Barcoding Populations of Pseudomonas fluorescens SBW25

Theodosiou, Loukas; Farr, Andrew D; Rainey, Paul B.

doi:10.1007/s00239-023-10103-6

Cited by 3 publications

(3 citation statements)

References 42 publications

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“…To test this model empirically we designed a system in which 5 P. aeruginosa PA14 strains, each representing 1 of the 5 genotypes in the deterministic model, were edited to include a unique DNA sequence in their genome—genetically tagging them, meaning the relative proportion of each strain in the overall population could be quantified by qPCR amplification. This quantification technique has previously been used to follow selection in mixed species populations without the addition of unique sequences [ 32 ] and follows a larger trend of barcoding and sequencing techniques as a useful tool for tracking populations during evolution experiments (reviewed in [ 41 ]). The selection experiment was restricted to 24 hours of co-culturing, reducing the potential for the emergence of newly evolved phenotypes during the experiment, as barcoding and qPCR quantification allows only the measurement of selection for/against each pre-assigned genotype as a stand-in for phage resistance/sensitivity phenotypes, not the measurement phenotype frequencies directly.…”

Section: Discussionmentioning

confidence: 99%

Conditions for the spread of CRISPR-Cas immune systems into bacterial populations

Elliott,

McLeod,

Taylor

et al. 2024

The ISME Journal

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Bacteria contain a wide variety of innate and adaptive immune systems which provide protection to the host against invading genetic material, including bacteriophages (phages). It is becoming increasingly clear that bacterial immune systems are frequently lost and gained through horizontal gene transfer (HGT). However, how and when new immune systems can become established in a bacterial population has remained largely unstudied. We developed a joint epidemiological and evolutionary model that predicts the conditions necessary for the spread of a CRISPR-Cas immune system into a bacterial population lacking this system. We find that whether bacteria carrying CRISPR-Cas will spread (increase in frequency) into a bacterial population depends on the abundance of phages and the difference in the frequency of phage resistance mechanisms between bacteria carrying a CRISPR-Cas immune system, and those not (denoted as ${f}_{\Delta }$). Specifically, the abundance of cells carrying CRISPR-Cas will increase if there is a higher proportion of phage resistance (either via CRISPR-Cas immunity or surface modification) in the CRISPR-Cas possessing population than in the cells lacking CRISPR-Cas. We experimentally validated these predictions using Pseudomonas aeruginosa PA14 and phage DMS3vir as a model. Specifically, by varying the initial ratios of different strains of bacteria that carry alternative forms of phage resistance we confirmed that the spread of cells carrying CRISPR-Cas through a population can be predicted based on phage density and the relative frequency of resistance phenotypes. Understanding which conditions promote the spread of CRISPR-Cas systems helps to predict when and where these defences can establish in bacterial populations after a horizontal gene transfer event, both in ecological and clinical contexts.

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Section: Discussionmentioning

confidence: 99%

Conditions for the spread of CRISPR-Cas immune systems into bacterial populations

Elliott,

McLeod,

Taylor

et al. 2024

The ISME Journal

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“…Construction of the 'rpoSp-kan reporter' and fluorescent strains with this reporter were also constructed using 'NEBuilder HiFi DNA assembly' (NEB). To construct the 'rpoSp-kan reporter', 401 bp of nlpD (with the C565T mutation situated centrally) was amplified along with a kanamycin resistance gene (kanR) and a backbone plasmid (extracted from MPB15151, see (Theodosiou, Farr, & Rainey, 2023)) encoding Tn7 elements, an origin of replication and a tetracycline resistance enzyme. These three fragments were assembled using 'NEBuilder HiFi DNA assembly Master Mix' (NEB) (see above), and transformed into top10 chemically competent cells.…”

Section: Reconstruction Of Mutations and Insertion Of Genesmentioning

confidence: 99%

An extreme mutational hotspot innlpDdepends on transcriptional induction ofrpoS

Farr,

Vasileiou,

Lind

et al. 2024

Preprint

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Mutation rate varies within and between genomes. Within genomes, tracts of nucleotides, including short sequence repeats and palindromes, can cause localised elevation of mutation rate. Additional mechanisms remain poorly understood. Here we report an instance of extreme mutational bias inPseudomonas fluorescensSBW25 associated with a single base-pair change innlpD.These mutants frequently evolve in static microcosms, and have a cell-chaining (CC) phenotype. Analysis of 153 replicate populations revealed 137 independent instances of a C565T loss-of-function mutation at codon 189 (CAG to TAG (Q189*)). Fitness measures of alternativenlpDmutants showed molecular parallelism to be unconnected to selective advantage. Recognising that transcription can be mutagenic, and that codon 189 overlaps with a predicted promoter (rpoSp) for the adjacent stationary phase sigma factor,rpoS, transcription across this promoter region was measured. This confirmedrpoSpis induced in stationary phase and that C565T mutation caused significant elevation of transcription. The latter provided opportunity to determine the C565T mutation rate using a reporter-gene fused torpoSp. Fluctuation assays demonstrate the C565T mutation rate to be 5,700-fold higher than expected. InPseudomonas, transcription ofrpoSrequires the positive activator PsrA, which we show also holds for SBW25. Fluctuation assays performed in a ΔpsrAbackground showed a 60-fold reduction in mutation rate confirming that the elevated rate of mutation at C565T mutation rate is dependent on induction of transcription. This hotspot suggests a generalisable phenomenon where the induction of transcription causes elevated mutation rates within defining regions of promoters.

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“…The introduction of unique DNA barcodes at neutral chromosomal locations of bacterial individuals offers an opportunity to track bacterial cell lineage dynamics in a high-resolution manner 23 – 25 . The unique barcodes constitute DNA tags that can be tracked via amplification and sequencing of the genetic sequence where they were initially inserted in the organisms.…”

Section: Introductionmentioning

confidence: 99%

Effects of plant tissue permeability on invasion and population bottlenecks of a phytopathogen

Jiang,

Zhang,

Chen

et al. 2024

Nat Commun

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Pathogen genetic diversity varies in response to environmental changes. However, it remains unclear whether plant barriers to invasion could be considered a genetic bottleneck for phytopathogen populations. Here, we implement a barcoding approach to generate a pool of 90 isogenic and individually barcoded Ralstonia solanacearum strains. We used 90 of these strains to inoculate tomato plants with different degrees of physical permeability to invasion (intact roots, wounded roots and xylem inoculation) and quantify the phytopathogen population dynamics during invasion. Our results reveal that the permeability of plant roots impacts the degree of population bottleneck, genetic diversity, and composition of Ralstonia populations. We also find that selection is the main driver structuring pathogen populations when barriers to infection are less permeable, i.e., intact roots, the removal of root physical and immune barriers results in the predominance of stochasticity in population assembly. Taken together, our study suggests that plant root permeability constitutes a bottleneck for phytopathogen invasion and genetic diversity.

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Barcoding Populations of Pseudomonas fluorescens SBW25

Cited by 3 publications

References 42 publications

Conditions for the spread of CRISPR-Cas immune systems into bacterial populations

Conditions for the spread of CRISPR-Cas immune systems into bacterial populations

An extreme mutational hotspot innlpDdepends on transcriptional induction ofrpoS

Effects of plant tissue permeability on invasion and population bottlenecks of a phytopathogen

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