Low-iron Conditions Research Articles

Iron-dependent regulation of diphtheria toxin and siderophore expression by the cloned Corynebacterium diphtheriae repressor gene dtxR in C. diphtheriae C7 strains

A regulatory gene (dtxR) responsible for iron-dependent repression of the toxin (tox) and siderophore genes in Corynebacterium diphtheriae was cloned and characterized. A DNA fragment carrying dtxR repressed expression of a tox-lacZ gene fusion in Escherichia coli DH5 alpha in a high-iron environment but not under low-iron conditions. A protein with mobility corresponding to approximately 28 to 29 kDa was identified as the product of the dtxR gene by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A shuttle vector designated pCM2.6 was constructed which carries the origin of replication from C. diphtheriae plasmid pNG2 and confers resistance to chloramphenicol in E. coli and C. diphtheriae. DNA fragments carrying dtxR were cloned into pCM2.6, and the hybrid shuttle plasmids were transformed by electroporation into wild-type C. diphtheriae C7(beta) and the regulatory mutant C7(beta)hm723, which produces toxin and siderophore constitutively under high-iron conditions. Expression of the cloned dtxR determinant did not affect the phenotype of C. diphtheriae C7(beta). In C. diphtheriae C7(beta)hm723, expression of cloned dtxR restored full repression of siderophore production and partial repression of diphtheria toxin production during growth in a high-iron environment.

Positive transcriptional regulation of an iron-regulated virulence gene in Vibrio cholerae.

We have previously described a virulence gene in Vibrio cholerae (irgA) that is more than 850-fold regulated in response to iron. Negative regulation of irgA by iron occurred at the transcriptional level, and there was a dyad symmetric nucleotide sequence in the vicinity of the irgA promoter homologous to the Fur binding site in Escherichia coli. When irgA was cloned into E. coli, we showed that transcription of irgA required 900 base pairs of DNA upstream of the irgA promoter that contained an open reading frame in inverse orientation to irgA. In the present study, we show that this upstream region of DNA encodes a gene in inverse orientation to irgA (named irgB) that is also negatively regulated by iron. Insertional inactivation of irgB on the V. cholerae chromosome leads to loss of expression of a chromosomal irgA'-'phoA fusion (in which the primes indicate truncated genes), which is restored to normal by provision of irgB on a plasmid in trans. DNA sequencing of irgB shows that the protein product (IrgB) is homologous to the LysR family of positive transcriptional activators, and secondary structure analysis of IrgB predicts a helix-turn-helix DNA binding motif. The promoters of irgB and irgA are divergent but overlap each other and the previously defined Fur-binding site. We propose a model for iron regulation of irgA expression in V. cholerae. In the presence of sufficient iron, transcription of both irgA and irgB is negatively regulated by a Fur-like protein. In low iron conditions, negative regulation of transcription is removed, and production of IrgB leads to positive transcriptional activation of irgA. It seems likely that the high induction ratio of irgA expression under low- and high-iron conditions (850-fold) relates to the fact that its cognate positive transcriptional activator (irgB) is itself negatively regulated by iron.

Molecular analysis of iron transport in plant growth-promotingPseudomonas putida WCS358

Root-colonizingPseudomonas putida WCS358 enhances growth of potato in part by producing under iron-limiting conditions a yellow-green, fluorescent siderophore designated pseudobactin 358. This siderophore efficiently complexes iron(III) in the rhizosphere, making it less available to certain endemic microorganisms, including phytopathogens, thus inhibiting their growth. At least 15 genes distributed over five gene clusters are required for the biosynthesis of pseudobactin 358. High-affinity iron(III) transport in strain WCS358 is initiated by an 86-kDa outer membrane receptor protein (PupA) which appears to be specific for ferric pseudobactin 358. PupA shares strong similarity with TonB-dependent receptor proteins ofEscherichia coli, which suggests a TonB-like protein in strain WCS358 is required for iron(III) transport. Strain WCS358 possesses a second uptake system for ferric pseudobactin 358 and structurally diverse ferric siderophores produced by other microorganisms. A second receptor gene (pupB) responsible for iron transport from pseudobactin BN7 or pseudobactin BN8 has been identified. The production of this and certain other ferric siderophore receptor proteins requires that strain WCS358 be grown in the presence of these siderophores. An apparent regulatory gene required for the expression ofpupB is located adjacent topupB. Two positive regulatory genes have been identified which can independently activate, under low-iron(III) conditions, transcription of genes coding for the biosynthesis of pseudobactin 358.

Response of Haemophilus somnus to iron limitation: expression and identification of a bovine-specific transferrin receptor

Nine clinical isolates of Haemophilus somnus were screened for their ability to use different transferrins as a source of iron growth. All nine strains were capable of using bovine but not porcine, human or chicken transferrin. A screening assay for siderophore production did not show any evidence of siderophore production by these strains. When iron-deficient cells from these strains were screened for their ability to bind perixodase-conjugated transferrin, binding was detected with conjugated bovine, but not human or porcine transferrin. Competition binding studies demonstrated that the binding of peroxidase-conjugated bovine transferrin was competitively inhibited by unconjugated bovine transferrin but not transferrin from other species. The induction of receptor expression by low iron conditions was inhibited by chloramphenicol and rifampicin but not ampicillin indicating that new protein and mRNA synthesis was required for expression of receptor activity. Affinity isolation of receptor proteins with biotinylated bovine transferrin, but not human or porcine transferrin, yielded three proteins from H. somnus strain H74. Two of the proteins were identified as 105 kDa and 73 kDa iron-regulated outer membrane proteins. A third protein of 85 kDa that was isolated did not co-migrate with any iron-regulated outer membrane protein. Affinity isolation of receptor proteins from other strains of H. somnus yielded a 73 kDa protein from all strains and a 105 kDa and 85 kDa protein in four of the six strains analysed.

Consequences of the iron-dependent formation of ferredoxin and flavodoxin on photosynthesis and nitrogen fixation on Anabaena strains

Iron-dependent formation of ferredoxin and flavodoxin was determined in Anabaena ATCC 29413 and ATCC 29211 by a FPLC procedure. In the first species ferredoxin is replaced by flavodoxin at low iron levels in the vegetative cells only. In the heterocysts from Anabaena ATCC 29151, however, flavodoxin is constitutively formed regardless of the iron supply.Replacement of ferredoxin by flavodoxin had no effect on photosynthetic electron transport, whereas nitrogen fixation was decreased under low iron conditions. As ferredoxin and flavodoxin exhibited the same Km values as electron donors to nitrogenase, an iron-limited synthesis of active nitrogenase was assumed as the reason for inhibited nitrogen fixation. Anabaena ATCC 29211 generally lacks the potential to synthesize flavodoxin. Under iron-starvation conditions, ferredoxin synthesis is limited, with a negative effect on photosynthetic oxygen evolution.

Coordinate regulation of siderophore and diphtheria toxin production by iron in Corynebacterium diphtheriae

Iron is an environmental signal which regulates the coordinate expression of genes associated with virulence in many pathogenic bacteria. In response to iron-deprivation, lysogenic Corynebacterium diphtheriae C7(β) synthesizes and secretes diphtheria toxin and siderophore and induces a high-affinity iron uptake system. Diphtheria toxin is encoded by β phage, but genes for siderophore production are encoded on the bacterial chromosome. Diphtheria toxin and siderophore production were shown to be coordinately induced during late logarithmic phase growth of wild-type C7(β) in iron-limited medium. C. diphtheriae mutant C7hm723 produced siderophore and toxin constitutively under low-iron and high-iron conditions, but in mutants HC1, HC3, HC4, and HC5 their synthesis was partially repressed under high-iron conditions. The phenotypes of HC1, HC3, HC4, and HC5 are consistent with their severe defects in iron uptake, but the phenotype of C7hm723 is more likely to be explained by inactivation of the repressor for the iron regulon of C. diphtheriae.

Activation of expression of the Escherichia coli cir gene by an iron-independent regulatory mechanism involving cyclic AMP-cyclic AMP receptor protein complex

Synthesis of the colicin I receptor protein, encoded by the cir gene, was determined to be sensitive to control by the catabolite repression regulatory system. Under both high- and low-iron conditions for growth, mutants unable to produce cyclic AMP (cAMP) (cya) or functional cAMP receptor protein (crp) exhibited decreased membrane levels of the receptor relative to those of the wild-type strain. Exogenous addition of cAMP to the cya mutant restored maximal expression. cAMP-dependent changes in steady-state levels of cir mRNA suggested that the effect is mediated by control of transcript synthesis or stability. Potential mechanisms for regulation were examined by deletion and sequence analysis.

Identification of an iron-regulated virulence determinant in Vibrio cholerae, using TnphoA mutagenesis

Several virulence determinants of bacteria are regulated by the concentration of iron in the medium, with increased expression occurring under low-iron conditions. Iron-regulated virulence factors have not been previously described in Vibrio cholerae. We used the transposon vector Tn5 IS50L::phoA (TnphoA) to obtain insertion mutations in iron-regulated genes of V. cholerae 0395. One strain, carrying an insertion mutation in iron-regulated gene irgA, had reduced virulence in an animal model and had lost the 77-kilodalton major iron-regulated outer membrane protein. The 50% lethal dose of this mutant strain (MBG40) in suckling mice was 3 x 10(5) bacteria compared with 4 x 10(3) bacteria for 0395 wild type. In an in vivo competition assay, the wild-type strain out competed the mutant almost 10-fold, suggesting a possible colonization defect in MBG40. The in vitro competitive index and in vitro growth curves in low- and high-iron media showed that the mutant strain had no discernable growth defect. These data suggest that the 77-kilodalton iron-regulated outer membrane protein may play a role in the pathogenesis of V. cholerae infection. It is not yet clear whether the reduction of virulence in MBG40, as assessed by 50% lethal dose analysis, is explained by a colonization defect or whether it relates to another defect associated with loss of the IrgA protein.

Effects of iron binding agents on Saccharomyces cerevisiae growth and cytochrome P450 content

The yeast Saccharomyces cerevisiae Y222 was studied in the presence of the following iron-binding agents: Desferal, dipyridyl, and human and bovine transferrins. We report that cell growth and lanosterol 14 alpha-demethylase cytochrome P450 are not affected by Desferal but that dipyridyl and serum transferrins decrease the cytochrome P450 content of the yeast. Paradoxically, while both human and bovine transferrins reduce cytochrome P450 content, only bovine transferrin appears to affect cell growth in this strain. No evidence for siderophore production by this strain was found under low iron conditions.

Purification of a Specific Repressor of Ferritin mRNA Translation from Rabbit Liver

The synthesis of ferritin is regulated at the translation level in coordination with iron availability. Under conditions of low iron, translation of ferritin mRNA is repressed and the majority of ferritin mRNA is non-polysomal. Upon an increase in iron, translation of ferritin mRNA is derepressed resulting in as much as a 50-100-fold increase in the rate of ferritin synthesis. This regulation is mediated at least in part by a specific translational repressor which binds to a conserved sequence, the iron responsive element, located in the 5'-untranslated region of ferritin mRNA. In this communication we report the purification of such a repressor from rabbit liver. This repressor, which we call the "ferritin repressor protein," has an apparent molecular mass of 90 kDa when analyzed by gel filtration chromatography. It inhibits translation of ferritin mRNA in a highly specific fashion when added to a wheat germ lysate programmed with liver poly(A+) mRNA. In addition, it binds specifically to sequences contained within the first 92 nucleotides of ferritin mRNA, most likely the iron responsive element. Analysis of highly purified repressor by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that it is composed primarily of a single polypeptide of approximately 90 kDa. Elution of this 90-kDa polypeptide from a sodium dodecyl sulfate gel followed by renaturation and analysis for repressor activity shows that it both binds to the 5'-untranslated region of ferritin mRNA and represses its translation in vitro.

Demonstration of ferric L-parabactin-binding activity in the outer membrane of Paracoccus denitrificans.

Under low-iron conditions, Paracoccus denitrificans excretes a catecholamine siderophore, L-parabactin, to sequester and utilize iron. In this report, we demonstrate the presence of stereospecific high-affinity ferric L-parabactin-binding activity associated with P. denitrificans membranes grown in low-iron medium. Isolated outer membrane components were shown to be three to four times higher in specific activity for ferric L-parabactin. The same amount of binding activity existed whether or not the radiolabel was present in the metal (55Fe) or the ligand (3H) portion of ferric parabactin chelate, suggesting that binding was to the intact complex. Ion-exchange chromatography of a Triton X-100-solubilized outer membrane mixture on DEAE-cellulose resulted in a 10-fold increase in binding activity relative to that present in whole membranes. Polypeptide profiles by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the products of each stage of the purification showed that binding activity copurified with one or more of the low-iron-induced outer membrane proteins in the 80-kilodalton (kDa) region. Membrane proteins and [55Fe]ferric L-parabactin electrophoresed in nondenaturing gels demonstrated the presence of membrane component(s) which stereo-specifically bound ferric L-parabactin, thus providing independent confirmation of the binding assay results. Moreover, when the band labeled by [55Fe]ferric L-parabactin was excised and profiled by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 80-kDa polypeptides were the major components present. These results demonstrate the presence of a high-affinity ferric L-parabactin receptor in P. denitrificans membranes and suggest that one or more of the 80-kDa low-iron-induced polypeptides are components of the ferric L-parabactin receptor.

Participation of iron on the growth inhibition of pathogenic strains of mycobacterium avium and M. paratuberculosis in serum

Serum, which is low in freely-available iron, was bacteriostatic for M. avium and M. paratuberculosis irrespective of the ability of these organisms to produce the iron-chelators mycobactin and exochelin for iron acquisition. Bacteriostasis was overcome when the serum was supplemented with exochelin or mycobactin, but only if the mycobacteria had previously adapted to growth in a low iron environment. The results imply that for studies on mycobacterial pathogenicity the test cultures should be grown under low iron conditions before being used. The role of the exochelins, mycobactin and iron in the development of in vivo growth is discussed.

Regulation of toxinogenesis in Corynebacterium diphtheriae: mutations in the bacterial genome that alter the effects of iron on toxin production.

Mutants of Corynebacterium diphtheriae C7(beta) that are resistant to the inhibitory effects of iron on toxinogenesis were identified by their ability to form colonies surrounded by toxin-antitoxin halos on agar medium containing both antitoxin and a high concentration of iron. Chromosomal mutations were essential for the altered phenotypes of four independently isolated mutant strains. During growth in deferrated liquid medium containing various amounts of added iron, these mutants differed from wild-type C. diphtheriae C7(beta) in several ways. Their growth rates were slower under low-iron conditions and were stimulated to various degrees under high-iron conditions. The concentrations of iron at which optimal toxin production occurred were higher for the mutants than for wild-type C. diphtheriae C7(beta). Toxin production by the mutants during growth in low-iron medium occurred throughout the period of exponential growth at nearly constant rates that were proportional to the bacterial growth rates. In contrast, toxin production by wild-type C. diphtheriae C7(beta) in similar low-iron cultures occurred predominantly during the late exponential phase, when iron was a growth-limiting nutrient. Additional studies demonstrated that these mutants had severe defects in their transport systems for ferric iron. We propose that the altered regulation of toxinogenesis by iron in our mutants was caused by the severe defects in their iron transport systems. As a consequence, the mutants exhibited a low-iron phenotype during growth under conditions that permitted wild-type C. diphtheriae C7(beta) to exhibit a high-iron phenotype.

Active transport of ferric schizokinen in Anabaena sp.

The cyanobacterium Anabaena sp. strain ATCC 27898 was found to utilize the siderophore schizokinen to accumulate iron from the environment. This organism had previously been shown to produce schizokinen under low-iron conditions, and we observed that the iron-transport capability is also increased in response to iron limitation. Uptake activity was specific for ferric schizokinen displayed kinetics typical of a protein-mediated process with an apparent Km of 0.04 microM and saturation at high concentrations of substrate. Light-driven transport was blocked by uncouplers and by ATPase inhibitors. Transport in dark-adapted cells was additionally blocked by inhibitors of respiration. We conclude that ATP serves as an energy source for the cellular uptake of ferric schizokinen.

Iron Transport in Microalgae: the Isolation and Biological Activity of a Hydroxamate Siderophore from the Blue-Green Alga Agmenellum quadruplicatum

Summary: Supernatants from iron-deficient cultures of the marine, coccoid blue-green alga Agmenellum quadruplicatum strain PR-6 contained a chloroform-soluble iron-binding hydroxamate which was stimulatory for the growth of Arthrobacter jlavescens JG-9. Production of this material was maximal at 1 μM-Fe, was temperature dependent, and was increased by adding NH4 +. At the absorption maximum of 430 nm the At of the purified ferric complex was 32. The iron-free compound yielded nitroso dimers on periodate oxidation and was 60% as active on a weight basis for growth of Arth. flavescens as ferrioxamine B. At concentrations as low as 1 ng ml−l the purified hydroxamate stimulated growth of iron-depleted Agm. quadruplicatum in a chemically defined medium. Growth factor activity for Arth. flavescens JG-9 was also found in the supernatants and/or cell extracts of other blue-green algae and several diatoms grown under low-iron conditions.

Enzymatic release of iron from sideramines in fungi. NADH: Sideramine oxidoreductase in Neurospora crassa

Young mycelia of the fungus Neurospora crassa contain a soluble NADH-linked sideramine reductase, which may be responsible for liberating iron in vivo from accumulated sideramines during iron-deficient cultivation. The enzymes can be assayed using a soluble supernatant fraction, EDTA, and an atmosphere of pure nitrogen. The enzyme is stable without loss of activity up to 45°C and has an optimum of activity at pH 7.0. Besides coprogen ( K m = 100 μM, V = 2.8 nmol/min. per mg protein), some other ferrichrome-type compounds are reduced. However, ferrichrome, ferrirubin, coprogen B and ferrioxamine are poor substrates. When the mucelia were grown in a medium containing 10 −5 M ferric iron, the activity of the reductase was found to be only 30% of that found under low iron conditions. The enzyme is inhibited by oxygen, SH-alkylating agents and partly by some detergents. Unlike the reductase of N. crassa, the corresponding enzyme from Aspergillus fumigatus revealed low reduction of coprogen and high reduction of ferrichrome, indicating genus-dependent specificities of sideramine reduction enzymes in fungi. The participation of acids of the citric acid cycle as natural iron acceptors during strong iron deficiency is studied and confirmed by iron uptake measurements on isolated mitochondria.

Purification and properties of a photosynthetic reaction center isolated from various chromatophore fractions of Rhodopseudomonas spheroides Y

1. 1. Crude chromatophores, ribosome-free chromatophores and light particles, isolated from Rhodopseudomonas spheroides Y cells deficient or not deficient in iron, were used as source material for the preparation of reaction centers. Isolation was performed by incubation with cetyltrimethylammonium bromide and differential centrifugation; the purification was based upon either centrifugation or on (NH 4) 2SO 4 precipitation and gel filtration. 2. 2. In all cases, the purified reaction center contained the photo-active bacteriochlorophylls P 865 and P 800 (in a 1:2 ratio) without any light-harvesting bacteriochlorophyll; carotenoids and bacteriopheophytin were present. The yield (P 865 recovered from 100 bacteriochlorophylls) and the P 865 content (expressed per g protein) varied with the source material; the highest values were obtained when starting from low-iron light particles, the lowest when starting from low-iron crude chromatophores. 3. 3. The size of the particle bearing the reaction center was independent of the source material; the particle weight, determined by gel filtration as well as by equilibrium sedimentation, was about 150000. The midpoint potential of the P 865 species was 525 mV at pH 7.5. 4. 4. Some components of the purified reaction center from crude chromatophores, and their ratios, were: in low iron conditions, P 865:ubiquinone: c-type heme:non-heme iron, 1:8.6:0.12:0.2; in normal conditions, P 865:ubiquinone: c-type heme: non-heme iron, 1:8:1.5:0.5.

Enterobactin, an iron transport compound from [formula omitted

Enterobactin, a physiologically active iron sequestering agent, is excreted into the medium by Salmonella typhimurium under low iron conditions. It is a cyclic polyester consisting of three residues of 2,3-dihydroxy-N-benzoylserine.