A High-Throughput Method for Screening for Genes Controlling Bacterial Conjugation of Antibiotic Resistance

Alalam, Hanna; F, Graf; Palm, Martin; Abadikhah, Marie; Zackrisson, Martin; Boström, Jonas; Fransson, Alfred; Hadjineophytou, Chris; Persson, Leif G.; Stenberg, Simon; Mattsson, Matilda; Ghiaci, Payam; Sunnerhagen, Per; Warringer, Jonas; Farewell, Anne

doi:10.1128/msystems.01226-20

Cited by 17 publications

(17 citation statements)

References 42 publications

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“…We have identified four chromosomally-encoded gene products that are involved in the E. coli donor ability of the broad-host-range conjugative plasmid pKJK10. In this respect, our work complements previous screenings searching for conserved targets for the development of COINs, which ultimately can be administered during treatment with antibiotics to halt the spread of antibiotic resistance [ 21 , 22 , 24 ]. Further work will elucidate the mechanisms whereby these gene products affect conjugation and explore whether their roles extend across a broader diversity of donors, recipients, plasmids and environmental conditions.…”

Section: Discussionmentioning

confidence: 73%

“…Furthermore, a transposon sequencing (Tn-Seq) screen has revealed the role of phospholipid biosynthesis in the transfer of the integrative and conjugative element ICE Bs1 in Bacillus subtilis [ 22 , 23 ]. Additionally, high-throughput monitoring of transconjugant growth, in which E. coli single-gene deletion mutants served as donors of the F plasmid, has revealed a role in the donor cell for DNA replication, chaperones or protein folding, and lipopolysaccharide core biosynthesis [ 24 ]. Finally, host factors have been described to interfere with the regulation of the transfer region of the conjugative elements [ 25 ].…”

Section: Introductionmentioning

confidence: 99%

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Genome-Wide Association Study Reveals Host Factors Affecting Conjugation in Escherichia coli

et al. 2022

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The emergence and dissemination of antibiotic resistance threaten the treatment of common bacterial infections. Resistance genes are often encoded on conjugative elements, which can be horizontally transferred to diverse bacteria. In order to delay conjugative transfer of resistance genes, more information is needed on the genetic determinants promoting conjugation. Here, we focus on which bacterial host factors in the donor assist transfer of conjugative plasmids. We introduced the broad-host-range plasmid pKJK10 into a diverse collection of 113 Escherichia coli strains and measured by flow cytometry how effectively each strain transfers its plasmid to a fixed E. coli recipient. Differences in conjugation efficiency of up to 2.7 and 3.8 orders of magnitude were observed after mating for 24 h and 48 h, respectively. These differences were linked to the underlying donor strain genetic variants in genome-wide association studies, thereby identifying candidate genes involved in conjugation. We confirmed the role of fliF, fliK, kefB and ucpA in the donor ability of conjugative elements by validating defects in the conjugation efficiency of the corresponding lab strain single-gene deletion mutants. Based on the known cellular functions of these genes, we suggest that the motility and the energy supply, the intracellular pH or salinity of the donor affect the efficiency of plasmid transfer. Overall, this work advances the search for targets for the development of conjugation inhibitors, which can be administered alongside antibiotics to more effectively treat bacterial infections.

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Section: Discussionmentioning

confidence: 73%

Section: Introductionmentioning

confidence: 99%

Genome-Wide Association Study Reveals Host Factors Affecting Conjugation in Escherichia coli

et al. 2022

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“…Likewise, as our populations are asexual, we fail to capture any evolvability effects of mating or inbreeding or of genes increasing meiotic (or mitotic in the case of parasexual fungal pathogens) recombination rates. Finally, transformation or conjugation promoting factors that enhance the horizontal transmission of genes, which at least in bacteria can have large effects on adaptation rates (Alalam et al, 2020; Graf et al, 2019), will be overlooked here. Bearing these caveats in mind, our finding that the >4.600 probed genes possessed no functions with a substantial impact on adaption rate is nevertheless quite remarkable and calls for caution when considering the evolutionary importance of evolvability in general.…”

Section: Discussionmentioning

confidence: 99%

Adaptation of the yeast gene knockout collection is near-perfectly predicted by fitness and diminishing return epistasis

Persson

Stenberg

Tamás

et al. 2022

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Adaptive evolution of clonally dividing cells and microbes is the ultimate cause of cancer and infectious diseases. The possibility of constraining the adaptation of cell populations, by inhibiting proteins that enhance their evolvability has therefore attracted substantial interest. However, our current understanding of how individual genes influence the speed of adaptation is limited, partly because accurately tracking adaptation for many experimental cell populations in parallel is challenging. Here we use a high throughput artificial laboratory evolution (ALE) platform to track the adaptation of >18.000 cell populations corresponding to single gene deletion strains in the haploid yeast deletion collection. We report that the fitness of gene knockout near-perfectly (R2=0.91) predicts their adaptation dynamics under arsenic exposure, leaving virtually no role for dedicated evolvability functions in the corresponding proteins. We tracked the adaptation of another >23.000 yeast gene knockout populations to a diverse range of selection pressures and generalised the almost perfect (R2=0.72 to 0.98) capacity of initial fitness to predict the rate of adaptation. Finally, we reconstruct mutations in the genes FPS1, ASK10, and ARR3, which together account for almost all arsenic adaptation in wildtype cells, in gene deletions covering a broad fitness range. We show that the predictability of arsenic adaptation can be understood almost entirely as a global epistasis phenomenon where excluding arsenic from cells, through these mutations, is more beneficial in cells with low arsenic fitness regardless of what causes the arsenic defects. The lack of genes with a meaningful effect on the adaptation dynamics of clonally reproducing cell populations diminishes the prospects of developing adjuvant drugs aiming to slow antimicrobial and chemotherapy resistance.

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“…Hence, not only does it bring evolutionary engineering into the realm of high-throughput science but it also opens a new fast-track lane for optimizing microbes for industry-desired traits. And because the platform works well with microbes covering broad swaths of the tree of life, including bacteria 96 , we expect it to be of substantial value across many industrial sectors.…”

Section: Discussionmentioning

confidence: 99%

“…Hence, not only does it bring evolutionary engineering into the realm of high-throughput science but it also opens a new fasttrack lane for optimizing microbes for industry-desired traits. And because the platform works well with microbes covering broad swaths of the tree of life, including bacteria 96 , we expect it to be of substantial value across many industrial sectors. S1) commercial and pre-commercial wine yeasts as replicated colony populations on each of eight synthetic grape must conditions designed to select for traits desired by the wine industry (Table S2 and S3).…”

Section: Parallelization Improves Ale Outcomes By Broadening the Evol...mentioning

confidence: 99%

Highly parallelized laboratory evolution of wine yeasts for enhanced metabolic phenotypes

Ghiaci

Jouhten

Martyushenko

et al. 2022

Preprint

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Adaptive Laboratory Evolution (ALE) of microbes can improve the efficiency of sustainable industrial processes important to the global economy, but chance and genetic background effects often lead to suboptimal outcomes. Here we report an ALE platform to circumvent these flaws through parallelized clonal evolution at an unprecedented scale. Using this platform, we clonally evolved 104 yeast populations in parallel from many strains for eight desired wine production traits. Expansions of both ALE replicates and lineage numbers broadened the evolutionary search spectrum and increased the chances of evolving improved wine yeasts unencumbered by unwanted side effects. ALE gains often coincided with distinct aneuploidies and the emergence of semi-predictable side effects that were characteristic of each selection niche. Many high performing ALE strains retained their desired traits upon transfer to industrial conditions and produced high quality wine. Overall, our ALE platform brings evolutionary engineering into the realm of high throughput science and opens opportunities for rapidly optimizing microbes for use in many industrial sectors which otherwise could take many years to accomplish.

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A High-Throughput Method for Screening for Genes Controlling Bacterial Conjugation of Antibiotic Resistance

Cited by 17 publications

References 42 publications

Genome-Wide Association Study Reveals Host Factors Affecting Conjugation in Escherichia coli

Genome-Wide Association Study Reveals Host Factors Affecting Conjugation in Escherichia coli

Adaptation of the yeast gene knockout collection is near-perfectly predicted by fitness and diminishing return epistasis

Highly parallelized laboratory evolution of wine yeasts for enhanced metabolic phenotypes

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