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

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.

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Molecular Characterization of a Flagellar Export Locus of Helicobacter pylori

Infection and Immunity ◽

10.1128/iai.67.5.2060-2070.1999 ◽

1999 ◽

Vol 67 (5) ◽

pp. 2060-2070 ◽

Cited By ~ 31

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).

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

Biochemical Journal ◽

10.1042/bj20020656 ◽

2003 ◽

Vol 370 (1) ◽

pp. 195-203 ◽

Cited By ~ 79

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.

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Characterization of Carbonic Anhydrase In Vivo Using Magnetic Resonance Spectroscopy

International Journal of Molecular Sciences ◽

10.3390/ijms21072442 ◽

2020 ◽

Vol 21 (7) ◽

pp. 2442

Author(s):

Jyoti Singh Tomar ◽

Jun Shen

Keyword(s):

Magnetic Resonance ◽

Carbonic Anhydrase ◽

Magnetic Resonance Spectroscopy ◽

Resonance Spectroscopy ◽

Carbonic Anhydrases ◽

Magnetization Saturation ◽

Wide Range ◽

In Vivo Characterization

Carbonic anhydrase is a ubiquitous metalloenzyme that catalyzes the reversible interconversion of CO2/HCO3−. Equilibrium of these species is maintained by the action of carbonic anhydrase. Recent advances in magnetic resonance spectroscopy have allowed, for the first time, in vivo characterization of carbonic anhydrase in the human brain. In this article, we review the theories and techniques of in vivo 13C magnetization (saturation) transfer magnetic resonance spectroscopy as they are applied to measuring the rate of exchange between CO2 and HCO3− catalyzed by carbonic anhydrase. Inhibitors of carbonic anhydrase have a wide range of therapeutic applications. Role of carbonic anhydrases and their inhibitors in many diseases are also reviewed to illustrate future applications of in vivo carbonic anhydrase assessment by magnetic resonance spectroscopy.

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

Applied and Environmental Microbiology ◽

10.1128/aem.65.11.4781-4787.1999 ◽

1999 ◽

Vol 65 (11) ◽

pp. 4781-4787 ◽

Cited By ~ 100

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.

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Characterization of a Second Ornithine Decarboxylase Isolated from Morganella morganii

Journal of Food Protection ◽

10.4315/0362-028x-71.3.657 ◽

2008 ◽

Vol 71 (3) ◽

pp. 657-661 ◽

Cited By ~ 15

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.

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Cloning and Expression of Brassica napus β-Carbonic Anhydrase cDNA

Zeitschrift für Naturforschung C ◽

10.1515/znc-2009-11-1220 ◽

2009 ◽

Vol 64 (11-12) ◽

pp. 875-881 ◽

Cited By ~ 2

Author(s):

Qiu-Hong Deng ◽

Mao-Teng Li ◽

Long-Jiang Yu

Keyword(s):

Brassica Napus ◽

Carbonic Anhydrase ◽

Expression Vector ◽

Open Reading Frame ◽

Cloning And Expression ◽

Carbonic Anhydrases ◽

Reading Frame ◽

Calculated Molecular Weight ◽

Calculated Mass ◽

Homologous Cloning

A new full-length β-carbonic anhydrase cDNA was obtained from Brassica napus by homologous cloning. The cDNA has an open-reading frame of 996 nucleotides, encoding 331 amino acids with a calculated molecular weight of 35,692 Da and an estimated pI value of 5.459. The deduced amino acid sequence of β-carbonic anhydrase from Brassica napus shared significant identity with β-carbonic anhydrases from Brassica carinata, Arabidopsis thaliana, and Thlaspi caerulescens (97.9%, 94%, and 93.5% identity, respectively). This cDNA was expressed in Escherichia coli BL21 (DE3) using the expression vector pET-32a(+). The expression band corresponded to the calculated mass plus the N-terminal fusion protein derived from the vector

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A Carbonic Anhydrase Pseudogene Sensitizes Select Brucella Lineages to Low CO2 Tension

Journal of Bacteriology ◽

10.1128/jb.00509-19 ◽

2019 ◽

Vol 201 (22) ◽

Cited By ~ 6

Author(s):

Lydia M. Varesio ◽

Jonathan W. Willett ◽

Aretha Fiebig ◽

Sean Crosson

Keyword(s):

Gene Expression ◽

Carbonic Anhydrase ◽

Transcriptional Activation ◽

Carbonic Anhydrases ◽

Loss Of Function ◽

Content Type ◽

Reading Frame ◽

Animal Host ◽

Standard Atmosphere ◽

Carbonic Anhydrase Gene

ABSTRACT Brucella spp. are intracellular pathogens that cause a disease known as brucellosis. Though the genus is highly monomorphic at the genetic level, species have animal host preferences and some defining physiologic characteristics. Of note is the requirement for CO2 supplementation to cultivate particular species, which confounded early efforts to isolate B. abortus from diseased cattle. Differences in the capacity of Brucella species to assimilate CO2 are determined by mutations in the carbonic anhydrase gene, bcaA. Ancestral single-nucleotide insertions in bcaA have resulted in frameshifted pseudogenes in B. abortus and B. ovis lineages, which underlie their inability to grow under the low CO2 tension of a standard atmosphere. Incubation of wild-type B. ovis in air selects for mutations that “rescue” a functional bcaA reading frame, which enables growth under low CO2 and enhances the growth rate under high CO2. Accordingly, we show that heterologous expression of functional Escherichia coli carbonic anhydrases enables B. ovis growth in air. Growth of B. ovis is acutely sensitive to a reduction in CO2 tension, while frame-rescued B. ovis mutants are insensitive to CO2 shifts. B. ovis initiates a gene expression program upon CO2 downshift that resembles the stringent response and results in transcriptional activation of its type IV secretion system. Our study provides evidence that loss-of-function insertion mutations in bcaA sensitize the response of B. ovis and B. abortus to reduced CO2 tension relative to that of other Brucella lineages. CO2-dependent starvation and virulence gene expression programs in these species may influence persistence or transmission in natural hosts. IMPORTANCE Brucella spp. are highly related, but they exhibit differences in animal host preference that must be determined by genome sequence differences. B. ovis and the majority of B. abortus strains require high CO2 tension to be cultivated in vitro and harbor conserved insertional mutations in the carbonic anhydrase gene, bcaA, which underlie this trait. Mutants that grow in a standard atmosphere, first reported nearly a century ago, are easily selected in the laboratory. These mutants harbor varied indel polymorphisms in bcaA that restore its consensus reading frame and rescue its function. Loss of bcaA function has evolved independently in the B. ovis and B. abortus lineages and results in a dramatically increased sensitivity to CO2 limitation.

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