scholarly journals Identification and Heterologous Expression of Genes Involved in Anaerobic Dissimilatory Phosphite Oxidation by Desulfotignum phosphitoxidans

2010 ◽  
Vol 192 (19) ◽  
pp. 5237-5244 ◽  
Author(s):  
Diliana Dancheva Simeonova ◽  
Marlena Marie Wilson ◽  
William W. Metcalf ◽  
Bernhard Schink
Keyword(s):  
Sequence Analysis ◽  
Gene Cluster ◽  
Electron Donor ◽  
Genomic Library ◽  
Amino Acid Sequences ◽  
Strictly Anaerobic ◽  
Oxidation Activity ◽  
Cell Extracts ◽  
Expression Of Genes ◽  
Phosphorus Source

ABSTRACT Desulfotignum phosphitoxidans is a strictly anaerobic, Gram-negative bacterium that utilizes phosphite as the sole electron source for homoacetogenic CO2 reduction or sulfate reduction. A genomic library of D. phosphitoxidans, constructed using the fosmid vector pJK050, was screened for clones harboring the genes involved in phosphite oxidation via PCR using primers developed based on the amino acid sequences of phosphite-induced proteins. Sequence analysis of two positive clones revealed a putative operon of seven genes predicted to be involved in phosphite oxidation. Four of these genes (ptxD-ptdFCG) were cloned and heterologously expressed in Desulfotignum balticum, a related strain that cannot use phosphite as either an electron donor or as a phosphorus source. The ptxD-ptdFCG gene cluster was sufficient to confer phosphite uptake and oxidation ability to the D. balticum host strain but did not allow use of phosphite as an electron donor for chemolithotrophic growth. Phosphite oxidation activity was measured in cell extracts of D. balticum transconjugants, suggesting that all genes required for phosphite oxidation were cloned. Genes of the phosphite gene cluster were assigned putative functions on the basis of sequence analysis and enzyme assays.

scholarly journals Identification and Expression of Genes Involved in Biosynthesis of l-Oleandrose and Its Intermediatel-Olivose in the Oleandomycin ProducerStreptomyces antibioticus

2000 ◽  
Vol 44 (5) ◽  
pp. 1266-1275 ◽  
Author(s):  
Ignacio Aguirrezabalaga ◽  
Carlos Olano ◽  
Nerea Allende ◽  
Leticia Rodriguez ◽  
Alfredo F. Braña ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Gene Cluster ◽  
Enzyme Activities ◽  
Open Reading Frames ◽  
Streptomyces Antibioticus ◽  
Recombinant Strains ◽  
Expression Of Genes ◽  
Streptomyces Albus ◽  
Erythronolide B ◽  
Reading Frames

ABSTRACT A 9.8-kb DNA region from the oleandomycin gene cluster inStreptomyces antibioticus was cloned. Sequence analysis revealed the presence of 8 open reading frames encoding different enzyme activities involved in the biosynthesis of one of the two 2,6-deoxysugars attached to the oleandomycin aglycone:l-oleandrose (the oleW, oleV,oleL, and oleU genes) andd-desosamine (the oleNI and oleTgenes), or of both (the oleS and oleE genes). AStreptomyces albus strain harboring the oleG2glycosyltransferase gene integrated into the chromosome was constructed. This strain was transformed with two different plasmid constructs (pOLV and pOLE) containing a set of genes proposed to be required for the biosynthesis of dTDP-l-olivose and dTDP-l-oleandrose, respectively. Incubation of these recombinant strains with the erythromycin aglycon (erythronolide B) gave rise to two new glycosylated compounds, identified asl-3-O-olivosyl- andl-3-O-oleandrosyl-erythronolide B, indicating that pOLV and pOLE encode all enzyme activities required for the biosynthesis of these two 2,6-dideoxysugars. A pathway is proposed for the biosynthesis of these two deoxysugars in S. antibioticus.

scholarly journals Novel Acetone Metabolism in a Propane-Utilizing Bacterium, Gordonia sp. Strain TY-5

2006 ◽  
Vol 189 (3) ◽  
pp. 886-893 ◽  
Author(s):  
Tetsuya Kotani ◽  
Hiroya Yurimoto ◽  
Nobuo Kato ◽  
Yasuyoshi Sakai
Keyword(s):  
Gene Cluster ◽  
Methyl Acetate ◽  
Amino Acid Sequences ◽  
Gene Products ◽  
Cyclic Ketones ◽  
Oxidation Activity ◽  
E Coli ◽  
Polycistronic Transcription ◽  
Terminal Amino ◽  
Induced Proteins

ABSTRACT In the propane-utilizing bacterium Gordonia sp. strain TY-5, propane was shown to be oxidized to 2-propanol and then further oxidized to acetone. In this study, the subsequent metabolism of acetone was studied. Acetone-induced proteins were found in extracts of cells induced by acetone, and a gene cluster designated acmAB was cloned on the basis of the N-terminal amino acid sequences of acetone-induced proteins. The acmA and acmB genes encode a Baeyer-Villiger monooxygenase (BVMO) and esterase, respectively. The BVMO encoded by acmA was purified from acetone-induced cells of Gordonia sp. strain TY-5 and characterized. The BVMO exhibited NADPH-dependent oxidation activity for linear ketones (C3 to C10) and cyclic ketones (C4 to C8). Escherichia coli expressing the acmA gene oxidized acetone to methyl acetate, and E. coli expressing the acmB gene hydrolyzed methyl acetate. Northern blot analyses revealed that polycistronic transcription of the acmAB gene cluster was induced by propane, 2-propanol, and acetone. These results indicate that the acmAB gene products play an important role in the metabolism of acetone derived from propane oxidation and clarify the propane metabolism pathway of strain TY-5 (propane → 2-propanol → acetone → methyl acetate → acetic acid + methanol). This paper provides the first evidence for BVMO-dependent acetone metabolism.

scholarly journals A Novel p-Nitrophenol Degradation Gene Cluster from a Gram-Positive Bacterium, Rhodococcus opacus SAO101

2004 ◽  
Vol 186 (15) ◽  
pp. 4894-4902 ◽  
Author(s):  
Wataru Kitagawa ◽  
Nobutada Kimura ◽  
Yoichi Kamagata
Keyword(s):  
Gene Cluster ◽  
Ralstonia Eutropha ◽  
Amino Acid Sequences ◽  
Expression Vectors ◽  
Rhodococcus Opacus ◽  
Positive Bacterium ◽  
Gram Positive ◽  
Crude Cell Extract ◽  
Cell Extracts ◽  
Degrading Bacteria

ABSTRACT p-Nitrophenol (4-NP) is recognized as an environmental contaminant; it is used primarily for manufacturing medicines and pesticides. To date, several 4-NP-degrading bacteria have been isolated; however, the genetic information remains very limited. In this study, a novel 4-NP degradation gene cluster from a gram-positive bacterium, Rhodococcus opacus SAO101, was identified and characterized. The deduced amino acid sequences of npcB, npcA, and npcC showed identity with phenol 2-hydroxylase component B (reductase, PheA2) of Geobacillus thermoglucosidasius A7 (32%), with 2,4,6-trichlorophenol monooxygenase (TcpA) of Ralstonia eutropha JMP134 (44%), and with hydroxyquinol 1,2-dioxygenase (ORF2) of Arthrobacter sp. strain BA-5-17 (76%), respectively. The npcB, npcA, and npcC genes were cloned into pET-17b to construct the respective expression vectors pETnpcB, pETnpcA, and pETnpcC. Conversion of 4-NP was observed when a mixture of crude cell extracts of Escherichia coli containing pETnpcB and pETnpcA was used in the experiment. The mixture converted 4-NP to hydroxyquinol and also converted 4-nitrocatechol (4-NCA) to hydroxyquinol. Furthermore, the crude cell extract of E. coli containing pETnpcC converted hydroxyquinol to maleylacetate. These results suggested that npcB and npcA encode the two-component 4-NP/4-NCA monooxygenase and that npcC encodes hydroxyquinol 1,2-dioxygenase. The npcA and npcC mutant strains, SDA1 and SDC1, completely lost the ability to grow on 4-NP as the sole carbon source. These results clearly indicated that the cloned npc genes play an essential role in 4-NP mineralization in R. opacus SAO101.

scholarly journals Glyoxylate Metabolism Is a Key Feature of the Metabolic Degradation of 1,4-Dioxane by Pseudonocardia dioxanivorans Strain CB1190

2012 ◽  
Vol 78 (9) ◽  
pp. 3298-3308 ◽  
Author(s):  
Ariel Grostern ◽  
Christopher M. Sales ◽  
Wei-Qin Zhuang ◽  
Onur Erbilgin ◽  
Lisa Alvarez-Cohen
Keyword(s):  
Energy Source ◽  
Gene Cluster ◽  
Gene Clusters ◽  
Glycolate Oxidase ◽  
Content Type ◽  
Cell Extracts ◽  
Expression Of Genes ◽  
Monooxygenase Gene ◽  
Glyoxylate Metabolism ◽  
Metabolic Degradation

ABSTRACTThe groundwater contaminant 1,4-dioxane (dioxane) is transformed by several monooxygenase-expressing microorganisms, but only a few of these, includingPseudonocardia dioxanivoransstrain CB1190, can metabolize the compound as a sole carbon and energy source. However, nothing is yet known about the genetic basis of dioxane metabolism. In this study, we used a microarray to study differential expression of genes in strain CB1190 grown on dioxane, glycolate (a previously identified intermediate of dioxane degradation), or pyruvate. Of eight multicomponent monooxygenase gene clusters carried by the strain CB1190 genome, only the monooxygenase gene cluster located on plasmid pPSED02 was upregulated with dioxane relative to pyruvate. Plasmid-borne genes for putative aldehyde dehydrogenases, an aldehyde reductase, and an alcohol oxidoreductase were also induced during growth with dioxane. With both dioxane and glycolate, a chromosomal gene cluster encoding a putative glycolate oxidase was upregulated, as were chromosomal genes related to glyoxylate metabolism through the glyoxylate carboligase pathway. Glyoxylate carboligase activity in cell extracts from cells pregrown with dioxane and inRhodococcus jostiistrain RHA1 cells expressing the putative strain CB1190 glyoxylate carboligase gene further demonstrated the role of glyoxylate metabolism in the degradation of dioxane. Finally, we used13C-labeled dioxane amino acid isotopomer analysis to provide additional evidence that metabolites of dioxane enter central metabolism as three-carbon compounds, likely as phosphoglycerate. The routing of dioxane metabolites via the glyoxylate carboligase pathway helps to explain how dioxane is metabolized as a sole carbon and energy source for strain CB1190.

scholarly journals Phenol Degradation in the Strictly Anaerobic Iron-Reducing Bacterium Geobacter metallireducens GS-15

2009 ◽  
Vol 75 (12) ◽  
pp. 3912-3919 ◽  
Author(s):  
Kathleen M. Schleinitz ◽  
Sirko Schmeling ◽  
Nico Jehmlich ◽  
Martin von Bergen ◽  
Hauke Harms ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Posttranscriptional Regulation ◽  
Phenol Degradation ◽  
Regulation Mechanism ◽  
Strictly Anaerobic ◽  
Carboxylase Activity ◽  
Transcription Analysis ◽  
Geobacter Metallireducens ◽  
Isotope Exchange Reaction ◽  
Cell Extracts

ABSTRACT Information on anaerobic phenol metabolism by physiological groups of bacteria other than nitrate reducers is scarce. We investigated phenol degradation in the strictly anaerobic iron-reducing deltaproteobacterium Geobacter metallireducens GS-15 using metabolite, transcriptome, proteome, and enzyme analyses. The results showed that the initial steps of phenol degradation are accomplished by phenylphosphate synthase (encoded by pps genes) and phenylphosphate carboxylase (encoded by ppc genes) as known from Thauera aromatica, but they also revealed some distinct differences. The pps-ppc gene cluster identified in the genome is functional, as shown by transcription analysis. In contrast to T. aromatica, transcription of the pps- and ppc-like genes was induced not only during growth on phenol, but also during growth on benzoate. In contrast, proteins were detected only during growth on phenol, suggesting the existence of a posttranscriptional regulation mechanism for these initial steps. Phenylphosphate synthase and phenylphosphate carboxylase activities were detected in cell extracts. The carboxylase does not catalyze an isotope exchange reaction between 14CO2 and 4-hydroxybenzoate, which is characteristic of the T. aromatica enzyme. Whereas the enzyme of T. aromatica is encoded by ppcABCD, the pps-ppc gene cluster of G. metallireducens contains only a ppcB homologue. Nearby, but oriented in the opposite direction, is a ppcD homologue that is transcribed during growth on phenol. Genome analysis did not reveal obvious homologues of ppcA and ppcC, leaving open the question of whether these genes are dispensable for phenylphosphate carboxylase activity in G. metallireducens or are quite different from the Thauera counterparts and located elsewhere in the genome.

scholarly journals Murine erythropoietin gene: cloning, expression, and human gene homology.

1986 ◽  
Vol 6 (3) ◽  
pp. 849-858 ◽  
Author(s):  
C B Shoemaker ◽  
L D Mitsock
Keyword(s):  
Amino Acid ◽  
Genomic Library ◽  
Cdna Probe ◽  
Amino Acid Sequences ◽  
Nucleotide Sequence Analysis ◽  
Coding Region ◽  
Transcription Control ◽  
Erythroid Progenitor ◽  
Human Genes ◽  
Cell Expression

The gene for murine erythropoietin (EPO) was isolated from a mouse genomic library with a human EPO cDNA probe. Nucleotide sequence analysis permitted the identification of the murine EPO coding sequence and the prediction of the encoded amino acid sequence based on sequence conservation between the mouse and human EPO genes. Both the coding DNA and the amino acid sequences were 80% conserved between the two species. Transformation of COS-1 cells with a mammalian cell expression vector containing the murine EPO coding region resulted in secretion of murine EPO with biological activity on both murine and human erythroid progenitor cells. The transcription start site for the murine EPO gene in kidneys was determined. This permitted tentative identification of the transcription control region. The region included 140 base pairs upstream of the cap site which was over 90% conserved between the murine and human genes. Surprisingly, the first intron and much of the 5'- and 3'-untranslated sequences were also substantially conserved between the genes of the two species.

scholarly journals Factor D̄ of the alternative pathway of human complement. Purification, alignment and N-terminal amino acid sequences of the major cyanogen bromide fragments, and localization of the serine residue at the active site

1980 ◽  
Vol 187 (3) ◽  
pp. 863-874 ◽  
Author(s):  
D M Johnson ◽  
J Gagnon ◽  
K B Reid
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Amino Acid Sequence ◽  
Alternative Pathway ◽  
Amino Acid Sequences ◽  
Serine Residue ◽  
Amino Acid Sequence Analysis ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

The serine esterase factor D of the complement system was purified from outdated human plasma with a yield of 20% of the initial haemolytic activity found in serum. This represented an approx. 60 000-fold purification. The final product was homogeneous as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (with an apparent mol.wt. of 24 000), its migration as a single component in a variety of fractionation procedures based on size and charge, and its N-terminal amino-acid-sequence analysis. The N-terminal amino acid sequence of the first 36 residues of the intact molecule was found to be homologous with the N-terminal amino acid sequences of the catalytic chains of other serine esterases. Factor D showed an especially strong homology (greater than 60% identity) with rat ‘group-specific protease’ [Woodbury, Katunuma, Kobayashi, Titani, & Neurath (1978) Biochemistry 17, 811-819] over the first 16 amino acid residues. This similarity is of interest since it is considered that both enzymes may be synthesized in their active, rather than zymogen, forms. The three major CNBr fragments of factor D, which had apparent mol.wts. of 15 800, 6600 and 1700, were purified and then aligned by N-terminal amino acid sequence analysis and amino acid analysis. By using factor D labelled with di-[1,3-14C]isopropylphosphofluoridate it was shown that the CNBr fragment of apparent mol.wt. 6600, which is located in the C-terminal region of factor D, contained the active serine residue. The amino acid sequence around this residue was determined.

scholarly journals Structure of the rat platelet factor 4 gene: a marker for megakaryocyte differentiation.

1987 ◽  
Vol 7 (2) ◽  
pp. 898-904 ◽  
Author(s):  
T Doi ◽  
S M Greenberg ◽  
R D Rosenberg
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Genomic Library ◽  
Transcriptional Start Site ◽  
Platelet Factor 4 ◽  
Start Codon ◽  
Cdna Expression Library ◽  
Amino Acid Residues ◽  
Platelet Factor ◽  
Nuclease Mapping

A rat platelet factor 4 (PF4) cDNA has been isolated by immunoscreening a g lambda 11 rat megakaryocyte cDNA expression library. Sequence analysis of the rat PF4 cDNA revealed that this megakaryocyte protein is composed of a leader sequence of 29 amino acid residues and a mature protein sequence of 76 amino acid residues. The structure of rat PF4 derived from its cDNA shows a marked homology with the amino acid sequence of human PF4 obtained by classical protein chemistry techniques. This observation is particularly striking with regard to the carboxy-terminal region of rat and human PF4, where 28 of the last 31 C-terminal residues are identical. The rat PF4 gene was obtained from a rat genomic library by using rat PF4 cDNA as a hybridization probe. Sequence analysis showed that the gene is constructed of three exons and two short introns. The transcriptional start site is located 73 base pairs upstream of the translational start codon as judged by S1 nuclease mapping and primer extension. The 5' noncoding region of the gene also exhibited a sequence homologous to the TATA box at -31, as well as a series of direct and inverted repeat sequences and a cluster of 26 T residues at -155 to -218. This latter domain may be involved in regulating PF4 gene expression during megakaryocytopoiesis.

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