Influence of Iron Regulation on the Metabolome of Cryptococcus neoformans

Iron is an essential nutrient for virtually all organisms and acts as a cofactor for many key enzymes of major metabolic pathways. Furthermore, iron plays a critical role in pathogen-host interactions. In this study, we analyzed metabolomic changes associated with iron availability and the iron regulatory protein Cir1 in a human fungal pathogen Cryptococcus neoformans. Our metabolite analysis revealed that Cir1 influences the glycolytic pathway, ergosterol biosynthesis and inositol metabolism, which require numerous iron-dependent enzymes and play important roles in pathogenesis and antifungal sensitivity of the fungus. Moreover, we demonstrated that increased cellular iron content and altered gene expression in the cir1 mutant contributed to metabolite changes. Our study provides a new insight into iron regulation and the role of Cir1 in metabolome of C. neoformans.     

Iron-regulated synthesis and uptake of colicin V

Abstract Virulent strains of Escherichia coli frequently contain ColV plasmids. It is shown that synthesis of the marker protein, colicin V, is regulated by iron via the iron repressor encoded by the fur gene. Mutants in the Cir outer membrane protein, in tonB , as well as in the exbB gene are resistant to colicin V suggesting all three functions to be required for colicin V uptake.     

The colicin Ia receptor,Cir, is also the translocator for colicin Ia

Colicin Ia, a channel‐forming bactericidal protein, uses the outer membrane protein, Cir, as its primary receptor. To kill Escherichia coli, it must cross this membrane. The crystal structure of Ia receptor‐binding domain bound to Cir, a 22‐stranded plugged β‐barrel protein, suggests that the plug does not move. Therefore, another pathway is needed for the colicin to cross the outer membrane, but no ‘second receptor’ has ever been identified for TonB‐dependent colicins, such as Ia. We show that if the receptor‐binding domain of colicin Ia is replaced by that of colicin E3, this chimera effectively kills cells, provided they have the E3 receptor (BtuB), Cir, and TonB. This is consistent with wild‐type Ia using one Cir as its primary receptor (BtuB in the chimera) and a second Cir as the translocation pathway for its N‐terminal translocation (T) domain and its channel‐forming C‐terminal domain. Deletion of colicin Ia's receptor‐binding domain results in a protein that kills E. coli, albeit less effectively, provided they have Cir and TonB. We show that purified T domain competes with Ia and protects E. coli from being killed by it. Thus, in addition to binding to colicin Ia's receptor‐binding domain, Cir also binds weakly to its translocation domain.     

Kinetics of biosynthesis of iron-regulated membrane proteins in Escherichia coli

Using biological iron chelators to control specifically iron availability to Escherichia coli K-12 in conjunction with radioactive pulse-labels, we examined the biosynthesis of six iron-regulated membrane proteins. Iron deprivation induced the synthesis of five proteins, which had molecular weights of 83,000 (83K), 81K (Fep), 78K (TonA), 74K (Cir), and 25K. The kinetics of induction were the same in entA and entA⁺ strains, but were affected by the initial iron availability in the media. Iron-poor cells induced rapidly (half-time, 10 min), whereas iron-rich cells began induction after a lag and showed a slower induction half-time (30 min). Within this general pattern of induction after iron deprivation, several different kinetic patterns were apparent. The 83K, 81K, and 74K proteins were coordinately controlled under all of the conditions examined. The 78K and 25K proteins were regulated differently. The synthesis of a previously unrecognized 90K inner membrane protein was inhibited by iron deprivation and stimulated by iron repletion. Both ferrichrome and ferric enterobactin completely repressed 81K and 74K synthesis when the siderophores were supplied at concentrations of 5 μM in vivo (half-time, 2.5 min). At concentrations less than 5 μM, however, both siderophores repressed synthesis only temporarily; the duration of repression was proportional to the amount of ferric siderophore added. The half-lives of the 81K and 74K mRNAs, as measured by rifampin treatment, were 1.2 and 1.6 min, respectively. The results of this study suggest that enteric bacteria are capable of instantaneously detecting and reacting to fluctuations in the extracellular iron concentration and that they store iron during periods of iron repletion for utilization during periods of iron stress. Neither iron storage nor iron regulation of envelope protein synthesis is dependent on the ability of the bacteria to form heme.     

Dihydroxybenzolyserine—a siderophore for E. coli

Dihydroxybenzoylserine, the breakdown product of enterochelin, was able to stimulate growth of Escherichia coli under iron limiting conditions by acting as a siderophore. The dihydroxybenzoylserine-iron complex was taken up via the outer membrane receptor proteins Fiu, FepA and to a minor extent via Cir. Transport of Fe3(+)-dihydroxybenzoylserine across the cytoplasmic membrane was only dependent on genes from the fep region. In addition, it was shown that dihydroxybenzoate was taken up via Fiu and Cir and less efficiently by FepA.     

Global analysis of cellular protein translation by pulsed SILAC

Current methods for system‐wide gene expression analysis detect changes in mRNA abundance, but neglect regulation at the level of translation. Pulse labeling with stable isotopes has been used to measure protein turnover rates, but this does not directly provide information about translation rates. Here, we developed pulsed stable isotope labeling by amino acids in cell culture (pSILAC) with two heavy isotope labels to directly quantify protein translation on a proteome‐wide scale. We applied the method to cellular iron homeostasis as a model system and demonstrate that it can confidently identify proteins that are translationally regulated by iron availability.     

Response of the cyanobacterium Gloeotrichia echinulata to iron and boron additions – an experiment from Lake Erken

1. This study considers whether the availability of iron and boron are important influences on the development of the cyanobacterium Gloeotrichia echinulata in Lake Erken, Sweden.
2. In an in situ experiment, phosphate and nitrate were added to all enclosures, but pelagic colonies of G. echinulata only increased in abundance in enclosures to which iron had also been added.
3. An even greater increase in the abundance of G. echinulata occured in enclosures in which the additions of phosphate, nitrate and iron were complemented by additions of boron.
4. Boron additions, together with phosphate and nitrate but without iron, did not stimulate the growth of G. echinulata .     

Ferric citrate transport in Escherichia coli requires outer membrane receptor protein fecA.

Mutants of Escherichia coli K-12 AB2847 and of E. coli K-12 AN92 were isolated which were unable to grow on ferric citrate as the sole iron source. Of 22 mutants, 6 lacked an outer membrane protein, designated FecA protein, which was expressed by growing cells in the presence of 1 mM citrate. Outer membranes showed an enhanced binding of radioactive iron, supplied as a citrate complex, depending on the amount of FecA protein. The FecA protein was the most resistant of the proteins involved in ferric irion iron translocation across the outer membrane (FhuA = TonA, FepA, Cir, or 83K proteins) to the action of pronase P. It is also shown that previously isolated fec mutants (G. C. Woodrow et al., J. Bacteriol. 133:1524-1526, 1978) which are cotransducible with argF all lack the FecA protein. They were termed fecA to distinguish them from the other ferric citrate transport mutants, now designated fecB, which mapped in the same gene region at 7 min but were not cotransducible with ArgF. E. coli W83-24 and Salmonella typhimurium, which are devoid of a citrate-dependent iron transport system, lacked the FecA protein. It is proposed that the FecA protein participates in the transport of ferric citrate.     

Iron-responsive degradation of iron-regulatory protein 1 does not require the Fe-S cluster

The generally accepted role of iron-regulatory protein 1 (IRP1) in orchestrating the fate of iron-regulated mRNAs depends on the interconversion of its cytosolic aconitase and RNA-binding forms through assembly/disassembly of its Fe-S cluster, without altering protein abundance. Here, we show that IRP1 protein abundance can be iron-regulated. Modulation of IRP1 abundance by iron did not require assembly of the Fe-S cluster, since a mutant with all cluster-ligating cysteines mutated to serine underwent iron-induced protein degradation. Phosphorylation of IRP1 at S138 favored the RNA-binding form and promoted iron-dependent degradation. However, phosphorylation at S138 was not required for degradation. Further, degradation of an S138 phosphomimetic mutant was not blocked by mutation of cluster-ligating cysteines. These findings were confirmed in mouse models with genetic defects in cytosolic Fe-S cluster assembly/disassembly. IRP1 RNA-binding activity was primarily regulated by IRP1 degradation in these animals. Our results reveal a mechanism for regulating IRP1 action relevant to the control of iron homeostasis during cell proliferation, inflammation, and in response to diseases altering cytosolic Fe-S cluster assembly or disassembly.     

Characterization of a Vibrio cholerae virulence factor homologous to the family of TonB-dependent proteins

IrgA is an iron-regulated virulence factor for infection in an animal model with classical Vibrio cholerae strain 0395. We detected gene sequences hybridizing to irgA at high stringency in clinical isolates in addition to 0395, including another classical strain of V. cholerae, three V. cholerae strains of the El Tor biotype, three non-O1 isolates of V. cholerae, and individual isolates of Vibrio parahaemolyticus, Vibrio fluvialis, and Vibrio alginolyticus. No hybridization to irgA was seen with chromosomal DNA from Vibrio vulnificus or Aeromonas hydrophila. To verify that irgA is the structural gene for the major iron-regulated outer membrane protein of V. cholerae, we determined the amino-terminal sequence of this protein recovered after gel electrophoresis and demonstrated that it corresponds to the amino acid sequence of IrgA deduced from the nucleotide sequence. Gel electrophoresis showed that two El Tor strains of V. cholerae had a major iron-regulated outer membrane protein identical in size and appearance to IrgA in strain 0395, consistent with the findings of DNA hybridization. We have previously suggested that IrgA might be the outer membrane receptor for the V. cholerae siderophore, vibriobactin. Biological data presented here, however, show that a mutation in irgA had no effect on the transport of vibriobactin and produced no defect in the utilization of iron from ferrichrome, ferric citrate, haemin or haemoglobin. The complete deduced amino acid sequence of IrgA demonstrated homology to the entire class of Escherichia coli TonB-dependent proteins, particularly Cir. Unlike the situation with Cir, however, we were unable to demonstrate a role for IrgA as a receptor for catechol-substituted cephalosporins. The role of IrgA in the pathogenesis of V. cholerae infection, its function as an outer membrane receptor, and its potential interaction with a TonB-like protein in V. cholerae remain to be determined.     

Utilization of enterobactin and other exogenous iron sources by Haemophilus influenzae, H. parainfluenzae and H. paraphrophilus

The ability of Haemophilus influenzae, H. parainfluenzae and H. paraphrophilus to utilize iron complexes, iron-proteins and exogenous microbial siderophores was evaluated. In a plate bioassay, all three species used not only ferric nitrate but also the iron chelates ferric citrate, ferric nitrilotriacetate and ferric 2,3-dihydroxybenzoate. Each Haemophilus species examined also used haemin, haemoglobin and haem-albumin as iron sources although only H. influenzae could acquire iron from transferrin or from haemoglobin complexed with haptoglobin. None of the haemophili obtained iron from ferritin or lactoferrin or from the microbial siderophores aerobactin or desferrioxamine B. However, the phenolate siderophore enterobactin supplied iron to both H. parainfluenzae and H. paraphrophilus, and DNA isolated from both organisms hybridized with a DNA probe prepared from the Escherichia coli ferric enterobactin receptor gene fepA. In addition, a monospecific polyclonal antiserum raised against the E. coli 81 kDa ferric enterobactin receptor (FepA) recognized an iron-repressible outer membrane protein (OMP) in H. parainfluenzae of between 80 and 82 kDa (depending on the strain). This anti-FepA serum did not cross-react with any of the OMPs of H. paraphrophilus or H. influenzae. The OMPs of each Haemophilus species were also probed with antisera raised against the 74 kDa Cir or 74 kDa IutA (aerobactin receptor) proteins of E. coli. Apart from one H. parainfluenzae strain (NCTC 10665), in which an OMP of about 80 kDa cross-reacted with the anti-IutA sera, no cross-reactivity was observed between Cir, IutA and the OMPs of H. influenzae, H. parainfluenzae or H. paraphrophilus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Iron metabolism in the yeast

Current data concerning transport, storage and utilization of iron in the yeast cells, particularly Saccharomyces cerevisiae are summarized in the paper. It has been marked that iron uptake in the cells provides by high affinity system, it function is carried out by protein complex Fet3-Ftr1, and Fet4, protein with low affinity to iron ion. The both systems utilize Fe(II). Furthermore, the active site of the protein Fre1 is exposed on the outer side of plasmalemma. This protein, due to ferrireductase activity, provides availability of Fe(III) to the cell. The information regards to participation of siderophores and metal-proton plasma membrane exchangers Smf1 in iron transport is brought. Particular attention is given to regulation of expression of the genes, coding the iron metabolic systems. Some aspects of iron utilization for Fe-S-containing enzymes synthesis are lighted. It has been concluded that the yeast is a perspective subject for studying balance of living organisms between iron essentiality and its ability to trigger free radical reactions.     

SORTING NEXIN1 Is Required for Modulating the Trafficking and Stability of the Arabidopsis IRON-REGULATED TRANSPORTER1

Dicotyledonous plants growing under limited iron availability initiate a response resulting in the solubilization, reduction, and uptake of soil iron. The protein factors responsible for these steps are transmembrane proteins, suggesting that the intracellular trafficking machinery may be involved in iron acquisition. In search for components involved in the regulation of Arabidopsis thaliana iron deficiency responses, we identified the members of the SORTING NEXIN (SNX) protein family. SNX loss-of-function plants display enhanced susceptibility to iron deficiency in comparison to the wild type. The absence of SNX led to reduced iron import efficiency into the root. SNX1 showed partial colocalization with the principal root iron importer IRON-REGULATED TRANSPORTER1 (IRT1). In SNX loss-of-function plants, IRT1 protein levels were decreased compared with the wild type due to enhanced IRT1 degradation. This resulted in diminished amounts of the IRT1 protein at the plasma membrane. snx mutants exhibited enhanced iron deficiency responses compared with the wild type, presumably due to the lower iron uptake through IRT1. Our results reveal a role of SNX1 for the correct trafficking of IRT1 and, thus, for modulating the activity of the iron uptake machinery.