scholarly journals Functional Characterization of pGKT2, a 182-Kilobase Plasmid Containing the xplAB Genes, Which Are Involved in the Degradation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine by Gordonia sp. Strain KTR9

2010 ◽  
Vol 76 (19) ◽  
pp. 6329-6337 ◽  
Author(s):  
Karl J. Indest ◽  
Carina M. Jung ◽  
Hao-Ping Chen ◽  
Dawn Hancock ◽  
Christine Florizone ◽  
...  
Keyword(s):  
Amino Acid Sequence Identity ◽  
Inorganic Nitrogen ◽  
Cytochromes P450 ◽  
Functional Characterization ◽  
Protein A ◽  
Nitrogen Sources ◽  
Pcr Analysis ◽  
Bacterial Cells ◽  
Glutamine Synthase

ABSTRACT Several microorganisms have been isolated that can transform hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), a cyclic nitramine explosive. To better characterize the microbial genes that facilitate this transformation, we sequenced and annotated a 182-kb plasmid, pGKT2, from the RDX-degrading strain Gordonia sp. KTR9. This plasmid carries xplA, encoding a protein sharing up to 99% amino acid sequence identity with characterized RDX-degrading cytochromes P450. Other genes that cluster with xplA are predicted to encode a glutamine synthase-XplB fusion protein, a second cytochrome P450, Cyp151C, and XplR, a GntR-type regulator. Rhodococcus jostii RHA1 expressing xplA from KTR9 degraded RDX but did not utilize RDX as a nitrogen source. Moreover, an Escherichia coli strain producing XplA degraded RDX but a strain producing Cyp151C did not. KTR9 strains cured of pGKT2 did not transform RDX. Physiological studies examining the effects of exogenous nitrogen sources on RDX degradation in strain KTR9 revealed that ammonium, nitrite, and nitrate each inhibited RDX degradation by up to 79%. Quantitative real-time PCR analysis of glnA-xplB, xplA, and xplR showed that transcript levels were 3.7-fold higher during growth on RDX than during growth on ammonium and that this upregulation was repressed in the presence of various inorganic nitrogen sources. Overall, the results indicate that RDX degradation by KTR9 is integrated with central nitrogen metabolism and that the uptake of RDX by bacterial cells does not require a dedicated transporter.

scholarly journals Identification and Functional Characterization of the CVOMT and EOMT Genes Promoters from Ocimum Basilicum L

2021 ◽  
Author(s):  
Fatemeh Khakdan ◽  
Zahra Shirazi ◽  
Mojtaba Ranjbar
Keyword(s):  
Water Deficit ◽  
Transcriptional Control ◽  
Functional Characterization ◽  
Pcr Analysis ◽  
Water Deficit Stress ◽  
Specific Expression ◽  
Stress Dependent ◽  
First Time ◽  
Dependent Mechanism

Abstract Methyl chavicol and methyl eugenol are important phenylpropanoid compounds previously purified from basil. These compounds are significantly enhanced by the water deficit stress-dependent mechanism. Here, for the first time, pObCVOMT and pObEOMT promoters were extracted by the genome walking method. They were then cloned into the upstream of the β-glucuronidase (GUS) reporter gene to identify the pattern of GUS water deficit stress-specific expression. Histochemical GUS assays showed in transgenic tobacco lines bearing the GUS gene driven by pObCVOMT and pObEOMT promoters, GUS was strongly expressed under water deficit stress. qRT-PCR analysis of pObCVOMT and pObEOMT transgenic plants confirmed the histochemical assays, indicating that the GUS expression is also significantly induced and up-regulated by increasing density of water deficit stress. This indicates these promoters are able to drive inducible expression. The cis-acting elements analysis showed that the pObCVOMT and pObEOMT promoters contained dehydration or water deficit-related transcriptional control elements.

THE ISOLATION AND CHARACTERIZATION OF METHANOMONAS METHANOOXIDANS BROWN AND STRAWINSKI

1964 ◽  
Vol 10 (5) ◽  
pp. 791-799 ◽  
Author(s):  
L. R. Brown ◽  
R. J. Strawinski ◽  
C. S. McCleskey
Keyword(s):  
Formic Acid ◽  
Inorganic Nitrogen ◽  
Nitrogen Sources ◽  
Energy Sources ◽  
Resting Cells ◽  
Gram Negative ◽  
Oxidation Of Methane ◽  
Isolation And Characterization ◽  
Trapping Agent

Procedures for the isolation and characterization of Metkanomonas methanooxidans Brown and Strawinski are described. Isolates from varied sources are alike in cellular morphology, inasmuch as they form only microcolonies, and in their dependence on methane or methanol as carbon and energy sources for growth. Both organic and inorganic nitrogen sources are used. The organism is a Gram negative non-sporeforming rod, 1.5 to 3.0 μ by 1.0 μ in size, and motile by means of a single polar flagellum. In growing cultures the oxygen/methane ratio was approximately 1.1 and in resting cells 1.7. The R.Q. for methane with resting cells was 0.43. Resting cells were unable to oxidize organic compounds other than methane, methanol, formaldehyde, and formate. Formic acid was detected in test solutions after cell suspensions had metabolized methane, methanol, and formaldehyde. Using sodium sulphite as trapping agent for formaldehyde, it was found that 60 to 70% of the methane or methanol consumed was converted to formaldehyde. In the presence of iodoacetate, 70% of the methane consumed was present terminally as methanol. Thus it was shown that methanol, formaldehyde, and formic acid are sequential intermediates in the oxidation of methane by these organisms.

Four novel splice variants of sulfonylurea receptor 1

2002 ◽  
Vol 283 (2) ◽  
pp. C587-C598 ◽  
Author(s):  
Annette Hambrock ◽  
Regina Preisig-Müller ◽  
Ulrich Russ ◽  
Anke Piehl ◽  
Peter J. Hanley ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Splice Variants ◽  
Functional Characterization ◽  
Pcr Analysis ◽  
Sulfonylurea Receptor ◽  
Transmembrane Segments ◽  
Sulfonylurea Receptor 1 ◽  
Green Fluorescent ◽  
Splice Forms

ATP-sensitive K+ (KATP) channels are composed of pore-forming Kir6.x subunits and regulatory sulfonylurea receptor (SUR) subunits. SURs are ATP-binding cassette proteins with two nucleotide-binding folds (NBFs) and binding sites for sulfonylureas, like glibenclamide, and for channel openers. Here we report the identification and functional characterization of four novel splice forms of guinea pig SUR1. Three splice forms originate from alternative splicing of the region coding for NBF1 and lack exons 17 (SUR1Δ17), 19 (SUR1Δ19), or both (SUR1Δ17Δ19). The fourth (SUR1C) is a COOH-terminal SUR1-fragment formed by exons 31–39 containing the last two transmembrane segments and the COOH terminus of SUR1. RT-PCR analysis showed that these splice forms are expressed in several tissues with strong expression of SUR1C in cardiomyocytes. Confocal microscopy using enhanced green fluorescent protein-tagged SUR or Kir6.x did not provide any evidence for involvement of these splice forms in the mitochondrial KATP channel. Only SUR1 and SUR1Δ17 showed high-affinity binding of glibenclamide ( K d≈ 2 nM in the presence of 1 mM ATP) and formed functional KATPchannels upon coexpression with Kir6.2.

scholarly journals A recently evolved diflavin-containing monomeric nitrate reductase is responsible for highly efficient bacterial nitrate assimilation

2020 ◽  
Vol 295 (15) ◽  
pp. 5051-5066 ◽  
Author(s):  
Wei Tan ◽  
Tian-Hua Liao ◽  
Jin Wang ◽  
Yu Ye ◽  
Yu-Chen Wei ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
Biochemical Characterization ◽  
Catalytic Domain ◽  
Inorganic Nitrogen ◽  
Nitrate Assimilation ◽  
Nitrogen Sources ◽  
Electron Transfer Mechanism ◽  
Cofactor Binding ◽  
Environmental Mycobacteria

Nitrate is one of the major inorganic nitrogen sources for microbes. Many bacterial and archaeal lineages have the capacity to express assimilatory nitrate reductase (NAS), which catalyzes the rate-limiting reduction of nitrate to nitrite. Although a nitrate assimilatory pathway in mycobacteria has been proposed and validated physiologically and genetically, the putative NAS enzyme has yet to be identified. Here, we report the characterization of a novel NAS encoded by Mycolicibacterium smegmatis Msmeg_4206, designated NasN, which differs from the canonical NASs in its structure, electron transfer mechanism, enzymatic properties, and phylogenetic distribution. Using sequence analysis and biochemical characterization, we found that NasN is an NADPH-dependent, diflavin-containing monomeric enzyme composed of a canonical molybdopterin cofactor-binding catalytic domain and an FMN–FAD/NAD-binding, electron-receiving/transferring domain, making it unique among all previously reported hetero-oligomeric NASs. Genetic studies revealed that NasN is essential for aerobic M. smegmatis growth on nitrate as the sole nitrogen source and that the global transcriptional regulator GlnR regulates nasN expression. Moreover, unlike the NADH-dependent heterodimeric NAS enzyme, NasN efficiently supports bacterial growth under nitrate-limiting conditions, likely due to its significantly greater catalytic activity and oxygen tolerance. Results from a phylogenetic analysis suggested that the nasN gene is more recently evolved than those encoding other NASs and that its distribution is limited mainly to Actinobacteria and Proteobacteria. We observed that among mycobacterial species, most fast-growing environmental mycobacteria carry nasN, but that it is largely lacking in slow-growing pathogenic mycobacteria because of multiple independent genomic deletion events along their evolution.

scholarly journals Genetic Characterization of vanG, a Novel Vancomycin Resistance Locus of Enterococcus faecalis

2000 ◽  
Vol 44 (11) ◽  
pp. 3224-3228 ◽  
Author(s):  
Stuart J. McKessar ◽  
Anne M. Berry ◽  
Jan M. Bell ◽  
John D. Turnidge ◽  
James C. Paton
Keyword(s):  
Enterococcus Faecalis ◽  
Amino Acid Sequence Identity ◽  
Vancomycin Resistance ◽  
Open Reading Frames ◽  
Pcr Analysis ◽  
Resistance Locus ◽  
Gene Products ◽  
The Individual ◽  
Reading Frames

ABSTRACT Enterococcus faecalis strain WCH9 displays a moderate level of resistance to vancomycin (MIC = 16 μg/ml) and full susceptibility to teicoplanin but is negative by PCR analysis using primers specific for all known enterococcal vancomycin resistance genotypes (vanA, vanB,vanC, vanD, and vanE). We have isolated and sequenced a novel putative vancomycin resistance locus (designated vanG), which contains seven open reading frames, from this strain. These are organized differently from those of all the other enterococcal vanloci, and, furthermore, the individual vanG gene products exhibit less than 50% amino acid sequence identity to othervan gene products.

scholarly journals Serotonin and Melatonin Biosynthesis in Plants: Genome-Wide Identification of the Genes and Their Expression Reveal a Conserved Role in Stress and Development

2021 ◽  
Vol 22 (20) ◽  
pp. 11034
Author(s):  
Bidisha Bhowal ◽  
Annapurna Bhattacharjee ◽  
Kavita Goswami ◽  
Neeti Sanan-Mishra ◽  
Sneh L. Singla-Pareek ◽  
...  
Keyword(s):  
Target Genes ◽  
Functional Characterization ◽  
Gene Families ◽  
Pcr Analysis ◽  
Tryptophan Decarboxylase ◽  
Cellular Processes ◽  
Genome Wide ◽  
A Genome ◽  
Suitable Target

Serotonin (Ser) and melatonin (Mel) serve as master regulators of plant growth and development by influencing diverse cellular processes. The enzymes namely, tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H) catalyse the formation of Ser from tryptophan. Subsequently, serotonin N-acetyl transferase (SNAT) and acetyl-serotonin methyltransferase (ASMT) form Mel from Ser. Plant genomes harbour multiple genes for each of these four enzymes, all of which have not been identified. Therefore, to delineate information regarding these four gene families, we carried out a genome-wide analysis of the genes involved in Ser and Mel biosynthesis in Arabidopsis, tomato, rice and sorghum. Phylogenetic analysis unravelled distinct evolutionary relationships among these genes from different plants. Interestingly, no gene family except ASMTs showed monocot- or dicot-specific clustering of respective proteins. Further, we observed tissue-specific, developmental and stress/hormone-mediated variations in the expression of the four gene families. The light/dark cycle also affected their expression in agreement with our quantitative reverse transcriptase-PCR (qRT-PCR) analysis. Importantly, we found that miRNAs (miR6249a and miR-1846e) regulated the expression of Ser and Mel biosynthesis under light and stress by influencing the expression of OsTDC5 and OsASMT18, respectively. Thus, this study may provide opportunities for functional characterization of suitable target genes of the Ser and Mel pathway to decipher their exact roles in plant physiology.

scholarly journals Functional Characterization of Cytochromes P450 2B from Woodrats

2012 ◽  
Vol 26 (S1) ◽  
Author(s):  
P Ross Wilderman ◽  
Hyun-Hee Jang ◽  
Elizabeth Angermeier ◽  
Jael R. Malenke ◽  
M. Denise Dearing ◽  
...  
Keyword(s):  
Cytochromes P450 ◽  
Functional Characterization

scholarly journals Genome-wide analysis and expression profiles of PdeMYB transcription factors in colored-leaf poplar (Populus deltoids)

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Weibing Zhuang ◽  
Xiaochun Shu ◽  
Xinya Lu ◽  
Tao Wang ◽  
Fengjiao Zhang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Expression Profiles ◽  
Functional Characterization ◽  
Green Leaf ◽  
Myb Transcription Factor ◽  
Pcr Analysis ◽  
Genome Wide ◽  
Populus Deltoids

Abstract Background MYB transcription factors, comprising one of the largest transcription factor families in plants, play many roles in secondary metabolism, especially in anthocyanin biosynthesis. However, the functions of the PdeMYB transcription factor in colored-leaf poplar remain elusive. Results In the present study, genome-wide characterization of the PdeMYB genes in colored-leaf poplar (Populus deltoids) was conducted. A total of 302 PdeMYB transcription factors were identified, including 183 R2R3-MYB, five R1R2R3-MYB, one 4R-MYB, and 113 1R-MYB transcription factor genes. Genomic localization and paralogs of PdeMYB genes mapped 289 genes on 19 chromosomes, with collinearity relationships among genes. The conserved domain, gene structure, and evolutionary relationships of the PdeMYB genes were also established and analyzed. The expression levels of PdeMYB genes were obtained from previous data in green leaf poplar (L2025) and colored leaf poplar (QHP) as well as our own qRT-PCR analysis data in green leaf poplar (L2025) and colored leaf poplar (CHP), which provide valuable clues for further functional characterization of PdeMYB genes. Conclusions The above results provide not only comprehensive insights into the structure and functions of PdeMYB genes but also provide candidate genes for the future improvement of leaf colorization in Populus deltoids.

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