Genetics and environmental regulation of Shigella iron transport systems

Shigella spp. have transport systems for both ferric and ferrous iron. The iron can be taken up as free iron or complexed to a variety of carriers. All Shigella species have both the Feo and Sit systems for acquisition of ferrous iron, and all have at least one siderophore-mediated system for transport of ferric iron. Several of the transport systems, including Sit, Iuc/IutA (aerobactin synthesis and transport), Fec (ferric di-citrate uptake), and Shu (heme transport) are encoded within pathogenicity islands. The presence and the genomic locations of these islands vary considerably among the Shigella species, and even between isolates of the same species. The expression of the iron transport systems is influenced by the concentration of iron and by environmental conditions including the level of oxygen. ArcA and FNR regulate iron transport gene expression as a function of oxygen tension, with the sit and iuc promoters being highly expressed in aerobic conditions, while the feo ferrous iron transporter promoter is most active under anaerobic conditions. The effects of oxygen are also seen in infection of cultured cells by Shigella flexneri; the Sit and Iuc systems support plaque formation under aerobic conditions, whereas Feo allows plaque formation anaerobically.     

Iron transport in Escherichia coli K-12

The study of iron uptake promoted by 2,3-dihydroxybenzoate (DHB) into Escherichia coli K-12 aroB mutants allowed some dissection of outer and cytoplasmic membrane functions. These strains are unable to produce the iron-transporting chelate enterochelin, unless fed with a precursor such as DHB. When added to the medium, enterochelin and its natural breakdown products, the linear dimer and trimer of 2,3-dihydroxybenzoylserine (DBS), efficiently transported iron via the feuB, tonB and fep gene products. Thus mutants in these genes were defective in transport of the above chelates. However, feuB and tonB mutants were able to take up iron when DHB was added to the medium. Thus DHB-promoted iron uptake bypassed two functions required for the transport of ferric-enterochelin from the medium. One of these functions, feuB, has been shown to be an outer membrane protein. In contrast to three other iron transport systems including ferric-enterochelin uptake, DHB-promoted iron uptake was little affected by the uncoupler 2,4-dinitrophenol. Dissipation of the energized state of the cytoplasmic membrane apparently only affects those iron transport systems which require an outer membrane protein. Since DHB-promoted iron uptake bypasses the feuB outer membrane protein and the tonB function, it is concluded that, in ferricenterochelin transport, the tonB gene may function in coupling the energized state of the cytoplasmic membrane to the protein-dependent outer membrane permeability. DHB-promoted iron uptake required the synthesis and enzymatic breakdown of enterochelin as judged by the effects of the entF and fesB mutations. A fep mutant was not only deficient in the transport of the ferric chelates of enterochelin and its breakdown products, but was also deficient in DHB-promoted iron uptake. A scheme is presented in which iron diffuses as DHB-complex through the outer membrane, and is subsequently captured by enterochelin or DBS dimer or trimer and translocated across the cytoplasmic membrane.List of Abbreviations DHB 2,3-dihydroxybenzoate - DBS 2,3-dihydroxybenzoylserine - NTA nitrilotriacetate - DNP 2,4-dinitrophenol     

Response of the cyanobacterium,Oscillatoria tenuis,to low iron environments: the effect on growth rate and evidence for siderophore production

Cyanobacteria vary in their ability to grow in media contaning low amounts of biologically available iron. Some strains, such as Oscillatoria tenuis, are well adapted to thrive in low-iron environments. We investigated the mechanism of iron scavenging in O. tenuis and found that this cyanobacterium has a siderophore-mediated iron transport system that differs significantly from the traditional hydroxamate-siderophore transport system reported from other cyanobacteria. Unlike other cyanobacteria, this strain produces two types of siderophores, a hydroxamate-type siderophore and a catechol-type siderophore. Production of these two siderophores is expressed at two different iron levels in the medium, suggesting two different iron regulated uptake systems. We compared the production of each siderophore with the growth rate of the culture and found that the production of the catechol siderophore enhances the growth rate of the cyanobacterium, whereas the cells maintain lower than maximal growth rates when only the hydroxamate-type siderophore is being produced.Abbreviation EDDA ethylene diamine di-(o-hydroxyphenylacetic acid)     

Iron acquisition in Vibrio cholerae

Vibrio cholerae, the causative agent of cholera, has an absolute requirement for iron and must obtain this element in the human host as well as in its varied environmental niches. It has multiple systems for iron acquisition, including the TonB-dependent transport of heme, the endogenous siderophore vibriobactin and several siderophores that are produced by other microorganisms. There is also a Feo system for the transport of ferrous iron and an ABC transporter, Fbp, which transports ferric iron. There appears to be at least one additional high affinity iron transport system that has not yet been identified. In iron replete conditions, iron acquisition genes are repressed by Fur. Fur also represses the synthesis of a small, regulatory RNA, RyhB, which negatively regulates genes for iron-containing proteins involved in the tricarboxylic acid cycle and respiration as well as genes for motility and chemotaxis. The redundancy in iron transport systems has made it more difficult to determine the role of individual systems in vivo and in vitro, but it may reflect the overall importance of iron in the growth and survival of V. cholerae.     

Iron Transport Systems in Neisseria meningitidis

Acquisition of iron and iron complexes has long been recognized as a major determinant in the pathogenesis of Neisseria meningitidis. In this review, high-affinity iron uptake systems, which allow meningococci to utilize the human host proteins transferrin, lactoferrin, hemoglobin, and haptoglobin-hemoglobin as sources of essential iron, are described. Classic features of bacterial iron transport systems, such as regulation by the iron-responsive repressor Fur and TonB-dependent transport activity, are discussed, as well as more specific features of meningococcal iron transport. Our current understanding of how N. meningitidis acquires iron from the human host and the vaccine potentials of various components of these iron transport systems are also reviewed.     

Power plays: iron transport and energy transduction in pathogenic vibrios

The Vibrios are a unique group of bacteria inhabiting a vast array of aquatic environments. Many Vibrio species are capable of infecting a wide assortment of hosts. Some of these species include V. parahaemolyticus, V. alginolyticus, V. vulnificus, V. anguillarum, and V. cholerae. The ability of these organisms to utilize iron is essential in establishing both an infection in their hosts as well as surviving in the environment. Bacteria are able to sequester iron through the secretion of low molecular weight iron chelators termed siderophores. The iron-siderophore complexes are bound by specific outer membrane receptors and are brought through both the outer and inner membranes of the cell. The energy needed to drive this active transport is achieved through the TonB energy transduction system. When first elucidated in E. coli, the TonB system was shown to be a three protein complex consisting of TonB, ExbB and ExbD. Most Vibrio species carry two TonB systems. The second TonB system includes a fourth protein; TtpC, which is essential for TonB2 mediated iron transport. Some Vibrio species have been shown to carry a third TonB system that also includes a TtpC protein.     

Iron Acquisition and Transport in Staphylococcus aureus

Pathogenic Gram-positive bacteria encounter many obstacles in route to successful invasion and subversion of a mammalian host. As such, bacterial species have evolved clever ways to prevent the host from clearing an infection, including the production of specialized virulence systems aimed at counteracting host defenses or providing protection from host immune mechanisms. Positioned at the interface of bacteria/host interactions is the bacterial cell wall, a dynamic surface organelle that serves a multitude of functions, ranging from physiologic processes such as structural scaffold and barrier to osmotic lysis to pathogenic properties, for example the deposition of surface molecules and the secretion of cytotoxins. In order to succeed in a battle with host defenses, invading bacteria need to acquire the nutrient iron, which is sequestered within host tissues. A cell-wall based iron acquisition and import pathway was uncovered in Staphylococcus aureus. This pathway, termed the isd or iron-responsive surface determinant locus, consists of a membrane transporter, cell wall anchored heme-binding proteins, heme/haptoglobin receptors, two heme oxygenases, and sortase B, a transpeptidase that anchors substrate proteins to the cell wall. Identification of the isd pathway provides an additional function to the already bountiful roles the cell wall plays in bacterial pathogenesis and provides new avenues for therapeutics to combat the rise of antimicrobial resistance in S. aureus. This review focuses on the molecular attributes of this locus, with emphasis placed on the mechanism of iron transport and the role of such a system during infection.     

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.     

Iron transport in the genus Marinobacter

Marinobacter belong to the class of Gammaproteobacteria and these motile, halophilic or halotolerent bacteria are widely distributed throughout the world’s oceans having been isolated from a wide variety of marine environments. They have also been identified as members of the bacterial flora associated with other marine organisms. Here, using a combination of natural products chemistry and genomic analysis, we assess the nature of the siderophores produced by this genus and their potential relationship to phylogeny and lifestyle/ecological niche of this diverse group of organisms. Our analysis shows a wide level of diversity in siderophore based iron uptake systems among this genus with three general strategies: (1) production and utilization of native siderophores in addition to utilization of a variety of exogenous ones, (2) production and utilization of native siderophores only, (3) lack of siderophore production but utilization of exogenous ones. They all share the presence of at least one siderophore-independent iron uptake ABC transport systems of the FbpABC iron metal type and lack the ability for direct transport of ferrous iron. Siderophore production and utilization can be correlated with phylogeny and thus it forms a type of chemotaxonomic marker for this genus.     

Epidemiology and molecular mechanism of integron-mediated antibiotic resistance in <Emphasis Type="Italic">Shigella</Emphasis>

Integrons are gene capture and expression systems that are characterized by the presence of an integrase gene. This encodes an integrase, a recombined site, and a promoter. They are able to capture gene cassettes from the environment and incorporate them using site-specific recombination. The role of integrons and gene cassettes in the dissemination of multidrug resistance in Gram-negative bacteria is significant. In Shigella species, antimicrobial resistance is often associated with the presence of class 1 and class 2 integrons that contain resistance gene cassettes. Multiple and complex expression regulation mechanisms involving mobile genetic elements in integrons have been developed in the evolution of Shigella strains. Knowledge of the epidemiology and molecular mechanisms of antimicrobial resistance in this important pathogen is essential for the implementation of intervention strategies. This review was conducted to introduce the structures and functions of integrons in Shigella species and mechanisms that control integron-mediated events linked to antibiotic resistance.     

Iron Acquisition by Legionella pneumophila

For nearly 20 years, it was believed that Legionella pneumophila does not produce siderophores. Yet, we have now determined that L. pneumophila secretes a siderophore (legiobactin) that is detectable by the CAS assay. We have optimized conditions for legiobactin expression, shown its biological activity, and found genes (lbtAB) involved in its production and secretion. LbtA is homologous with siderophore synthetases from E. coli (aerobactin), Sinorhizobium (rhizobactin), and Bordetella (alcaligin), while LbtB is a member of the major facilitator superfamily of multidrug efflux pumps. Mutants lacking lbtAB produce 40–70% less CAS reactivity. The lbtA mutant is also defective for growth in deferrated media containing citrate, indicating that legiobactin is required in conditions of severe iron limitation. lbtAB mutants grow normally in macrophages and amoebae host cells as well as within the lungs of mice. L. pneumophila does express lbtA in macrophages, suggesting that legiobactin has a dispensable role in infection. Legiobactin is iron repressed and does not react in the Csáky and Arnow assays. Anion-exchange HPLC has been used to purify legiobactin, and thus far, structural analysis suggests that the molecule is similar but not identical to rhizobactin, rhizoferrin, and alcaligin. The residual CAS reactivity present in supernatants of the lbtAB mutants suggests that L. pneumophila might produce a second siderophore. Besides siderophores, we have determined that ferrous iron transport, encoded by feoB, is critical for L. pneumophila growth in low-iron conditions, in host cells, and in the mammalian lung. Some of our other studies have discovered a critical, yet undefined, role for the L. pneumophila cytochrome c maturation locus in low-iron growth, intracellular infection, and virulence.     

Revisiting the Molecular Evolutionary History of <Emphasis Type="Italic">Shigella</Emphasis> spp.

The theory that Shigella is derived from multiple independent origins of Escherichia coli (Pupo et al. 2000) has been challenged by recent findings that the virulence plasmids (VPs) and the chromosomes share a similar evolutionary history (Escobar-Paramo et al. 2003), which suggests that an ancestral VP entered an E. coli strain only once, which gave rise to Shigella spp. In an attempt to resolve these conflicting theories, we constructed three phylogenetic trees in this study: a robust chromosomal tree using 23 housekeeping genes from 46 strains of Shigella and enteroinvasive E. coli (EIEC), a chromosomal tree using 4 housekeeping genes from 19 EcoR strains and 46 Shigella/EIEC strains, and a VP tree using 5 genes outside of the VP cell-entry region from 38 Shigella/EIEC strains. Both chromosomal trees group Shigella into three main clusters and five outliers, and strongly suggest that Shigella has multiple origins within E. coli. Most strikingly, the VP tree shows that the VPs from two main Shigella clusters, C1 and C2, are more closely related, which contradicts the chromosomal trees that place C2 and C3 next to each other but C1 at a distance. Additionally, we have identified a complete tra operon of the F-plasmid in the genome sequence of an EIEC strain and found that two other EIEC strains are also likely to possess a complete tra operon. All lines of evidence support an alternative multiorigin theory that transferable diverse ancestral VPs entered diverse origins of E. coli multiple times during a prolonged period of time, resulting in Shigella species with diverse genomes but similar pathogenic properties. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Martin Kreitman] Jian Yang and Huan Nie contributed equally to this work.     

Iron uptake regulation in Pseudomonas aeruginosa

The Pseudomonas genus belongs to the γ division of Proteobacteria and many species produce the characteristic yellow–green siderophore pyoverdine, and often a second siderophore, of lower affinity for iron. These bacteria are known for their ability to colonize different ecological niches and for their versatile metabolism. It is therefore not surprising that they are endowed with the capacity to take up exogenous xenosiderophores via different TonB-dependent receptors. Uptake of iron is controlled by the central regulator Fur, and via extracytoplasmic sigma factors or other types of regulators (two-component systems, AraC regulators). In this review the Fur regulon (experimentally proven and/or predicted) of P. aeruginosa will be presented. An interesting feature revealed by this analysis of Fur-regulated genes is the overlap between the iron and the sulfur regulons as well with the quorum sensing system.     

Hydroxamate Production as a High Affinity Iron Acquisition Mechanism in Paracoccidioides Spp

Iron is a micronutrient required by almost all living organisms, including fungi. Although this metal is abundant, its bioavailability is low either in aerobic environments or within mammalian hosts. As a consequence, pathogenic microorganisms evolved high affinity iron acquisition mechanisms which include the production and uptake of siderophores. Here we investigated the utilization of these molecules by species of the Paracoccidioides genus, the causative agents of a systemic mycosis. It was demonstrated that iron starvation induces the expression of Paracoccidioides ortholog genes for siderophore biosynthesis and transport. Reversed-phase HPLC analysis revealed that the fungus produces and secretes coprogen B, which generates dimerumic acid as a breakdown product. Ferricrocin and ferrichrome C were detected in Paracoccidioides as the intracellular produced siderophores. Moreover, the fungus is also able to grow in presence of siderophores as the only iron sources, demonstrating that beyond producing, Paracoccidioides is also able to utilize siderophores for growth, including the xenosiderophore ferrioxamine. Exposure to exogenous ferrioxamine and dimerumic acid increased fungus survival during co-cultivation with macrophages indicating that these molecules play a role during host-pathogen interaction. Furthermore, cross-feeding experiments revealed that Paracoccidioides siderophores promotes growth of Aspergillus nidulans strain unable to produce these iron chelators. Together, these data denote that synthesis and utilization of siderophores is a mechanism used by Paracoccidioides to surpass iron limitation. As iron paucity is found within the host, siderophore production may be related to fungus pathogenicity.     

Genomic insights into the iron uptake mechanisms of the biomining microorganism Acidithiobacillus ferrooxidans

Commercial bioleaching of copper and the biooxidation of gold is a cost-effective and environmentally friendly process for metal recovery. A partial genome sequence of the acidophilic, bioleaching bacterium Acidithiobacillus ferrooxidans is available from two public sources. This information has been used to build preliminary models that describe how this microorganism confronts unusually high iron loads in the extremely acidic conditions (pH 2) found in natural environments and in bioleaching operations. A. ferrooxidans contains candidate genes for iron uptake, sensing, storage, and regulation of iron homeostasis. Predicted proteins exhibit significant amino acid similarity with known proteins from neutrophilic organisms, including conservation of functional motifs, permitting their identification by bioinformatics tools and allowing the recognition of common themes in iron transport across distantly related species. However, significant differences in amino acid sequence were detected in pertinent domains that suggest ways in which the periplasmic and outer membrane proteins of A. ferrooxidans maintain structural integrity and relevant protein-protein contacts at low pH. Unexpectedly, the microorganism also contains candidate genes, organized in operon-like structures that potentially encode at least 11 siderophore systems for the uptake of Fe(III), although it does not exhibit genes that could encode the biosynthesis of the siderophores themselves. The presence of multiple Fe(III) uptake systems suggests that A. ferrooxidans can inhabit aerobic environments where iron is scarce and where siderophore producers are present. It may also help to explain why it cannot tolerate high Fe(III) concentrations in bioleaching operations where it is out-competed by Leptospirillum species.

     

Iron transport in Francisella in the absence of a recognizable TonB protein still requires energy generated by the proton motive force

The mechanism of iron transport in Francisella is still a puzzle since none of the sequenced Francisella strains appears to encode a TonB protein, the energy transducer of the proton motive force necessary to act on the bacterial outer membrane siderophore receptor to allow the internalization of iron. In this work we demonstrate using kinetic experiments of radioactive Fe³⁺ utilization, that iron uptake in Francisella novicida, although with no recognizable TonB protein, is indeed dependent on energy generated by the proton motive force. Moreover, mutants of a predicted outer membrane receptor still transport iron and are sensitive to the iron dependent antimicrobial compound streptonigrin. Our studies suggest that alternative pathways to internalize iron might exist in Francisella.     

ThenadB gene ofSalmonella typhimurium complements the nicotinic acid auxotrophy ofShigella flexneri

Shigella species are characteristically nicotinic acid (NA) auxotrophs. The invasiveS. flexneri strain M90T, transformed with the multicopy plasmid pZT349 encoding thenadB gene ofSalmonella typhimurium, can grow in minimal glucose medium without exogenous NA, whereas, M90T containing the control vector, pUC18 does not, suggesting that this species lacksl-aspartic acid oxidase, the first enzyme in the de novo NAD biosynthetic pathway. The estimated growth rate of strain M90T (pZT349) in HeLa cells was identical to that of M90T (pUC18), indicating the available intracellular concentration of NA is not limiting for bacterial growth.     

Iron and carbon metabolism by a mineral-oxidizing Alicyclobacillus-like bacterium

A novel iron-oxidizing, moderately thermophilic, acidophilic bacterium (strain “GSM”) was isolated from mineral spoil taken from a gold mine in Montana. Biomolecular analysis showed that it was most closely related to Alicyclobacillus tolerans, although the two bacteria differed in some key respects, including the absence (in strain GSM) of ϖ-alicyclic fatty acids and in their chromosomal base compositions. Isolate GSM was able to grow in oxygen-free media using ferric iron as terminal electron acceptor confirming that it was a facultative anaerobe, a trait not previously described in Alicyclobacillus spp.. The acidophile used both organic and inorganic sources of energy and carbon, although growth and iron oxidation by isolate GSM was uncoupled in media that contained both fructose and ferrous iron. Fructose utilization suppressed iron oxidation, and oxidation of ferrous iron occurred only when fructose was depleted. In contrast, fructose catabolism was suppressed when bacteria were harvested while actively oxidizing iron, suggesting that both ferrous iron- and fructose-oxidation are inducible in this acidophile. Isolate GSM accelerated the oxidative dissolution of pyrite in liquid media either free of, or amended with, organic carbon, although redox potentials were significantly different in these media. The potential of this isolate for commercial mineral processing is discussed.