Mapping Transposon Insertions in Bacterial Genomes by Arbitrarily Primed PCR

Saavedra, José T.; Schwartzman, Julia; Gilmore, Michael S.

doi:10.1002/cpmb.38

Cited by 21 publications

(16 citation statements)

References 63 publications

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“…No antibiotics were present in the growth medium for the aggregation assays. Transposon insertion sites were determined using arbitrary PCR (Saavedra et al, 2017). Images of colony opacity were obtained after 24 h of growth of colonies on LB plates at 37°C using a stereomicroscope (Leica, Wetzlar, Germany; M125; 20X zoom) equipped with a Leica MC170 HD camera and using a gooseneck light source to provide oblique sample illumination.…”

Section: Methodsmentioning

confidence: 99%

Quorum sensing controls Vibrio cholerae multicellular aggregate formation

Jemielita

Wingreen

Bassler

2018

eLife

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Bacteria communicate and collectively regulate gene expression using a process called quorum sensing (QS). QS relies on group-wide responses to signal molecules called autoinducers. Here, we show that QS activates a new program of multicellularity in Vibrio cholerae. This program, which we term aggregation, is distinct from the canonical surface-biofilm formation program, which QS represses. Aggregation is induced by autoinducers, occurs rapidly in cell suspensions, and does not require cell division, features strikingly dissimilar from those characteristic of V. cholerae biofilm formation. Extracellular DNA limits aggregate size, but is not sufficient to drive aggregation. A mutagenesis screen identifies genes required for aggregate formation, revealing proteins involved in V. cholerae intestinal colonization, stress response, and a protein that distinguishes the current V. cholerae pandemic strain from earlier pandemic strains. We suggest that QS-controlled aggregate formation is important for V. cholerae to successfully transit between the marine niche and the human host.

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Section: Methodsmentioning

confidence: 99%

Quorum sensing controls Vibrio cholerae multicellular aggregate formation

Jemielita

Wingreen

Bassler

2018

eLife

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“…Transposon insertion sites in bacterial chromosomes were determined by arbitrarily primed PCR, in which transposon junctions were amplified in two steps [ 5 , 39 ]. Bacterial cells were resuspended in 10–20 μl of deionized water and 1 μl was used directly as the PCR template.…”

Section: Methodsmentioning

confidence: 99%

Efficient transposon mutagenesis mediated by an IPTG-controlled conditional suicide plasmid

et al. 2018

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BackgroundTransposon mutagenesis is highly valuable for bacterial genetic and genomic studies. The transposons are usually delivered into host cells through conjugation or electroporation of a suicide plasmid. However, many bacterial species cannot be efficiently conjugated or transformed for transposon saturation mutagenesis. For this reason, temperature-sensitive (ts) plasmids have also been developed for transposon mutagenesis, but prolonged incubation at high temperatures to induce ts plasmid loss can be harmful to the hosts and lead to enrichment of mutants with adaptive genetic changes. In addition, the ts phenotype of a plasmid is often strain- or species-specific, as it may become non-ts or suicidal in different bacterial species.ResultsWe have engineered several conditional suicide plasmids that have a broad host range and whose loss is IPTG-controlled. One construct, which has the highest stability in the absence of IPTG induction, was then used as a curable vector to deliver hyperactive miniTn5 transposons for insertional mutagenesis. Our analyses show that these new tools can be used for efficient and regulatable transposon mutagenesis in Escherichia coli, Acinetobacter baylyi and Pseudomonas aeruginosa. In P. aeruginosa PAO1, we have used this method to generate a Tn5 insertion library with an estimated diversity of ~ 108, which is ~ 2 logs larger than the best transposon insertional library of PAO1 and related Pseudomonas strains previously reported.ConclusionWe have developed a number of IPTG-controlled conditional suicide plasmids. By exploiting one of them for transposon delivery, a highly efficient and broadly useful mutagenesis system has been developed. As the assay condition is mild, we believe that our methodology will have broad applications in microbiology research.Electronic supplementary materialThe online version of this article (10.1186/s12866-018-1319-0) contains supplementary material, which is available to authorized users.

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“…Mutants that displayed biofilm growth at the 8 h timepoint but failed to disperse by the 13 h timepoint were subcultured, grown overnight, and subsequently reimaged using the time-lapse approach described above to assess their biofilm lifecycles in real time. Mutants that exhibited biofilm dispersal defects after this reassessment step were analyzed for the locations of transposon insertions using arbitrary PCR ( 44 ).…”

Section: Methodsmentioning

confidence: 99%

Identification of signaling pathways, matrix-digestion enzymes, and motility components controlling Vibrio cholerae biofilm dispersal

Bridges

Fei

Bassler

2020

Proc. Natl. Acad. Sci. U.S.A.

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Bacteria alternate between being free-swimming and existing as members of sessile multicellular communities called biofilms. The biofilm lifecycle occurs in three stages: cell attachment, biofilm maturation, and biofilm dispersal.Vibrio choleraebiofilms are hyperinfectious, and biofilm formation and dispersal are considered central to disease transmission. While biofilm formation is well studied, almost nothing is known about biofilm dispersal. Here, we conducted an imaging screen forV. choleraemutants that fail to disperse, revealing three classes of dispersal components: signal transduction proteins, matrix-degradation enzymes, and motility factors. Signaling proteins dominated the screen and among them, we focused on an uncharacterized two-component sensory system that we term DbfS/DbfR for dispersal of biofilm sensor/regulator. Phospho-DbfR represses biofilm dispersal. DbfS dephosphorylates and thereby inactivates DbfR, which permits dispersal. Matrix degradation requires two enzymes: LapG, which cleaves adhesins, and RbmB, which digests matrix polysaccharides. Reorientation in swimming direction, mediated by CheY3, is necessary for cells to escape from the porous biofilm matrix. We suggest that these components act sequentially: signaling launches dispersal by terminating matrix production and triggering matrix digestion, and subsequent cell motility permits escape from biofilms. This study lays the groundwork for interventions aimed at modulatingV. choleraebiofilm dispersal to ameliorate disease.

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Mapping Transposon Insertions in Bacterial Genomes by Arbitrarily Primed PCR

Cited by 21 publications

References 63 publications

Quorum sensing controls Vibrio cholerae multicellular aggregate formation

Quorum sensing controls Vibrio cholerae multicellular aggregate formation

Efficient transposon mutagenesis mediated by an IPTG-controlled conditional suicide plasmid

Identification of signaling pathways, matrix-digestion enzymes, and motility components controlling Vibrio cholerae biofilm dispersal

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