Ferrous Iron Transport Research Articles

Experimental determination of stability constant of ferrous iron borate complex [FeB(OH)4+] at 25 °C from solubility measurements: Implications for transport of iron in reducing environments

In this study, the formation constant (log10β10) for FeB(OH)4+ as expressed in the following reaction,Fe2++BOH4−⇌FeBOH4+is determined as 3.70 ± 0.10 (2σ) at 25 °C based on our long-term solubility experiments up to 2121 days on Fe(OH)2(cr) in the presence of borate ranging from 0.01 to 0.03 mol·kg−1. In our solubility measurements, the equilibrium was attained from the direction of supersaturation. In the experimental design, we used Fe2(OH)3Cl (pure iron end member of hibbingite) as the starting material. When Fe2(OH)3Cl was in contact with solutions without background concentrations of chloride, it was completely converted to Fe(OH)2(cr).The stability constant of FeB(OH)4+ is expected to find applications in many areas of study. For instance, FeB(OH)4+ may have played an important role in transport of ferrous iron in primitive oceans at the surface of the Earth. In the near-field of geological repositories, the formation of FeB(OH)4+ can sequestrate soluble borate, lowering borate concentrations available to the formation of the Am(III)-borate aqueous complex.

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.

Toward a mechanistic understanding of Feo-mediated ferrous iron uptake.

Virtually all organisms require iron and have evolved to obtain this element in free or chelated forms. Under anaerobic or low pH conditions commonly encountered by numerous pathogens, iron predominantly exists in the ferrous (Fe2+) form. The ferrous iron transport (Feo) system is the only widespread mechanism dedicated solely to bacterial ferrous iron import, and this system has been linked to pathogenic virulence, bacterial colonization, and microbial survival. The canonical feo operon encodes for three proteins that comprise the Feo system: FeoA, a small cytoplasmic β-barrel protein; FeoB, a large, polytopic membrane protein with a soluble G-protein domain capable of hydrolyzing GTP; and FeoC, a small, cytoplasmic protein containing a winged-helix motif. While previous studies have revealed insight into soluble and fragmentary domains of the Feo system, the chief membrane-bound component FeoB remains poorly studied. However, recent advances have demonstrated that large quantities of intact FeoB can be overexpressed, purified, and biophysically characterized, revealing glimpses into FeoB function. Two models of full-length FeoB have been published, providing starting points for hypothesis-driven investigations into the mechanism of FeoB-mediated ferrous iron transport. Finally, in vivo studies have begun to shed light on how this system functions as a unique multicomponent complex. In light of these new data, this review will summarize what is known about the Feo system, including recent advancements in FeoB structure and function.

Bacterial cyclic β-(1,2)-glucans sequester iron to protect against iron-induced toxicity.

Cellular iron homeostasis is critical for survival and growth. Bacteria employ a variety of strategies to sequester iron from the environment and to store intracellular iron surplus that can be utilized in iron-restricted conditions while also limiting the potential for the production of iron-induced reactive oxygen species (ROS). Here, we report that membrane-derived oligosaccharide (mdo) glucan, an intrinsic component of Gram-negative bacteria, sequesters the ferrous form of iron. Iron-binding, uptake, and localization experiments indicated that both secreted and periplasmic β-(1,2)-glucans bind iron specifically and promote growth under iron-restricted conditions. Xanthomonas campestris and Escherichia coli mutants blocked in the production of β-(1,2)-glucan accumulate low amounts of intracellular iron under iron-restricted conditions, whereas they exhibit elevated ROS production and sensitivity under iron-replete conditions. Our results reveal a critical role of glucan in intracellular iron homeostasis conserved in Gram-negative bacteria.

Expression and purification of functionally active ferrous iron transporter FeoB from Klebsiella pneumoniae

The acquisition of ferrous iron (Fe2+) is an important virulence factor utilized by several hospital-acquired (nosocomial) pathogens such as Klebsiella pneumoniae to establish infection within human hosts. Virtually all bacteria use the ferrous iron transport system (Feo) to acquire ferrous iron from their environments, which are often biological niches that stabilize Fe2+ relative to Fe3+. However, the details of this process remain poorly understood, likely owing to the few expression and purification systems capable of supplying sufficient quantities of the chief component of the Feo system, the integral membrane GTPase FeoB. This bottleneck has undoubtedly hampered efforts to understand this system in order to target it for therapeutic intervention. In this study, we describe the expression, solubilization, and purification of the Fe2+ transporter from K. pneumoniae, KpFeoB. We show that this protein may be heterologously overexpressed in Escherichia coli as the host organism. After testing several different commercially-available detergents, we have developed a solubilization and purification protocol that produces milligram quantities of KpFeoB with sufficient purity for enzymatic and biophysical analyses. Importantly, we demonstrate that KpFeoB displays robust GTP hydrolysis activity (kcatGTP of ∼10−1 s−1) in the absence of any additional stimulatory factors. Our findings suggest that K. pneumoniae may be capable of using its Feo system to drive Fe2+ import in an active manner.

TonB-Dependent Utilization of Dihydroxamate Xenosiderophores in Synechocystis sp. PCC 6803.

In Gram-negative bacteria, transport of ferric siderophores through outer membrane is a complex process that requires specific outer membrane transporters and energy-transducing TonB-ExbB-ExbD system in the cytoplasmic membrane. The genome of the non-siderophore-producing cyanobacterium Synechocystis sp. PCC 6803 encodes all putative components of the siderophore-mediated iron uptake system. So far, there has been no experimental evidence for the existence of such a pathway in this organism. On the contrary, its reductive iron uptake pathway has been studied in detail. We demonstrate that Synechocystis sp. PCC 6803 is capable of using dihydroxamate xenosiderophores, either ferric schizokinen (FeSK) or a siderophore of the filamentous cyanobacterium Anabaena variabilis ATCC 29413 (SAV), as the sole source of iron. Inactivation of the tonB gene or the exbB1-exbD1 gene cluster resulted in an inability to utilize these siderophores. At the same time, the inactivation of the feoB gene encoding FeoB plasma membrane ferrous iron transporter, or one of the futB or futC genes encoding permease and ATPase subunit of FutABC ferric iron transporter, did not impair the ability of cells to utilize FeSK or SAV as the sole source of iron for growth. Our data suggest that cyanobacterium Synechocystis sp. PCC 6803 is capable of acquiring iron-siderophore complexes in a TonB-dependent manner without iron reduction in the periplasm.

Large scale expression and purification of secreted mouse hephaestin.

Hephaestin is a large membrane-anchored multicopper ferroxidase involved in mammalian iron metabolism. Newly absorbed dietary iron is exported across the enterocyte basolateral membrane by the ferrous iron transporter ferroportin, but hephaestin increases the efficiency of this process by oxidizing the transported iron to its ferric form and promoting its release from ferroportin. Deletion or mutation of the hephaestin gene leads to systemic anemia with iron accumulation in the intestinal epithelium. The crystal structure of human ceruloplasmin, another multicopper ferroxidase with 50% sequence identity to hephaestin, has provided a framework for comparative analysis and modelling. However, detailed structural information for hephaestin is still absent, leaving questions relating to metal coordination and binding sites unanswered. To obtain structural information for hephaestin, a reliable protocol for large-scale purification is required. Here, we present an expression and purification protocol of soluble mouse hephaestin, yielding milligram amounts of enzymatically active, purified protein using the baculovirus/insect cell system.

Genome-Wide Search for Genes Required for Bifidobacterial Growth under Iron-Limitation.

Bacteria evolved over millennia in the presence of the vital micronutrient iron. Iron is involved in numerous processes within the cell and is essential for nearly all living organisms. The importance of iron to the survival of bacteria is obvious from the large variety of mechanisms by which iron may be acquired from the environment. Random mutagenesis and global gene expression profiling led to the identification of a number of genes, which are essential for Bifidobacterium breve UCC2003 survival under iron-restrictive conditions. These genes encode, among others, Fe-S cluster-associated proteins, a possible ferric iron reductase, a number of cell wall-associated proteins, and various DNA replication and repair proteins. In addition, our study identified several presumed iron uptake systems which were shown to be essential for B. breve UCC2003 growth under conditions of either ferric and/or ferrous iron chelation. Of these, two gene clusters encoding putative iron-uptake systems, bfeUO and sifABCDE, were further characterised, indicating that sifABCDE is involved in ferrous iron transport, while the bfeUO-encoded transport system imports both ferrous and ferric iron. Transcription studies showed that bfeUO and sifABCDE constitute two separate transcriptional units that are induced upon dipyridyl-mediated iron limitation. In the anaerobic gastrointestinal environment ferrous iron is presumed to be of most relevance, though a mutation in the sifABCDE cluster does not affect B. breve UCC2003's ability to colonise the gut of a murine model.

Vibrio cholerae VciB Mediates Iron Reduction.

Vibrio cholerae is the causative agent of the severe diarrheal disease cholera. V. cholerae thrives within the human host, where it replicates to high numbers, but it also persists within the aquatic environments of ocean and brackish water. To survive within these nutritionally diverse environments, V. cholerae must encode the necessary tools to acquire the essential nutrient iron in all forms it may encounter. A prior study of systems involved in iron transport in V. cholerae revealed the existence of vciB, which, while unable to directly transport iron, stimulates the transport of iron through ferrous (Fe2+) iron transport systems. We demonstrate here a role for VciB in V. cholerae in which VciB stimulates the reduction of Fe3+ to Fe2+, which can be subsequently transported into the cell with the ferrous iron transporter Feo. Iron reduction is independent of functional iron transport but is associated with the electron transport chain. Comparative analysis of VciB orthologs suggests a similar role for other proteins in the VciB family. Our data indicate that VciB is a dimer located in the inner membrane with three transmembrane segments and a large periplasmic loop. Directed mutagenesis of the protein reveals two highly conserved histidine residues required for function. Taken together, our results support a model whereby VciB reduces ferric iron using energy from the electron transport chain.IMPORTANCEVibrio cholerae is a prolific human pathogen and environmental organism. The acquisition of essential nutrients such as iron is critical for replication, and V. cholerae encodes a number of mechanisms to use iron from diverse environments. Here, we describe the V. cholerae protein VciB that increases the reduction of oxidized ferric iron (Fe3+) to the ferrous form (Fe2+), thus promoting iron acquisition through ferrous iron transporters. Analysis of VciB orthologs in Burkholderia and Aeromonas spp. suggest that they have a similar activity, allowing a functional assignment for this previously uncharacterized protein family. This study builds upon our understanding of proteins known to mediate iron reduction in bacteria.

Iron and Virulence in Francisella tularensis.

Francisella tularensis, the causative agent of tularemia, is a Gram-negative bacterium that infects a variety of cell types including macrophages, and propagates with great efficiency in the cytoplasm. Iron, essential for key enzymatic and redox reactions, is among the nutrients required to support this pathogenic lifestyle and the bacterium relies on specialized mechanisms to acquire iron within the host environment. Two distinct pathways for iron acquisition are encoded by the F. tularensis genome- a siderophore-dependent ferric iron uptake system and a ferrous iron transport system. Genes of the Fur-regulated fslABCDEF operon direct the production and transport of the siderophore rhizoferrin. Siderophore biosynthesis involves enzymes FslA and FslC, while export across the inner membrane is mediated by FslB. Uptake of the rhizoferrin- ferric iron complex is effected by the siderophore receptor FslE in the outer membrane in a TonB-independent process, and FslD is responsible for uptake across the inner membrane. Ferrous iron uptake relies largely on high affinity transport by FupA in the outer membrane, while the Fur-regulated FeoB protein mediates transport across the inner membrane. FslE and FupA are paralogous proteins, sharing sequence similarity and possibly sharing structural features as well. This review summarizes current knowledge of iron acquisition in this organism and the critical role of these uptake systems in bacterial pathogenicity.

Ferrous Iron Up-regulation in Fibroblasts of Patients with Beta Propeller Protein-Associated Neurodegeneration (BPAN).

Mutations in WDR45 gene, coding for a beta-propeller protein, have been found in patients affected by Neurodegeneration with Brain Iron Accumulation, NBIA5 (also known as BPAN). BPAN is a movement disorder with Non Transferrin Bound Iron (NTBI) accumulation in the basal ganglia as common hallmark between NBIA classes (Hayflick et al., 2013). WDR45 has been predicted to have a role in autophagy, while the impairment of iron metabolism in the different NBIA subclasses has not currently been clarified. We found the up-regulation of the ferrous iron transporter (-)IRE/Divalent Metal Transporter1 and down-regulation of Transferrin receptor in the fibroblasts of two BPAN affected patients with splicing mutations 235+1G>A (BPAN1) and 517_519ΔVal 173 (BPAN2). The BPAN patients showed a concomitant increase of intracellular ferrous iron after starvation. An altered pattern of iron transporters with iron overload is highlighted in BPAN human fibroblasts, supporting for a role of DMT1 in NBIA. We here present a novel element, about iron accumulation, to the existing knowledge in field of NBIA. Attention is focused to a starvation-dependent iron overload, possibly accounting for iron accumulation in the basal ganglia. Further investigation could clarify iron regulation in BPAN.

Effects of geodynamic setting on the redox state of fluids released by subducted mantle lithosphere

Magnetite breakdown during subduction of serpentinised ultramafic rocks may produce oxidised fluids that oxidise the deep Earth and/or the sub-arc mantle, either via direct transport of ferric iron, or via redox reactions between ferric iron and other elements, such as sulfur. However, so far, there is no consensus on the oxidation state of fluids released during subduction of ultramafic rocks, or the factors that control this oxidation state.Subducted samples from a magma-poor rifted margin and a supra-subduction zone geodynamic setting were compared to examine evidence of changes in opaque phase assemblage and ferric iron content as a consequence of subduction, and as a function of geodynamic setting. Thermodynamic calculations in the system Fe–Ni–O–H–S and Fe–Ni–O–S at the pressures and temperatures of interest were used to constrain oxygen activities and fluid compositions.Samples from New Caledonia, which exemplify supra-subduction zone mantle, contain awaruite (FeNi3) and equilibrated with hydrogen-bearing fluids at oxygen activity less than the FMQ (fayalite–magnetite–quartz) buffer. In contrast, samples from the Zermatt Saas Zone ophiolite, Western Alps, which are thought to represent mantle from a subducted magma-poor rifted margin, contain magnetite plus sulfur-rich phases such as pyrite (FeS2), and are inferred to have equilibrated with hydrogen-poor fluids at oxygen activity greater than FMQ. This major difference is independent of differences in subduction pressure–temperature conditions, variation in peridotite protolith composition, or the nature of adjacent units. We propose that the Zermatt Saas Zone samples would have undergone more complete serpentinisation prior to subduction than the supra-subduction zone (SSZ) New Caledonian samples. This difference explains the different fluid compositions, because incompletely serpentinised rocks containing olivine and brucite retain or evolve awaruite-bearing assemblages that buffer fluid compositions to high hydrogen activity (aH2).Ultramafic rocks are associated with two distinctly different fluid compositions during pre-subduction and subduction serpentinisation. Initially, while olivine is in equilibrium with infiltrating fluid, mineral assemblages that include awaruite in the rocks buffer fluids to H2-bearing, low aO2 compositions. Deserpentinisation of incompletely serpentinised rocks in which awaruite is present also produces H2-bearing fluids. Once awaruite is exhausted, H2-poor, high aO2 fluids co-exist with awaruite-absent assemblages, and deserpentinisation of such rocks would produce H2O-rich fluids.Thus, deserpentinisation of ultramafic rocks could produce either hydrogen-bearing fluids that could infiltrate and reduce the sub-arc mantle, or more oxidised fluids, which could transfer redox budget to other geochemical reservoirs such as the sub-arc mantle. Therefore, the redox contribution of subducted ultramafic rocks to the deep Earth and sub-arc mantle depends on the extent of protolith serpentinisation. Pre-subduction settings that promote extensive serpentinisation by oxidised fluids at high fluid:rock ratios in open systems, such as slow and ultraslow spreading ridges, transform faults, oceanic core complexes, and exhumed mantle at rifted continental margins, may produce more oxidised fluids than those associated with less pervasive serpentinisation and fluids that may be rock-buffered to a reduced state.

The extracellular proteome of two Bifidobacterium species reveals different adaptation strategies to low iron conditions

BackgroundBifidobacteria are among the first anaerobic bacteria colonizing the gut. Bifidobacteria require iron for growth and their iron-sequestration mechanisms are important for their fitness and possibly inhibit enteropathogens. Here we used combined genomic and proteomic analyses to characterize adaptations to low iron conditions of B. kashiwanohense PV20-2 and B. pseudolongum PV8-2, 2 strains isolated from the feces of iron-deficient African infants and selected for their high iron-sequestering ability.ResultsAnalyses of the genome contents revealed evolutionary adaptation to low iron conditions. A ferric and a ferrous iron operon encoding binding proteins and transporters were found in both strains. Remarkably, the ferric iron operon of B. pseudolongum PV8-2 is not found in other B. pseudolongum strains and likely acquired via horizontal gene transfer. The genome B. kashiwanohense PV20-2 harbors a unique region encoding genes putatively involved in siderophore production.Additionally, the secretomes of the two strains grown under low-iron conditions were analyzed using a combined genomic-proteomic approach. A ferric iron transporter was found in the secretome of B. pseudolongum PV8-2, while ferrous binding proteins were detected in the secretome of B. kashiwanohense PV20-2, suggesting different strategies to take up iron in the strains. In addition, proteins such as elongation factors, a glyceraldehyde-3-phosphate dehydrogenase, and the stress proteins GroEL and DnaK were identified in both secretomes. These proteins have been previously associated with adhesion of lactobacilli to epithelial cells.ConclusionAnalyses of the genome and secretome of B. kashiwanohense PV20-2 and B. pseudolongum PV8-2 revealed different adaptations to low iron conditions and identified extracellular proteins for iron transport. The identified extracellular proteins might be involved in competition for iron in the gastrointestinal tract.

Concentration-dependent roles of DMT1 and ZIP14 in cadmium absorption in Caco-2 cells.

Intestinal absorption of cadmium (Cd) is considered to be mediated mainly by the ferrous iron transporter DMT1, or the calcium transporter CaT1. The roles of zinc transporters such as ZIP8 and ZIP14 remain unclear, and the roles of these four transporters in the intestinal uptake of Cd under physiological conditions have not been compared. Here, we used a trans-well cell culture system to investigate the effects of the down-regulation of these four transporters on the uptake of Cd from the apical side of enterocytes. We used a Caco-2-kh cell line that can form tight junctions within a few days. The transfection of DMT1 siRNA significantly decreased the Cd uptake from the apical side at 5 μM, but not at 0.1 or 1 μM. The transfection of ZIP14 siRNA markedly decreased the Cd uptake at 0.1 and 1 μM, but not at 5 μM. The transfection of siRNA of CaT1 or ZIP8 did not alter the Cd uptake at any concentrations of Cd examined. These results suggest that DMT1 and ZIP14 play different roles in intestinal Cd absorption depending on the concentration of Cd.

Siderophore Cephalosporin Cefiderocol Utilizes Ferric Iron Transporter Systems for Antibacterial Activity against Pseudomonas aeruginosa.

Cefiderocol (S-649266) is a novel parenteral siderophore cephalosporin conjugated with a catechol moiety at the third-position side chain. The in vitro activity of cefiderocol against Pseudomonas aeruginosa was enhanced under iron-depleted conditions, whereas that of ceftazidime was not affected. The monitoring of [thiazole-14C]cefiderocol revealed the increased intracellular accumulation of cefiderocol in P. aeruginosa cells incubated under iron-depleted conditions compared with those incubated under iron-sufficient conditions. Cefiderocol was shown to have potent chelating activity with ferric iron, and extracellular iron was efficiently transported into P. aeruginosa cells in the presence of cefiderocol as well as siderophores, while enhanced transport of extracellular ferric iron was not observed when one of the hydroxyl groups of the catechol moiety of cefiderocol was replaced with a methoxy group. We conclude that cefiderocol forms a chelating complex with iron, which is actively transported into P. aeruginosa cells via iron transporters, resulting in potent antibacterial activity of cefiderocol against P. aeruginosa.

Studies on the X-Ray and Solution Structure of FeoB from Escherichia coli BL21

The ferrous iron transporter FeoB is an important factor in the iron metabolism of many bacteria. Although several structural studies have been performed on its cytosolic GTPase domain (NFeoB), the full-length structure of FeoB remains elusive. Based on a crystal packing analysis that was performed on crystals of NFeoB, a trimeric structure of the FeoB channel was proposed, where the transport pore runs along the trimer axis. Because this trimer has not been observed in some subsequently solved structures of NFeoB homologs, it remains unclear whether or not the trimer is indeed functionally relevant. Here, pulsed electron-electron double resonance spectroscopy, negative stain electron microscopy, and native mass spectrometry are used to analyze the oligomeric state of different soluble and full-length FeoB constructs. The results show that the full-length protein is predominantly monomeric, whereas dimers and trimers are formed to a small percentage. Furthermore, the solution structure of the switch I region is analyzed by pulsed electron-electron double resonance spectroscopy and a new, to our knowledge, crystal structure of NFeoB from Escherichia coli BL21 is presented.

Transcriptome analysis reveals physiological characteristics required for magnetosome formation in Magnetospirillum gryphiswaldense MSR-1.

Magnetosome synthesis ability of Magnetospirillum gryphiswaldense MSR-1 in an autofermentor can be precisely controlled through strict control of dissolved oxygen concentration. In this study, using transcriptome data we discovered gene transcriptional differences and compared physiological characteristics of MSR-1 cells cultured under aerobic (high-oxygen) and micro-aerobic (low-oxygen) conditions. The results showed that 77 genes were up-regulated and 95 genes were down-regulated significantly under micro-aerobic situation. These genes were involved primarily in the categories of cell metabolism, transport, regulation and unknown-function proteins. The nutrient transport and physiological metabolism were slowed down under micro-aerobic condition, whereas dissimilatory denitrification pathways were activated and it may supplemental energy was made available for magnetosome synthesis. The result suggested that the genes of magnetosome membrane proteins (Mam and Mms) are not directly regulated by oxygen level, or are constitutively expressed. A proposed regulatory network of differentially expressed genes reflects the complexity of physiological metabolism in MSR-1, and suggests that some yet-unknown functional proteins play important roles such as ferric iron uptake and transport during magnetosome synthesis. The transcriptome data provides a holistic view of the responses of MSR-1 cells to differing oxygen levels. This approach will give new insights into general principles of magnetosome formation.

Two parallel pathways for ferric and ferrous iron acquisition support growth and virulence of the intracellular pathogen Francisella tularensis Schu S4.

Iron acquisition mechanisms in Francisella tularensis, the causative agent of tularemia, include the Francisella siderophore locus (fsl) siderophore operon and a ferrous iron–transport system comprising outer‐membrane protein FupA and inner‐membrane transporter FeoB. To characterize these mechanisms and to identify any additional iron uptake systems in the virulent subspecies tularensis, single and double deletions were generated in the fsl and feo iron acquisition systems of the strain Schu S4. Deletion of the entire fsl operon caused loss of siderophore production that could be restored by complementation with the biosynthetic genes fslA and fslC and Major Facilitator Superfamily (MFS) transporter gene fslB. 55Fe‐transport assays demonstrated that siderophore‐iron uptake required the receptor FslE and MFS transporter FslD. A ΔfeoB′ mutation resulted in loss of ability to transport ferrous iron (55Fe2+). A ΔfeoB′ ΔfslA mutant that required added exogenous siderophore for growth in vitro was unable to grow within tissue culture cells and was avirulent in mice, indicating that no compensatory cryptic iron uptake systems were induced in vivo. These studies demonstrate that the fsl and feo pathways function independently and operate in parallel to effectively support virulence of F. tularensis.

Highly expressed amino acid biosynthesis genes revealed by global gene expression analysis of Salmonella enterica serovar Enteritidis during growth in whole egg are not essential for this growth

Salmonella enterica serovar Enteritidis (S. Enteritidis) is the most common cause of egg borne salmonellosis in many parts of the world. This study analyzed gene expression of this bacterium during growth in whole egg, and whether highly expressed genes were essential for the growth. High quality RNA was extracted from S. Enteritidis using a modified RNA-extraction protocol. Global gene expression during growth in whole egg was compared to growth in LB-medium using DNA array method. Twenty-six genes were significantly upregulated during growth in egg; these belonged to amino acid biosynthesis, di/oligopeptide transport system, biotin synthesis, ferrous iron transport system, and type III secretion system. Significant downregulation of 15 genes related to formate hydrogenlyase (FHL) and trehalose metabolism was observed. The results suggested that S. Enteritidis is starved for amino-acids, biotin and iron when growing in egg. However, site specific mutation of amino acid biosynthesis genes asnA (17.3 fold upregulated), asnB (18.6 fold upregulated), asnA/asnB and, serA (12.0 fold upregulated) and gdhA (3.7 fold upregulated), did not result in growth attenuation, suggesting that biosynthesis using the enzymes encoded from these genes may represent the first choice for S. Enteritidis when growing in egg, but when absent, the bacterium could use alternative ways to obtain the amino acids.

Vibrio cholerae FeoA, FeoB, and FeoC Interact To Form a Complex.

Feo is the major ferrous iron transport system in prokaryotes. Despite having been discovered over 25 years ago and found to be widely distributed among bacteria, Feo is poorly understood, as its structure and mechanism of iron transport have not been determined. The feo operon in Vibrio cholerae is made up of three genes, encoding the FeoA, FeoB, and FeoC proteins, which are all required for Feo system function. FeoA and FeoC are both small cytoplasmic proteins, and their function remains unclear. FeoB, which is thought to function as a ferrous iron permease, is a large integral membrane protein made up of an N-terminal GTPase domain and a C-terminal membrane-spanning region. To date, structural studies of FeoB have been carried out using a truncated form of the protein encompassing only the N-terminal GTPase region. In this report, we show that full-length FeoB forms higher-order complexes when cross-linked in vivo in V. cholerae. Our analysis of these complexes revealed that FeoB can simultaneously associate with both FeoA and FeoC to form a large complex, an observation that has not been reported previously. We demonstrate that interactions between FeoB and FeoA, but not between FeoB and FeoC, are required for complex formation. Additionally, we identify amino acid residues in the GTPase region of FeoB that are required for function of the Feo system and for complex formation. These observations suggest that this large Feo complex may be the active form of Feo that is used for ferrous iron transport. The Feo system is the major route for ferrous iron transport in bacteria. In this work, the Vibrio cholerae Feo proteins, FeoA, FeoB, and FeoC, are shown to interact to form a large inner membrane complex in vivo. This is the first report showing an interaction among all three Feo proteins. It is also determined that FeoA, but not FeoC, is required for Feo complex assembly.