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Pumping iron: mechanisms for iron uptake by Campylobacter

Department of Genetics, University of Leicester, Leicester LE1 7RH, UK

Correspondence
Claire E. Miller
cem32{at}leicester.ac.uk

	ABSTRACT

TOP ABSTRACT Introduction Genomics, transcriptomics and... Iron uptake by siderophores Iron uptake from host... Exbbd-Tonb energy transduction... Uptake of ferrous iron Concluding remarks REFERENCES

 
Campylobacter requires iron for successful colonization of the host. In the last 7 years, a wealth of data has been generated allowing detailed molecular characterization of Campylobacter iron-uptake systems. Several exogenous siderophores have been identified as sources of ferric iron for Campylobacter. Ferri-enterochelin uptake requires both the outer-membrane receptor protein CfrA and the inner-membrane ABC transporter system CeuBCDE. Ferrichrome has been shown to support growth of some Campylobacter jejuni strains and the presence of homologues of Escherichia coli fhuABD genes was proposed; the Cj1658–Cj1663 system appears to be involved in the uptake of ferri-rhodotorulic acid. In addition to siderophores, the importance of host iron sources was highlighted by recent studies demonstrating that C. jejuni can exploit haem compounds and the transferrins using ChuABCDZ and Cj0173c–Cj0178, respectively. An additional putative receptor, Cj0444, present in some, but not all, strains has not yet been characterized. Following diffusion through the outer membrane, inner-membrane transport of ferrous iron can occur via the FeoB protein. While it may be assumed that all systems are not essential, there is growing evidence supporting the need for multiple iron-uptake systems for successful host colonization by Campylobacter. In light of this, comparative molecular characterization of iron systems in all Campylobacter strains is necessary to gain further insight into the pathogenesis of members of this genus.

	Introduction

TOP ABSTRACT Introduction Genomics, transcriptomics and... Iron uptake by siderophores Iron uptake from host... Exbbd-Tonb energy transduction... Uptake of ferrous iron Concluding remarks REFERENCES

 
For many micro-organisms, iron acquisition is essential for colonization and infection of the host. Iron is a cofactor in many proteins involved in metabolism and basic cellular pathways in both pathogens and their hosts. Under aqueous aerobic conditions at physiological pH, iron is predominantly present as Fe³⁺, forming insoluble ferric oxide hydrates that are poorly available for use by cells (solubility 1.4x10^–9 M). Many host iron-binding and transport processes deliver Fe³⁺ to cells; in turn these processes can be hijacked by pathogens.

Unlike Fe²⁺, which can passively diffuse through the outer-membrane porins (limit 600 Da), Fe³⁺ uptake using siderophores (low-molecular-mass, high-affinity iron-binding compounds) by Gram-negative pathogens begins by binding of the iron source to a specific iron-regulated ligand-gated outer-membrane receptor protein (Miethke & Marahiel, 2007). Following binding, a conformational change facilitates transport through the receptor pore using energy transduced from the cytoplasmic membrane to the outer membrane by the ExbBD-TonB protein complex (Chakraborty et al., 2007). Once within the periplasm, ferri-siderophore or free iron is sequestered by a periplasmic binding protein and delivered to an inner-membrane ATP-binding cassette (ABC) transporter system consisting of permease protein subunits, which form an inner-membrane pore, coupled with an ATP-binding protein (Davidson & Maloney, 2007). ATP is hydrolysed to provide the energy to drive transport across the inner membrane into the cytoplasm, where iron bound to siderophores may be liberated by reduction or by siderophore degradation (Faraldo-Gómez & Sansom, 2003). Host iron-binding or transport proteins such as haem-containing proteins or ferri-transferrins can also be used as Fe³⁺ sources. Haem compounds are bound directly by specific outer-membrane receptors or delivered to the receptors by haemophores (Tong & Guo, 2009). Transferrin-bound iron may be acquired by receptor-mediated release, proteolytic cleavage or reduction to Fe²⁺, which dissociates from the protein (Cornelissen, 2003). Following transport across the outer membrane, uptake of haem or iron from the transferrins proceeds as described above.

Since the 1980s Campylobacter spp. have emerged as the most prevalent food-borne enteric pathogens in the Western world, constituting a major economic burden. Campylobacter is commensal in many animals, notably birds, but colonization of the human intestinal tract results in disease (Skirrow, 1994; Humphrey, 2006). Enteritis is the most frequent outcome of human infection. Campylobacter pathogenesis, recently reviewed by Poly & Guerry (2008), is not as well understood as the pathogenesis of other bacteria, but motility, chemotaxis, adhesion, invasion, toxin production and the acquisition of iron have all been demonstrated to be important. In addition, aspects of Campylobacter virulence, including growth, invasion of Caco-2 cells, disruption of cultured Caco-2 cell monolayers and cellular tight junction breakdown, are enhanced in the presence of the catecholamine host stress hormone noradrenaline (Cogan et al., 2006). The observed modulation of virulence in a stressed host would be expected to lead to increased colonization and disease (Cogan et al., 2006).

	Genomics, transcriptomics and the regulation of iron uptake by C. jejuni

TOP ABSTRACT Introduction Genomics, transcriptomics and... Iron uptake by siderophores Iron uptake from host... Exbbd-Tonb energy transduction... Uptake of ferrous iron Concluding remarks REFERENCES

 
Campylobacter jejuni uses highly adapted specific mechanisms for scavenging iron from a range of sources in the human host to enable its survival and proliferation (Palyada et al., 2004; Naikare et al., 2006). The importance of iron is suggested by the large number of genes dedicated to iron uptake, regulation and homeostasis in the relatively small genomes of C. jejuni strains NCTC 11168 (Parkhill et al., 2000), 81-176 (Hofreuter et al., 2006) and 81116 (Pearson et al., 2007). Moreover, comparative genomics indicates variation in iron-associated genes among the sequenced strains. A representation of the structure of all known Campylobacter iron-uptake systems, their components and sources is shown in Fig. 1; the chromosomal organization of iron-uptake genes is represented in Fig. 2, and Table 1 provides a directory of genes associated with iron uptake in C. jejuni.

View larger version (35K): [in this window] [in a new window]   Fig. 1. Iron-uptake systems of C. jejuni. Ferric iron sources are typically bound by a surface ligand-gated porin, which transports the iron source into the periplasm using energy derived from proton-motive force transferred from the cytoplasmic membrane (CM) to the outer membrane (OM) by the energy transduction system ExbBD-TonB. Once within the periplasm, iron sources are delivered to the CM ABC transporter systems by the periplasmic binding proteins. Ferrous iron diffuses through the OM, only requiring transport across the CM. See Fig. 2 for colour key.

View larger version (30K): [in this window] [in a new window]   Fig. 2. Genes involved in iron uptake in C. jejuni. The diagrams indicate the genomic organization and functions of the components of systems involved in the uptake of iron from (a) ferri-enterochelin, (b) ferrichrome (hypothetical), (c) ferri-rhodotorulic acid, (d) haem and (e) ferri-transferrins, and (f) in the uptake of ferrous iron; (g) represents the outer-membrane receptor gene cj0444. Gene names, sizes and context are based on NCTC 11168; when information from other strains is included, this is indicated in the figure. Diagrams are to scale and are based on the genomic structure taken from http://xbase.bham.ac.uk/campydb/. Dashed lines indicate a lack of close linkage; solid lines indicate the presence of an intergenic spacer region of greater than 10 bp. Genes with increased relative transcript levels under iron limitation are indicated by the fold change shown beneath the gene; none of the genes demonstrated decreased relative transcript levels when iron was limited (Holmes et al., 2005). Where the change is not known or has not been reported, the spaces beneath the genes are blank.

	Iron uptake by siderophores

TOP ABSTRACT Introduction Genomics, transcriptomics and... Iron uptake by siderophores Iron uptake from host... Exbbd-Tonb energy transduction... Uptake of ferrous iron Concluding remarks REFERENCES

 
C. jejuni was long thought to be incapable of synthesizing siderophores (Field et al., 1986; Pickett et al., 1992), and the failure to identify any siderophore biosynthesis genes within the genome sequences of C. jejuni NCTC 11168, RM1221 and 81-176 supports this view (Parkhill et al., 2000; Fouts et al., 2005; Hofreuter et al., 2006). The organism is, however, capable of utilizing exogenous siderophores as sources of iron; one early study indicated that the Escherichia coli siderophore enterochelin and the fungal siderophore ferrichrome were able to promote growth in iron-restricted conditions (Field et al., 1986).

Enterochelin
C. jejuni acquires iron from ferri-enterochelin using the receptor CfrA, which shows homology with other outer-membrane ferric siderophore receptor proteins (Palyada et al., 2004), and the binding-protein-dependent inner-membrane ABC transporter system encoded by the ceuBCDE genes (Parkhill et al., 2000). Genes encoding parts of the enterochelin transport system were first identified in Campylobacter coli (Richardson & Park, 1995). A cfrA mutant of C. jejuni NCTC 11168 was unable to grow when supplied with ferri-enterochelin as the only iron source (Palyada et al., 2004), although, inexplicably, a site-specific cfrA mutant of C. coli was apparently as capable of utilizing ferri-enterochelin as were wild-type cells (Guerry et al., 1997). The crystal structure of CfrA indicates that it is a classical β-barrel ligand-gated porin, but homology modelling indicates that some regions thought to be important for ligand binding differ significantly from the corresponding regions of the E. coli enterochelin receptor FepA (Carswell et al., 2008).

The ceuBCDE operon is very similar in C. jejuni and C. coli; ceuBC encode the inner-membrane permease proteins, ceuD an ATPase and ceuE the periplasmic binding lipoprotein (Parkhill et al., 2000; Palyada et al., 2004). A signature sequence that is conserved in siderophore-binding periplasmic proteins was identified at amino acids 137–152 of CeuE (Park & Richardson, 1995). A ceuE mutant of C. jejuni was slightly impaired in its ability to grow when provided with ferri-enterochelin, indicating that CeuE is involved in, but not essential for, utilizing iron from ferri-enterochelin; this suggests redundancy among inner-membrane transport systems, an observation supported by other studies described below. The potential significance of ferri-enterochelin uptake in vivo is highlighted by the fact that cfrA or ceuE mutants were markedly impaired in colonization of the avian gastrointestinal tract (Palyada et al., 2004). In addition, the growth increase seen when NCTC 11168 was incubated under iron-limited conditions in the presence of noradrenaline was lost in a cfrA mutant strain, suggesting that noradrenaline increases growth by enhancing CfrA-dependent iron uptake (Haigh et al., 2008).

Ferrichrome
C. jejuni strain M129 carries the cfhuABD operon encoding proteins homologous with the ferrichrome-uptake systems of E. coli and Pseudomonas aeruginosa (Galindo et al., 2001). This operon, which has not been found in any of the sequenced strains of C. jejuni (Parkhill et al., 2000; Fouts et al., 2005; Hofreuter et al., 2006; Pearson et al., 2007), is surprisingly GC rich – 65 mol%, compared with the average 30–35 % of the C. jejuni genome – but there was no suggestion that the region is a genomic island. CFhuA is an 80 kDa receptor, which is 33 % identical to the E. coli ferrichrome receptor FhuA and is expressed under low-iron conditions. Southern blot analysis demonstrated that only six of eleven C. jejuni isolates tested had a fhuA homologue (Galindo et al., 2001). CFhuB and CFhuD demonstrate homology with the permease FhuB (26 % identity) and the periplasmic binding protein FhuD (25 % identity), respectively, of the E. coli ferrichrome-uptake system. Of all iron sources tested, ferrichrome most effectively promoted growth of strain M129, but experimental confirmation of direct roles for CFhuA in the uptake of ferrichrome and for cfhuABD in C. jejuni pathogenesis are still required.

Rhodotorulic acid
Recent data indicate a role for the cj1658–cj1663 locus of the C. jejuni NCTC 11168 genome in the uptake of iron from the fungal hydroxamate siderophore ferri-rhodotorulic acid. A 19 kDa protein designated P19, first purified from C. jejuni 81-176 by anion-exchange chromatography (Janvier et al., 1998), is a periplasmic binding protein. Gene cj1658, which is located immediately upstream of the p19 gene, encodes a membrane-associated protein that does not resemble a classical TonB-dependent receptor and may be a permease; cj1661–cj1663 encode proteins that resemble an inner-membrane ABC transporter system (Parkhill et al., 2000). The potential involvement of this region in iron uptake was inferred from the observation that cj1658 and p19 homologues are found on a Yersinia pestis iron-uptake pathogenicity island (Carniel, 2001). C. jejuni NCTC 11168 cj1658 or p19 mutants were unable to utilize iron supplied as ferri-rhodotorulic acid for growth (Stintzi et al., 2008); the ability to acquire a siderophore that may not commonly be encountered during transmission either may not be physiologically relevant or may reflect an association with a relevant siderophore with a similar structure.

	Iron uptake from host sources

TOP ABSTRACT Introduction Genomics, transcriptomics and... Iron uptake by siderophores Iron uptake from host... Exbbd-Tonb energy transduction... Uptake of ferrous iron Concluding remarks REFERENCES

 
Haem
C. jejuni can use iron derived from haem-containing compounds for growth, including haemin, haemoglobin, haemin-haemopexin and haemoglobin-haptoglobin (Pickett et al., 1992). Mutations in an iron-regulated gene named chuA, which encodes a 70 kDa receptor with similarity to other iron-regulated bacterial receptors (van Vliet et al., 1998), resulted in failure of cells to grow when supplied with haemin or haem-containing compounds as sole iron sources (Pickett et al., 1992). In C. jejuni NCTC 11168, chuA is part of an operon, chuABCD. ChuB is predicted to be a permease, ChuC the ATPase and ChuD the periplasmic binding protein of the inner-membrane ABC transporter system (Parkhill et al., 2000; Ridley et al., 2006). A chuA mutant strain grew significantly less well than the wild-type with haemin or haemoglobin as iron sources, implying an important role for ChuA in haem uptake. A slight, but statistically significant, reduction in growth of chuB, chuC or chuD mutant strains was also observed, implying that the ABC transport system is involved in, but not essential for, haem uptake, suggesting redundancy among the inner-membrane transport systems. Upstream of chuA, and separated by a region containing divergent promoters, is chuZ, which encodes an iron-responsive cellular haem oxygenase (Ridley et al., 2006), a homologue of the haem-uptake protein HugZ in Plesiomonas shigelloides (Henderson et al., 2001). Expression of chu is not essential for colonization of the caecal lumen model, but the chu genes are upregulated in both the rabbit ileal loop model and the chick caecum (Palyada et al., 2004; Stintzi et al., 2005).

Transferrin proteins
C. jejuni NCTC 11168 cells can utilize iron bound to the iron-transport glycoproteins human ferri-transferrin (Tf), human ferri-lactoferrin (Lf), and ferri-ovotransferrin for growth (Miller et al., 2008). The association of human ferri-Lf with the C. jejuni cell surface appears to be iron-responsive, with the majority (>90 %) of released iron found in the cell cytoplasm and periplasm. Partitioning of C. jejuni from the iron source by dialysis membrane reduced levels of growth significantly, and also reduced the levels of internalized iron; internalization of iron was saturable. The uptake of iron from the transferrin family was recently attributed to genes in the cj0173c–cj0178 region of the C. jejuni NCTC 11168 genome (Miller et al., 2008). Organized as two operons separated by divergent promoters, cj0176c–cj0173c and cj0177–cj0178 demonstrate homology with known siderophore-dependent iron-uptake systems (Parkhill et al., 2000). Genes cj0175c–cj0173c were originally identified as a Fe³⁺-transporting inner-membrane system similar to one described for Serratia marcescens (Zimmermann et al., 1989). The operon, named cfbpABC, demonstrates homology with hitABC of Haemophilus influenzae and Y. pestis (van Vliet et al., 2002). CfbpC (Cj0173c) shows similarity to an ATPase, with CfbpB (Cj0174c) annotated as the permease; CfbpA (Cj0175c) is the periplasmic binding protein. The crystal structure of CfbpA has been solved and shown to preferentially bind free Fe²⁺ (which is then oxidized to Fe³⁺) or Fe³⁺ chelated to oxalate (Tom-Yew et al., 2005). Requirement for a synergistic anion, as seen with other ferric-transport proteins, was not observed, indicating that this is a novel type of binding protein. CfbpA was proposed to be similar to neisserial FbpA, a component of the FbpABC transport system thought to transport Tf-derived iron into the cytoplasm (Tom-Yew et al., 2005). Upstream of cj0175c and transcribed in the same orientation is gene cj0176c, which encodes an uncharacterized lipoprotein (Parkhill et al., 2000; van Vliet et al., 2002).

Cj0177 and Cj0178 have been suggested to function as a haem-uptake system in addition to ChuABCDZ (Chan et al., 2006); the authors propose that multiple haem-uptake systems may be required since available haem sources are diverse. The lipoprotein Cj0177 is homologous with the inner-membrane protein PhuW from P. aeruginosa, which is co-expressed with a haem-uptake ABC transporter system and is necessary, but not essential, for fully efficient haem uptake (Ochsner et al., 2000). The crystal structure of Cj0177 showed two bound cofacial haem groups in a pocket formed by the Cj0177 dimer (Chan et al., 2006). Cj0178 shows homology with the haemin receptor PhuR of P. aeruginosa (Ochsner et al., 2000), but experimental evidence for such a role in C. jejuni is lacking. Interestingly, Cj0178 is required for both chick and rabbit ileal loop colonization, suggesting the importance of this receptor in vivo (Palyada et al., 2004; Stintzi et al., 2005). More recently, mutation of cj0178 was shown to cause decreased binding and acquisition of iron from Tfs, and reduced growth in the presence of human ferri-Lf compared to wild-type (Miller et al., 2008). Moreover, a cj0178 mutant was not affected in either haem acquisition (Miller et al., 2008) or ferri-enterochelin utilization (Palyada et al., 2004). A decrease in growth was also seen when a cfrA mutant strain was incubated with ferri-Lf, but the difference was not as marked as that seen with the loss of cj0178; mutation of chuA had no effect on iron acquisition from Tfs (Miller et al., 2008). Deletion of the gene for the permease Cj0174c resulted in a small but significant decrease in growth with ferri-Lf (Miller et al., 2008).

In addition to these major iron receptors, the C. jejuni NCTC 11168 genome also contains the pseudogene cj0444. Interestingly, in strains where cj0444 is a pseudogene, genes cfrA and cj0178 are functional (NCTC 11168, RM1221), while in C. jejuni strains 81-176 and 81116, which contain a functional copy of cj0444, the receptors CfrA and Cj0178 are absent (Parkhill et al., 2000; Fouts et al., 2005; Hofreuter et al., 2006; Pearson et al., 2007). Cj0444, annotated as CJJ81176-0471 in strain 81-176, shares 34 %, 32 % and 24 % identity with CfrA, ChuA and Cj0178 from NCTC 11168, respectively (http://xbase.bham.ac.uk/campydb/blast.pl), which may suggest a role for Cj0444 in ferri-enterochelin uptake. As yet there are no experimental data to confirm that functional Cj0444 substitutes for CfrA or Cj0178, but immune cross-reactivity has been observed between Cj0444 and CfrA (Zeng & Lin, 2008).

	Exbbd-Tonb energy transduction systems

TOP ABSTRACT Introduction Genomics, transcriptomics and... Iron uptake by siderophores Iron uptake from host... Exbbd-Tonb energy transduction... Uptake of ferrous iron Concluding remarks REFERENCES

 
Located downstream of cj0178 are cj0179–cj0181, which encode ExbB, ExbD and TonB homologues (Parkhill et al., 2000). There are two additional ExbBD-TonB systems, encoded by cj1628–30 and by cj0109–10 and cj0753c in the C. jejuni NCTC 11168 genome (Parkhill et al., 2000). Note that in two of the ExbBD-TonB systems, the exbBD genes are transcriptionally coupled to a tonB gene, while cj0109–10 are coupled to the ATP synthase operon (Parkhill et al., 2000; van Vliet et al., 2002). The TonB proteins may function with specific uptake systems; for example cj0753c is found adjacent to (but divergently transcribed from) cfrA, and cj0181 is immediately downstream of cj0173c–cj0178. Alternatively, the different TonB proteins may function with any of the uptake systems (Parkhill et al., 2000; van Vliet et al., 2002).

	Uptake of ferrous iron

TOP ABSTRACT Introduction Genomics, transcriptomics and... Iron uptake by siderophores Iron uptake from host... Exbbd-Tonb energy transduction... Uptake of ferrous iron Concluding remarks REFERENCES

 
The C. jejuni NCTC 11168 genome contains a homologue of the E. coli feoB gene (cj1398, 29 % identity) adjacent to a smaller gene (cj1397) with limited identity (16 %) to the feoA gene of E. coli, but lacks a feoC homologue (Parkhill et al., 2000). The putative feoB and feoA orthologues form an operon (Naikare et al., 2006). Previous work with C. jejuni strains M129 and F38011 suggested that feoB mutants did not differ from the wild-type in the amount of Fe²⁺ taken up, and the authors therefore concluded that FeoB is not required for Fe²⁺ uptake in C. jejuni (Raphael & Joens, 2003). However, more recently it was shown that mutation of feoB in NCTC 11168 reduced Fe²⁺ transport by half, and the Fe²⁺ that was taken up remained in the periplasm (Naikare et al., 2006). Moreover, wild-type cells out-competed the feoB mutant in survival and colonization studies in the rabbit ileal loop model. Colonization of the chick caecum was also compromised in a feoB mutant, and wild-type cells out-competed the mutant in an intestinal infection model using colostrum-deprived piglets (Naikare et al., 2006). The feoB gene is functional in NCTC 11168 and potentially so in 81116 (Parkhill et al., 2000; Pearson et al., 2007), but non-functional in 81-176 and RM1221 due to frameshift mutations (Fouts et al., 2005; Hofreuter et al., 2006); identification of candidate genes for Fe²⁺ uptake in addition to feoB in the latter strains is therefore required along with experimental confirmation of their role, if any, in Fe²⁺ transport. The full genome sequences of strains M129 and F38011 are not yet known; they might contain additional feoB homologues or genes for alternative Fe²⁺ uptake systems.

	Concluding remarks

TOP ABSTRACT Introduction Genomics, transcriptomics and... Iron uptake by siderophores Iron uptake from host... Exbbd-Tonb energy transduction... Uptake of ferrous iron Concluding remarks REFERENCES

 
The acquisition of iron by Campylobacter spp. relies upon outer-membrane receptor proteins that appear almost exclusively specific for particular iron sources. The outer-membrane receptors, some of which are essential for colonization, appear to be non-redundant, and so it is interesting that only a subset of these receptors may be present or functional in some strains. The combinations of receptors in different strains presumably enable exploitation of iron sources characteristic of different host environments, but no epidemiological studies have yet been performed. The requirement for FeoB, CfrA and Cj0178 for successful host colonization demonstrated by multiple animal models supports the suggestion that several iron-uptake systems are essential for survival and colonization in the host by Campylobacter. The importance of these systems for host colonization highlights the need for the detailed molecular characterization of each, but the high level of strain conservation found among the iron-uptake proteins of the Chu (Ridley et al., 2006) and P19 systems, which currently do not appear to be essential for host colonization, seems counterintuitive and also requires clarification. The role of Cj0444 needs elucidating; identification of the iron source this receptor binds and transports will assist in this process. In contrast to the specificity of the outer-membrane receptors, the inner-membrane ABC transporter systems show redundancy, with a single system apparently capable of transporting iron derived from a number of sources.

Over recent years there have been major advances in our understanding of the methods by which C. jejuni acquires iron. The requirement for many sources, lack of redundancy in the outer-membrane proteins and requirement for components of the major iron-uptake systems for colonization indicate the importance of iron in the progression of disease in the human host. A link has been shown between the production of stress hormones, iron availability and an increase in pathogenicity of other organisms (Freestone et al., 2008). The stress-hormone-induced increases in C. jejuni pathogenesis (Cogan et al., 2006) impact not only on colonization and disease in humans, but also on colonization of chickens (Humphrey, 2006). A reduction in intensive poultry production coupled with an increase in awareness of poultry stress and welfare may reduce stress-hormone-enhanced Campylobacter colonization of chickens, and in turn reduce the risk of human infection (Humphrey, 2006).

Further characterization of a number of the iron-uptake systems, comparisons between strains with different system content and comparative analysis of regulation of the systems are all required for complete understanding of the role of iron during colonization and infection by campylobacters. Characterization of how Campylobacter improves its physique by pumping iron (including hormonal enhancement?) will build a powerful body of knowledge to aid our understanding of intestinal colonization and disease.

	ACKNOWLEDGEMENTS

 
Work in the authors' laboratory is funded by the BBSRC. The authors thank Richard Haigh for critically reading the manuscript and offering valuable input.

	REFERENCES

TOP ABSTRACT Introduction Genomics, transcriptomics and... Iron uptake by siderophores Iron uptake from host... Exbbd-Tonb energy transduction... Uptake of ferrous iron Concluding remarks REFERENCES

 
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