Transposition of the Autonomous Fot1 Element in the Filamentous Fungus Fusarium oxysporum

Abstract Autonomous mobility of different copies of the Fot1 element was determined for several strains of the fungal plant pathogen Fusarium oxysporum to develop a transposon tagging system. Two Fot1 copies inserted into the third intron of the nitrate reductase structural gene (niaD) were separately introduced into two genetic backgrounds devoid of endogenous Fot1 elements. Mobility of these copies was observed through a phenotypic assay for excision based on the restoration of nitrate reductase activity. Inactivation of the Fot1 transposase open reading frame (frameshift, deletion, or disruption) prevented excision in strains free of Fot1 elements. Molecular analysis of the Nia+ revertant strains showed that the Fot1 element reintegrated frequently into new genomic sites after excision and that it can transpose from the introduced niaD gene into a different chromosome. Sequence analysis of several Fot1 excision sites revealed the socalled footprint left by this transposable element. Three reinserted Fot1 elements were cloned and the DNA sequences flanking the transposon were determined using inverse polymerase chain reaction. In all cases, the transposon was inserted into a TA dinucleotide and created the characteristic TA target site duplication. The availability of autonomous Fot1 copies will now permit the development of an efficient two-component transposon tagging system comprising a trans-activator element supplying transposase and a cis-responsive marked element.

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The genes YNI1 and YNR1, encoding nitrite reductase and nitrate reductase respectively in the yeast Hansenula polymorpha, are clustered and co-ordinately regulated

Biochemical Journal ◽

10.1042/bj3170089 ◽

1996 ◽

Vol 317 (1) ◽

pp. 89-95 ◽

Cited By ~ 30

Author(s):

Nélida BRITO ◽

Julio AVILA ◽

M. Dolores PEREZ ◽

Celedonio GONZALEZ ◽

José M. SIVERIO

Keyword(s):

Nitrate Reductase ◽

Nitrite Reductase ◽

Reductase Activity ◽

Hansenula Polymorpha ◽

Enzymic Activity ◽

Wild Type ◽

Reading Frame ◽

Dna Library ◽

Genomic Dna Library ◽

Nitrate And Nitrite

The nitrite reductase-encoding gene (YNI1) from the yeast Hansenula polymorpha was isolated from a lambda EMBL3 H. polymorpha genomic DNA library, using as a probe a 481 bp DNA fragment from the gene of Aspergillus nidulans encoding nitrite reductase (niiA). An open reading frame of 3132 bp, encoding a putative protein of 1044 amino acids with high similarity with nitrite reductases from fungi, was located by DNA sequencing in the phages λNB5 and λJA13. Genes YNI1 and YNR1 (encoding nitrate reductase) are clustered, separated by 1700 bp. Northern blot analysis showed that expression of YNI1 and YNR1 is co-ordinately regulated; induced by nitrate and nitrite and repressed by sources of reduced nitrogen, even in the presence of nitrate. A mutant lacking nitrite reductase activity was obtained by deletion of the chromosomal copy of YNI1. The mutant does not grow in nitrate or in nitrite; it exhibits a similar level of transcription of YNR1 to the wild type, but the nitrate reductase enzymic activity is only about 50% of the wild type. In the presence of nitrate the Δyni1::URA3 mutant extrudes approx. 24 nmol of nitrite/h per mg of yeast (wet weight), about five times more than the wild type.

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Modulation of Anaerobic Energy Metabolism of Bacillus subtilis by arfM(ywiD)

Journal of Bacteriology ◽

10.1128/jb.183.23.6815-6821.2001 ◽

2001 ◽

Vol 183 (23) ◽

pp. 6815-6821 ◽

Cited By ~ 20

Author(s):

Marco Marino ◽

Hugo Cruz Ramos ◽

Tamara Hoffmann ◽

Philippe Glaser ◽

Dieter Jahn

Keyword(s):

Bacillus Subtilis ◽

Nitrate Reductase ◽

Reductase Activity ◽

Transcriptional Start Site ◽

Anaerobic Respiration ◽

Regulatory System ◽

Reading Frame ◽

Regulatory Cascade ◽

Anaerobic Induction ◽

Respiratory Nitrate Reductase

ABSTRACT Bacillus subtilis grows under anaerobic conditions utilizing nitrate ammonification and various fermentative processes. The two-component regulatory system ResDE and the redox regulator Fnr are the currently known parts of the regulatory system for anaerobic adaptation. Mutation of the open reading frame ywiDlocated upstream of the respiratory nitrate reductase operonnarGHJI resulted in elimination of the contribution of nitrite dissimilation to anaerobic nitrate respiratory growth. Significantly reduced nitrite reductase (NasDE) activity was detected, while respiratory nitrate reductase activity was unchanged. Anaerobic induction of nasDE expression was found to be significantly dependent on intact ywiD, while anaerobicnarGHJI expression was ywiD independent. Anaerobic transcription of hmp, encoding a flavohemoglobin-like protein, and of the fermentative operonslctEP and alsSD, responsible for lactate and acetoin formation, was partially dependent on ywiD. Expression of pta, encoding phosphotransacetylase involved in fermentative acetate formation, was not influenced byywiD. Transcription of the ywiD gene was anaerobically induced by the redox regulator Fnr via the conserved Fnr-box (TGTGA-6N-TCACT) centered 40.5 bp upstream of the transcriptional start site. Anaerobic induction of ywiDby resDE was found to be indirect viaresDE-dependent activation of fnr. TheywiD gene is subject to autorepression and nitrite repression. These results suggest a ResDE → Fnr → YwiD regulatory cascade for the modulation of genes involved in the anaerobic metabolism of B. subtilis. Therefore,ywiD was renamed arfM for anaerobic respiration and fermentation modulator.

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Recovery of Mutants Impaired in Pathogenicity After Transposition of Impala in Fusarium oxysporum f. sp. melonis

Phytopathology ◽

10.1094/phyto.2000.90.11.1279 ◽

2000 ◽

Vol 90 (11) ◽

pp. 1279-1284 ◽

Cited By ~ 24

Author(s):

Quirico Migheli ◽

Christian Steinberg ◽

Jean-Michel Davière ◽

Chantal Olivain ◽

Catherine Gerlinger ◽

...

Keyword(s):

Nitrate Reductase ◽

Fusarium Oxysporum ◽

Nitrate Reductase Activity ◽

Reductase Activity ◽

Symptom Development ◽

Pathogenic Potential ◽

Class 1 ◽

First Results ◽

Wilt Symptom ◽

Kinetics Of

The ability of transposon impala to inactivate genes involved in pathogenicity was tested in Fusarium oxysporum f. sp. melonis. Somatic excision of an impala copy inserted in the nitrate reductase-encoding niaD gene was positively selected through a phenotypic assay based on the restoration of nitrate reductase activity. Independent excision events were analyzed molecularly and shown to carry reinsertedimpala in more than 70% of the cases. Mapping of reinserted impala elements on large NotI-restriction fragments showed that impala transposes randomly. By screening 746 revertants on plants, a high proportion (3.5%) of mutants impaired in their pathogenic potential was recovered. According to the kinetics of wilt symptom development, the strains that were impaired in pathogenicity were clustered in three classes: class 1 grouped two strains that never induced Fusarium wilt symptoms on the host plant; class 2 and class 3 grouped 15 and 9 revertants which caused symptoms more than 50 and 30 days after inoculation, respectively. The first results demonstrate the efficiency of transposition in generating mutants affected in pathogenicity, which are usually difficult to obtain by classical mutagenesis, and open the possibility to clone the altered genes with impala as a tag.

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