scholarly journals Simultaneous Identification of Two Cyclohexanone Oxidation Genes from an Environmental Brevibacterium Isolate Using mRNA Differential Display

2000 ◽  
Vol 182 (15) ◽  
pp. 4241-4248 ◽  
Author(s):  
Patricia C. Brzostowicz ◽  
Katharine L. Gibson ◽  
Stuart M. Thomas ◽  
Mary Sue Blasko ◽  
Pierre E. Rouvière
Keyword(s):  
Escherichia Coli ◽  
Differential Display ◽  
Reverse Transcription ◽  
Mrna Differential Display ◽  
Environmental Isolate ◽  
Dna Fragments ◽  
Reverse Transcription Pcr ◽  
Metabolic Genes ◽  
Mrna Population ◽  
Simultaneous Identification

ABSTRACT The technique of mRNA differential display was used to identify simultaneously two metabolic genes involved in the degradation of cyclohexanone in a new halotolerant Brevibacteriumenvironmental isolate. In a strategy based only on the knowledge that cyclohexanone oxidation was inducible in this strain, the mRNA population of cells exposed to cyclohexanone was compared to that of control cells using reverse transcription-PCR reactions primed with a collection of 81 arbitrary oligonucleotides. Three DNA fragments encoding segments of flavin monooxygenases were isolated with this technique, leading to the identification of the genes of two distinct cyclohexanone monooxygenases, the enzymes responsible for the oxidation of cyclohexanone. Each monooxygenase was expressed in Escherichia coli and characterized. This work validates the application of mRNA differential display for the discovery of new microbial metabolic genes.

scholarly journals mRNA Differential Display in a Microbial Enrichment Culture: Simultaneous Identification of Three Cyclohexanone Monooxygenases from Three Species

2003 ◽  
Vol 69 (1) ◽  
pp. 334-342 ◽  
Author(s):  
Patricia C. Brzostowicz ◽  
Dana M. Walters ◽  
Stuart M. Thomas ◽  
Vasantha Nagarajan ◽  
Pierre E. Rouvière
Keyword(s):  
Differential Display ◽  
Enrichment Culture ◽  
Bacterial Species ◽  
Degradation Pathway ◽  
Mrna Differential Display ◽  
Dna Fragments ◽  
Genes Encoding ◽  
Repeated Sampling ◽  
Mixed Microbial Community ◽  
Simultaneous Identification

ABSTRACT mRNA differential display has been used to identify cyclohexanone oxidation genes in a mixed microbial community derived from a wastewater bioreactor. Thirteen DNA fragments randomly amplified from the total RNA of an enrichment subculture exposed to cyclohexanone corresponded to genes predicted to be involved in the degradation of cyclohexanone. Nine of these DNA fragments are part of genes encoding three distinct Baeyer-Villiger cyclohexanone monooxygenases from three different bacterial species present in the enrichment culture. In Arthrobacter sp. strain BP2 and Rhodococcus sp. strain Phi2, the monooxygenase is part of a gene cluster that includes all the genes required for the degradation of cyclohexanone, while in Rhodococcus sp. strain Phi1 the genes surrounding the monooxygenase are not predicted to be involved in this degradation pathway but rather seem to belong to a biosynthetic pathway. Furthermore, in the case of Arthrobacter strain BP2, three other genes flanking the monooxygenase were identified by differential display, demonstrating that the repeated sampling of bacterial operons shown earlier for a pure culture (D. M. Walters, R. Russ, H. Knackmuss, and P. E. Rouvière, Gene 273:305-315, 2001) is also possible for microbial communities. The activity of the three cyclohexanone monooxygenases was confirmed and characterized following their expression in Escherichia coli.

scholarly journals Arr-cb Is a Rifampin Resistance Determinant Found Active or Cryptic in Clostridiumbolteae Strains

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Jean-Christophe Marvaud ◽  
Thierry Lambert
Keyword(s):  
Escherichia Coli ◽  
Gut Microbiota ◽  
Reverse Transcription ◽  
Recent Analysis ◽  
Pcr Analysis ◽  
Directed Mutagenesis ◽  
Human Gut ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Rifampin Resistance

ABSTRACT Clostridium bolteae, which belongs to the Clostridium clostridioforme complex, is a member of the human gut microbiota. Recent analysis of seven genomes of C. bolteae revealed the presence of an arr-like gene. Among these strains, only 90A7 was found to be resistant to rifampin in the absence of alteration of RpoB. Cloning of arr-cb from 90A7 in Escherichia coli combined with directed mutagenesis demonstrated that Arr-cb was functional but that a Q127→R variant present in 90A9 and 90B3 was inactive. Quantitative reverse transcription-PCR analysis indicated that arr-cb was silent in the four remaining strains because of defective transcription. Thus, two independent mechanisms can make the probably intrinsic arr-cb gene of C. bolteae cryptic.

scholarly journals mkp-1 Encoding Mitogen-Activated Protein Kinase Phosphatase 1, a Verotoxin 1 Responsive Gene, Detected by Differential Display Reverse Transcription-PCR in Caco-2 Cells

2000 ◽  
Vol 68 (5) ◽  
pp. 2791-2796 ◽  
Author(s):  
Shihoko Kojima ◽  
Itaru Yanagihara ◽  
Gengo Kono ◽  
Tomomi Sugahara ◽  
Hatsumi Nasu ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Differential Display ◽  
Reverse Transcription ◽  
Mitogen Activated Protein Kinase ◽  
28S Rrna ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Mitogen Activated Protein ◽  
A Subunit ◽  
Differential Display Reverse Transcription

ABSTRACT The major cytotoxic effect of the verotoxins (VTs) produced by strains of VT-producing Escherichia coli is the inhibition of host-cell protein synthesis, but VTs are also suspected to play a role in apoptotic cell signaling and cytokine release. Four differentially expressed genes, including mkp-1 (encoding mitogen-activated protein kinase phospatase 1), were detected by differential display reverse transcription-PCR (DD RT-PCR) stimulated by VT1 in Caco-2 cells. Northern blot analysis showed the induction ofmkp-1 mRNA 6 h after VT1 stimulation. Neither mutant VT1 (mutVT1), harboring two mutations in the A subunit (E167Q-R170L), nor cycloheximide induced mkp-1 mRNA, but mkp-1mRNA was detected with both wild-type VT1 (wtVT1) and anisomycin, a 28S rRNA inhibitor. Therefore, we concluded that the A subunit of VT1 was essential for mkp-1 induction. Increased amounts of phosphorylated c-Jun protein were also found with wtVT1 and anisomycin. Although the precise mechanism of induction of MKP-1 is unknown, we hypothesized that 28S rRNA not only was a sensor for ribotoxic stress, but also was involved in the signal cascade of MKP-1. This is the first report of detection by DD RT-PCR of cellular genes induced by bacterial toxins.

scholarly journals Applications of Differential-Display Reverse Transcription-PCR to Molecular Pathogenesis and Medical Mycology

2000 ◽  
Vol 13 (3) ◽  
pp. 408-427 ◽  
Author(s):  
Joy Sturtevant
Keyword(s):  
Gene Expression ◽  
Differential Display ◽  
Reverse Transcription ◽  
Drug Targets ◽  
Cell Activation ◽  
Histoplasma Capsulatum ◽  
Medical Mycology ◽  
Reverse Transcription Pcr ◽  
Differential Display Reverse Transcription ◽  
Plant Pathogenesis

SUMMARY The host-fungus interaction is characterized by changes in gene expression in both host and pathogen. Differential-display reverse transcription PCR (DDRT-PCR) is a PCR-based method that allows extensive analysis of gene expression among several cell populations. Several limitations and drawbacks to this procedure have now been addressed, including the large number of false-positive results and the difficulty in confirming differential expression. Modifications that simplify the reaction time, allow the use of minute quantities of RNA, or address unusual species- or gene-specific sequences have been reported. DDRT-PCR has been used to address biological questions in mammalian systems, including cell differentiation, cell activation, cell stress, and identification of drug targets. In microbial pathogenesis and plant pathogenesis, DDRT-PCR has allowed the identification of virulence factors, genes involved in cell death, and signaling genes. In Candida albicans, DDRT-PCR studies identified TIF-2, which may play a role in the upregulation of phospholipases, and the stress-related genes, CIP1 and CIP2. In Histoplasma capsulatum and C. albicans, genes involved in the host-pathogen interaction, including a member of the 100-kDa family in Histoplasma and an ALS and 14-3-3 gene in Candida, were potentially identified by DDRT-PCR. Although very few reports have been published in medical mycology, studies in mammalian, nonfungal microbial, and plant pathogen systems are easily applied to basic questions in fungal pathogenesis and antifungal therapeutics.

scholarly journals The Properties of 5-Methyltetrahydrofolate Dehydrogenase (MetF1) and Its Role in the Tetrahydrofolate-Dependent Dicamba Demethylation System inRhizorhabdus dicambivoransNdbn-20

2019 ◽  
Vol 201 (17) ◽  
Author(s):  
Shigang Yao ◽  
Le Chen ◽  
Zhou Yang ◽  
Li Yao ◽  
Jianchun Zhu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cultured Cells ◽  
Reductase Activity ◽  
Gene Clusters ◽  
Biochemical Properties ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Metabolic Genes ◽  
Methyltransferase Gene

ABSTRACTThe herbicide dicamba is initially degraded via the tetrahydrofolate (THF)-dependent demethylation system inRhizorhabdus dicambivoransNdbn-20. Two THF-dependent dicamba methyltransferase gene clusters, scaffold 50 and scaffold 66, were found in the genome of strain Ndbn-20. Each cluster contains a dicamba methyltransferase gene and three THF metabolism-related genes, namely,metF(coding for 5,10-CH2-THF reductase),folD(coding for 5,10-CH2-THF dehydrogenase–5,10-methenyl-THF cyclohydrolase), andpurU(coding for 10-formyl-THF deformylase). In this study, reverse transcription-PCR (RT-PCR) results showed that only genes in scaffold 66, not those in scaffold 50, were transcribed in dicamba-cultured cells. ThemetFgene of scaffold 66 (metF1) was expressed inEscherichia coliBL21(DE3), and the product was purified as a His6-tagged protein. Purified MetF1 was found to be a monomer and exhibited 5-CH3-THF dehydrogenase activityin vitro. ThekcatandKmfor 5-CH3-THF were 0.23 s−1and 16.48 μM, respectively. However, 5,10-CH2-THF reductase activity was not detected for MetF1 under the conditions tested. Gene disruption results showed thatmetF1is essential for dicamba degradation, whereasfolD1is dispensable.IMPORTANCEThere are several THF-dependent methyltransferase genes and THF-metabolic genes in the genome ofR. dicambivoransNdbn-20; however, which genes are involved in dicamba demethylation and the mechanism underlying THF regeneration remain unknown. This study revealed that scaffold 66 is responsible for dicamba demethylation and that MetF1 physiologically catalyzes the dehydrogenation of 5-CH3-THF to 5,10-CH2-THF in the THF-dependent dicamba demethylation system inR. dicambivoransNdbn-20. Furthermore, the results showed that MetF1 differs from previously characterized MetF in phylogenesis, biochemical properties, and catalytic activity; e.g., MetF1in vitrodid not show 5,10-CH2-THF reductase activity, which is the physiological function ofEscherichia coliMetF. This study provides new insights into the mechanism of the THF-dependent methyltransferase system.

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