scholarly journals RegA Control of Bacteriochlorophyll and Carotenoid Synthesis in Rhodobacter capsulatus

2007 ◽  
Vol 189 (21) ◽  
pp. 7765-7773 ◽  
Author(s):  
Jonathan Willett ◽  
James L. Smart ◽  
Carl E. Bauer
Keyword(s):  
Rhodobacter Capsulatus ◽  
Protoporphyrin Ix ◽  
Carotenoid Biosynthesis ◽  
Divergent Promoters ◽  
Anaerobic Induction ◽  
Single Region ◽  
Phytoene Dehydrogenase ◽  
Carotenoid Synthesis

ABSTRACT We provide in vivo genetic and in vitro biochemical evidence that RegA directly regulates bacteriochlorophyll and carotenoid biosynthesis in Rhodobacter capsulatus. β-Galactosidase expression assays with a RegA-disrupted strain containing reporter plasmids for Mg-protoporphyrin IX monomethyl ester oxidative cyclase (bchE), Mg-protoporphyrin IX chelatase (bchD), and phytoene dehydrogenase (crtI) demonstrate RegA is responsible for fourfold anaerobic induction of bchE, threefold induction of bchD, and twofold induction of crtI. Promoter mapping studies, coupled with DNase I protection assays, map the region of RegA binding to three sites in the bchE promoter region. Similar studies at the crtA and crtI promoters indicate that RegA binds to a single region equidistant from these divergent promoters. These results demonstrate that RegA is directly responsible for anaerobic induction of bacteriochlorophyll biosynthesis genes bchE, bchD, bchJ, bchI, bchG, and bchP and carotenoid biosynthesis genes crtI, crtB, and crtA.

scholarly journals Mg-protoporphyrin IX monomethyl ester cyclase from Rhodobacter capsulatus: radical SAM-dependent synthesis of the isocyclic ring of bacteriochlorophylls

2020 ◽  
Vol 477 (23) ◽  
pp. 4635-4654
Author(s):  
Milan Wiesselmann ◽  
Stefanie Hebecker ◽  
José M. Borrero-de Acuña ◽  
Manfred Nimtz ◽  
David Bollivar ◽  
...  
Keyword(s):  
Rhodobacter Capsulatus ◽  
Protoporphyrin Ix ◽  
Three Dimensional ◽  
Homology Model ◽  
Subcellular Fractionation ◽  
Site Directed Mutagenesis ◽  
Bioinformatics Analyses ◽  
Monomethyl Ester

During bacteriochlorophyll a biosynthesis, the oxygen-independent conversion of Mg-protoporphyrin IX monomethyl ester (Mg-PME) to protochlorophyllide (Pchlide) is catalyzed by the anaerobic Mg-PME cyclase termed BchE. Bioinformatics analyses in combination with pigment studies of cobalamin-requiring Rhodobacter capsulatus mutants indicated an unusual radical S-adenosylmethionine (SAM) and cobalamin-dependent BchE catalysis. However, in vitro biosynthesis of the isocyclic ring moiety of bacteriochlorophyll using purified recombinant BchE has never been demonstrated. We established a spectroscopic in vitro activity assay which was subsequently validated by HPLC analyses and H218O isotope label transfer onto the carbonyl-group (C-131-oxo) of the isocyclic ring of Pchlide. The reaction product was further converted to chlorophyllide in the presence of light-dependent Pchlide reductase. BchE activity was stimulated by increasing concentrations of NADPH or SAM, and inhibited by S-adenosylhomocysteine. Subcellular fractionation experiments revealed that membrane-localized BchE requires an additional, heat-sensitive cytosolic component for activity. BchE catalysis was not sustained in chimeric experiments when a cytosolic extract from E. coli was used as a substitute. Size-fractionation of the soluble R. capsulatus fraction indicated that enzymatic activity relies on a specific component with an estimated molecular mass between 3 and 10 kDa. A structure guided site-directed mutagenesis approach was performed on the basis of a three-dimensional homology model of BchE. A newly established in vivo complementation assay was used to investigate 24 BchE mutant proteins. Potential ligands of the [4Fe-4S] cluster (Cys204, Cys208, Cys211), of SAM (Phe210, Glu308 and Lys320) and of the proposed cobalamin cofactor (Asp248, Glu249, Leu29, Thr71, Val97) were identified.

The protective properties of synthetic porphyrin tin complexes in toxic hyperbilirubinemia

2000 ◽  
Vol 04 (03) ◽  
pp. 243-247 ◽  
Author(s):  
T. O. PHILIPPOVA ◽  
B. N. GALKIN ◽  
N. YA. GOLOVENKO ◽  
Z. I. ZHILINA ◽  
S. V. VODZINSKII
Keyword(s):  
Heme Oxygenase ◽  
Serum Bilirubin ◽  
Protoporphyrin Ix ◽  
Serum Bilirubin Level ◽  
Oxygenase Activity ◽  
Tetraphenyl Porphyrin ◽  
Tin Complexes ◽  
Cytochrome P 450

Tin complexes of meso-substituted synthetic porphyrins, namely Sn 4+-meso-tetraphenyl- porphyrin ( Sn - TPP ) and Sn 4+-meso-tetrakis(N-methyl-3-pyridyl)porphyrin tetratosylate ( Sn - TMe -3- PyP ), efficiently decrease the serum bilirubin level when injected subcutaneously at a dose of 100 μM kg−1 body weight into mice. These compounds are active during hyperbilirubinemia, induced by phenylhydrazine, hemin and tetrachloromethane, and also during autoimmune hemolytic anemia. In the latter case a decrease in serum bilirubin content was observed, as well as a decrease in the amount of blood reticulocytes which reflects a milder course of the disease. The Sn complexes under study induce, in vivo, cytochrome P-450, inhibit microsomal heme oxygenase and decrease the intensity of lipid peroxidation. At the same time, in vitro the hepatic and splenic heme oxygenase activity is blocked only when a 0.1 μM concentration of Sn - TMe -3- PyP or Sn -protoporphyrin IX is added to the incubation mixture. Sn - TPP does not affect the activity of this enzyme in vitro.

In vitro and in vivo effects of HAL on porphyrin production in rat bladder cancer cells (AY27)

2019 ◽  
Vol 23 (07n08) ◽  
pp. 813-820
Author(s):  
Odrun A. Gederaas ◽  
Harald Husebye ◽  
Anders Johnsson ◽  
Susan Callaghan ◽  
Anders Brunsvik
Keyword(s):  
Bladder Cancer ◽  
Cancer Cells ◽  
Aminolevulinic Acid ◽  
Protoporphyrin Ix ◽  
Active Agent ◽  
Laser Scanning Microscopy ◽  
Rat Bladder ◽  
Bladder Cancer Cells

Aminolevulinic acid and hexyl-aminolevulinate serve as biological precursors to produce photosensitive porphyrins in cells via the heme biosynthetic pathway. This pathway is integral to porphyrin-based photodynamic diagnosis and therapy. By adding exogenous hexyl-aminolevulinate to rat bladder cancer cells (AY27, in vitro) and an animal bladder cancer model (in vivo), fluorescent endogenous porphyrin production was stimulated. Lipophilic protoporphyrin IX was identified as the dominant species by reverse high-pressure liquid chromatography. Subcellular porphyrin localization in the AY27 cells was evaluated by confocal laser scanning microscopy and showed almost quantitative bleaching after 20 s. From this study, we ascertained that the protocol described herein is suitable for hexyl-aminolevulinate-mediated photodynamic therapy and diagnosis when protoporphyrin IX is the active agent.

scholarly journals RNase III Processing of Intervening Sequences Found in Helix 9 of 23S rRNA in the Alpha Subclass of Proteobacteria

2000 ◽  
Vol 182 (17) ◽  
pp. 4719-4729 ◽  
Author(s):  
Elena Evguenieva-Hackenberg ◽  
Gabriele Klug
Keyword(s):  
Rhodobacter Capsulatus ◽  
23S Rrna ◽  
Recent Attempt ◽  
Rnase Iii ◽  
E Coli ◽  
Rdna Sequences ◽  
Intervening Sequences ◽  
Rna Maturation

ABSTRACT We provide experimental evidence for RNase III-dependent processing in helix 9 of the 23S rRNA as a general feature of many species in the alpha subclass of Proteobacteria(alpha-Proteobacteria). We investigated 12Rhodobacter, Rhizobium,Sinorhizobium, Rhodopseudomonas, andBartonella strains. The processed region is characterized by the presence of intervening sequences (IVSs). The 23S rDNA sequences between positions 109 and 205 (Escherichia coli numbering) were determined, and potential secondary structures are proposed. Comparison of the IVSs indicates very different evolutionary rates in some phylogenetic branches, lateral genetic transfer, and evolution by insertion and/or deletion. We show that the IVS processing inRhodobacter capsulatus in vivo is RNase III-dependent and that RNase III cleaves additional sites in vitro. While all IVS-containing transcripts tested are processed in vitro by RNase III from R. capsulatus, E. coli RNase III recognizes only some of them as substrates and in these substrates frequently cleaves at different scissile bonds. These results demonstrate the different substrate specificities of the two enzymes. Although RNase III plays an important role in the rRNA, mRNA, and bacteriophage RNA maturation, its substrate specificity is still not well understood. Comparison of the IVSs of helix 9 does not hint at sequence motives involved in recognition but reveals that the “antideterminant” model, which represents the most recent attempt to explain the E. coli RNase III specificity in vitro, cannot be applied to substrates derived from alpha-Proteobacteria.

Redox-controlled, in vivo and in vitro phosphorylation of the ? subunit of the light-harvesting complex I in Rhodobacter capsulatus

1992 ◽  
Vol 158 (5) ◽  
pp. 315-319 ◽  
Author(s):  
Nestor Cortez ◽  
Augusto F. Garcia ◽  
Monier H. Tadros ◽  
Nasser Gad'on ◽  
Emil Schiltz ◽  
...  
Keyword(s):  
Complex I ◽  
Rhodobacter Capsulatus ◽  
Light Harvesting ◽  
Light Harvesting Complex

scholarly journals The CckA-ChpT-CtrA phosphorelay controlling Rhodobacter capsulatus gene transfer agent (RcGTA) production is bi-directional and regulated by cyclic-di-GMP

2020 ◽  
Author(s):  
Reynold G. Farrera-Calderon ◽  
Purvikalyan Pallegar ◽  
Alexander B. Westbye ◽  
Christina Wiesmann ◽  
Andrew S. Lang ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Phosphatase Activity ◽  
Regulatory Networks ◽  
Rhodobacter Capsulatus ◽  
Response Regulator ◽  
Evolutionary Significance ◽  
Secondary Messenger ◽  
Gene Transfer Agent

Protein phosphorylation is a universal mechanism for transducing cellular signals in prokaryotes and eukaryotes. The histidine kinase CckA, histidine phosphotransferase ChpT and response regulator CtrA are conserved throughout the alphaproteobacteria. In Rhodobacter capsulatus these proteins are key regulators of the gene transfer agent (RcGTA), which is present in several alphaproteobacteria. Using purified recombinant R. capsulatus proteins, we show in vitro autophosphorylation of CckA protein, and phosphotransfer to ChpT and thence to CtrA to biochemically demonstrate that they form a phosphorelay. The secondary messenger cyclic-di-GMP changed CckA from a kinase to a phosphatase resulting in reversal of the phosphotransfer flow in the relay. The substitutions of two residues in CckA greatly affected the kinase or phosphatase activity of the protein in vitro, and production of mutant CckA proteins in vivo confirmed the importance of kinase but not phosphatase activity for lytic release of RcGTA. The binding of cyclic-di-GMP to the wild type and mutant CckA proteins was evaluated directly using a pull-down assay based on biotinylated cyclic-di-GMP and streptavidin-linked beads. IMPORTANCE The CckA, ChpT and CtrA phosphorelay proteins are widespread in the alphaproteobacteria, and there are two groups of organisms that differ in terms of whether this pathway is essential for cell viability. Little is known about the biochemical function of these proteins in organisms where the pathway is not essential, a group that includes Rhodobacter capsulatus. This work biochemically demonstrates that CckA, ChpT and CtrA also form a functional phosphorelay in this latter group, and that the direction of phosphotransfer is reversed by cyclic-di-GMP. It is important to improve the understanding of more representatives of this pathway to obtain a deeper insight into the function, composition, and evolutionary significance of a wider range of bacterial regulatory networks.

scholarly journals Effector-Mediated Interaction of CbbRI and CbbRII Regulators with Target Sequences in Rhodobacter capsulatus

2004 ◽  
Vol 186 (23) ◽  
pp. 8026-8035 ◽  
Author(s):  
Padungsri Dubbs ◽  
James M. Dubbs ◽  
F. Robert Tabita
Keyword(s):  
Rhodobacter Capsulatus ◽  
Energy Charge ◽  
Dnase I ◽  
Pentose Phosphate ◽  
In Vivo Studies ◽  
Mobility Shift ◽  
Fructose 1,6 Bisphosphate ◽  
Target Promoters

ABSTRACT In Rhodobacter capsulatus, genes encoding enzymes of the Calvin-Benson-Bassham reductive pentose phosphate pathway are located in the cbbI and cbbII operons. Each operon contains a divergently transcribed LysR-type transcriptional activator (CbbRI and CbbRII) that regulates the expression of its cognate cbb promoter in response to an as yet unidentified effector molecule(s). Both CbbRI and CbbRII were purified, and the ability of a variety of potential effector molecules to induce changes in their DNA binding properties at their target promoters was assessed. The responses of CbbRI and CbbRII to potential effectors were not identical. In gel mobility shift assays, the affinity of both CbbRI and CbbRII for their target promoters was enhanced in the presence of ribulose-1,5-bisphosphate (RuBP), phosphoenolpyruvate, 3-phosphoglycerate, 2-phosphoglycolate. ATP, 2-phosphoglycerate, and KH2PO4 were found to enhance only CbbRI binding, while fructose-1,6-bisphosphate enhanced the binding of only CbbRII. The DNase I footprint of CbbRI was reduced in the presence of RuBP, while reductions in the CbbRII DNase I footprint were induced by fructose-1,6-bisphosphate, 3-phosphoglycerate, and KH2PO4. The current in vitro results plus recent in vivo studies suggest that CbbR-mediated regulation of cbb transcription is controlled by multiple metabolic signals in R. capsulatus. This control reflects not only intracellular levels of Calvin-Benson-Bassham cycle metabolic intermediates but also the fixed (organic) carbon status and energy charge of the cell.

scholarly journals Regulation and Characterization of Two Nitroreductase Genes, nprA and nprB, of Rhodobacter capsulatus

2005 ◽  
Vol 71 (12) ◽  
pp. 7643-7649 ◽  
Author(s):  
Eva Pérez-Reinado ◽  
Rafael Blasco ◽  
Francisco Castillo ◽  
Conrado Moreno-Vivián ◽  
M. Dolores Roldán
Keyword(s):  
Gene Expression ◽  
Salicylic Acid ◽  
Rhodobacter Capsulatus ◽  
Sequence Data ◽  
Regulatory System ◽  
Wide Range ◽  
Nitrofuran Derivatives ◽  
Response To Stress

ABSTRACT Among photosynthetic bacteria, strains B10 and E1F1 of Rhodobacter capsulatus photoreduce 2,4-dinitrophenol (DNP), which is stoichiometrically converted into 2-amino-4-nitrophenol by a nitroreductase activity. The reduction of DNP is inhibited in vivo by ammonium, which probably acts at the level of the DNP transport system and/or physiological electron transport to the nitroreductase, since this enzyme is not inhibited by ammonium in vitro. Using the complete genome sequence data for strain SB1003 of R. capsulatus, two putative genes coding for possible nitroreductases were isolated from R. capsulatus B10 and disrupted. The phenotypes of these mutant strains revealed that both genes are involved in the reduction of DNP and code for two major nitroreductases, NprA and NprB. Both enzymes use NAD(P)H as the main physiological electron donor. The nitroreductase NprA is under ammonium control, whereas the nitroreductase NprB is not. In addition, the expression of the nprB gene seems to be constitutive, whereas nprA gene expression is inducible by a wide range of nitroaromatic and heterocyclic compounds, including several dinitroaromatics, nitrofuran derivatives, CB1954, 2-aminofluorene, benzo[a]pyrene, salicylic acid, and paraquat. The identification of two putative mar/sox boxes in the possible promoter region of the nprA gene and the induction of nprA gene expression by salicylic acid and 2,4-dinitrophenol suggest a role in the control of the nprA gene for the two-component MarRA regulatory system, which in Escherichia coli controls the response to some antibiotics and environmental contaminants. In addition, upregulation of the nprA gene by paraquat indicates that this gene is probably a member of the SoxRS regulon, which is involved in the response to stress conditions in other bacteria.

scholarly journals Corynebacterium glutamicum CrtR and Its Orthologs in Actinobacteria: Conserved Function and Application as Genetically Encoded Biosensor for Detection of Geranylgeranyl Pyrophosphate

2020 ◽  
Vol 21 (15) ◽  
pp. 5482 ◽  
Author(s):  
Nadja A. Henke ◽  
Sophie Austermeier ◽  
Isabell L. Grothaus ◽  
Susanne Götker ◽  
Marcus Persicke ◽  
...  
Keyword(s):  
Deletion Mutant ◽  
Carotenoid Biosynthesis ◽  
Sequence Motif ◽  
Transcriptional Fusion ◽  
Fluorescent Signal ◽  
Geranylgeranyl Pyrophosphate ◽  
Target Dna ◽  
Genetically Encoded Biosensor

Carotenoid biosynthesis in Corynebacteriumglutamicum is controlled by the MarR-type regulator CrtR, which represses transcription of the promoter of the crt operon (PcrtE) and of its own gene (PcrtR). Geranylgeranyl pyrophosphate (GGPP), and to a lesser extent other isoprenoid pyrophosphates, interfere with the binding of CrtR to its target DNA in vitro, suggesting they act as inducers of carotenoid biosynthesis. CrtR homologs are encoded in the genomes of many other actinobacteria. In order to determine if and to what extent the function of CrtR, as a metabolite-dependent transcriptional repressor of carotenoid biosynthesis genes responding to GGPP, is conserved among actinobacteria, five CrtR orthologs were characterized in more detail. EMSA assays showed that the CrtR orthologs from Corynebacteriumcallunae, Acidipropionibacteriumjensenii, Paenarthrobacternicotinovorans, Micrococcusluteus and Pseudarthrobacterchlorophenolicus bound to the intergenic region between their own gene and the divergently oriented gene, and that GGPP inhibited these interactions. In turn, the CrtR protein from C. glutamicum bound to DNA regions upstream of the orthologous crtR genes that contained a 15 bp DNA sequence motif conserved between the tested bacteria. Moreover, the CrtR orthologs functioned in C. glutamicum in vivo at least partially, as they complemented the defects in the pigmentation and expression of a PcrtE_gfpuv transcriptional fusion that were observed in a crtR deletion mutant to varying degrees. Subsequently, the utility of the PcrtE_gfpuv transcriptional fusion and chromosomally encoded CrtR from C. glutamicum as genetically encoded biosensor for GGPP was studied. Combined FACS and LC-MS analysis demonstrated a correlation between the sensor fluorescent signal and the intracellular GGPP concentration, and allowed us to monitor intracellular GGPP concentrations during growth and differentiate between strains engineered to accumulate GGPP at different concentrations.

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