Involvement of outer membrane proteins in enterochelin-mediated iron uptake in Escherichia coli.

Escherichia coli K-12 grown in iron-deficient media contained a large amount of outer membrane proteins O-2a, O-2b, and O-3, while cells grown in iron-supplemented media contained far smaller amounts of these proteins. The iron uptake by the iron-deficient cells was significantly stimulated in the presence of enterochelin, while that by the iron-rich cells was not. The outer membrane isolated from cells grown in the iron-deficient media showed enterochelin-stimulated binding of iron, while the outer membrane from iron-rich cells and cytoplasmic membranes from both types of cells did not show such binding activity. The amount of iron bound by the outer membrane was almost equivalent to the amount of O-2a, O2b, or O-3, irrespective of the amount of these proteins in the outer membrane, which is controlled by the amount of iron in the medium. Small particles rich in these proteins were prepared from cells by EDTA extraction. The particles were active in enterochelin-mediated iron binding and the amount of iron bound was equivalent to the amount of each of these proteins in the particles. Although the outer membrane of E. coli B was as active in iron binding as that of E. coli K-12, it did not possess an appreciable amount of O-2a. Gel electrophoretic analysis revealed that 9-2b and 9-3 were identical with the proteins missing mutants feuB and feuA, respectively.     

Fuctional organization of the outer membrane of escherichia coli: Phage and colicin receptors as components of iron uptake systems

The functional interaction of outer memberane proteins of E. coli can be studied using phage and colicin receptors which are essential components of penetration systems. The uptake of ferric iron in the form of the ferrichrome complex requires the ton A and ton B functions in the outer membrane of E. coli. The ton A gene product is the receptor protein for phage T5 and is required together with the ton B function by the phages T1 anf ?80 to infect cells and by colicin M and the antibiotic albomycin, a structural analogue of ferrichrome, to kill cells. The ton B function is necessary for the uptake of ferric iron complexed by citrate. Iron complexed by enterochelin is only transported in the presence of the ton B and feu functions. Cells which have lost the feu function are resistant to the colicins B, I or V while ton B mutants are resistant to all colicins. The interaction of the ton A, Ton B, and feu functions apparently permits quite different “substrates” to overcome the permeablility barrier of the outer membrane. It was shown for ferrichrome dependent iron uptake that the complexing agent was not altered and could be used repeatedly. Only very low amounts of ³H-labeled ferrichrome were found in the cell. It is possible that the iron is mobilized in the membrane and that desferriferrichrome is released into the medium without having entered the cytoplasm. Growth on ferrichrome as the sole iron source waw used to select revertants of T5 resistant ton A mutants. All revertants exhibited wild-type properties with the exception of partial revertants. In these 4 strains, as in the ton A mutants, the ton A protein was not detectable by SDS polyacrylamide gel electrophoreses of outer membranes. Albomycin resistant mutants were selected and shown to fall into 5 categories: (1) ton A; (2) ton B mutants; (3) mutants with no iron transport defects and normal ton A/ton B functions, which might be target site mutants; (4) mutants which were deficient in ferrichrome-mediated iron uptake but had normal ton A/ton B functions. We tentatively consider that the defect might be located in the active transport system of the cytoplasmic membrane; (5) a variety of mutants with the following general properties: most of them were resistant to colicin M, transported iron poorly, and, like ton B mutants, contained additional proteins in the outer membrane. The outer membrane protein patterns of wild-type and ton B mutant strains were compared by slab gel electrophoresis in an attempt to identify a ton B protein. It was observed that under most growth conditions, ton B mutants overproduced 3 proteins of molecular weights 74,000–83,000. In extracted, iron-deficient medium, both the wild-type and ton B mutant strains had similar large amounts of these proteins in their outer membranes. The appearance of these proteins was suppressed by excess iron in both wild-type and mutant. From this evidence it is apparent that the proteins appear as a response to low intracellular iron rather than being controlled by the ton B gene. The nature of these proteins and their possible role in iron transport is disussed.     

Influence of growth rate and iron limitation on the expression of outer membrane proteins and enterobactin by Klebsiella pneumoniae grown in continuous culture.

The influence of the growth rate on outer membrane protein composition and enterobactin production was studied with Klebsiella pneumoniae grown under conditions of iron limitation in chemostats. More enterobactin was produced at fast (D = 0.4 h-1) and slow (D = 0.1 h-1) growth rates in continuous cultures than in either logarithmic- or stationary-phase batch cultures. When the growth rate was controlled under conditions of carbon limitation and the iron level was reduced to 0.5 microM, the iron-regulated outer membrane proteins and enterobactin were induced at the fast growth rate. At the slow growth rate, although the iron-regulated outer membrane proteins were barely visible, a significant level of enterobactin was still produced. These results suggest that under conditions of either carbon or iron limitation, the growth rate can influence the induction of the high-affinity iron uptake system of K. pneumoniae. Other outer membrane proteins, including a 39-kilodalton peptidoglycan-associated protein, were found to vary with the growth rate and nutrient limitation.     

Role of siderophore in iron uptake in cowpea Rhizobium GN1 (peanut isolate): Possible involvement of iron repressible outer membrane proteins

Abstract Siderophore produced by cowpea Rhizobium GN1 (Peanut isolate) was shown to be involved in iron uptake by this organism. Siderophore enhanced iron uptake in iron-starved cells. SDS-PAGE analysis of the outer membrane proteins showed two iron repressible outer membrane proteins with approximate molecular mass of 80 kDa and 76 kDa. A siderophore non-producing mutant, which was unable to grow on a medium containing synthetic iron chelators unless and until iron was added exogenously in the medium, could use siderophore of the wild-type for iron uptake indicating that the receptor for Fe-siderophore complex was intact in the mutant.     

Identification of iron-regulated outer membrane proteins in uropathogenic Proteus mirabilis and its relationship with heme uptake

The effect of iron deprivation on the expression of outer membrane proteins and the ability to use heme as an iron source by uropathogenic Proteus mirabilis , Pr 6515, was studied. Examination of iron-restricted bacteria showed three outer membrane proteins ranging from 66 to 75 kDa to be affected by iron restriction, as well as a newly expressed 64-kDa protein. These proteins were induced within 15 minutes of iron-deprivation. The strain grew in the presence of ferric citrate, hemin and hemoglobin as iron sources, but could not use transferrin, lactoferrin or siderophores from exogenous sources. The 64- and 66-kDa proteins showed hemin-binding activity by affinity chromatography, and both reacted in Western blots with sera from mice transurethrally infected with the same strain. We suggest that P. mirabilis expresses iron-regulated outer membrane proteins that could be involved in heme uptake and may have a role in pathogenesis.     

Iron transport systems of Serratia marcescens.

Serratia marcescens W225 expresses an unconventional iron(III) transport system. Uptake of Fe3+ occurs in the absence of an iron(III)-solubilizing siderophore, of an outer membrane receptor protein, and of the TonB and ExbBD proteins involved in outer membrane transport. The three SfuABC proteins found to catalyze iron(III) transport exhibit the typical features of periplasmic binding-protein-dependent systems for transport across the cytoplasmic membrane. In support of these conclusions, the periplasmic SfuA protein bound iron chloride and iron citrate but not ferrichrome, as shown by protection experiments against degradation by added V8 protease. The cloned sfuABC genes conferred upon an Escherichia coli aroB mutant unable to synthesize its own enterochelin siderophore the ability to grow under iron-limiting conditions (in the presence of 0.2 mM 2.2'-dipyridyl). Under extreme iron deficiency (0.4 mM 2.2'-dipyridyl), however, the entry rate of iron across the outer membrane was no longer sufficient for growth. Citrate had to be added in order for iron(III) to be translocated as an iron citrate complex in a FecA- and TonB-dependent manner through the outer membrane and via SfuABC across the cytoplasmic membrane. FecA- and TonB-dependent iron transport across the outer membrane could be clearly correlated with a very low concentration of iron in the medium. Expression of the sfuABC genes in E. coli was controlled by the Fur iron repressor gene. S. marcescens W225 was able to synthesize enterochelin and take up iron(III) enterochelin. It contained an iron(III) aerobactin transport system but lacked aerobactin synthesis. This strain was able to utilize the hydroxamate siderophores ferrichrome, coprogen, ferrioxamine B, rhodotorulic acid, and schizokinen as sole iron sources and grew on iron citrate as well. In contrast to E. coli K-12, S. marcescens could utilize heme. DNA fragments of the E. coli fhuA, iut, exbB, and fur genes hybridized with chromosomal S. marcescens DNA fragments, whereas no hybridization was obtained between S. marcescens chromosomal DNA and E. coli fecA, fhuE, and tonB gene fragments. The presence of multiple iron transport systems was also indicated by the increased synthesis of at least five outer membrane proteins (in the molecular weight range of 72,000 to 87,000) after growth in low-iron media. Serratia liquefaciens and Serratia ficaria produced aerobactin, showing that this siderophore also occurs in the genus Serratia.     

Localization of cytochromes in the outer membrane of Desulfovibrio vulgaris (Hildenborough) and their role in anaerobic biocorrosion

A protocol was developed whereby the outer and cytoplasmic membranes of the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) were isolated and partially characterized. The isolated outer membrane fractions from cultures grown under high (100 ppm) and low (5 ppm) Fe2+ conditions were compared by SDS-PAGE electrophoresis, and showed that several protein bands were derepressed under the low iron conditions, most notably at 50 kDa, and 77.5 kDa. Outer membrane isolated from low iron cultured cells was found to contain two proteins, 77.5 kDa and 62.5 kDa in size, that reacted with a heme-specific stain and were referred to as high molecular weight cytochromes. Studies conducted on the low iron isolated outer membrane by a phosphate/mild steel hydrogen evolution system showed that addition of the membrane fraction caused an immediate acceleration in H2 production. A new model for the anaerobic biocorrosion of mild steel is proposed.     

Hydroxamate production by Aquaspirillum magnetotacticum.

Spent culture fluids from Aquaspirillum magnetotacticum MS-1 grown at high (20 microM) but not low (5 microM) iron concentration contained material yielding a positive hydroxamate test. Cells possessed six major outer membrane proteins. Three outer membrane proteins ranging from 72,000 to 85,000 daltons were coordinately produced at iron concentrations conducive to hydroxamate production. A 55,000-dalton iron-repressible outer membrane protein was also present in strain MS-1 cultured at low but not high ferric quinate concentration. Culture fluids from strain MS-1 which were hydroxamate positive augmented growth of a Salmonella typhimurium siderophore-deficient (enb-7) mutant in low-iron medium, suggesting a role of hydroxamate in uptake of iron by the cell.     

Iron-regulated outer membrane proteins and non-siderophore-mediated iron acquisition by Paracoccus denitrificans

Abstract Deprivation of Paracoccus denitrificans of iron in sodium molybdate-containing medium caused a slower rate of growth and lower final cell yield, in contrast to our previous studies in non-sodium molybdate-containing medium, where iron deprivation had little effect on growth rate. Five high M _r outer membrane proteins and catechol production were induced in iron-deprived cultures. The fifth protein, M _r 72 000, was produced later than the others. Growth of iron-deprived cells in medium containing 20 μM ferric citrate repressed siderophore and iron deprivation-induced protein production, and led to production of an M _r 23 000 outer membrane protein (half maximum production after 5 h). Synthesis of the M _r 23 000 and high M _r proteins appeared to be mutally exclusive, and to be regulated by the cell's iron status. Cells inoculated into medium containing 20 μM ferric citrate took up 92% of the iron within 1 h, suggesting the occurrence of a nonsiderophore mediated, 'low affinity' iron uptake pathway.     

Influence of iron on growth and extracellular products of Acinetobacter baumannii

Iron is an important nutrient required by bacteria for optimal growth. Acquisition of iron from the host where iron is restricted is an important mediator of bacterial pathogenesis. In iron deplete chemically defined medium (CDM-Fe) growth of Acinetobacter baumannii was restricted as compared to iron replete medium (CDM + Fe). Bacteria developed four high molecular weight outer membrane proteins (OMPs) of 88, 84, 80 and 77 kDa in CDM-Fe medium which were absent in CDM + Fe medium, and are known iron regulated outer membrane proteins (IROMPs). A. baumannii secreted siderophores extracellularly into the medium which act as iron chelators which had been demonstrated in the supernatants of CDM-Fe media. The siderophore was of catechol type. This shows that A. baumannii under iron restricted conditions express IROMPs along with production of catechol type siderophore in order to acquire iron from the external milieu.     

Localization and Solubilization of the Iron(III) Reductase of Geobacter sulfurreducens

The iron(III) reductase activity of Geobacter sulfurreducens was determined with the electron donor NADH and the artificial electron donor horse heart cytochrome c. The highest reduction rates were obtained with Fe(III) complexed by nitrilotriacetic acid as an electron acceptor. Fractionation experiments indicated that no iron(III) reductase activity was present in the cytoplasm, that approximately one-third was found in the periplasmic fraction, and that two-thirds were associated with the membrane fraction. Sucrose gradient separation of the outer and cytoplasmic membranes showed that about 80% of the iron(III) reductase was present in the outer membrane. The iron(III) reductase could be solubilized from the membrane fraction with 0.5 M KCl showing that the iron(III) reductase was weakly bound to the membranes. In addition, solubilization of the iron(III) reductase from whole cells with 0.5 M KCl, without disruption of cells, indicated that the iron(III) reductase is a peripheral protein on the outside of the outer membrane. Redox difference spectra of KCl extracts showed the presence of c-type cytochromes which could be oxidized by ferrihydrite. Only one activity band was observed in native polyacrylamide gels stained for the iron(III) reductase activity. Excision of the active band from a preparative gel followed by extraction of the proteins and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of high-molecular-mass, cytochrome-containing proteins in this iron(III) reductase activity band. From these experimental data it can be hypothesized that the iron(III) reductase of G. sulfurreducens is a peripheral outer membrane protein that might contain a c-type cytochrome.     

Mechanisms and regulation of iron and heme utilization in Gram-negative bacteria

Iron and heme are essential nutrients for most pathogenic microorganisms and play a pivotal role in microbial pathogenesis. To survive within the iron-limited environment of the host, bacteria utilize iron-siderophore complexes, iron-binding proteins (transferrin, lactoferrin), free heme and heme bound to hemoproteins (hemoglobin, haptoglobin, hemopexin). A mechanism of iron and heme transport depends on the structures of Gram-negative bacterial membranes. Siderophores, hemophores and outer membrane receptors take part in iron or heme binding. The transport of these ligands across the outer membrane involves outer membrane receptors. The energy for this transport is delivered from the inner membrane by a TonB-ExbB-ExbD complex. The transport across the cytoplasmic membrane involves periplasmic and inner membrane proteins comprising the ABC systems, which utilize the energy derived from ATP hydrolysis. The major regulatory role in iron homeostasis plays a Fur-Fe2+ repressor.     

Outer membrane proteome of Prevotella intermedia 17: identification of thioredoxin and iron-repressible hemin uptake loci

Although hemin is an indispensable nutrient for the oral pathogen Prevotella intermedia, not much is known regarding the molecular mechanisms of hemin acquisition. The availability of the genomic sequence of the bacterium allowed us to apply proteomic approaches to identify proteins that may be mediating the hemin acquisition process. As hemin acquisition mechanisms have been shown to be induced in iron-depleted conditions, we applied proteomic approaches to detect those proteins whose expressions were affected by iron. We analyzed 40 protein spots and identified 19 such proteins. Interestingly, two proteins drastically upregulated in iron-depleted conditions, PIN0009 and PINA0611, are homologs of hemin uptake receptors in other bacteria. PIN0009 is predicted to be an outer membrane lipoprotein. It is encoded by a gene that is the first of a seven-gene genomic locus encoding proteins of a novel hemin acquisition system. The second protein, PINA0611, is a homolog of numerous TonB-dependent outer membrane receptors including outer membrane iron uptake receptors of various Gram-negative bacteria. There was also another protein, regulated by iron, that was previously demonstrated to bind hemoglobin in P. intermedia. Finally, we identified a thioredoxin-like protein that has a novel outer membrane location.     

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.     

Structural biology of bacterial iron uptake

To fulfill their nutritional requirement for iron, bacteria utilize various iron sources which include the host proteins transferrin and lactoferrin, heme, and low molecular weight iron chelators termed siderophores. The iron sources are transported into the Gram-negative bacterial cell via specific uptake pathways which include an outer membrane receptor, a periplasmic binding protein (PBP), and an inner membrane ATP-binding cassette (ABC) transporter. Over the past two decades, structures for the proteins involved in bacterial iron uptake have not only been solved, but their functions have begun to be understood at the molecular level. However, the elucidation of the three dimensional structures of all components of the iron uptake pathways is currently limited. Despite the low sequence homology between different bacterial species, the available three-dimensional structures of homologous proteins are strikingly similar. Examination of the current three-dimensional structures of the outer membrane receptors, PBPs, and ABC transporters provides an overview of the structural biology of iron uptake in bacteria.     

Studies on siderophore production and effect of iron deprivation on the outer membrane proteins of Madurella mycetomatis

The purpose of this investigation was to determine whether Madurella mycetomatis, the most frequent agent of eumycotic mycetomas, produces siderophores and synthesizes new outer membrane proteins under iron-starvation conditions. Siderophore production, only of the hydroxamate type, was demonstrated in all nine strains tested. It was regulated by extracellular iron concentrations. Under iron-restricted conditions, M. mycetomatis expressed various outer membrane iron-regulated proteins, particularly of 24-kilodalton, that may participate in iron metabolism.     

Increased production of the outer membrane receptors for colicins B, D and M by Escherichia coli under iron starvation.

Growth of $\text{E. coli}$ K-12 under severe iron stress results in increased production of the outer membrane receptors for colicins B, D, Ib and M. The increase in colicin receptor activity coincides with the appearance of large amounts of two high molecular weight proteins in the outer membrane of the cells. These proteins are identified as the outer membrane receptors for colicins B and D and for colicin M. Mutants lacking a functional outer membrane receptor for colicins B and D are defective in the uptake of iron complexed with the siderochrome enterochelin, and are thus comparable with $\text{tonA}$ mutants which lack a functional receptor for colicin M and are defective in the uptake of iron complexed with ferrichrome (6). The colicin B and D receptor may therefore function in the uptake of ferri-enterochelin.     

Structural biology of bacterial iron uptake

To fulfill their nutritional requirement for iron, bacteria utilize various iron sources which include the host proteins transferrin and lactoferrin, heme, and low molecular weight iron chelators termed siderophores. The iron sources are transported into the Gram-negative bacterial cell via specific uptake pathways which include an outer membrane receptor, a periplasmic binding protein (PBP), and an inner membrane ATP-binding cassette (ABC) transporter. Over the past two decades, structures for the proteins involved in bacterial iron uptake have not only been solved, but their functions have begun to be understood at the molecular level. However, the elucidation of the three dimensional structures of all components of the iron uptake pathways is currently limited. Despite the low sequence homology between different bacterial species, the available three-dimensional structures of homologous proteins are strikingly similar. Examination of the current three-dimensional structures of the outer membrane receptors, PBPs, and ABC transporters provides an overview of the structural biology of iron uptake in bacteria.