Enhancing multiplex genome editing by natural transformation (MuGENT) via inactivation of ssDNA exonucleases

Dalia, Triana N.; Yoon, Soo Hun; Galli, Elisa; Barre, François Xavier; Waters, Christopher M.

doi:10.1093/nar/gkx496

Cited by 40 publications

(43 citation statements)

References 44 publications

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“…Our system was developed without strain engineering, however rational removal of single-strand exonucleases could potentially enhance efficiency as shown in V . cholerae 57 and E . coli 49,56,58 .…”

Section: Discussionmentioning

confidence: 99%

A new recombineering system for precise genome-editing in Shewanella oneidensis strain MR-1 using single-stranded oligonucleotides

Corts

Thomason

Gill

et al. 2019

Sci Rep

107

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Shewanella oneidensis MR-1 is an invaluable host for the discovery and engineering of pathways important for bioremediation of toxic and radioactive metals and understanding extracellular electron transfer. However, genetic manipulation is challenging due to the lack of genetic tools. Previously, the only reliable method used for introducing DNA into Shewanella spp. at high efficiency was bacterial conjugation, enabling transposon mutagenesis and targeted knockouts using suicide vectors for gene disruptions. Here, we describe development of a robust and simple electroporation method in S. oneidensis that allows an efficiency of ~4.0 x 106 transformants/µg DNA. High transformation efficiency is maintained when cells are frozen for long term storage. In addition, we report a new prophage-mediated genome engineering (recombineering) system using a λ Red Beta homolog from Shewanella sp. W3-18-1. By targeting two different chromosomal alleles, we demonstrate its application for precise genome editing using single strand DNA oligonucleotides and show that an efficiency of ~5% recombinants among total cells can be obtained. This is the first effective and simple strategy for recombination with markerless mutations in S. oneidensis. Continued development of this recombinant technology will advance high-throughput and genome modification efforts to engineer and investigate S. oneidensis and other environmental bacteria.

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Section: Discussionmentioning

confidence: 99%

A new recombineering system for precise genome-editing in Shewanella oneidensis strain MR-1 using single-stranded oligonucleotides

Corts

Thomason

Gill

et al. 2019

Sci Rep

107

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“…Mutants were constructed by MuGENT, Exo-MuGENT, and/or natural transformation exactly as previously described 35,37 . Briefly, mutant constructs for deletions or fluorescent fusions were generated via splicing-by-overlap (SOE) PCR to stitch (1) the upstream region of homology (aka the UP arm), (2) the mutation (aka the MIDDLE arm, which could represent an antibiotic resistance cassette or a fluorescent gene), and (3) the downstream region of homology (aka the DOWN arm).…”

Section: Methodsmentioning

confidence: 99%

Retraction of DNA-bound type IV competence pili initiates DNA uptake during natural transformation in Vibrio cholerae

et al. 2018

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Natural transformation is a broadly conserved mechanism of horizontal gene transfer in bacterial species that can shape evolution and foster the spread of antibiotic resistance determinants, promote antigenic variation and lead to the acquisition of novel virulence factors. Surface appendages called competence pili promote DNA uptake during the first step of natural transformation ; however, their mechanism of action has remained unclear owing to an absence of methods to visualize these structures in live cells. Here, using the model naturally transformable species Vibrio cholerae and a pilus-labelling method, we define the mechanism for type IV competence pilus-mediated DNA uptake during natural transformation. First, we show that type IV competence pili bind to extracellular double-stranded DNA via their tip and demonstrate that this binding is critical for DNA uptake. Next, we show that type IV competence pili are dynamic structures and that pilus retraction brings tip-bound DNA to the cell surface. Finally, we show that pilus retraction is spatiotemporally coupled to DNA internalization and that sterically obstructing pilus retraction prevents DNA uptake. Together, these results indicate that type IV competence pili directly bind to DNA via their tip and mediate DNA internalization through retraction during this conserved mechanism of horizontal gene transfer.

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“…In order to induce competence in V. cholerae strains, the master competence regulator TfoX was overexpressed using an IPTG-inducible or constitutive P tac promoter and the cells were genetically locked in a state of high cell density via deletion of luxO [41, 44-48]. Chitin-independent transformation assays were performed exactly as previously described [42]. Briefly, strains were grown overnight rolling at 30 °C with 100 µM IPTG then ∼10 8 colony forming units (CFU) were subcultured into 3mL of LB + 100 µM IPTG + 20 mM MgCl 2 + 10 mM CaCl 2 and grown to late log.…”

Section: Methodsmentioning

confidence: 99%

PilT and PilU are homohexameric ATPases that coordinate to retract type IVa pili

Chlebek

Hughes

Ratkiewicz

et al. 2019

Preprint

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19Bacterial type IV pili are critical for diverse biological processes including horizontal gene 20 transfer, surface sensing, biofilm formation, adherence, motility, and virulence. These dynamic 21 appendages extend and retract from the cell surface. In many type IVa pilus systems, extension 22 occurs through the action of an extension ATPase, often called PilB, while optimal retraction 23 requires the action of a retraction ATPase, PilT. Many type IVa systems also encode a homolog 24 of PilT called PilU. However, the function of this protein has remained unclear because pilU 25 mutants exhibit inconsistent phenotypes among type IV pilus systems and because it is 26 relatively understudied compared to PilT. Here, we study the type IVa competence pilus of 27Vibrio cholerae as a model system to define the role of PilU. We show that the ATPase activity 28 of PilU is critical for pilus retraction in PilT Walker A and/or Walker B mutants. PilU does not, 29 however, contribute to pilus retraction in ΔpilT strains. Thus, these data suggest that PilU is a 30 bona fide retraction ATPase that supports pilus retraction in a PilT-dependent manner. We also 31 found that a ΔpilU mutant exhibited a reduction in the force of retraction suggesting that PilU is 32 important for generating maximal retraction forces. Additional in vitro and in vivo data show that 33PilT and PilU act as independent homo-hexamers that may form a complex to facilitate pilus 34 retraction. Finally, we demonstrate that the role of PilU as a PilT-dependent retraction ATPase 35 is conserved in Acinetobacter baylyi, suggesting that the role of PilU described here may be 36 broadly applicable to other type IVa pilus systems. 37 38Author Summary 39 Almost all bacterial species use thin surface appendages called pili to interact with their 40 environments. These structures are critical for the virulence of many pathogens and represent 41 one major way that bacteria share DNA with one another, which contributes to the spread of 42 antibiotic resistance. To carry out their function, pili dynamically extend and retract from the 43 bacterial surface. Here, we show that retraction of pili in some systems is determined by the 44 combined activity of two motor ATPase proteins. 45 46Type IV pili are ubiquitous surface appendages in Gram-negative bacteria that promote diverse 48 activities including attachment, virulence, biofilm formation, horizontal gene transfer, and 49 twitching motility [1][2][3][4][5]. These structures can dynamically extend and retract from the cell 50 surface, which is often critical for their function. A detailed mechanistic understanding of pilus 51 dynamic activity, however, remains lacking. 52Type IV pili are composed almost exclusively of a single protein called the major pilin, which 53 forms a helical fiber that extends from the cell surface [6]. Extension and retraction of the pilus is 54 hypothesized to occur through the interaction of cytoplasmic hexameric ATPases with the inner 55 membrane platform of the pilus machine, whereby ATP ...

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Enhancing multiplex genome editing by natural transformation (MuGENT) via inactivation of ssDNA exonucleases

Cited by 40 publications

References 44 publications

A new recombineering system for precise genome-editing in Shewanella oneidensis strain MR-1 using single-stranded oligonucleotides

A new recombineering system for precise genome-editing in Shewanella oneidensis strain MR-1 using single-stranded oligonucleotides

Retraction of DNA-bound type IV competence pili initiates DNA uptake during natural transformation in Vibrio cholerae

PilT and PilU are homohexameric ATPases that coordinate to retract type IVa pili

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