Conditions Of Iron Deprivation Research Articles

Succinate Dehydrogenase b mRNA of Drosophila melanogaster Has a Functional Iron-responsive Element in Its 5′-Untranslated Region

Iron-responsive elements (IREs) are cis-acting mRNA stem-loop structures that specifically bind cytoplasmic iron regulatory proteins (IRPs). IRP-IRE interactions mediate the coordinate post-transcriptional regulation of key proteins in iron metabolism, such as ferritin, transferrin receptor, and erythroid 5-aminolevulinic acid synthase. Depending on whether the IRE is located in the 5'- or 3'-untranslated region (UTR), binding of IRP will inhibit mRNA translation or degradation, respectively. Here we describe a new IRE in the 5'-UTR of succinate dehydrogenase subunit b (SDHb) mRNA of Drosophila melanogaster. The SDHb IRE binds in vitro to vertebrate and insect IRPs with a high affinity equal to that of human ferritin H chain IRE. Under conditions of iron deprivation, SDHb mRNA of Drosophila SL-2 cells shifts to a non-polysome-bound pool. Moreover, translation of a human growth hormone mRNA with the SDHb IRE in its 5'-UTR is iron-dependent in stably transfected L cells. We conclude that the SDHb IRE mediates translational inhibition both in insect and vertebrate cells. This constitutes the first identification of a functional IRE in insects. Furthermore, Drosophila SDHb represents the second example, after porcine mitochondrial aconitase, of an enzyme of the citric acid cycle whose mRNA possesses all necessary features for translational regulation by cellular iron levels.

Cross-Protection Studies with Pasteurella multocida Bacterins Prepared from Bacteria Propagated in Iron-Depleted Medium

Strains X-73 (serotype 1) and P-1059 (serotype 3) of Pasteurella multocida, avian origin, expressed additional membrane proteins (MPs) when grown in brain-heart infusion (BHI) broth containing the iron chelator dipyridyl and when grown in BHI broth treated with the iron chelator Chelex 100. These additional MPs were not detected when both strains were grown in BHI broth. Chickens and turkeys were vaccinated twice with inactivated oil-emulsion vaccines containing bacterial cells expressing these MPs or with vaccines containing bacterial cells grown in BHI broth. Two weeks after the final vaccination, all birds were challenged to determine whether bacterins made from P. multocida that had been propagated in conditions of iron deprivation would induce heterologous serotype immunity. The bacterins produced in medium low in iron did not consistently induce significant protection against heterologous challenge.

Regulation of transferrin receptors by iron in human erythroblasts.

We investigated the regulatory mechanism of human erythroblast transferrin receptors (Tf.R) under conditions of iron deprivation and iron loading. Treatment of erythroblasts with an iron chelator, desferrioxamine, induced an increase in surface Tf.R number associated with an elevation of biosynthetic rate and the mRNA level of Tf.R. Reduced cellular iron pool increased the Tf.R number by altering the level of mRNA, as in nonhemoglobin-producing cells. Although treatment of erythroblasts with hemin induced a decrease in the biosynthetic rate and in the level of mRNA, the number of surface Tf.R did not decrease. This phenomenon may explain the fact that a high level of serum iron has no influence on the surface Tf.R number in vivo, as we reported previously. We suggest the existence of a regulatory mechanism specific for hemoglobin-producing cells that keeps surface Tf.R expression constant despite iron loading.

Utilization of transferrin-bound iron byHaemophilusspecies of human and porcine origins

Haemophilus influenzae and H. haemolyticus acquired iron bound to human transferrin but not to human lactoferrin, ovo- or porcine transferrins. Conversely the swine pathogens H. pleuropneumoniae and H. parasuis used iron bound only to porcine transferrin. Growth under conditions of iron deprivation induced the production of siderophores and iron-repressible outer membrane proteins in H. parainfluenzae, H. paraphrophilus and H. parasuis but not in H. influenzae, H. haemolyticus or H. pleuropneumoniae. The latter 3 Haemophilus species appear to sequester transferrin bound iron via a siderophore-independent mechanism. However, the ability to produce iron chelating compounds did not enable H. parainfluenzae or H. paraphrophilus to utilize transferrin bound iron.

Antigenically related iron-regulated outer membrane proteins produced by different somatic serotypes of Pasteurella multocida.

An 84-kilodalton outer membrane protein was expressed when Pasteurella multocida, somatic serotype 3, was grown in brain-heart infusion broth containing the iron chelator dipyridyl but not in brain-heart infusion broth alone. Antigenically related outer membrane proteins of various molecular masses were also expressed by P. multocida strains belonging to all of the other 15 somatic serotypes (somatic serotype 12 being the possible exception) as well as by isolates expressing somatic antigens representative of multiple somatic serotypes when grown under the same conditions of iron deprivation.

Effect of iron on accumulation of exotoxin A-specific mRNA in Pseudomonas aeruginosa

A DNA probe from an internal fragment of the exotoxin A structural gene was used to study the effects of selected culture conditions on steady-state levels of exotoxin-specific mRNA in Pseudomonas aeruginosa. Cells grown under conditions of iron deprivation began to synthesize and excrete the exotoxin A polypeptide during the late exponential phase of growth and throughout the stationary phase of growth, concomitant with a sharp increase in exotoxin A mRNA pools in P. aeruginosa cells. The addition of iron to the medium resulted in the failure of these cells to synthesize exotoxin A mRNA, despite significantly enhanced growth. The inhibition of the production of exotoxin A and the accumulation of its mRNA by iron was dose dependent, with a half-maximal inhibitory concentration of FeSO4 of 5 to 10 microM. A blockade of the initiation of transcription by rifampin resulted in the decay of exotoxin A mRNA, with a half-life of approximately 8 to 10 min, depending on the media used for growth. The addition of iron to cells actively engaged in exotoxin A synthesis also resulted in a gradual decrease in the amount of this mRNA in bacteria. However, the rate of decline of mRNA induced by iron was relatively slow (half-life, 90 min), with a considerable lag time between the iron addition and the first detectable effect on mRNA. While iron clearly appears to influence the production of exotoxin A at the transcriptional level, the molecular basis of this effect may involve several interacting factors affecting the initiation of transcription and perhaps mRNA turnover.

Siderophore production by Pasteurella multocida.

Pasteurella multocida grown under conditions of iron deprivation secreted into the culture medium a growth-enhancing factor which functioned as a siderophore. The siderophore was found to be neither a phenolate nor a hydroxamate by chemical tests and bioassays and was given the trivial name multocidin. Multocidin was partially purified and found to be a highly polar, nonaromatic, and dialyzable compound. This is the first report demonstrating the production of a siderophore by P. multocida.

Genetic and biochemical evidence for a siderophore-dependent iron transport system in Corynebacterium diphtheriae.

During growth under conditions of iron deprivation, Corynebacterium diphtheriae secreted a siderophore into the culture medium. This extracellular siderophore was necessary for rates of iron uptake at pH 8.0 by C. diphtheriae C7 and related strains. We isolated a mutant of C. diphtheriae C7(beta), strain HC6, which did not make the corynebacterial siderophore. Strain HC6 grew very poorly, even under high-iron conditions, and had a severe defect in iron transport. Both growth and iron uptake by strain HC6 were greatly stimulated by the corynebacterial siderophore. We used strain HC6 to develop a bioassay for the corynebacterial siderophore and to look for other potential siderophores for C. diphtheriae. Among the purified phenolate and hydroxamate siderophores tested, only aerobactin was able to stimulate the growth of strain HC6. Partial purification of the corynebacterial siderophore was achieved. The siderophore did not give positive reactions in the Arnow test for phenolates or the Csaky test for hydroxamates and may have a novel chemical structure.

ColV plasmid-specific aerobactin synthesis by invasive strains of Escherichia coli.

Certain strains of Escherichia coli associated with bacteremia of humans and domestic animals harbor plasmids that promote efficient iron uptake. The mechanism, which is an important component of the virulence of invasive strains, is independent of the enterobactin system for iron uptake. Plasmid-specified siderophore was assayed by its ability to support the growth of a chelator-deficient mutant in conditions of iron deprivation. The chelator, which was chemically determined to be a hydroxamate compound, was identical on the basis of field desorption mass spectrometry with aerobactin, a siderophore synthesized by Aerobacter aerogenes. In conditions of iron stress, aerobactin is secreted into the culture medium of plasmid-bearing E. coli strains. Reconstruction experiments involving a chelator-deficient mutant growing with exogenous chelator suggested that association of a small fraction of the total siderophore synthesized with cellular material is due to transient binding of aerobactin to membrane receptors during active bacterial growth.

Control of Haemoglobin Synthesis

The distribution of ribosomes along the mRNAs for α and β chains was measured in cells which had achieved a steady state of protein synthesis after incubation with haem and iron or with the iron‐chelating agent 2,2′‐dipyridyl. The distribution was found to be uniform under both conditions for the α and β chain mRNAs, despite the dramatic breakdown of polysomes observed in the presence of dipyridyl. Since a uniform distribution of ribosomes along an mRNA indicates the absence of rate‐limiting steps in chain elongation or chain termination, we conclude that the slowing of protein synthesis during iron deprivation is not caused by the introduction of rate‐limiting steps into these processes. In the presence of dipyridyl the total numbers of ribosomes synthesising α and β chains were reduced to roughly the same extent indicating that iron starvation inhibits the synthesis of both α and β chains. In order to confirm that the rate chain elongation was not altered during iron starvation, we attempted to compare the rates of translation of the two mRNAs in the presence and absence of iron. Some of the difficulties in measuring the translation times for the α and β chain mRNAs under conditions of iron deprivation are discussed. Under conditions where a partial effect of iron deprivation was evident, we showed that the rate of translation of both α and β chain mRNAs was essentially unchanged. Both sets of findings support the hypothesis that, in vivo, the availability of haem controls the process of chain initiation.