Efficient Dual-Negative Selection for Bacterial Genome Editing

Cianfanelli, Francesca Romana; Cunrath, Olivier; Bumann, Dirk

doi:10.1101/2020.03.03.974816

Cited by 3 publications

(7 citation statements)

References 35 publications

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“…The BZB1011 ΔbtuB sfgfp::Tn7 or mrfp1::Tn7 strains were created by using the method described by Cianfanelli et al 60 . Briefly, the regions (700 base pairs) upstream and downstream of the btuB gene were amplified by PCR and joined together in the pFOK plasmid using the NEB® HiFi DNA Assembly Master Mix.…”

Section: Construction Of Bacterial Strainsmentioning

confidence: 99%

Droplet printing reveals the importance of micron-scale structure for bacterial ecology

Kumar

Meiller-Legrand

Alcinesio

et al. 2020

Preprint

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Bacteria often live in diverse communities where the spatial arrangement of strains and species is considered critical for their ecology, including whether strains can coexist, which are ecologically dominant, and how productive they are as a community. However, a test of the importance of spatial structure requires manipulation at the fine scales at which this structure naturally occurs. Here we develop a droplet-based printing method to arrange different bacterial genotypes across a sub-millimetre array. We use this to test the importance of fine-scale spatial structure by printing strains of the gut bacterium Escherichia coli that naturally compete with one another using protein toxins. This reveals that the spatial arrangement of bacterial genotypes is important for ecological outcomes. Toxin-producing strains largely eliminate susceptible non-producers when genotypes are well-mixed. However, printing strains side-by-side creates an ecological refuge such that susceptible strains can coexist with toxin producers, even to the extent that a susceptible strain outnumbers the toxin producer. Head-to-head competitions between toxin producers also reveals strong effects, where spatial structure can make the difference between one strain winning and mutual destruction. Finally, we print different potential barriers between two competing strains to understand why space is so important. This reveals the importance of processes that limit the free diffusion of molecules. Specifically, we show that cells closest to a toxin producer bind to and capture toxin molecules, which creates a refuge for their clonemates. Our work provides a new method to generate customised bacterial communities with defined spatial distributions, and reveals that micron-scale changes in these distributions can drive major shifts in their ecology.

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Section: Construction Of Bacterial Strainsmentioning

confidence: 99%

Droplet printing reveals the importance of micron-scale structure for bacterial ecology

Kumar

Meiller-Legrand

Alcinesio

et al. 2020

Preprint

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“…We used ampicillin selection for plasmid cloning and propagation, and the other markers only for positive selection of trans-conjugants, thus limiting the use of toxic or expensive selection agents. Vector construction and mutagenesis of target strains was performed using E. coli strain Jke201 -a pir-carrying, diaminopimelic acid (DAP)-auxotrophic donor strain that allows for replication of plasmids containing an R6K origin and the subsequent removal of donor strain on any growth medium not supplemented with DAP [Harms et al, 2017;Cianfanelli et al, 2020].…”

Section: Resultsmentioning

confidence: 99%

“…Once the pALFI1 plasmid was synthesized, pALFI2 was generated by replacing hygR with tetA with its native regulator tetR from A. baumannii AB0057. pALFI3 was generated by replacing hygR with tpm ( thiopurine-S-methyltranferase) conferring resistance to tellurite from pFOKT [Cianfanelli et al, 2020] (Supplementary material S2). The TcR and Tpm markers were cloned using the SwaI/ScaI restriction sites.…”

Section: Construction Of Suicide Vectorsmentioning

confidence: 99%

“…The majority of suicide vectors used in Gram-negative bacteria include a sacB gene from Bacillus subtilis encoding levansucrase for counter-selection. Levansucrase is an enzyme that confers sucrose sensitivity in Gram-negative bacteria by catalysing the hydrolysis of sucrose into glucose and the toxic product levan in the periplasm [Hmelo et al, 2015;Cianfanelli et al, 2020;Biswas, 2015]. Double crossover mutants are isolated by growing the single crossover mutants in the presence of sucrose, as only those bacteria that have lost the sacB gene can grow under this condition.…”

Section: Introductionmentioning

confidence: 99%

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A suite of modular, all-synthetic suicide vectors for allelic exchange mutagenesis in multidrug resistant Acinetobacter strains

Pokhrel

Short

et al. 2022

Preprint

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Background: Acinetobacter baumannii is an opportunistic human pathogen that causes a variety of infections in immunosuppressed individuals and patients in intensive care units. The success of this pathogen in nosocomial settings can be directly attributed to its persistent nature and its ability to rapidly acquire multidrug resistance. It is now considered to be one of the top priority pathogens for development of novel therapeutic approaches. Several high-throughput techniques have been utilised to identify the genetic determinants contributing to the success of A. baumannii as a global pathogen. However, targeted gene-function studies remain challenging due to the lack of appropriate genetic tools. Results: Here, we have constructed a series of all-synthetic allelic exchange vectors – pALFI1, pALFI2 and pALFI3 – with suitable selection markers for targeted genetic studies in highly drug resistant A. baumannii isolates. The vectors follow the Standard European Vector Architecture (SEVA) framework for easy replacement of components. This method allows for rapid plasmid construction with the mutant allele, efficient conjugational transfer using a diaminopimelic acid-dependent Escherichia coli donor strain, efficient positive selection using the suitable selection markers and finally, sucrose-dependent counter-selection to obtain double-crossovers. Conclusions: We have used this method to create scar-less deletion mutants in three different strains of A. baumannii, which resulted in up to 75% deletion frequency of the targeted gene. We believe this method can be effectively used to perform genetic manipulation studies in multidrug resistant Gram-negative bacterial strains.

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“…Reporter strains carried pSC101 derivatives with constitutive expression of mCherry or mtagbfp2 from the P ybaJ promoter and transcriptional fusions of candidate promoters to gfp-ova coding for degradable fluorescent protein variants (half-life in the range of 30 min) (133). Salmonella mutants with gene deletions were obtained by two consecutive single crossovers with positive selection for resistance to kanamycin and negative selection for levan sucrase-mediated sensitivity to sucrose (134).…”

Section: Methodsmentioning

confidence: 99%

Tissue compartmentalization enablesSalmonellapersistence during chemotherapy

Claudi

Fanous

et al. 2021

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

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Antimicrobial chemotherapy can fail to eradicate the pathogen, even in the absence of antimicrobial resistance. Persisting pathogens can subsequently cause relapsing diseases. In vitro studies suggest various mechanisms of antibiotic persistence, but their in vivo relevance remains unclear because of the difficulty of studying scarce pathogen survivors in complex host tissues. Here, we localized and characterized rare surviving Salmonella in mouse spleen using high-resolution whole-organ tomography. Chemotherapy cleared >99.5% of the Salmonella but was inefficient against a small Salmonella subset in the white pulp. Previous models could not explain these findings: drug exposure was adequate, Salmonella continued to replicate, and host stresses induced only limited Salmonella drug tolerance. Instead, antimicrobial clearance required support of Salmonella-killing neutrophils and monocytes, and the density of such cells was lower in the white pulp than in other spleen compartments containing higher Salmonella loads. Neutrophil densities declined further during treatment in response to receding Salmonella loads, resulting in insufficient support for Salmonella clearance from the white pulp and eradication failure. However, adjunctive therapies sustaining inflammatory support enabled effective clearance. These results identify uneven Salmonella tissue colonization and spatiotemporal inflammation dynamics as main causes of Salmonella persistence and establish a powerful approach to investigate scarce but impactful pathogen subsets in complex host environments.

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Efficient Dual-Negative Selection for Bacterial Genome Editing

Cited by 3 publications

References 35 publications

Droplet printing reveals the importance of micron-scale structure for bacterial ecology

Droplet printing reveals the importance of micron-scale structure for bacterial ecology

A suite of modular, all-synthetic suicide vectors for allelic exchange mutagenesis in multidrug resistant Acinetobacter strains

Tissue compartmentalization enablesSalmonellapersistence during chemotherapy

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