1-Deoxy-
            <scp>d</scp>
            -Xylulose 5-Phosphate Synthase, the Gene Product of Open Reading Frame (ORF) 2816 and ORF 2895 in
            <i>Rhodobacter capsulatus</i>

Hahn, Frederick M.; Eubanks, Lisa M.; Testa, Charles A.; Blagg, Brian S. J.; Baker, Jonathan A.; Poulter, C. Dale

doi:10.1128/jb.183.1.1-11.2001

Cited by 85 publications

(74 citation statements)

References 52 publications

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“…The bla is a single-copy gene of pBR322 (Watson, 1988), and the dxs is a singlecopy gene of E. coli chromosomal DNA (Hahn et al, 2001). Consequently, quantification of sample with the bla-and dxs-set represents corresponding amount of pBR322 and E. coli chromosomal DNA.…”

Section: Design Of Primer Sets For Real-time Qpcrmentioning

confidence: 99%

Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli

Lee

Kim

Shin

et al. 2006

Journal of Biotechnology

571

401

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Real-time QPCR based methods for determination of plasmid copy number in recombinant Escherichia coli cultures are presented. Two compatible methods based on absolute and relative analyses were tested with recombinant E. coli DH5␣ harboring pBR322, which is a common bacterial cloning vector. The separate detection of the plasmid and the host chromosomal DNA was achieved using two separate primer sets, specific for the plasmid ␤-lactamase gene (bla) and for the chromosomal d-1-deoxyxylulose 5-phosphate synthase gene (dxs), respectively. Since both bla and dxs are single-copy genes of pBR322 and E. coli chromosomal DNA, respectively, the plasmid copy number can be determined as the copy ratio of bla to dxs.These methods were successfully applied to determine the plasmid copy number of pBR322 of E. coli host cells. The results of the absolute and relative analyses were identical and highly reproducible with coefficient of variation (CV) values of 2.8-3.9% and 4.7-5.4%, respectively. The results corresponded to the previously reported values of pBR322 copy number within E. coli host cells, 15-20.The methods introduced in this study are convenient to perform and cost-effective compared to the traditionally used Southern blot method. The primer sets designed in this study can be used to determine plasmid copy number of any recombinant E. coli with a plasmid vector having bla gene.

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Section: Design Of Primer Sets For Real-time Qpcrmentioning

confidence: 99%

Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli

Lee

Kim

Shin

et al. 2006

Journal of Biotechnology

571

401

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“…Both RMC26 and RMC29 are viable when supplemented with ME through use of the MEP pathway genes downstream of ispC which biosynthesize IPP and DMAPP without the necessity of IPP isomerase activity. ME complements disruptions in dxs and dxr (9,38). The alcohol is imported and phosphorylated before it enters the MEP pathway.…”

Section: Figurementioning

confidence: 99%

Identification of an Archaeal Type II Isopentenyl Diphosphate Isomerase in Methanothermobacter thermautotrophicus

2004

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Isopentenyl diphosphate (IPP):dimethylallyl diphosphate isomerase catalyzes the interconversion of the fundamental five-carbon homoallylic and allylic diphosphate building blocks required for biosynthesis of isoprenoid compounds. Two different isomerases have been reported. The type I enzyme, first characterized in the late 1950s, is widely distributed in eukaryota and eubacteria. The type II enzyme was recently discovered in Streptomyces sp. strain CL190. Open reading frame 48 (ORF48) in the archaeon Methanothermobacter thermautotrophicus encodes a putative type II IPP isomerase. A plasmid-encoded copy of the ORF complemented IPP isomerase activity in vivo in Salmonella enterica serovar Typhimurium strain RMC29, which contains chromosomal knockouts in the genes for type I IPP isomerase (idi) and 1-deoxy-D-xylulose 5-phosphate (dxs). The dxs gene was interrupted with a synthetic operon containing the Saccharomyces cerevisiae genes erg8, erg12, and erg19 allowing for the conversion of mevalonic acid to IPP by the mevalonate pathway. His 6 -tagged M. thermautotrophicus type II IPP isomerase was produced in Escherichia coli and purified by Ni The isoprenoid biosynthetic pathway is ubiquitous across all three kingdoms of life. Over 36,000 natural products derived from the fundamental five-carbon isoprenoid building blocks isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) have now been identified (3). Many of these molecules have important biological functions, including glycoprotein synthesis (dolichols), inter-and intracellular signaling (prenylated proteins and steroidal hormones), membrane structure (steroids), electron carriers during redox reactions (ubiquinones), photoprotection (carotenoids), photosynthesis (chlorophyll), and defense against predators (sesquiterpenes and pyrethrins).There are two independent biosynthetic routes to IPP and DMAPP (Fig. 1). In the mevalonate (MVA) pathway, first discovered in the late 1950s (4, 6), IPP is synthesized from mevalonic acid by the consecutive action of mevalonate kinase (MVA kinase), phosphomevalonate kinase (PMVA kinase), and mevalonate diphosphate decarboxylase (DPMVA decarboxylase). The isomerization of IPP to DMAPP is a mandatory step needed to create the electrophilic allylic diphosphates needed for subsequent prenyl transfer reactions. IPP isomerase is an essential enzyme in archaea, eukaryota, and some gram-positive eubacteria, where IPP is synthesized by the MVA pathway. More recently, it was discovered that IPP and DMAPP are synthesized from D-glyceraldehyde phosphate and pyruvate by the methyl erythritol phosphate (MEP) pathway found in many eubacteria, cyanobacteria, and plant chloroplasts (18,28). In the final step of the MEP pathway, the ispH gene product synthesizes IPP and DMAPP from 4-hydroxydimethylallyl diphosphate (Fig. 1) (26). In organisms that utilize the MEP pathway, idi is not essential or does not exist (10).In 1997, Methanothermobacter thermautotrophicus was the first archaeon to have its genome fully sequenced (34). At th...

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“…Similarly, dxsA (RSP0254) located at the other end of the PS cluster, downstream of the puf operon, must be included in the PS gene cluster. dxsA encodes deoxyxylulose-5-phosphate synthase (15), whose product is a common precursor to isoprenoids in the methylerythritol phos- FIG. 7.…”

Section: Vol 186 2004mentioning

confidence: 99%

Construction and Validation of the Rhodobacter sphaeroides 2.4.1 DNA Microarray: Transcriptome Flexibility at Diverse Growth Modes

et al. 2004

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A high-density oligonucleotide DNA microarray, a genechip, representing the 4.6-Mb genome of the facultative phototrophic proteobacterium, Rhodobacter sphaeroides 2.4.1, was custom-designed and manufactured by Affymetrix, Santa Clara, Calif. The genechip contains probe sets for 4,292 open reading frames (ORFs), 47 rRNA and tRNA genes, and 394 intergenic regions. The probe set sequences were derived from the genome annotation generated by Oak Ridge National Laboratory after extensive revision, which was based primarily upon codon usage characteristic of this GC-rich bacterium. As a result of the revision, numerous missing ORFs were uncovered, nonexistent ORFs were deleted, and misidentified start codons were corrected. To evaluate R. sphaeroides transcriptome flexibility, expression profiles for three diverse growth modes-aerobic respiration, anaerobic respiration in the dark, and anaerobic photosynthesis-were generated. Expression levels of onefifth to one-third of the R. sphaeroides ORFs were significantly different in cells under any two growth modes. Pathways involved in energy generation and redox balance maintenance under three growth modes were reconstructed. Expression patterns of genes involved in these pathways mirrored known functional changes, suggesting that massive changes in gene expression are the major means used by R. sphaeroides in adaptation to diverse conditions. Differential expression was observed for genes encoding putative new participants in these pathways (additional photosystem genes, duplicate NADH dehydrogenase, ATP synthases), whose functionality has yet to be investigated. The DNA microarray data correlated well with data derived from quantitative reverse transcription-PCR, as well as with data from the literature, thus validating the R. sphaeroides genechip as a powerful and reliable tool for studying unprecedented metabolic versatility of this bacterium.Rhodobacter sphaeroides belongs to the facultatively phototrophic anoxygenic proteobacteria, which are known for their broad range of energetic and metabolic capabilities. To derive energy for growth, R. sphaeroides uses aerobic respiration in the presence of oxygen, whereas in the absence of oxygen it can either use anoxygenic photosynthesis (in the presence of light), anaerobic respiration (in the dark, in the presence of appropriate electron acceptors), or fermentation (with appropriate substrates) (23,26,38). The metabolic capabilities of R. sphaeroides include, but are not limited to, dinitrogen fixation, utilization of single-carbon compounds (carbon dioxide or methanol), and utilization or production of molecular hydrogen, as well as detoxification of metal oxides and oxyanions.In R. sphaeroides, transitions from one growth mode to another are affected by diverse environmental factors. Oxygen tension is among the most influential factors that dictate the mode of energy generation. At high oxygen tension, R. sphaeroides lacks a photosynthetic apparatus. Synthesis of the photosystem (PS) is controlled by oxygen levels and is ...

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1-Deoxy- d -Xylulose 5-Phosphate Synthase, the Gene Product of Open Reading Frame (ORF) 2816 and ORF 2895 in Rhodobacter capsulatus

Cited by 85 publications

References 52 publications

Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli

Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli

Identification of an Archaeal Type II Isopentenyl Diphosphate Isomerase in Methanothermobacter thermautotrophicus

Construction and Validation of the Rhodobacter sphaeroides 2.4.1 DNA Microarray: Transcriptome Flexibility at Diverse Growth Modes

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