Induction of coproporphyrinogen oxidase in Chlamydomonas chloroplasts occurs via transcriptional regulation of Cpx1 mediated by copper response elements and increased translation from a copper deficiency-specific form of the transcript. (1/92)

Coproporphyrinogen III oxidase, encoded by a single nuclear gene in Chlamydomonas reinhardtii, produces three distinct transcripts. One of these transcripts is greatly induced in copper-deficient cells by transcriptional activation, whereas the other forms are copper-insensitive. The induced form of the transcript was expressed coordinately with the cytochrome c6-encoding (Cyc6) gene, which is known to be transcriptionally regulated in copper-deficient cells. The sequence GTAC, which forms the core of a copper response element associated with the Cyc6 gene, is also essential for induction of the Cpx1 gene, suggesting that both are targets of the same signal transduction pathway. The constitutive and induced Cpx1 transcripts have the same half-lives in vivo, and all encode the same polypeptide with a chloroplast-targeting transit sequence, but the shortest one representing the induced form is a 2-4-fold better template for translation than are either of the constitutive forms. The enzyme remains localized to a soluble compartment in the chloroplast even in induced cells, and its abundance is not affected when the tetrapyrrole pathway is manipulated either genetically or by gabaculine treatment.  (+info)

Transcriptional control of Bacillus subtilis hemN and hemZ. (2/92)

Previous characterization of Bacillus subtilis hemN, encoding a protein involved in oxygen-independent coproporphyrinogen III decarboxylation, indicated the presence of a second hemN-like gene (B. Hippler, G. Homuth, T. Hoffmann, C. Hungerer, W. Schumann, and D. Jahn, J. Bacteriol. 179:7181-7185, 1997). The corresponding hemZ gene was found to be split into the two potential open reading frames yhaV and yhaW by a sequencing error of the genome sequencing project. The hemZ gene, encoding a 501-amino-acid protein with a calculated molecular mass of 57,533 Da, complemented a Salmonella typhimurium hemF hemN double mutant under aerobic and anaerobic growth conditions. A B. subtilis hemZ mutant accumulated coproporphyrinogen III under anaerobic growth conditions. A hemN hemZ double mutant exhibited normal aerobic and anaerobic growth, indicating the presence of a third alternative oxygen-independent enzymatic system for coproporphyrinogen III oxidation. The hemY gene, encoding oxygen-dependent protoporphyrinogen IX oxidase with coproporphyrinogen III oxidase side activity, did not significantly contribute to this newly identified system. Growth behavior of hemY mutants revealed the presence of an oxygen-independent protoporphyrinogen IX oxidase in B. subtilis. A monocistronic hemZ mRNA, starting 31 bp upstream of the translational start codon, was detected. Reporter gene fusions of hemZ and hemN demonstrated a fivefold anaerobic induction of both genes under nitrate ammonifying growth conditions. No anaerobic induction was observed for fermentatively growing B. subtilis. The B. subtilis redox regulatory systems encoded by resDE, fnr, and ywiD were indispensable for the observed transcriptional induction. A redox regulation cascade proceeding from an unknown sensor via resDE, through fnr and ywiD to hemN/hemZ, is suggested for the observed coregulation of heme biosynthesis and the anaerobic respiratory energy metabolism. Finally, only hemZ was found to be fivefold induced by the presence of H(2)O(2), indicating further coregulation of heme biosynthesis with the formation of the tetrapyrrole enzyme catalase.  (+info)

Coordinate copper- and oxygen-responsive Cyc6 and Cpx1 expression in Chlamydomonas is mediated by the same element. (3/92)

Chlamydomonas reinhardtii activates the transcription of the Cyc6 and the Cpx1 genes (encoding cytochrome c(6) and coprogen oxidase) in response to copper deficiency. Mutational analysis of promoter regions of the Cyc6 and Cpx1 genes revealed a four nucleotide sequence, GTAC, which was absolutely essential for copper responsiveness. The Cyc6 promoter contains two copper response elements, each with a functionally important GTAC sequence, whereas the Cpx1 promoter contains only one. This may contribute to the stronger and more tightly regulated expression of the Cyc6 gene. Mutation or deletion of sequences flanking the GTACs implicates additional nucleotides contributing to copper-responsive expression, but none are absolutely essential. Metal ion selectivity of Cpx1 expression is identical to that described previously for Cyc6 and is restricted to the copper deficiency-induced Cpx1 transcript. The Cyc6 and Cpx1 genes are also induced by oxygen deficiency. Reporter gene constructs indicate that the induction occurs at the level of transcription and requires the same GTAC sequence that is critical for copper responsiveness. We suggest that components of the copper-responsive signal transduction pathway are used for some of the changes in gene expression in hypoxic cells.  (+info)

Alcohol and porphyrin metabolism. (4/92)

Alcohol is a porphyrinogenic agent which may cause disturbances in porphyrin metabolism in healthy persons as well as biochemical and clinical manifestations of acute and chronic hepatic porphyrias. After excessive consumption of alcohol, a temporary, clinically asymptomatic secondary hepatic coproporphyrinuria is observable, which can become persistent in cases of alcohol-induced liver damage. Nowadays, the alcohol-liver-porphyrinuria syndrome is the first to be mentioned in secondary hepatic disturbances of porphyrin metabolism. Acute hepatic porphyrias (acute intermittent porphyria, variegate porphyria and hereditary coproporphyria) are considered to be molecular regulatory diseases, in contrast to non-acute, chronic hepatic porphyria, clinically appearing as porphyria cutanea tarda (PCT). Porphyrins do not accumulate in the liver in acute porphyrias, whereas in chronic hepatic porphyrias they do. Thus, chronic hepatic porphyria is a porphyrin-accumulation disease, whereas acute hepatic porphyrias are haem-pathway-dysregulation diseases, characterized in general by induction of delta-aminolevulinic acid synthase in the liver and excessive stimulation of the pathway without storage of porphyrins in the liver. The clinical expression of acute hepatic porphyrias can be triggered by alcohol, because alcohol augments the inducibility of delta-aminolevulinic acid synthase. In chronic hepatic porphyrias, however, which are already associated with liver damage, alcohol potentiates the disturbance of the decarboxylation of uro- and heptacarboxyporphyrinogen, which is followed by a hepatic accumulation of uro- and heptacarboxyporphyrin and their sometimes extreme urinary excretion. Especially in persons with a genetic deficiency of uroporphyrinogen decarboxylase, but also in patients with the so-called sporadic variety of PCT, alcohol is able to transform an asymptomatic coproporphyrinuria into PCT. Alcohol has many biochemical and clinical effects on porphyrin and haem synthesis both in humans and laboratory animals. Ethanol suppresses the activity of porphobilinogen synthase (synonym: delta-aminolevulinic acid dehydratase), uroporphyrinogen decarboxylase, coproporphyrinogen oxidase and ferrochelatase, whereas it induces the first and rate-limiting enzyme in the pathway, delta-aminolevulinic acid synthase and also porphobilinogen deaminase. Therefore, teetotalism is a therapeutically and prophylactically important measure in all types of hepatic porphyrias.  (+info)

Interacting regulatory circuits involved in orderly control of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1. (5/92)

FnrL, the homolog of the global anaerobic regulator Fnr, is required for the induction of the photosynthetic apparatus in Rhodobacter sphaeroides 2.4.1. Thus, the precise role of FnrL in photosynthesis (PS) gene expression and its interaction(s) with other regulators of PS gene expression are of considerable importance to our understanding of the regulatory circuitry governing spectral complex formation. Using a CcoP and FnrL double mutant strain, we obtained results which suggested that FnrL is not involved in the transduction of the inhibitory signal, by which PS gene expression is "silenced," emanating from the cbb(3) oxidase encoded by the ccoNOQP operon under aerobic conditions. The dominant effect of the ccoP mutation in the FnrL mutant strain with respect to spectral complex formation under aerobic conditions and restoration of a PS-positive phenotype suggested that inactivation of the cbb(3) oxidase to some extent bypasses the requirement for FnrL in the formation of spectral complexes. Additional analyses revealed that anaerobic induction of the bchE, hemN, and hemZ genes, which are involved in the tetrapyrrole biosynthetic pathways, requires FnrL. Thus, FnrL appears to be involved at multiple loci involved in the regulation of PS gene expression. Additionally, bchE was also shown to be regulated by the PrrBA two-component system, in conjunction with hemN and hemZ. These and other results to be discussed permit us to more accurately describe the role of FnrL as well as the interactions between the FnrL, PrrBA, and other regulatory circuits in the regulation of PS gene expression.  (+info)

The Crd1 gene encodes a putative di-iron enzyme required for photosystem I accumulation in copper deficiency and hypoxia in Chlamydomonas reinhardtii. (6/92)

Chlamydomonas reinhardtii adapts to copper deficiency by degrading apoplastocyanin and inducing Cyc6 and Cpx1 encoding cytochrome c(6) and coproporphyrinogen oxidase, respectively. To identify other components in this pathway, colonies resulting from insertional mutagenesis were screened for copper- conditional phenotypes. Twelve crd (copper response defect) strains were identified. In copper-deficient conditions, the crd strains fail to accumulate photosystem I and light-harvesting complex I, and they contain reduced amounts of light-harvesting complex II. Cyc6, Cpx1 expression and plastocyanin accumulation remain copper responsive. The crd phenotype is rescued by a similar amount of copper as is required for repression of Cyc6 and Cpx1 and for maintenance of plastocyanin at its usual stoichiometry, suggesting that the affected gene is a target of the same signal transduction pathway. The crd strains represent alleles at a single locus, CRD1, which encodes a 47 kDa, hydrophilic protein with a consensus carboxylate-bridged di-iron binding site. Crd1 homologs are present in the genomes of photosynthetic organisms. In Chlamydomonas, Crd1 expression is activated in copper- or oxygen-deficient cells, and Crd1 function is required for adaptation to these conditions.  (+info)

Regulation of the expression of human ferrochelatase by intracellular iron levels. (7/92)

Mammalian ferrochelatase, the terminal enzyme of the heme biosynthetic pathway, catalyzes the insertion of a ferrous ion into protoporphyrin and contains a labile [2Fe-2S] cluster center at the C-terminus. To clarify the roles of the iron-sulfur cluster in the expression of mammalian ferrochelatase, enzyme activity in human erythroleukemia K562 cells under iron-depleted conditions was examined. Treatment of cells with an iron chelator, desferrioxamine, resulted in a decrease in enzyme activity, in a dose- and time-dependent manner. Heme content decreased during desferrioxamine treatment of the cells. Addition of ferric ion-nitrilotriacetate [Fe (III)NTA] to desferrioxamine-containing cultures led to restoration of the reduction in the enzyme activity. While RNA blots showed that the amount of ferrochelatase mRNA remained unchanged during these treatments, the amount of ferrochelatase decreased with a concomitant decrease in enzyme activity. When full-length human ferrochelatase was expressed in Cos7 cells, the activity was found mainly in the mitochondria and was decreased markedly by treatment with desferrioxamine. The activity in Cos7 cells expressing human ferrochelatase in cytoplasm decreased with desferrioxamine, but to a lesser extent. When Escherichia coli ferrochelatase, which lacks the iron-sulfur cluster, was expressed in Cos7 cells, the activity did not change following any treatment. Conversely, the addition of Fe (III)NTA to the culture of K562 and Cos7 cells led to an increase in ferrochelatase activity. These results indicate that the expression of mammalian ferrochelatase is regulated by intracellular iron levels, via the iron-sulfur cluster center at the C-terminus, and this contributes to the regulation of the biosynthesis of heme at the terminal step.  (+info)

One of two hemN genes in Bradyrhizobium japonicum is functional during anaerobic growth and in symbiosis. (8/92)

Previously, we screened the symbiotic gene region of the Bradyrhizobium japonicum chromosome for new NifA-dependent genes by competitive DNA-RNA hybridization (A. Nienaber, A. Huber, M. Gottfert, H. Hennecke, and H. M. Fischer, J. Bacteriol. 182:1472-1480, 2000). Here we report more details on one of the genes identified, a hemN-like gene (now called hemN(1)) whose product exhibits significant similarity to oxygen-independent coproporphyrinogen III dehydrogenases involved in heme biosynthesis in facultatively anaerobic bacteria. In the course of these studies, we discovered that B. japonicum possesses a second hemN-like gene (hemN(2)), which was then cloned by using hemN(1) as a probe. The hemN(2) gene maps outside of the symbiotic gene region; it is located 1.5 kb upstream of nirK, the gene for a Cu-containing nitrite reductase. The two deduced HemN proteins are similar in size (445 and 450 amino acids for HemN(1) and HemN(2), respectively) and share 53% identical (68% similar) amino acids. Expression of both hemN genes was monitored with the help of chromosomally integrated translational lacZ fusions. No significant expression of either gene was detected in aerobically grown cells, whereas both genes were strongly induced (> or = 20-fold) under microaerobic or anaerobic conditions. Induction was in both cases dependent on the transcriptional activator protein FixK(2). In addition, maximal anaerobic hemN(1) expression was partially dependent on NifA, which explains why this gene had been identified by the competitive DNA-RNA hybridization approach. Strains were constructed carrying null mutations either in individual hemN genes or simultaneously in both genes. All mutants showed normal growth in rich medium under aerobic conditions. Unlike the hemN(1) mutant, strains lacking a functional hemN(2) gene were unable to grow anaerobically under nitrate-respiring conditions and largely failed to fix nitrogen in symbiosis with the soybean host plant. Moreover, these mutants lacked several c-type cytochromes which are normally detectable by heme staining of proteins from anaerobically grown wild-type cells. Taken together, our results revealed that B. japonicum hemN(2), but not hemN(1), encodes a protein that is functional under the conditions tested, and this conclusion was further corroborated by the successful complementation of a Salmonella enterica serovar Typhimurium hemF hemN mutant with hemN(2) only.  (+info)

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