High-efficiency transposon mutagenesis by electroporation of a Pseudomonas fluorescens strain

Artiguenave, François; Vilagines, R.; Danglot, C.

doi:10.1111/j.1574-6968.1997.tb12597.x

Cited by 22 publications

(6 citation statements)

References 15 publications

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“…Ligations were performed with the Fast‐link DNA Ligation Kit (Epicentre Technologies). Transformation by electroporation was carried out as described (17). Clones and genomic deletions were verified by sequencing PCR products (18).…”

Section: Methodsmentioning

confidence: 99%

Auxotrophic Markers pyrF and proC Can Replace Antibiotic Markers on Protein Production Plasmids in High-Cell-Density Pseudomonas fluorescens Fermentation

Schneider

Jenings

Mun

et al. 2008

Biotechnol Progress

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The use of antibiotic-resistance genes as selectable markers in transgenic organisms is coming under increased scrutiny, for fear that they may spread to human pathogens, thereby reducing the effectiveness of antibiotic therapy. A current Pseudomonas fluorescens protein expression system uses a tetracycline resistance gene (tetR/tetA) to maintain an expression plasmid under control of a repressible promoter and a kanamycin resistance gene (kanR) to maintain a plasmid carrying a repressor gene. We investigated using auxotrophic markers to replace these two antibiotic resistance genes: pyrF (encoding orotidine-5'-phosphate decarboxylase) in place of tetR/tetA and proC (encoding pyrroline-5-carboxylate reductase) in place of kanR, complementing their respective precise chromosomal deletions created by allele exchange using a suicide vector carrying pyrF as a counterselectable marker. The resulting strains, devoid of antibiotic-resistance genes, were shown to achieve high productivity of nitrilase and thermostable alpha-amylase equal to that of the former antibiotic-resistant production host. The production plasmids were stable. The pyrF (uracil-dependent) background of the production host strain also allows us to sequentially alter the genome to incorporate other desired genomic changes, deletions, or insertions using 5'-fluoroorotic acid counterselection, restoring the selectable marker after each step.

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Section: Methodsmentioning

confidence: 99%

Auxotrophic Markers pyrF and proC Can Replace Antibiotic Markers on Protein Production Plasmids in High-Cell-Density Pseudomonas fluorescens Fermentation

Schneider

Jenings

Mun

et al. 2008

Biotechnol Progress

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“…maculicola ES4326 and pv. tomato DC3000 by electroporation (Artiguenave et al, 1997). Briefly, 500 ml of King's B medium (50 lg ml )1 rifampicin) was inoculated with overnight culture of P. syringae and allowed to grow to OD 600 = 1.0.…”

Section: Discussionmentioning

confidence: 99%

High‐throughput quantitative luminescence assay of the growth in planta of Pseudomonas syringae chromosomally tagged with Photorhabdus luminescens luxCDABE

Fan¹,

Crooks

Lamb³

2007

The Plant Journal

131

157

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SummaryBioluminescent strains of the Arabidopsis thaliana pathogens Pseudomonas syringae pathovar (pv.) tomato and pv. maculicola were made by insertion of the luxCDABE operon from Photorhabdus luminescens into the P. syringae chromosome under the control of a constitutive promoter. Stable integration of luxCDABE did not affect bacterial fitness, growth in planta or disease outcome. Luminescence accurately and reliably reported bacterial growth in infected Arabidopsis leaves both with a fixed inoculum followed over time and with varying inocula assayed at a single time point. Furthermore, the bioluminescence assay could detect a small (1.3-fold) difference in bacterial growth between different plant genotypes with a precision comparable to that of the standard plate assay. Luminescence of luxCDABE-tagged P. syringae allows rapid and convenient quantification of bacterial growth without the tissue extraction, serial dilution, plating and manual scoring involved in standard assays of bacterial growth by colony formation in plate culture of samples from infected tissue. The utility of the bioluminescence assay was illustrated by surveying the 500-fold variation in growth of the universally virulent P. syringae pv. maculicola ES4326 among more than 100 Arabidopsis ecotypes and identification of two quantitative trait loci accounting for 48% and 16%, respectively, of the variance of basal resistance to P. syringae pv. tomato DC3000 in the Col-0 · Fl-1 F 2 population. Luminescence assay of bacteria chromosomally tagged with luxCDABE should greatly facilitate the genetic dissection of quantitative differences in gene-for-gene, basal and acquired disease resistance and other aspects of plant interactions with bacterial pathogens requiring high-throughput assays or large-scale quantitative screens.

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“…Preparation of Pseudomonas sp. B13 GFP1 cells for electroporation was carried out as described by Artiguenave et al (1997). Electroporation of Pseudomonas sp.…”

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confidence: 99%

Antibiotic multiresistance plasmid pRSB101 isolated from a wastewater treatment plant is related to plasmids residing in phytopathogenic bacteria and carries eight different resistance determinants including a multidrug transport system

et al. 2004

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Ten different antibiotic resistance plasmids conferring high-level erythromycin resistance were isolated from an activated sludge bacterial community of a wastewater treatment plant by applying a transformation-based approach. One of these plasmids, designated pRSB101, mediates resistance to tetracycline, erythromycin, roxythromycin, sulfonamides, cephalosporins, spectinomycin, streptomycin, trimethoprim, nalidixic acid and low concentrations of norfloxacin. Plasmid pRSB101 was completely sequenced and annotated. Its size is 47 829 bp. Conserved synteny exists between the pRSB101 replication/partition (rep/par) module and the pXAC33-replicon from the phytopathogen Xanthomonas axonopodis pv. citri. The second pRSB101 backbone module encodes a three-Mob-protein type mobilization (mob) system with homology to that of IncQ-like plasmids. Plasmid pRSB101 is mobilizable with the help of the IncP-1a plasmid RP4 providing transfer functions in trans. A 20 kb resistance region on pRSB101 is located within an integron-containing Tn402-like transposon. The variable region of the class 1 integron carries the genes dhfr1 for a dihydrofolate reductase, aadA2 for a spectinomycin/streptomycin adenylyltransferase and bla TLA-2 for a so far unknown Ambler class A extended spectrum b-lactamase. The integron-specific 39-segment (qacED1-sul1-orf5D) is connected to a macrolide resistance operon consisting of the genes mph(A) (macrolide 29-phosphotransferase I), mrx (hydrophobic protein of unknown function) and mphR(A) (regulatory protein). Finally, a putative mobile element with the tetracycline resistance genes tetA (tetracycline efflux pump) and tetR was identified upstream of the Tn402-specific transposase gene tniA. The second 'genetic load' region on pRSB101 harbours four distinct mobile genetic elements, another integron belonging to a new class and footprints of two more transposable elements. A tripartite multidrug (MDR) transporter consisting of an ATP-binding-cassette (ABC)-type ATPase and permease, and an efflux membrane fusion protein (MFP) of the RND-family is encoded between the replication/partition and the mobilization module. Homologues of the macrolide resistance genes mph(A), mrx and mphR(A) were detected on eight other erythromycin resistance-plasmids isolated from activated sludge bacteria. Plasmid pRSB101-like repA amplicons were also obtained from plasmid-DNA preparations of the final effluents of the wastewater treatment plant indicating that pRSB101-like plasmids are released with the final effluents into the environment.

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High-efficiency transposon mutagenesis by electroporation of a Pseudomonas fluorescens strain

Cited by 22 publications

References 15 publications

Auxotrophic Markers pyrF and proC Can Replace Antibiotic Markers on Protein Production Plasmids in High-Cell-Density Pseudomonas fluorescens Fermentation

Auxotrophic Markers pyrF and proC Can Replace Antibiotic Markers on Protein Production Plasmids in High-Cell-Density Pseudomonas fluorescens Fermentation

High‐throughput quantitative luminescence assay of the growth in planta of Pseudomonas syringae chromosomally tagged with Photorhabdus luminescens luxCDABE

Antibiotic multiresistance plasmid pRSB101 isolated from a wastewater treatment plant is related to plasmids residing in phytopathogenic bacteria and carries eight different resistance determinants including a multidrug transport system

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