Jean-Michel Camadro

Siderophore uptake and use by the yeast Saccharomyces cerevisiae.

The non-reductive uptake of several siderophores (ferrioxamine B, ferrichrome, triacetylfusarinine C and ferricrocin) by various strains of Saccharomyces cerevisiae was studied. Several aspects of siderophore transport were examined, including specificity of transport, regulation of transport and intracellular localization of the ferri-siderophores. Ferrioxamine B was taken up preferentially via the products of the SIT1 gene and triacetylfusarinine C by the TAF1 gene product, but the specificity was not absolute. Ferrichrome and ferricrocin uptake was not dependent on a single major facilitator superfamily (MFS) gene product. The apparent specificity of transport was strongly dependent on the genetic background of the cells. Non-reductive uptake of siderophores was induced under more stringent conditions (of iron deprivation) than was the reductive uptake of ferric citrate. Regulation of transport depended on the transcriptional factors Aft1 and Tup1/Ssn6. Cells disrupted for the TUP1 or SSN6 genes were constitutively derepressed for the uptake of ferrichrome, ferricrocin or ferrioxamine B, but not for the uptake of triacetylfusarinine C. Cells bearing the AFT1(up) mutation accumulated large amounts of ferric siderophores. Intracellular decomplexation of the siderophores occurred when transcription of the AFT1(up) gene was repressed. Ferrioxamine B and ferrichrome seemed to accumulate in an endosomal compartment, as shown by biochemical studies and by confocal microscopy study of cells loaded with a fluorescent derivative of ferrichrome. Endocytosis was, however, not involved in the non-reductive uptake of siderophores.

Protoporphyrinogen oxidase inhibition by three peroxidizing herbicides: Oxadiazon, LS 82-556 and M&B 39279

Three chemically unrelated peroxidizing molecules, namely oxadiazon [5‐(t‐butyl)‐3‐(2,4‐dichloro‐5‐isopropoxyphenyl)‐1,3,4‐oxadiazol‐2‐one], LS 82‐556 [(S)3‐N‐(methylbenzyl)carbamoyl‐5‐propionyl‐2,6‐lutidine] and M&B 39279 [5‐amino‐4‐cyano‐1‐(2,6‐dichloro‐4‐trifluoromethylphenyl)pyrazol], are potent inhibitors of plant, yeast and mouse protoporphyrinogen oxidase.

Siderophore uptake by Candida albicans: effect of serum treatment and comparison with Saccharomyces cerevisiae.

Iron uptake systems often function as virulence factors in pathogenic organisms. Candida albicans is a fungal pathogen that infects immunocompromised hosts, such as AIDS patients or granulocytopenic bone marrow transplant recipients. Here we show that iron uptake from siderophores occurs in C. albicans and is mediated by one or more high‐affinity transport systems. Iron carried on ferrioxamine B, triacethyl‐fusarinine, ferrichrome, or ferricrocin was actively taken up via a high‐affinity mechanism. The kinetic parameters of uptake were similar to those found in S. cerevisiae. Furthermore, for ferrichrome and ferrioxamine B, cellular uptake of fluorescent analogues was observed. In C. albicans, iron uptake from siderophores was regulated by iron availability, with iron deprivation inducing uptake. Serum exposure, which induces a morphogenic shift from yeast to filamentous forms known to be required for virulence, also resulted in induction of iron transport from ferrichrome‐type siderophores. In a tup1/tup1 strain which grows constitutively in the filamentous form, iron transport was derepressed for all siderophores tested. The genes mediating uptake and utilization of iron from siderophores in C. albicans have not been identified; however, the transcript abundance for CaSIT1 was regulated in a manner consistent with the pattern of iron uptake from ferrichrome‐type siderophores. Furthermore, CaSIT1 overexpression in S. cerevisiae resulted in inhibited siderophore iron uptake, suggesting that the expressed protein may interact with proteins of S. cerevisiae involved in iron uptake from siderophores. In summary, iron uptake from ferrichrome‐type siderophores was induced in filamentous C. albicans, and a potential role of this iron acquisition system in pathogenicity should be considered. Copyright

A new assay for protoporphyrinogen oxidase — Evidence for a total deficiency in that activity in a heme-less mutant of Saccharomyces cerevisiae

Abstract A new spectrophotometric assay for protoporphyrinogen oxidase activity has been developed, involving enzymatic generation of protoporphyrinogen in the incubation medium. This assay, more sensitive and reliable than those previously described, can be used to measure this activity in yeast mitochondrial membranes, rat liver mitochondria and E. coli membranes. By measuring protoporphyrinogen oxidase activity in different wild type and heme-mutant yeast strains, it was shown that 1) one heme-mutant was totally lacking this activity, 2) different factors might control its level in yeast.

Kinetic studies of ferrochelatase in yeast: Zinc or iron as competing substrates

Ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) has been studied in yeast mitochondrial membranes with special reference to zinc-chelatase and iron-chelatase activities. Using physiological substrates (protoporphyrin IX, Fe(II) and Zn(II), anaerobic conditions of incubation and direct spectrophotometric assay, apparent Km values smaller than those previously described were found for the membrane-bound enzyme. Fe(II) but not Fe(III) was a strong competitive inhibitor of zinc-chelatase activity, while Zn(II) was a slight competitive inhibitor of iron-chelatase activity. These results could point to modes of control of ferrochelatase activity in yeast. We suggest that reduced supply of Fe(II) may explain the in vivo accumulation of zinc-protoporphyrin in yeast cells incubated under resting conditions.

Effects of iron deficiency and chronic iron overloading on mitochondrial heme biosynthetic enzymes in rat liver.

The effects of iron deficiency and iron overloading on the mitochondrial enzymes involved in heme synthesis were studied in rat livers. The in vitro activities of several of the enzymes in this pathway were differentially influenced by the in vivo iron status of the animals. delta-Aminolevulinic acid synthase was slightly increased in iron-overloaded animals, but remained normal in iron-deficient animals (0.58 +/- 0.09, 0.91 +/- 0.19 and 0.61 +/- 0.12 nmol delta-aminolevulinic acid/mg per h). Copro- and protoporphyrinogen oxidase activities were increased (20 and 60% above controls) in iron-deficient animals. In contrast, coproporphyrinogen oxidase was decreased by 20%, while protoporphyrinogen oxidase remained unchanged in iron-overloaded rats. These variations of activities were not due to changes in the affinity of these enzymes toward their substrates, as coporphyrinogen had the same Km in each case (0.62 +/- 0.05 M) as did protoporphyrinogen (0.22 +/- 0.035 M). Thus, the Km did not vary with the treatment received by the animals. Ferrochelatase activity was measured by both the pyridine hemochromogen method and by measurement of zinc protoporphyrin with endogenous zinc as substrate. In all cases, ferrochelatase was found to be able to synthesize zinc protoporphyrin with endogenous zinc as substrate. However, the apparent Km of zinc chelatase for protoporphyrin was significantly different in the three groups of animals with Km,appProto, app = 2.4 +/- 0.1 10(-7), 4 +/- 0.3 10(-7) and 9.10 +/- 0.05 10(-7) M in iron-overloaded, control and iron-deficient animals, respectively. When ferrochelatase activity was measured by pyridine hemochromogen, identical results were observed in iron-deficient and control animals but decreased by 45% in iron-overloaded animals. The mitochondrial heme content was also decreased by 40% in iron-overloaded rats but unchanged in either iron-deficient or control rats.

bPeaks: a bioinformatics tool to detect transcription factor binding sites from ChIPseq data in yeasts and other organisms with small genomes

Peak calling is a critical step in ChIPseq data analysis. Choosing the correct algorithm as well as optimized parameters for a specific biological system is an essential task. In this article, we present an original peak‐calling method (bPeaks) specifically designed to detect transcription factor (TF) binding sites in small eukaryotic genomes, such as in yeasts. As TF interactions with DNA are strong and generate high binding signals, bPeaks uses simple parameters to compare the sequences (reads) obtained from the immunoprecipitation (IP) with those from the control DNA (input). Because yeasts have small genomes (<20u2009Mb), our program has the advantage of using ChIPseq information at the single nucleotide level and can explore, in a reasonable computational time, results obtained with different sets of parameter values. Graphical outputs and text files are provided to rapidly assess the relevance of the detected peaks. Taking advantage of the simple promoter structure in yeasts, additional functions were implemented in bPeaks to automatically assign the peaks to promoter regions and retrieve peak coordinates on the DNA sequence for further predictions of regulatory motifs, enriched in the list of peaks. Applications of the bPeaks program to three different ChIPseq datasets from Saccharomyces cerevisiae, Candida albicans and Candida glabrata are presented. Each time, bPeaks allowed us to correctly predict the DNA binding sequence of the studied TF and provided relevant lists of peaks. The bioinformatics tool bPeaks is freely distributed to academic users. Supplementary data, together with detailed tutorials, are available online: http://bpeaks.gene‐networks.net. Copyright

Characteristics of Protoporphyrinogen Oxidase

Protoporphyrinogen oxidase (EC 1.3.3.4) catalyzes the oxidative O2-dependent aromatization of the colorless protoporphyrinogen IX to the highly conjugated protoporphyrin IX, the precursor of both hemes and chlorophylls (Fig. 1). It is the final enzyme in the common branch of the heme and chlorophyll biosyn-thetic pathways in plants (Fig. 2).