Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange

Hmelo, Laura R.; Borlee, Bradley R.; Almblad, Henrik; Love, Michelle E.; Randall, Trevor E.; Tseng, Boo Shan; Lin, Chuyang; Irie, Yasuhiko; Storek, Kelly M.; Yang, Jaeun; Siehnel, Richard; Howell, P. Lynne; Singh, Pradeep Kumar; Tolker‐Nielsen, Tim; Parsek, Matthew R.; Schweizer, Herbert P.; Harrison, Joe J.

doi:10.1038/nprot.2015.115

Cited by 434 publications

(414 citation statements)

References 106 publications

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“…Deletion of the diol synthase operon was performed by allelic exchange using the suicide vector pEX100Tlink and a previously described method34 Briefly, a fragment of P. aeruginosa chromosome containing the genes PA2078 and PA2077 plus ∼300 bp of chromosomal flanking regions were amplified by PCR using the primers JCG1 (5′-ggcgaaagcttcgccttcctgccg-3′) and JCG2 (5′-ggcgggaattctggtcaccaccttct-3′). These primers introduced sites EcoRI and HindIII at the ends of the amplified fragment that were used for cloning into the suicide vector pEX100Tlink to obtain the plasmid pEX-PA77-78.…”

Section: Methodsmentioning

confidence: 99%

Oxylipins produced by Pseudomonas aeruginosa promote biofilm formation and virulence

Martínez

Campos-Gómez

2016

Nat Commun

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The oxygenation of unsaturated fatty acids by dioxygenases occurs in all kingdoms of life and produces physiologically important lipids called oxylipins. The biological roles of oxylipins have been extensively studied in animals, plants, algae and fungi, but remain largely unidentified in prokaryotes. The bacterium Pseudomonas aeruginosa displays a diol synthase activity that transforms several monounsaturated fatty acids into mono- and di-hydroxylated derivatives. Here we show that oxylipins derived from this activity inhibit flagellum-driven motility and upregulate type IV pilus-dependent twitching motility of P. aeruginosa. Consequently, these oxylipins promote bacterial organization in microcolonies, increasing the ability of P. aeruginosa to form biofilms in vitro and in vivo (in Drosophila flies). We also demonstrate that oxylipins produced by P. aeruginosa promote virulence in Drosophila flies and lettuce. Our study thus uncovers a role for prokaryotic oxylipins in the physiology and pathogenicity of bacteria.

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Section: Methodsmentioning

confidence: 99%

Oxylipins produced by Pseudomonas aeruginosa promote biofilm formation and virulence

Martínez

Campos-Gómez

2016

Nat Commun

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“…This may be particularly critical in situations where conditions required for lethal counterselection may interfere with the phenotype of the desired mutants (e.g., during the isolation of osmosensitive mutants with sacB-based systems; see reference 39). Finally, our mCherry-based screening method is less prone to the counterselection escape problem associated even with optimized sacB-based selection protocols (40) because no toxic effects are generated by mCherry expression.…”

Section: Resultsmentioning

confidence: 99%

Versatile Vectors for Efficient Mutagenesis of Bradyrhizobium diazoefficiens and Other Alphaproteobacteria

Ledermann

Strebel

Kampik

et al. 2016

Appl Environ Microbiol

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Analysis of bacterial gene function commonly relies on gene disruption or replacement followed by phenotypic characterization of the resulting mutant strains. Deletion or replacement of targeted regions is commonly achieved via two homologous recombination (HR) events between the bacterial genome and a nonreplicating plasmid carrying DNA fragments flanking the region to be deleted. The counterselection of clones that have integrated the entire plasmid in their genome via a single HR event is crucial in this procedure. Various genetic tools and well-established protocols are available for this type of mutagenesis in model bacteria; however, these methods are not always efficiently applicable in less established systems. Here we describe the construction and application of versatile plasmid vectors pREDSIX and pTETSIX for marker replacement and markerless mutagenesis, respectively. Apart from an array of restriction sites optimized for cloning of GC-rich DNA fragments, the vector backbone contains a constitutively expressed gene for mCherry, enabling the rapid identification of clones originating from single or double HR events by fluorescence-assisted cell sorting (FACS). In parallel, we constructed a series of plasmids from which gene cassettes providing resistance against gentamicin, kanamycin, hygromycin B, streptomycin and spectinomycin, or tetracycline were excised for use with pREDSIX-based marker replacement mutagenesis. In proof-of-concept mutagenesis experiments, we demonstrated the potential for the use of the developed tools for gene deletion mutagenesis in the nitrogen-fixing soybean symbiont Bradyrhizobium diazoefficiens (formerly Bradyrhizobium japonicum) and three additional members of the alphaproteobacteria. IMPORTANCEMutation and phenotypic analysis are essential to the study of gene function. Efficient mutagenesis protocols and tools are available for many bacterial species, including various model organisms; however, genetic analysis of less-well-characterized organisms is often impaired by the lack of efficient methods. Here we describe a set of novel genetic tools for facilitated mutagenesis of the nitrogen-fixing soybean symbiont Bradyrhizobium diazoefficiens and related alphaproteobacteria. We demonstrated their usefulness by generating several mutant strains lacking defined genes. Isolation of both antibiotic resistance gene-containing and markerless deletion mutants is greatly facilitated because undesired clones which contain the entire mutagenic plasmid integrated in the genome can be identified on the basis of their fluorescent phenotype derived from the mCherry gene carried by the vector backbone. The possibility to generate markerless mutants assists with the isolation of strains carrying multiple deletions, which can be crucial while studying functionally redundant genes. Many functional studies of biological phenomena rely on the isolation of mutants and the availability of the respective genetic tools. Mutants are preferably constructed in a targeted manner, which traditi...

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“…Gene deletions were performed as previously described in strain PA14 [49]. Briefly, 800-1200bp regions flanking the target deletion gene of interest were PCR amplified, stitched and recombined into the pEXG2 [49] plasmid containing GentR and SacB markers using Gateway Cloning.…”

Section: Methods Validation With Clean Gene Deletionsmentioning

confidence: 99%

“…Briefly, 800-1200bp regions flanking the target deletion gene of interest were PCR amplified, stitched and recombined into the pEXG2 [49] plasmid containing GentR and SacB markers using Gateway Cloning. Plasmids were conjugated into PA14 using the E. coli helper plasmid pRK2013 for 8 hours, followed by selection on LB agar containing 15ug/ml triclosan + 30ug/ml gentamicin.…”

Section: Methods Validation With Clean Gene Deletionsmentioning

confidence: 99%

Defining the core essential genome ofPseudomonas aeruginosa

Poulsen

Yang

Clatworthy

et al. 2018

Preprint

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Pseudomonas aeruginosa is a clinically significant pathogen that has alarming antibiotic resistance rates and very few candidate drugs in development. The challenges of novel drug discovery are exacerbated by incomplete knowledge of the essential genes across the species required for survival under infection conditions. We thus sought to define the core essential genome of P. aeruginosa by performing transposon insertion sequencing (Tn-Seq) on nine strains of P. aeruginosa isolated from different infection sites including wound, eye, lung, urinary tract, and blood, with an environmentally isolated strain for comparison, in five different media conditions: three were infection relevant media (serum, sputum, urine) and two were lab-based media (LB and M9 minimal). We developed a novel statistical model, FiTnEss, to classify genes as essential versus nonessential across all strain-media combinations. FiTnEss required minimal assumptions and had good predictive power in a limited set of validation studies with mutant strains of PA14 containing clean gene deletions. A core set of 321 essential genes emerged that are the highest probability targets for successful novel drug discovery against this important pathogen.

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Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange

Cited by 434 publications

References 106 publications

Oxylipins produced by Pseudomonas aeruginosa promote biofilm formation and virulence

Oxylipins produced by Pseudomonas aeruginosa promote biofilm formation and virulence

Versatile Vectors for Efficient Mutagenesis of Bradyrhizobium diazoefficiens and Other Alphaproteobacteria

Defining the core essential genome ofPseudomonas aeruginosa

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