scholarly journals Functional and transcriptional analyses of the initial oxygenase genes for acenaphthene degradation from Sphingomonas sp. strain A4

Microbiology ◽  
2006 ◽  
Vol 152 (8) ◽  
pp. 2455-2467 ◽  
Author(s):  
Atsushi Kouzuma ◽  
Onruthai Pinyakong ◽  
Hideaki Nojiri ◽  
Toshio Omori ◽  
Hisakazu Yamane ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Gene Disruption ◽  
Pcr Analysis ◽  
Nucleotide Sequence Analysis ◽  
Rt Pcr ◽  
Transcription Start ◽  
Ferredoxin Reductase ◽  
Genes Encoding ◽  
Dna Fragment ◽  
Putative Binding Site

Sphingomonas sp. strain A4 is capable of utilizing acenaphthene as its sole carbon and energy source. To isolate the genes responsible for acenaphthene degradation, transposon mutagenesis was performed on strain A4 and four mini-Tn5-inserted mutants lacking the ability to utilize acenaphthene were isolated. In three of the four mini-Tn5 inserted mutants, the mini-Tn5s were inserted into the same locus (within about 16 kb) as the arhA1A2 genes, which had previously been identified as the genes encoding the terminal oxygenase components for the initial oxygenation of acenaphthene. The nucleotide sequence analysis of the corresponding 16.4 kb DNA fragment revealed the existence of 16 ORFs and a partial ORF. From these ORFs, the genes encoding the ferredoxin (ArhA3) and ferredoxin reductase (ArhA4) complementary to ArhA1A2 were identified. RT-PCR analysis suggested that a 13.5 kb gene cluster, consisting of 13 ORFs and including all the arhA genes, forms an operon, although it includes several ORFs that are apparently unnecessary for acenaphthene degradation. Furthermore, using gene disruption and quantitative RT-PCR analyses, the LysR-type activator, ArhR, required for expression of the 13.5 kb gene cluster was also identified. Transcription of the gene cluster by ArhR was induced in the presence of acenaphthene (or its metabolite), and a putative binding site (T-N11-A motif) for ArhR was found upstream from the transcription start point of arhA3.

scholarly journals Genetic Investigation of the Catabolic Pathway for Degradation of Abietane Diterpenoids by Pseudomonas abietaniphilaBKME-9

2000 ◽  
Vol 182 (13) ◽  
pp. 3784-3793 ◽  
Author(s):  
Vincent J. J. Martin ◽  
William W. Mohn
Keyword(s):  
Gene Cluster ◽  
Sequence Similarity ◽  
Open Reading Frames ◽  
Ring Cleavage ◽  
Catabolic Pathway ◽  
Transcriptional Fusion ◽  
Abietane Diterpenoids ◽  
Genes Encoding ◽  
Dna Fragment ◽  
Reading Frames

ABSTRACT We have cloned and sequenced the dit gene cluster encoding enzymes of the catabolic pathway for abietane diterpenoid degradation by Pseudomonas abietaniphila BKME-9. Thedit gene cluster is located on a 16.7-kb DNA fragment containing 13 complete open reading frames (ORFs) and 1 partial ORF. The genes ditA1A2A3 encode the α and β subunits and the ferredoxin of the dioxygenase which hydroxylates 7-oxodehydroabietic acid to 7-oxo-11,12-dihydroxy-8,13-abietadien acid. The dioxygenase mutant strain BKME-941 (ditA1::Tn5) did not grow on nonaromatic abietanes, and transformed palustric and abietic acids to 7-oxodehydroabietic acid in cell suspension assays. Thus, nonaromatic abietanes are aromatized prior to further degradation. Catechol 2,3-dioxygenase activity of xylEtranscriptional fusion strains showed induction of ditA1and ditA3 by abietic, dehydroabietic, and 7-oxodehydroabietic acids, which support the growth of strain BKME-9, as well as by isopimaric and 12,14-dichlorodehydroabietic acids, which are diterpenoids that do not support the growth of strain BKME-9. In addition to the aromatic-ring-hydroxylating dioxygenase genes, thedit cluster includes ditC, encoding an extradiol ring cleavage dioxygenase, and ditR, encoding an IclR-type transcriptional regulator. Although ditR is not strictly required for the growth of strain BKME-9 on abietanes, aditR::Kmr mutation in aditA3::xylE reporter strain demonstrated that it encodes an inducer-dependent transcriptional activator of ditA3. An ORF with sequence similarity to genes encoding permeases (ditE) is linked with genes involved in abietane degradation.

scholarly journals microRNA and mRNA profiles in the amygdala are relevant to fear memory induced by physical or psychological stress

2019 ◽  
Vol 122 (3) ◽  
pp. 1002-1022 ◽  
Author(s):  
Yan Sun ◽  
Wei Lu ◽  
Kaixin Du ◽  
Jin-Hui Wang
Keyword(s):  
Psychological Stress ◽  
High Throughput Sequencing ◽  
Fear Memory ◽  
Physical Stress ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Genes Encoding ◽  
Reporter Assays ◽  
Molecular Profiles

Anxiety is presumably driven by fear memory. Molecular profiles in the amygdala of mice with fear memory induced by psychological and physical stresses remain to be elucidated. Fear memory in mice was induced by a paradigm of social defeat. Physical and psychological stresses (PPS) to an intruder were given by attacks from an aggressive resident. Psychological stress (PS) to an observer was given by the witnessing of aggressor attacks. Amygdala tissues from these mice showing fear memory and anxiety vs. tissues from control mice were harvested to analyze mRNA and microRNA profiles by high-throughput sequencing. In the amygdala of intruders and observers with fear memory, the genes encoding 5-HTR1b, 5-HTR2a, DAR2, AChRM3, and IP3R1 are upregulated, whereas genes encoding GPγ11, GPγ13, GPγT2, RasC3, and P450 are downregulated, indicating that these molecules are involved in fear memory induced by physical/psychological stresses. In the comparison of intruders with observers, the upregulation of genes encoding 5-HTR6, GPγ8, P2R7, NFκ2, CREB3/1, and Itgα9 as well as the downregulation of genes encoding DAR5, 5-HTR1a, and HSP1a are involved in fear memory induced by physical stress. The upregulation of genes encoding DAR1, 5-HTR5a and SSR2/3 as well as the downregulation of AdRα1, CREB3/1, GPγ13 and GPγ8 are involved in fear memory induced by psychological stress. Results obtained by sequencing mRNA and microRNA profiles are consistent with results of quantitative RT-PCR analysis and dual-luciferase reporter assays performed for validation. In conclusion, fear memories and anxiety induced by PPS vs. PS are caused by the imbalanced regulation of different synapses and signaling pathways in the amygdala. NEW & NOTEWORTHY The current study identifies the molecular mechanism underlying fear memory and anxiety induced by psychological stress vs. physical stress, in which the imbalanced expression of microRNA-regulated mRNAs relevant to dopaminergic, adrenergic, and serotonergic synapses in the amygdala plays an important role. This result reveals different molecular profiles for psychological and physical stresses.

Identification of a gene cluster for cell-surface genes of the SRS superfamily inNeospora caninumand characterization of the novelSRS9gene

Parasitology ◽  
2011 ◽  
Vol 138 (14) ◽  
pp. 1832-1842 ◽  
Author(s):  
V. RISCO-CASTILLO ◽  
V. MARUGÁN-HERNÁNDEZ ◽  
A. FERNÁNDEZ-GARCÍA ◽  
A. AGUADO-MARTÍNEZ ◽  
E. JIMÉNEZ-RUIZ ◽  
...  
Keyword(s):  
Western Blot ◽  
Gene Cluster ◽  
Neospora Caninum ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Specific Expression ◽  
Initial Characterization ◽  
Hydrophilic Region

SUMMARYHere we present the detection of a gene cluster forNeospora caninumsurface genes, similar to theToxoplasma gondiiSRS9 locus, and the cloning and characterization of the NcSRS9gene. PCR genome walking, using NcBSR4gene as a framework, allows the identification, upstream NcBSR4, of 2 sequences homologous to theSRS5and the Ubiquinol-cytochrome C reductase genes and, downstream NcBSR4, of an ORF of 1191 bp coding for a 396-amino acid polypeptide with 59% similarity to the TgSRS9 antigen. A putative 39-residue signal peptide was found at the NH2-terminus followed by a hydrophilic region, and a potential site for a glycosylphosphatidylinositol anchor at the COOH-terminus. A recombinant NcSRS9 protein was produced and was recognized on a Western blot by a low proportion of sera from a panel of naturally infected cows and calves. In addition, Western blot analysis using polyclonal anti-rNcSRS9 revealed stage-specific expression of NcSRS9 in bradyzoites but not in tachyzoites, and immunohistochemistry on brain from a congenitally infected calf showed NcSRS9 recognition in bradyzoites contained in tissue cysts. However, bradyzoite-specific expression of NcSRS9 could not be proven by immunofluorescence on bradyzoites obtainedin vitroand RT-PCR analysis showed no significant variations of NcSRS9transcripts duringin vitrotachyzoite-bradyzoite switch, probably due to incomplete maturity ofin vitrobradyzoites. Initial characterization of NcSRS9 in this study may lead to further studies for a better understanding ofN. caninumpersistence.

scholarly journals Transcriptional Profiling of Methyltransferase Genes during Growth of Methanosarcina mazei on Trimethylamine

2009 ◽  
Vol 191 (16) ◽  
pp. 5108-5115 ◽  
Author(s):  
Christian Krätzer ◽  
Paul Carini ◽  
Raymond Hovey ◽  
Uwe Deppenmeier
Keyword(s):  
Transcriptional Profiling ◽  
Expression Patterns ◽  
Pcr Analysis ◽  
Exponential Growth Phase ◽  
Methane Formation ◽  
Rt Pcr ◽  
Transcript Levels ◽  
Methanosarcina Mazei ◽  
Depth Analysis ◽  
Genes Encoding

ABSTRACT The genomic expression patterns of Methanosarcina mazei growing with trimethylamine were measured in comparison to those of cells grown with methanol. We identified a total of 72 genes with either an increased level (49 genes) or a decreased level (23 genes) of mRNA during growth on trimethylamine with methanol-grown cells as the control. Major differences in transcript levels were observed for the mta, mtb, mtt, and mtm genes, which encode enzymes involved in methane formation from methanol and trimethylamine, respectively. Other differences in mRNA abundance were found for genes encoding enzymes involved in isopentenyl pyrophosphate synthesis and in the formation of aromatic amino acids, as well as a number of proteins with unknown functions. The results were verified by in-depth analysis of methyltransferase genes using specific primers for real-time quantitative reverse transcription-PCR (RT-PCR). The monitored transcript levels of genes encoding corrinoid proteins involved in methyl group transfer from methylated C1 compounds (mtaC, mtbC, mttC, and mtmC) indicated increased amounts of mRNA from the mtaBC1, mtaBC2, and mtaBC3 operons in methanol-grown cells, whereas mRNA of the mtb1-mtt1 operon was found in high concentrations during trimethylamine consumption. The genes of the mtb1-mtt1 operon encode methyltransferases that are responsible for sequential demethylation of trimethylamine. The analysis of product formation of trimethylamine-grown cells at different optical densities revealed that large amounts of dimethylamine and monomethylamine were excreted into the medium. The intermediate compounds were consumed only in the very late exponential growth phase. RT-PCR analysis of key genes involved in methanogenesis led to the conclusion that M. mazei is able to adapt to changing trimethylamine concentrations and the consumption of intermediate compounds. Hence, we assume that the organism possesses a regulatory network for optimal substrate utilization.

Identification and mapping of the gene translation products involved in the first steps of the Comamonas testosteroni B-356 biphenyl/chlorobiphenyl biodegradation pathway

1994 ◽  
Vol 40 (9) ◽  
pp. 743-753 ◽  
Author(s):  
Janique Bergeron ◽  
Darakhshan Ahmad ◽  
Diane Barriault ◽  
Angèle Larose ◽  
Michel Sylvestre ◽  
...  
Keyword(s):  
Cleavage Product ◽  
Enzymatic Conversion ◽  
Comamonas Testosteroni ◽  
Gene Encoding ◽  
Ferredoxin Reductase ◽  
Oxidized Iron ◽  
Sds Page ◽  
Genes Encoding ◽  
Dna Fragment ◽  
T7 Polymerase

In this study, we have mapped Comamonas testosteroni B-356 genes encoding enzymes for the conversion of biphenyl and 4-chlorobiphenyl into the corresponding meta-cleavage compounds onto a 6.3-kb DNA fragment, and we have determined the subunit composition of the enzymes involved in this pathway. The various proteins encoded by this 6.3-kb DNA fragment and by subclones derived from it were overexpressed and selectively labelled using the T7 polymerase promoter system in Escherichia coli. They were then analyzed using SDS-PAGE, which allowed the encoding locus of each polypeptide to be mapped. Despite apparent dissimilarity in the congener selectivity patterns of the initial oxygenase of strain B-356 with those of Pseudomonas sp. strain LB400, the number and sizes of the polypeptides involved in the enzymatic conversion of biphenyl or 4-chlorobiphenyl into the meta-cleavage product appear to be similar in the two strains. In both strains, the bph operon encodes the following: the large (51-kDa polypeptide encoded by bphA) and the small (22-kDa polypeptide encoded by bphE) subunits of the iron sulphur protein, which is thought to interact directly with the substrate to introduce the oxygen molecule; the ferredoxin (12-kDa polypeptide encoded by bphF) involved in electron transfer from the reduced ferredoxin reductase to the oxidized iron sulphur protein; the 29-kDa polypeptide of the 2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase encoded by bphB; and the 32-kDa polypeptide of the 2,3-dihydroxybiphenyl-1,2-dioxygenase encoded by bphC, which catalyzes meta-1,2 fission of the aromatic ring. A major difference between strain B-356 and strain LB400 is that the bphG gene encoding biphenyl dioxygenase ferredoxin reductase is located outside the bph gene cluster in strain B-356. Several lines of evidence indicate that bphG is absent in clones carrying the bph operon from strain B-356.Key words: PCB, gene expression, biphenyl oxygenase, bph.

scholarly journals Identification and functional analysis of the genes for naphthalenesulfonate catabolism by Sphingomonas xenophaga BN6

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 1929-1940 ◽  
Author(s):  
Andreas Keck ◽  
Doris Conradt ◽  
Anette Mahler ◽  
Andreas Stolz ◽  
Ralf Mattes ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Functional Analysis ◽  
Degradative Pathway ◽  
Ferredoxin Reductase ◽  
Insertional Inactivation ◽  
Genes Encoding ◽  
Transcriptional Units ◽  
Dna Fragment ◽  
Sphingomonas Aromaticivorans ◽  
Sphingomonas Xenophaga

Sphingomonas xenophaga BN6 degrades various (substituted) naphthalenesulfonates to the corresponding (substituted) salicylates. A gene cluster was identified on the plasmid pBN6 which coded for several enzymes participating in the degradative pathway for naphthalenesulfonates. A DNA fragment of 16 915 bp was sequenced which contained 17 ORFs. The genes encoding the 1,2-dihydroxynaphthalene dioxygenase, 2-hydroxychromene-2-carboxylate isomerase, and 2′-hydroxybenzalpyruvate aldolase of the naphthalenesulfonate pathway were identified on the DNA fragment and the encoded proteins heterologously expressed in Escherichia coli. Also, the genes encoding the ferredoxin and ferredoxin reductase of a multi-component, ring-hydroxylating naphthalenesulfonate dioxygenase were identified by insertional inactivation. The identified genes generally demonstrated the highest degree of homology to enzymes encoded by the phenanthrene-degrading organism Sphingomonas sp. P2, or the megaplasmid pNL1 of the naphthalene- and biphenyl-degrading strain Sphingomonas aromaticivorans F199. The genes of S. xenophaga BN6 participating in the degradation of naphthalenesulfonates also shared the same organization in three different transcriptional units as the genes involved in the degradation of naphthalene, biphenyl, and phenanthrene previously found in Sphingomonas sp. P2 and S. aromaticivorans F199. The genes were flanked in S. xenophaga BN6 by ORFs which specify proteins that show the highest homologies to proteins of mobile genetic elements.

scholarly journals The fluorene catabolic linear plasmid in Terrabacter sp. strain DBF63 carries the β-ketoadipate pathway genes, pcaRHGBDCFIJ, also found in proteobacteria

Microbiology ◽  
2005 ◽  
Vol 151 (11) ◽  
pp. 3713-3722 ◽  
Author(s):  
Hiroshi Habe ◽  
Jin-Sung Chung ◽  
Ayako Ishida ◽  
Kano Kasuga ◽  
Kazuki Ide ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Tricarboxylic Acid Cycle ◽  
Bacterial Species ◽  
Linear Plasmid ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Gene Encoding ◽  
Positive Trait ◽  
Tricarboxylic Acid Cycle Intermediates

Terrabacter sp. strain DBF63 is capable of degrading fluorene (FN) to tricarboxylic acid cycle intermediates via phthalate and protocatechuate. Genes were identified for the protocatechuate branch of the β-ketoadipate pathway (pcaR, pcaHGBDCFIJ) by sequence analysis of a 70 kb DNA region of the FN-catabolic linear plasmid pDBF1. RT-PCR analysis of RNA from DBF63 cells grown with FN, dibenzofuran, and protocatechuate indicated that the pcaHGBDCFIJ operon was expressed during both FN and protocatechuate degradation in strain DBF63. The gene encoding β-ketoadipate enol-lactone hydrolase (pcaD) was not fused to the next gene, which encodes γ-carboxymuconolactone decarboxylase (pcaC), in strain DBF63, even though the presence of the pcaL gene (the fusion of pcaD and pcaC) within a pca gene cluster has been thought to be a Gram-positive trait. Quantitative RT-PCR analysis revealed that pcaD mRNA levels increased sharply in response to protocatechuate, and a biotransformation experiment with cis,cis-muconate using Escherichia coli carrying both catBC and pcaD indicated that PcaD exhibited β-ketoadipate enol-lactone hydrolase activity. The location of the pca gene cluster on the linear plasmid, and the insertion sequences around the pca gene cluster suggest that the ecologically important β-ketoadipate pathway genes, usually located chromosomally, may be spread widely among bacterial species via horizontal transfer or transposition events.

scholarly journals Liver receptor homologue-1 is expressed in the adrenal and can regulate transcription of 11 beta-hydroxylase

2001 ◽  
Vol 27 (2) ◽  
pp. 255-258 ◽  
Author(s):  
ZN Wang ◽  
M Bassett ◽  
WE Rainey
Keyword(s):  
Adrenal Glands ◽  
Pcr Analysis ◽  
Orphan Nuclear Receptor ◽  
Rt Pcr ◽  
Hormone Production ◽  
Mobility Shift ◽  
Gene Encoding ◽  
Liver And Pancreas ◽  
Genes Encoding ◽  
Mobility Shift Assay

Liver receptor homologue-1 (LRH-1, designated NR5A2) is a mammalian homologue of Drosophila fushi tarazu factor (dFTZ-F1) and structurally belongs to the orphan nuclear receptor superfamily. LRH-1 can recognize the DNA sequence 5'-AAGGTCA-3', the canonical recognition motif for steroidogenic factor 1 (SF-1). Herein, we hypothesized that LRH-1 might play a role in the regulation of human adrenal expression of steroidogenic enzymes. To test this hypothesis, LRH-1 expression in human adult and fetal adrenal glands was examined by RT-PCR analysis. The fetal and adult adrenal glands, as well as liver and pancreas, were observed to express LRH-1 mRNA using RT-PCR. The ability of LRH-1 to enhance transcription of the gene encoding human 11 beta- hydroxylase (hCYP11B1) was then examined using the H295R adrenal cell line. LRH-1 co-transfection with hCYP11B1 luciferase promoter constructs caused a 25-fold induction of luciferase activity. Furthermore, co-transfection of a hCYP11B1 reporter construct containing a mutation in the SF-1 binding cis-element abolished the stimulatory effect of both SF-1 and LRH-1. Electrophoretic mobility shift assay (EMSA) demonstrated that LRH-1 could bind to the SF-1 response element. Taken together, our data suggested that LRH-1 is expressed in the adrenal, and can substitute for SF-1 to enhance transcription of genes encoding certain of the steroid-metabolizing enzymes. A role for LRH-1 in the regulation of adrenal or gonadal steroid hormone production should be further studied.

scholarly journals Regulatory Effects of Nitric Oxide on Reproduction and Melanin Biosynthesis in Onion Pathogenic Fungus Stemphylium Eturmiunum

2020 ◽  
Author(s):  
Yanxia Zhao ◽  
Wenwen Yuan ◽  
Mengni Sun ◽  
Xiuguo Zhang ◽  
Weifa Zheng
Keyword(s):  
Nitric Oxide ◽  
Soluble Guanylate Cyclase ◽  
Pathogenic Fungus ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Melanin Biosynthesis ◽  
Synthetic Genes ◽  
Genes Encoding ◽  
Regulatory Effects ◽  
Dopa Melanin

Abstract The formation of propagules (conidia and ascospores) is the critical stage for the transmission of the pathogenic fungus Stemphylium eturmiunum. However, how the development of these propagules is regulated remains to be fully understood. Here, we show that nitric oxide (NO) is necessary for the formation of conidia and pseudothecia in S. eturmiunum. Application of NO scavenger carboxy-CPTIO (cPTIO) or soluble guanylate cyclase (sGC) inhibitor NS2028 abolishes the formation of conidia and pseudothecia. In the culture of S. eturmiunum, supplement of NO-releasing compound sodium nitroprusside (SNP) results in an increased formation of conidia at 0.2 mmol/L, and pseudothecia at 2 mmol/L. SNP supplement also triggered increased biosynthesis of melanin, which can be inhibited partially upon the addition of either arbutin or tricyclazole, the specific inhibitors for 3,4-dihydroxyphenylalanine (DOPA) and dihydroxynaphthalene (DHN) synthetic pathway, respectively. Intriguingly, enhanced melanin biosynthesis triggered an increased formation of propagules; while its inhibition impaired their formation. The SNP-induced increment in the formation of propagules can be also compromised upon supplement of cPTIO or NS-2028. RT-PCR analysis showed that SNP at 0.2 mmol/L promoted transcription of the genes encoding the conidiation co-regulators brlA, abA, and wetA, and inhibited at 2 mmol/L. In contrast, application of SNP at 2 mmol/L increased transcription of the genes encoding mat1, and mat2, the genes related to sexual reproduction, and the transcription of two DNH melanin synthetic genes pks1 and pks2, and the key gene tyr for DOPA melanin biosynthesis. However, the increased transcription of these genes is down-regulated or blocked upon supplement of cPTIO or NS-2028. Thus, NO regulates asexual and sexual development, as well as melanin synthesis in S. eturmiunum possibly through NO-sGC-GMP signaling pathway.

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