzymes from E. coli, S. flexneri and pColV-K30 (Payne & Mey, 2004; Supplementary Figs S2–S5). This suggests that upon acquisition of the pathogenicity island encoding the yersiniabactin iron transport system, the enteric aerobactin system became unnecessary for growth in iron-deficient environments. The iucA frameshift in Y. pestis strains may save energy by eliminating translation of the downstream ORFs.
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ACKNOWLEDGEMENTS |
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Edited by: S. C. Andrews
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Received 13 November 2006;
revised 5 February 2007;
accepted 22 February 2007.
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