Characterization of an Operon Encoding Two c-Type Cytochromes, an aa3-Type Cytochrome Oxidase, and Rusticyanin in Thiobacillus ferrooxidansATCC 33020

1999 ◽  
Vol 65 (11) ◽  
pp. 4781-4787 ◽  
Author(s):  
Corinne Appia-Ayme ◽  
Nicolas Guiliani ◽  
Jeanine Ratouchniak ◽  
Violaine Bonnefoy
Keyword(s):  
Electron Transfer ◽  
Thiobacillus Ferrooxidans ◽  
Energetic Metabolism ◽  
Reading Frame ◽  
Electron Transfer Proteins ◽  
Reverse Transcription Pcr ◽  
Genes Encoding ◽  
Transcriptional Start Sites ◽  
Transfer Chain

ABSTRACT Despite the importance of Thiobacillus ferrooxidans in bioremediation and bioleaching, little is known about the genes encoding electron transfer proteins implicated in its energetic metabolism. This paper reports the sequences of the fourcox genes encoding the subunits of anaa 3-type cytochrome c oxidase. These genes are in a locus containing four other genes:cyc2, which encodes a high-molecular-weight cytochromec; cyc1, which encodes ac 4-type cytochrome (c 552); open reading frame 1, which encodes a putative periplasmic protein of unknown function; and rus, which encodes rusticyanin. The results of Northern and reverse transcription-PCR analyses indicated that these eight genes are cotranscribed. Two transcriptional start sites were identified for this operon. Upstream from each of the start sites was a ς70-type promoter recognized in Escherichia coli. While transcription in sulfur-grown T. ferrooxidans cells was detected from the two promoters, transcription in ferrous-iron-grown T. ferrooxidans cells was detected only from the downstream promoter. The cotranscription of seven genes encoding redox proteins suggests that all these proteins are involved in the same electron transfer chain; a model taking into account the biochemistry and the genetic data is discussed.

scholarly journals Characterization of P450 FcpC, the Enzyme Responsible for Bioconversion of Diosgenone to Isonuatigenone in Streptomyces virginiae IBL-14

2009 ◽  
Vol 75 (12) ◽  
pp. 4202-4205 ◽  
Author(s):  
Wei Wang ◽  
Feng-Qing Wang ◽  
Dong-Zhi Wei
Keyword(s):  
Escherichia Coli ◽  
Electron Transfer ◽  
Cytochrome P450 ◽  
Cytochrome P450 Monooxygenase ◽  
P450 Monooxygenase ◽  
Whole Cell ◽  
Electron Transfer Chain ◽  
Streptomyces Virginiae ◽  
Transfer Chain

ABSTRACT A new cytochrome P450 monooxygenase, FcpC, from Streptomyces virginiae IBL-14 has been identified. This enzyme is found to be responsible for the bioconversion of a pyrano-spiro steroid (diosgenone) to a rare nuatigenin-type spiro steroid (isonuatigenone), which is a novel C-25-hydroxylated diosgenone derivative. A whole-cell P450 system was developed for the production of isonuatigenone via the expression of the complete three-component electron transfer chain in an Escherichia coli strain.

Characterization of Electron Transfer Proteins

1995 ◽  
pp. 113-149 ◽  
Author(s):  
Liang Chen ◽  
Ming-Y. Liu ◽  
Jean Le Gall
Keyword(s):  
Electron Transfer ◽  
Electron Transfer Proteins

scholarly journals Molecular Characterization of a Flagellar Export Locus of Helicobacter pylori

1999 ◽  
Vol 67 (5) ◽  
pp. 2060-2070 ◽  
Author(s):  
Steffen Porwollik ◽  
Brian Noonan ◽  
Paul W. O’Toole
Keyword(s):  
Helicobacter Pylori ◽  
Virulence Factor ◽  
Housekeeping Genes ◽  
Reading Frame ◽  
Reverse Transcription Pcr ◽  
Mutant Strains ◽  
H Pylori ◽  
Export Protein ◽  
Successful Colonization

ABSTRACT Motility of Helicobacter species has been shown to be essential for successful colonization of the host. We have investigated the organization of a flagellar export locus in Helicobacter pylori. A 7-kb fragment of the H. pylori CCUG 17874 genome was cloned and sequenced, revealing an operon comprising an open reading frame of unknown function (ORF03), essential housekeeping genes (ileS and murB), flagellar export genes (fliI and fliQ), and a homolog to a gene implicated in virulence factor transport in other pathogens (virB11). A promoter for this operon, showing similarity to the Escherichia coli ς70 consensus, was identified by primer extension. Cotranscription of the genes in the operon was demonstrated by reverse transcription-PCR, and transcription of virB11, fliI, fliQ, andmurB was detected in human or mouse biopsies obtained from infected hosts. The genetic organization of this locus was conserved in a panel of H. pylori clinical isolates. EngineeredfliI and fliQ mutant strains were completely aflagellate and nonmotile, whereas a virB11 mutant still produced flagella. The fliI and fliQ mutant strains produced reduced levels of flagellin and the hook protein FlgE. Production of OMP4, a member of the outer membrane protein family identified in H. pylori 26695, was reduced in both thevirB11 mutant and the fliI mutant, suggesting related functions of the virulence factor export protein (VirB11) and the flagellar export component (FliI).

scholarly journals Identification and Preliminary Characterization of Two cDNAs Encoding Unique Carbonic Anhydrases from the Marine Alga Emiliania huxleyi

2006 ◽  
Vol 72 (8) ◽  
pp. 5500-5511 ◽  
Author(s):  
Amelia R. Soto ◽  
Hong Zheng ◽  
Dorinda Shoemaker ◽  
Jason Rodriguez ◽  
Betsy A. Read ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Time Course ◽  
Dissolved Inorganic Carbon ◽  
Emiliania Huxleyi ◽  
Carbonic Anhydrases ◽  
Reading Frame ◽  
Preliminary Characterization ◽  
Reverse Transcription Pcr ◽  
Ecological Importance

ABSTRACT Marine coccolithophorid algae are thought to play a significant role in carbon cycling due to their ability to incorporate dissolved inorganic carbon (DIC) into both calcite and photosynthetic products. Among coccolithophorids, Emiliania huxleyi is the most prolific, forming massive blooms that affect the global environment. In addition to its ecological importance, the elaborate calcite structures (coccoliths) are being investigated for the design of potential materials for science and biotechnological devices. To date, most of the research focus in this organism has involved the partitioning of DIC between calcification and photosynthesis, primarily using measurements of an external versus internal carbonic anhydrase (CA) activity under defined conditions. The actual genes, proteins, and pathways employed in these processes have not been identified and characterized (see the work of Quinn et al. in this issue [P. Quinn, R. M. Bowers, X. Zhang, T. M. Wahlund, M. A. Fanelli, D. Olszova, and B. A. Read, Appl. Environ. Microbiol. 72:5512-5526, 2006]). In this study, the cloning and preliminary characterization of two genetically distinct carbonic anhydrase cDNAs are described. Phylogenetic analysis indicated that these two genes belonged to the gamma (γ-EhCA2) and delta (δ-EhCA1) classes of carbonic anhydrases. The deduced amino acid sequence of δ-EhCA1 revealed that it encodes a protein of 702 amino acids (aa) (ca. 77.3 kDa), with a transmembrane N-terminal region of 373 aa and an in-frame C-terminal open reading frame of 329 aa that defines the CA region. The γ-EhCA2 protein was 235 aa in length (ca. 24.9 kDa) and was successfully expressed in Escherichia coli BL21(DE3) and purified as an active recombinant CA. The expression levels of each transcript from quantitative reverse transcription-PCR experiments under bicarbonate limitation and over a 24-h time course suggest that these isozymes perform different functions in E. huxleyi.

scholarly journals Molecular cloning and characterization of CIDE-3, a novel member of the cell-death-inducing DNA-fragmentation-factor (DFF45)-like effector family

2003 ◽  
Vol 370 (1) ◽  
pp. 195-203 ◽  
Author(s):  
Liang LIANG ◽  
Mujun ZHAO ◽  
Zhenhua XU ◽  
Kazunari K. YOKOYAMA ◽  
Tsaiping LI
Keyword(s):  
Amino Acids ◽  
Cell Death ◽  
Small Intestine ◽  
Dna Fragmentation ◽  
Nuclear Dna ◽  
Fluorescent Protein ◽  
Reading Frame ◽  
Reverse Transcription Pcr ◽  
Green Fluorescent

DNA fragmentation is one of the critical steps in apoptosis, which is induced by DNA fragmentation factor (DFF). DFF is composed of two subunits, a 40kDa caspase-activated nuclease (DFF40) and a 45kDa inhibitor (DFF45). Recently a novel family of cell-death-inducing DFF45-like effectors (CIDEs) has been identified. Among CIDEs, two from human (CIDE-A and CIDE-B) and three from mouse (CIDE-A, CIDE-B and FSP27) have been reported. In this study human CIDE-3, a novel member of CIDEs, was identified upon sequence analysis of a previously unidentified cDNA that encoded a protein of 238 amino acids. It was shown to be a human homologue of mouse FSP27, and shared homology with the CIDE-N and CIDE-C domains of CIDEs. Apoptosis-inducing activity was clearly shown by DNA-fragmentation assay of the nuclear DNA of CIDE-3 transfected 293T cells. The expression pattern of CIDE-3 was different from that of CIDE-B. As shown by Northern-blot analysis, CIDE-3 was expressed mainly in human small intestine, heart, colon and stomach, while CIDE-B showed strong expression in liver and small intestine and at a lower level in colon, kidney and spleen. Green-fluorescent-protein-tagged CIDE-3 was revealed in some cytosolic corpuscles. Alternative splicing of the CIDE-3 gene was also identified by reverse transcription PCR, revealing that two transcripts, CIDE-3 and CIDE-3α, were present in HepG2 and A375 cells. CIDE-3 comprised a full-length open reading frame with 238 amino acids; in CIDE-3α exon 3 was deleted and it encoded a protein of 164 amino acids. Interestingly the CIDE-3α isoform still kept the apoptosis-inducing activity and showed the same pattern of subcellular localization as CIDE-3. Consistent with its chromosome localization at 3p25, a region associated with high frequency loss of heterozygosity in many tumours, CIDE-3 may play an important role in prevention of tumorigenesis.

Characterization of a Second Ornithine Decarboxylase Isolated from Morganella morganii

2008 ◽  
Vol 71 (3) ◽  
pp. 657-661 ◽  
Author(s):  
BLANCA DE LAS RIVAS ◽  
RAMÓN GONZÁLEZ ◽  
JOSÉ MARÍA LANDETE ◽  
ROSARIO MUÑOZ
Keyword(s):  
Ornithine Decarboxylase ◽  
Growth Conditions ◽  
Open Reading Frame ◽  
Morganella Morganii ◽  
Reading Frame ◽  
Reverse Transcription Pcr ◽  
Acid Protein ◽  
New Gene ◽  
Encoding Genes

The genes involved in the putrescine formation by Morganella morganii were investigated because putrescine is an indicator of food process deterioration. We report here on the existence of a new gene for ornithine decarboxylase (ODC) in M. morganii. The sequenced 5,311-bp DNA region showed the presence of four complete and one partial open reading frame. Putative functions have been assigned to several gene products by sequence comparison with the proteins included in the databases. The third open reading frame (speC ) encoded a 722–amino acid protein showing 70.9% identity to the M. morganii ODC previously characterized (SpeF ).The speC gene has been expressed in Escherichia coli, resulting in ODC activity. The presence of a functional promoter (PspeC ) located upstream of speC has been demonstrated. Quantitative real-time reverse transcription PCR assay was used to quantify expression of both M. morganii ODC-encoding genes, speC and speF, under different growth conditions. This assay allows us to identify SpeF as the inducible M. morganii ODC, since it was highly expressed in the presence of ornithine.

scholarly journals Identification and Characterization of EctR1, a New Transcriptional Regulator of the Ectoine Biosynthesis Genes in the Halotolerant Methanotroph Methylomicrobium alcaliphilum 20Z

2009 ◽  
Vol 192 (2) ◽  
pp. 410-417 ◽  
Author(s):  
Ildar I. Mustakhimov ◽  
Alexander S. Reshetnikov ◽  
Anatoly S. Glukhov ◽  
Valentina N. Khmelenina ◽  
Marina G. Kalyuzhnaya ◽  
...  
Keyword(s):  
Binding Site ◽  
Transcriptional Regulator ◽  
Negative Regulator ◽  
Reading Frame ◽  
Genes Encoding ◽  
Methylomicrobium Alcaliphilum ◽  
Translational Start ◽  
Identification And Characterization ◽  
Ectoine Biosynthesis

ABSTRACTGenes encoding key enzymes of the ectoine biosynthesis pathway in the halotolerant obligate methanotrophMethylomicrobium alcaliphilum20Z have been shown to be organized into anectABC-askoperon. Transcription of theectoperon is initiated from two promoters,ectAp1andectAp2(ectAp1p2), similar to the σ70-dependent promoters ofEscherichia coli. Upstream of the gene cluster, an open reading frame (ectR1) encoding a MarR-like transcriptional regulator was identified. Investigation of the influence of EctR1 on the activity of theectAp1p2promoters in wild-typeM. alcaliphilum20Z andectR1mutant strains suggested that EctR1 is a negative regulator of theectABC-askoperon. Purified recombinant EctR1-His6specifically binds as a homodimer to the putative −10 motif of theectAp1promoter. The EctR1 binding site contains a pseudopalindromic sequence (TATTTAGT-GT-ACTATATA) composed of 8-bp half-sites separated by 2 bp. Transcription of theectR1gene is initiated from a single σ70-like promoter. The location of the EctR1 binding site between the transcriptional and translational start sites of theectR1gene suggests that EctR1 may regulate its own expression. The data presented suggest that inMethylomicrobium alcaliphilum20Z, EctR1-mediated control of the transcription of theectgenes is not the single mechanism for the regulation of ectoine biosynthesis.

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