Mechanisms of iron and haem transport by Listeria monocytogenes

Listeria monocytogenes, the causative agent of listeriosis, is a virulent foodborne Gram-positive bacterial pathogen, with 20-30% mortality. It has a broad ability to transport iron, either in the form of ferric siderophores, or by extracting it from mammalian iron binding proteins. In this review we focus on the mechanisms of ferric siderophore and haem transport into the listerial cell. Despite the fact that it does not synthesize siderophores, L. monocytogenes transports ferric siderophores in the wild environment by the actions of cytoplasmic membrane ABC-transporter systems. The bacterium acquires haem, on the other hand, by two mechanisms. At low (nanomolar) concentrations, sortase B-dependent, peptidoglycan-anchored proteins scavenge the iron porphyrin in human or animal tissues, and transfer it to the underlying ABC-transporters in the cytoplasmic membrane for uptake. At concentrations at or above 50 nM, however, haem transport becomes sortase-independent, and occurs by direct interactions of the iron porphyrin with the same ABC-transporter complexes. The architecture of the Gram-positive cell envelope plays a fundamental role in these mechanisms, and the haem acquisition abilities of L. monocytogenes are an element of its ability to cause infectious disease.     

The Bradyrhizobium japonicum fsrB gene is essential for utilization of structurally diverse ferric siderophores to fulfill its nutritional iron requirement

In Bradyrhizobium japonicum, iron uptake from ferric siderophores involves selective outer membrane proteins and non-selective periplasmic and cytoplasmic membrane components that accommodate numerous structurally diverse siderophores. Free iron traverses the cytoplasmic membrane through the ferrous (Fe²⁺) transporter system FeoAB, but the other non-selective components have not been described. Here, we identify fsrB as an iron-regulated gene required for growth on iron chelates of catecholate- and hydroxymate-type siderophores, but not on inorganic iron. Utilization of the non-physiological iron chelator EDDHA as an iron source was also dependent on fsrB. Uptake activities of ⁵⁵Fe³⁺ bound to ferrioxamine B, ferrichrome or enterobactin were severely diminished in the fsrB mutant compared with the wild type. Growth of the fsrB or feoB strains on ferrichrome were rescued with plasmid-borne E. coli fhuCDB ferrichrome transport genes, suggesting that FsrB activity occurs in the periplasm rather than the cytoplasm. Whole cells of an fsrB mutant are defective in ferric reductase activity. Both whole cells and spheroplasts catalyzed the demetallation of ferric siderophores that were defective in an fsrB mutant. Collectively, the data support a model whereby FsrB is required for reduction of iron and its dissociation from the siderophore in the periplasm, followed by transport of the ferrous ion into the cytoplasm by FeoAB.     

Multiple outer membrane receptors for uptake of ferric pseudobactins inPseudomonas putida WCS358

Under iron limitationPseudomonas putida WCS358 produces a fluorescent siderophore, pseudobactin 358, which, after complexing iron, is transported back into the cell via the specific outer membrane receptor PupA. In addition, this strain has the capacity to take up iron via a large variety of siderophores produced by other fluorescent pseudomonads. Putative receptor genes for such siderophores were identified in the chromosome of strain WCS358 by PCR using primers matching two domains conserved in four ferric pseudobactin receptors, including PupA. Eleven amplification products within the expected size range were obtained. Sequence analysis confirmed that the products were derived from genes encoding outer membrane receptors. Two complete receptor genes were isolated from a genomic library ofP. putida WCS358. Both protein products are involved in the transport of a limited number of specific ferric pseudobactins. These results indicate that the ability ofP. putida WCS358 to exploit many different heterologous pseudobactins is related to the presence of multiple outer membrane receptor proteins.     

Ferric hydroxamate binding protein FhuD from Escherichia coli: mutants in conserved and non-conserved regions

Uptake of iron complexes into the Gram-negative bacterial cell requires highly specific outer membrane receptors and specific ATP-dependent (ATP-Binding-Cassette (ABC)) transport systems located in the inner membrane. The latter type of import system is characterized by a periplasmic binding protein (BP), integral membrane proteins, and membrane-associated ATP-hydrolyzing proteins. In Gram-positive bacteria lacking the periplasmic space, the binding proteins are lipoproteins tethered to the cytoplasmic membrane. To date, there is little structural information about the components of ABC transport systems involved in iron complex transport. The recently determined structure of the Escherichia coli periplasmic ferric siderophore binding protein FhuD is unique for an ABC transport system (Clarke et al. 2000). Unlike other BP's, FhuD has two domains connected by a long -helix. The ligand binds in a shallow pocket between the two domains. In vivo and in vitro analysis of single amino acid mutants of FhuD identified several residues that are important for proper functioning of the protein. In this study, the mutated residues were mapped to the protein structure to define special areas and specific amino acid residues in E. coli FhuD that are vital for correct protein function. A number of these important residues were localized in conserved regions according to a multiple sequence alignment of E. coli FhuD with other BP's that transport siderophores, heme, and vitamin B₁₂. The alignment and structure prediction of these polypeptides indicate that they form a distinct family of periplasmic binding proteins.     

Rapid evolution of a bacterial iron acquisition system

Under iron limitation, bacteria scavenge ferric (Fe³⁺) iron bound to siderophores or other chelates from the environment to fulfill their nutritional requirement. In gram‐negative bacteria, the siderophore uptake system prototype consists of an outer membrane transporter, a periplasmic binding protein and a cytoplasmic membrane transporter, each specific for a single ferric siderophore or siderophore family. Here, we show that spontaneous single gain‐of‐function missense mutations in outer membrane transporter genes of Bradyrhizobium japonicum were sufficient to confer on cells the ability to use synthetic or natural iron siderophores, suggesting that selectivity is limited primarily to the outer membrane and can be readily modified. Moreover, growth on natural or synthetic chelators required the cytoplasmic membrane ferrous (Fe²⁺) iron transporter FeoB, suggesting that iron is both dissociated from the chelate and reduced to the ferrous form within the periplasm prior to cytoplasmic entry. The data suggest rapid adaptation to environmental iron by facile mutation of selective outer membrane transporter genes and by non‐selective uptake components that do not require mutation to accommodate new iron sources.     

Iron-hydroxamate uptake systems in Bacillus subtilis: identification of a lipoprotein as part of a binding protein-dependent transport system

Bacillus subtilis was shown to utilize three types of hydroxamate siderophores, ferrichromes, ferrioxamines and shizokinen, each of which is taken up by different transport systems. Mutants deficient in the uptake of ferrichrome and/or ferrioxamine B were isolated. The gene fhuD, which was able to complement a mutant defective in ferrichrome uptake, was cloned. The deduced sequence of FhuD showed low but significant homology to the binding proteins FepB, FecB and FhuD of Escherichia coli, which are all components of binding protein-dependent, ferric siderophore transport systems. The first 23 amino acids of FhuD of B. subtilis possessed all characteristics of a lipoprotein signal sequence. The processing of FhuD in E. coli was inhibited by globomycin. Inhibition by globomycin indicated a lipid modification at the N-terminal cysteine in E. coli. It is highly likely that this step may also take place in B. subtilis. As in other binding protein-dependent transport systems of Gram-positive organisms it is proposed that the lack of a periplasm is compensated for by the lipid through which the binding protein is anchored to the cytoplasmic membrane.     

Ferric iron transport system of Pseudomonas aeruginosa PAO1 that functions as the uptake pathway of a novel catechol-substituted cephalosporin,S-9096

A novel catechol-substituted cephalosporin, S-9096, showed potent antibacterial activity against Pseudomonas aeruginosa under both iron-deficient and aerobic conditions. S-9096 and ferric iron formed a chelate complex at the molar ratio of 3 to 1, which could be incorporated into P. aeruginosa cells grown under such conditions. Incorporation decreased when the cells were grown under either iron-sufficient or anaerobic conditions, with a concomitant disappearance of iron-regulated outer membrane proteins that were considered to function as receptors for ferric siderophores. These results indicated that the ferric chelate of S-9096 was incorporated into P. aeruginosa cells via a ferric iron transport pathway, which caused the high antibacterial potency of S-9096. All of the S-9096-resistant mutants that were able to grow even under iron-deficient conditions lacked an iron-regulated outer membrane protein having an apparent molecular mass of 66 kDa, suggesting the role of this protein as a receptor for the ferric chelate of S-9096. Correspondence to: Y. Yamano     

Recent insights into iron import by bacteria

Bacteria are confronted with a low availability of iron owing to its insolubility in the Fe3+ form or its being bound to host proteins. The bacteria cope with the iron deficiency by using host heme or siderophores synthesized by themselves or other microbes. In contrast to most other nutrients, iron compounds are tightly bound to proteins at the cell surfaces, from which they are further translocated by highly specific proteins across the cell wall of gram-positive bacteria and the outer membrane of gram-negative bacteria. Once heme and iron siderophores arrive at the cytoplasmic membrane, they are taken up across the cytoplasmic membrane by ABC transporters. Here we present an outline of bacterial heme and iron siderophore transport exemplified by a few selected cases in which recent progress in the understanding of the transport mechanisms has been achieved.     

Iron acquisition by Streptococcus species: An updated review

Streptococcus is a genus of spherical Gram-positive bacteria responsible for many cases of meningitis, bacterial pneumonia, endocarditis, erysipelas, and necrotizing fasciitis. To survive in the host environment with limited free iron available, Streptococcus species have developed various mechanisms to uptake iron as an essential nutrient. They can directly extract the metal ions from host iron-containing proteins such as ferritin, transferrin, lactoferrin, and hemoproteins. Other iron-uptake strategies, which are broadly distributed in the strains, include the employment of specialized secreted hemophores to acquire heme and the usage of small molecules called siderophores as high-affinity ferric chelators. This review intends to discuss the most recent discoveries of these iron acquisition systems and their relevant regulators in Streptococcus species.     

Haemophore functions revisited

Haem is the major iron source for bacteria that develop in higher organisms. In these hosts, bacteria have to cope with nutritional immunity imposed by the host, since haem and iron are tightly bound to carrier and storage proteins. Siderophores were the first recognized fighters in the battle for iron between bacteria and host. They are non-proteinaceus organic molecules having an extremely high affinity for Fe(3+) and able to extract it from host proteins. Haemophores, that display functional analogy with siderophores, were more recently discovered. They are a class of secreted proteins with a high affinity for haem; they are able to extract haem from host haemoproteins and deliver it to specific receptors that internalize haem. In the past few years, a wealth of data has accumulated on haem acquisition systems that are dependent on surface exposed/secreted bacterial proteins. They promote haem transfer from its initial source (in most cases, a eukaryotic haem binding protein) to the transporter that carries out the membrane crossing step. Here we review recent discoveries in this field, with particular emphasis on similar and dissimilar mechanisms in haemophores and siderophores, from the initial host source to the binding protein/receptor at the cell surface.     

Sortase independent and dependent systems for acquisition of haem and haemoglobin in Listeria monocytogenes

We studied three Fur-regulated systems of Listeria monocytogenes: the srtB region, that encodes sortase-anchored proteins and a putative ABC transporter, and the fhu and hup operons, that produce putative ABC transporters for ferric hydroxamates and haemin (Hn)/haemoglobin (Hb) respectively. Deletion of lmo2185 in the srtB region reduced listerial [(59) Fe]-Hn transport, and purified Lmo2185 bound [(59) Fe]-Hn (K(D) = 12 nM), leading to its designation as a Hn/Hb binding protein (hbp2). Purified Hbp2 also acted as a haemophore, capturing and supplying Hn from the environment. Nevertheless, Hbp2 only functioned in [(59) Fe]-Hn transport at external concentrations less than 50 nM: at higher Hn levels its uptake occurred with equivalent affinity and rate without Hbp2. Similarly, deletion of sortase A had no effect on ferric siderophore or Hn/Hb transport at any concentration, and the srtA-independence of listerial Hn/Hb uptake distinguished it from comparable systems of Staphylococcus aureus. In the cytoplasmic membrane, the Hup transporter was specific for Hn: its lipoprotein (HupD) only showed high affinity for the iron porphyrin (K(D) = 26 nM). Conversely, the FhuD lipoprotein encoded by the fhu operon had broad specificity: it bound both ferric siderophores and Hn, with the highest affinity for ferrioxamine B (K(D) = 123 nM). Deletions of Hup permease components hupD, hupG or hupDGC reduced Hn/Hb uptake, and complementation of ΔhupC and ΔhupG by chromosomal integration of hupC(+) and hupG(+) alleles on pPL2 restored growth promotion by Hn/Hb. However, ΔhupDGC did not completely eliminate [(59) Fe]-Hn transport, implying the existence of another cytoplasmic membrane Hn transporter. The overall K(M) of Hn uptake by wild-type strain EGD-e was 1 nM, and it occurred at similar rates (V(max) = 23 pmol 10(9) cells(-1) min(-1)) to those of ferric siderophore transporters. In the ΔhupDGC strain uptake occurred at a threefold lower rate (V(max) = 7 pmol 10(9) cells(-1) min(-1)). The results show that at low (< 50 nM) levels of Hn, SrtB-dependent peptidoglycan-anchored proteins (e.g. Hbp2) bind the porphyrin, and HupDGC or another transporter completes its uptake into the cytoplasm. However, at higher concentrations Hn uptake is SrtB-independent: peptidoglycan-anchored binding proteins are dispensable because HupDGC directly absorbs and internalizes Hn. Finally, ΔhupDGC increased the LD(50) of L. monocytogenes 100-fold in the mouse infection model, reiterating the importance of this system in listerial virulence.     

Iron gathering by zoopathogenic fungi

Iron is a metal required by most microorganisms and is prominently used in the transfer of electrons during metabolism. The gathering of iron is, then, an essential process and its fulfillment becomes a crucial pathogenetic event for zoopathogenic fungi. Iron is rather unavailable because it occurs on the earth's surface in its insoluble ferric form in oxides and hydroxides. In the infected host iron is bound to proteins such as transferrin and ferritin. Solubilization of ferric iron is the major problem confronting microorganisms. This process is achieved by two major mechanisms: ferric reduction and siderophore utilization. Ferric reductase is frequently accompanied by a copper oxidase transport system. There is one example of direct ferric iron transport apparently without prior reduction. Ferric reduction may also be accomplished by low molecular mass compounds. Some fungi have evolved a process of iron acquisition involving the synthesis of iron-gathering compounds called siderophores. Even those fungi that do not synthesize siderophores have developed permeases for transport of such compounds formed by other organisms. Fungi can also reductively release iron from siderophores and transport the ferrous iron often by the copper oxidase transport system. There is a great diversity of iron-gathering mechanisms expressed by pathogenic fungi and such diversity may be found even in a single species.     

Identification of Iron Transporters Expressed in the Magnetotactic Bacterium <Emphasis Type="Italic">Magnetospirillum magnetotacticum</Emphasis>

The magnetotactic bacterium Magnetospirillum magnetotacticum MS-1 mineralizes the magnetite (Fe₃O₄) crystal and organizes a highly ordered intracellular structure, called the magnetosome. However, the iron transport system, which supports the biogenesis of magnetite, is not fully understood. In this study, we first identified the expressions of both the ferric and the ferrous iron transporter proteins in M. magnetotacticum. The cellular protein compositions of ferric and ferrous iron-rich cultures were examined using two-dimensional electrophoresis. According to the gel patterns, two outer-membrane ferric-siderophore receptor homologues were identified as proteins strongly induced in the ferrous iron-rich condition. Also, we identified for the first time that the ferrous iron transport protein, FeoB, is expressed in the M. magnetotacticum cytoplasmic membrane using immunoblotting.     

TonB or not TonB: is that the question?

Bacteria are able to survive in low-iron environments by sequestering this metal ion from iron-containing proteins and other biomolecules such as transferrin, lactoferrin, heme, hemoglobin, or other heme-containing proteins. In addition, many bacteria secrete specific low molecular weight iron chelators termed siderophores. These iron sources are transported into the Gram-negative bacterial cell through an outer membrane receptor, a periplasmic binding protein (PBP), and an inner membrane ATP-binding cassette (ABC) transporter. In different strains the outer membrane receptors can bind and transport ferric siderophores, heme, or Fe3+ as well as vitamin B12, nickel complexes, and carbohydrates. The energy that is required for the active transport of these substrates through the outer membrane receptor is provided by the TonB/ExbB/ExbD complex, which is located in the cytoplasmic membrane. In this minireview, we will briefly examine the three-dimensional structure of TonB and the current models for the mechanism of TonB-dependent energy transduction. Additionally, the role of TonB in colicin transport will be discussed.     

Iron limitation and its effect on membrane proteins and siderophore transport inNeurospora crassa

Summary Cells of the fungusNeurospora crassa were grown under iron-deficient and iron-sufficient conditions and their plasma membrane proteins were compared. Three strains were studied:N. crassa 74A (wild type), a siderophore-free mutantN. crassa (arg-5 ota aga) as well as a slime variant ofN. crassa which lacks a cell wall. Plasma membranes were purified, solubilized and analyzed by one-dimensional SDS/polyacrylamide gel electrophoresis yielding approximately 50 distinct protein bands with molecular masses in the range 14–160 kDa. Iron-sufficient and iron-deficient growth resulted in nearly identical plasma membrane protein profiles in all strains. Although minor alterations in the proportion of certain proteins could be detected, significant overproduction of certain membrane proteins during iron limitation could not be observed. Transport of⁵⁵ Fe-labeled siderophores seems to be correlated to the degree of iron limitation. For example, transport rates were enhanced five-fold after 16 h of growth in iron-deficient medium compared to growth in iron-sufficient medium. Extraction and HPLC measurement of siderophores from conidiospores yielded approximately 10^–15 mol/spore, indicating that germination tubes and young cells used for transport measurements are not iron-deficient. It is suggested that the putative transport systems for siderophores in fungal plasma membranes are constitutively expressed and enhanced uptake of siderophores during iron limitation is rather the result of cellular transport regulation mechanisms.