Co-ordination of iron acquisition, iron porphyrin chelation and iron–protoporphyrin export via the cytochrome c biogenesis protein CcmC in Pseudomonas fluorescens

Microbiology ◽  
2003 ◽  
Vol 149 (12) ◽  
pp. 3543-3552 ◽  
Author(s):  
Christine Baysse ◽  
Sandra Matthijs ◽  
Max Schobert ◽  
Gunhild Layer ◽  
Dieter Jahn ◽  
...  
Keyword(s):  
Pseudomonas Fluorescens ◽  
Aminolevulinic Acid ◽  
Light Exposure ◽  
Iron Acquisition ◽  
Protoporphyrin Ix ◽  
Indicator Strain ◽  
Proteinase K ◽  
Wild Type ◽  
Feeding Experiments ◽  
Iron Protoporphyrin

The cytoplasmic membrane protein CcmC is, together with other Ccm proteins, a component for the maturation of c-type cytochromes in Gram-negative bacteria. A Pseudomonas fluorescens ATCC 17400 ccmC mutant is cytochrome c-deficient and shows considerably reduced production of the two siderophores pyoverdine and quinolobactin, paralleled by a general inability to utilize various iron sources, with the exception of haem. The ccmC mutant accumulates in a 5-aminolevulinic acid-dependent synthesis a reddish, fluorescent pigment identified as protoporphyrin IX. As a consequence a visA phenotype similar to that of a ferrochelatase-deficient hemH mutant characterized by drastically reduced growth upon light exposure was observed for the ccmC mutant. The defect of iron–protoporphyrin formation was further demonstrated by the failure of ccmC cell-free proteinase K-treated extracts to stimulate the growth of a haem auxotrophic hemH indicator strain, compared to similarly prepared wild-type extracts. In addition, the ccmC mutant did not sustain hemH growth in cross-feeding experiments while the wild-type did. Significantly reduced resistance to oxidative stress mediated by haem-containing catalases was observed for the ccmC mutant. A double hemH ccmC mutant could not be obtained in the presence of external haem without the hemH gene in trans, indicating that the combination of the two mutations is lethal. It was concluded that CcmC, apart from its known function in cytochrome c biogenesis, plays a role in haem biosynthesis. A function in the regulatory co-ordination of iron acquisition via siderophores, iron insertion into porphyrin via ferrochelatase and iron–protoporphyrin export for cytochrome c formation is predicted.

scholarly journals Uterus-specific transcriptional regulation underlies eggshell pigment production in Japanese quail

2021 ◽  
Author(s):  
Satoshi Ishishita ◽  
Shumpei Kitahara ◽  
Mayuko Takahashi ◽  
Sakura Iwasaki ◽  
Shoji Tatsumoto ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Japanese Quail ◽  
Aminolevulinic Acid ◽  
Protoporphyrin Ix ◽  
Genomic Region ◽  
Pigment Production ◽  
Wild Type ◽  
Its Gene ◽  
Uterine Mucosa ◽  
In The Wild

The precursor of heme, protoporphyrin IX (PPIX), accumulates abundantly in the uterus of birds, such as Japanese quail, Coturnix japonica, resulting in brown-speckled eggshells. The molecular basis of PPIX production in the uterus remains largely unknown. Here, we investigated the cause of low PPIX production in a classical Japanese quail mutant exhibiting white eggshells by comparing its gene expression in the uterus with that of the wild type using transcriptome analysis and performed genetic linkage mapping to identify the causative genomic region of the white eggshell phenotype. We showed that 11 genes, including the 5-aminolevulinic acid synthase 1 (ALAS1) and ferroxidase hephaestin-like 1 (HEPHL1) genes, were specifically upregulated in the wild-type uterus and downregulated in the mutant. We mapped the 172 kb candidate genomic region on chromosome 6, which contains several genes, including a part of the paired-like homeodomain 3 (PITX3), which encodes a transcription factor. ALAS1, HEPHL1, and PITX3 were expressed in the apical cells of the luminal epithelium and lamina propria cells of the uterine mucosa of the wild-type quail, and their expression was downregulated in these cells of the mutant quail. Biochemical analysis using uterine homogenates indicated that the restricted availability of 5-aminolevulinic acid is the main cause of low PPIX production. These results suggest that uterus-specific transcriptional regulation of heme-biosynthesis-related genes is an evolutionarily acquired mechanism of eggshell pigment production in Japanese quail.

Rice FLUORESCENT1 Is Involved in the Regulation of Chlorophyll

2019 ◽  
Vol 60 (10) ◽  
pp. 2307-2318
Author(s):  
Zhiyun Li ◽  
Weiping Mo ◽  
Liqiang Jia ◽  
Yong-Chao Xu ◽  
Weijiang Tang ◽  
...  
Keyword(s):  
Singlet Oxygen ◽  
Aminolevulinic Acid ◽  
Light Exposure ◽  
Chlorophyll Biosynthesis ◽  
Expression Patterns ◽  
Light Illumination ◽  
Loss Of Function ◽  
Wild Type ◽  
Genes Encoding ◽  
Important Regulator

Abstract Chlorophyll biosynthesis plays essential roles in photosynthesis and plant growth in response to environmental conditions. The accumulation of excess chlorophyll biosynthesis intermediates under light results in the production of reactive oxygen species and oxidative stress. In this study, we identified a rice (Oryza sativa) mutant, oxidation under photoperiod (oxp), that displayed photobleached lesions on its leaves, reduced growth and decreased chlorophyll content during light/dark cycles or following a dark-to-light transition. The oxp mutant accumulated more chlorophyll precursors (5-aminolevulinic acid and protochlorophyllide) than the wild type in the dark, and more singlet oxygen following light exposure. Several singlet-oxygen-responsive genes were greatly upregulated in oxp, whereas the expression patterns of OsPORA and OsPORB, two genes encoding the chlorophyll biosynthesis enzyme NADPH:protochlorop hyllide oxidoreductase, were altered in de-etiolated oxp seedlings. Molecular and complementation studies revealed that oxp is a loss-of-function mutant in LOC_Os01g32730, a homolog of FLUORESCENT (FLU) in Arabidopsis thaliana. Rice PHYTOCHROME-INTERACTING FACTOR-LIKE14 (OsPIL14) transcription factor directly bound to the OsFLU1 promoter and activated its expression. Dark-grown transgenic rice seedlings overexpressing OsPIL14 accumulated more chlorophyll and turned green faster than the wild type upon light illumination. Thus, OsFLU1 is an important regulator of chlorophyll biosynthesis in rice.

scholarly journals A Dominant Mutation in Mitochondrial Unfoldase CLPX Results in Erythropoietic Protoporphyria

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 77-77
Author(s):  
Yvette Y Yien ◽  
Gael Nicolas ◽  
Lisa van der Vorm ◽  
Laurent Gouya ◽  
Hector Begonia ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Protein Stability ◽  
Pyridoxal Phosphate ◽  
Protein Quality ◽  
Aminolevulinic Acid ◽  
Conflicts Of Interest ◽  
Mitochondrial Protein ◽  
Protoporphyrin Ix ◽  
Wild Type ◽  
Heme Synthesis

Abstract Red cells synthesize large quantities of heme during terminal differentiation. Central to erythropoiesis is heme synthesis, which requires tight coordination between mitochondrial iron import and synthesis of protoporphyrin IX (PPIX). Most individuals with erythropoietic porphyria carry loss of function mutations in FECH, or gain of function mutations in ALAS2, resulting in protoporphyrin IX accumulation. We performed whole exome sequencing to identify novel mutations in an individual exhibiting symptoms in whom FECH and ALAS2 mutations were absent (Fig. A, asterisk). We identified a novel CLPX point mutation in this individual (III.2), her father (II.4) and her paternal uncle (II.2), who also exhibited increased porphyrin levels relative to healthy individuals. The individual's mother was healthy and had a wild-type CLPX genotype (Fig. A). CLPX encodes a mitochondrial protein unfoldase that partially unfolds ALA synthase (ALAS) to allow efficient incorporation of its cofactor, pyridoxal phosphate (Kardon et al. 2015 Cell). This greatly stimulates the synthesis of d-aminolevulinic acid (ALA), the first step in heme biosynthesis. To determine if the CLPX mutation was causative for porphyria, we expressed mutant CLPX in HEK293T embryonic kidney cells and Friend mouse erythroleukemia (MEL) cells. Mutant CLPX expression (MUT) caused a significant increase in ALAS1 (non-erythroid isoform) (Fig. B) and ALAS2 (erythroid isoform) (Fig. C) activity relative to control and wild-type CLPX expressing samples (WT). This increase in ALAS enzymatic activity translated to an increase in PPIX levels (Fig. D, E), consistent with the porphyria phenotype observed in the individuals in this study. We observed that MUT-expressing samples had increased levels of ALAS1 and ALAS2. To determine if mutant CLPX altered ALAS protein stability, we transfected WT or MUT CLPX into HEK293T and MEL cells. Cells were treated with cycloheximide to block translation. We quantitated degradation rate of ALAS by western blot analysis of cell lysates obtained at several time points after cycloheximide treatment (chase). Expression of MUT CLPX stabilized both ALAS1 and ALAS2, accounting for the increase in ALAS protein levels, activity and downstream production of PPIX. The control of ALAS enzymatic activity and protein stability by CLPX unveils a novel cause of protoporphyria and insights revealing the ways in which mitochondrial physiology and heme synthesis are interdependent. Our results reveal an important regulatory node where the mitochondrial protein quality control machinery intersects with a key step in heme synthesis and provides an important genetic tool for understanding the pathology of porphyrias. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Protoporphyrin IX Fluorescence Photobleaching and the Response of Rat Barrett's Esophagus Following 5-aminolevulinic Acid Photodynamic Therapy

2006 ◽  
Vol 82 (6) ◽  
pp. 1638 ◽  
Author(s):  
Ingrid A. Boere ◽  
Dominic J. Robinson ◽  
Henriette S. de Bruijn ◽  
Jolanda Kluin ◽  
Hugo W. Tilanus ◽  
...  
Keyword(s):  
Photodynamic Therapy ◽  
Barrett’S Esophagus ◽  
Barrett's Esophagus ◽  
Aminolevulinic Acid ◽  
Protoporphyrin Ix

Kinetics of protoporphyrin IX formation in rat oral mucosa and skin after application of 5-aminolevulinic acid and its methylester

2004 ◽  
Author(s):  
Stefan Kristiansson ◽  
Asta Juzeniene ◽  
Petras Juzenas ◽  
Vladimir Iani ◽  
Lennart Löfgren ◽  
...  
Keyword(s):  
Oral Mucosa ◽  
Aminolevulinic Acid ◽  
Protoporphyrin Ix ◽  
Kinetics Of

scholarly journals The Serratia marcescens Siderophore Serratiochelin Is Necessary for Full Virulence during Bloodstream Infection

2020 ◽  
Vol 88 (8) ◽  
Author(s):  
Danelle R. Weakland ◽  
Sara N. Smith ◽  
Bailey Bell ◽  
Ashootosh Tripathi ◽  
Harry L. T. Mobley
Keyword(s):  
Serratia Marcescens ◽  
Bloodstream Infection ◽  
Iron Acquisition ◽  
Gene Clusters ◽  
Clinical Isolates ◽  
Wild Type ◽  
Serratia Plymuthica ◽  
Content Type ◽  
Cas Assay ◽  
Essential Nutrient

ABSTRACT Serratia marcescens is a bacterium frequently found in the environment, but over the last several decades it has evolved into a concerning clinical pathogen, causing fatal bacteremia. To establish such infections, pathogens require specific nutrients; one very limited but essential nutrient is iron. We sought to characterize the iron acquisition systems in S. marcescens isolate UMH9, which was recovered from a clinical bloodstream infection. Using RNA sequencing (RNA-seq), we identified two predicted siderophore gene clusters (cbs and sch) that were regulated by iron. Mutants were constructed to delete each iron acquisition locus individually and in conjunction, generating both single and double mutants for the putative siderophore systems. Mutants lacking the sch gene cluster lost their iron-chelating ability as quantified by the chrome azurol S (CAS) assay, whereas the cbs mutant retained wild-type activity. Mass spectrometry-based analysis identified the chelating siderophore to be serratiochelin, a siderophore previously identified in Serratia plymuthica. Serratiochelin-producing mutants also displayed a decreased growth rate under iron-limited conditions created by dipyridyl added to LB medium. Additionally, mutants lacking serratiochelin were significantly outcompeted during cochallenge with wild-type UMH9 in the kidneys and spleen after inoculation via the tail vein in a bacteremia mouse model. This result was further confirmed by an independent challenge, suggesting that serratiochelin is required for full S. marcescens pathogenesis in the bloodstream. Nine other clinical isolates have at least 90% protein identity to the UMH9 serratiochelin system; therefore, our results are broadly applicable to emerging clinical isolates of S. marcescens causing bacteremia.

scholarly journals Two Subpopulations of Human Monocytes That Differ by Mitochondrial Membrane Potential

Biomedicines ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 153
Author(s):  
Nikita G. Nikiforov ◽  
Anastasia Ryabova ◽  
Marina V. Kubekina ◽  
Igor D. Romanishkin ◽  
Kirill A. Trofimov ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Primary Culture ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Function ◽  
Mitochondrial Membrane ◽  
Aminolevulinic Acid ◽  
Protoporphyrin Ix ◽  
Intima Media Thickness ◽  
Mtdna Mutations ◽  
Proinflammatory Activity

Atherosclerosis is associated with a chronic local inflammatory process in the arterial wall. Our previous studies have demonstrated the altered proinflammatory activity of circulating monocytes in patients with atherosclerosis. Moreover, atherosclerosis progression and monocyte proinflammatory activity were associated with mitochondrial DNA (mtDNA) mutations in circulating monocytes. The role of mitochondria in the immune system cells is currently well recognized. They can act as immunomodulators by releasing molecules associated with bacterial infection. We hypothesized that atherosclerosis can be associated with changes in the mitochondrial function of circulating monocytes. To test this hypothesis, we performed live staining of the mitochondria of CD14+ monocytes from healthy donors and atherosclerosis patients with MitoTracker Orange CMTMRos dye, which is sensitive to mitochondrial membrane potential. The intensity of such staining reflects mitochondrial functional activity. We found that parts of monocytes in the primary culture were characterized by low MitoTracker staining (MitoTracker-low monocytes). Such cells were morphologically similar to cells with normal staining and able to metabolize 5-aminolevulinic acid and accumulate the heme precursor protoporphyrin IX (PplX), indicative of partially preserved mitochondrial function. We assessed the proportion of MitoTracker-low monocytes in the primary culture for each study subject and compared the results with other parameters, such as monocyte ability to lipopolysaccharide (LPS)-induced proinflammatory activation and the intima-media thickness of carotid arteries. We found that the proportion of MitoTracker-low monocytes was associated with the presence of atherosclerotic plaques. An increased number of such monocytes in the primary culture was associated with a reduced proinflammatory activation ability of cells. The obtained results indicate the presence of circulating monocytes with mitochondrial dysfunction and the association of such cells with chronic inflammation and atherosclerosis development.

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