Oligonucleotide recombination: A hidden treasure

Swingle, Bryan; Markel, Eric; Cartinhour, Samuel

doi:10.4161/bbug.1.4.12098

Cited by 8 publications

(9 citation statements)

References 26 publications

(26 reference statements)

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“…1A). Similar results have been reported for P. syringae (Swingle et al ., 2010a). Therefore, we tested whether this slight increase in mutation frequency was either restricted to sequences encoded by the oligo, a general response to an influx of single‐stranded DNA, or a local but indirect effect.…”

Section: Resultssupporting

confidence: 91%

Oligonucleotides stimulate genomic alterations of Legionella pneumophila

Bryan

Swanson²

2011

Molecular Microbiology

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SummaryGenetic variation generates diversity in all kingdoms of life. The corresponding mechanisms can also be harnessed for laboratory studies of fundamental cellular processes. Here we report that oligonucleotides (oligos) generate mutations on the Legionella pneumophila chromosome by a mechanism that requires homologous DNA, but not RecA, RadA or any known phage recombinase. Instead we propose that DNA replication contributes, as oligo-induced mutagenesis required Ն 21 nucleotides of homology, was strand-dependent, and was most efficient in exponential phase. Mutagenesis did not require canonical 5Ј phosphate or 3Ј hydroxyl groups, but the primosomal protein PriA and DNA Pol I contributed. After electroporation, oligos stimulated excision of 2.1 kb of chromosomal DNA or insertion of 18 bp, and non-homologous flanking sequences were also processed. We exploited this endogenous activity to generate chromosomal deletions and to insert an epitope into a chromosomal coding sequence. Compared with Escherichia coli, L. pneumophila encodes fewer canonical single-stranded exonucleases, and the frequency of mutagenesis increased substantially when either its RecJ and ExoVII nucleases were inactivated or the oligos modified by nuclease-resistant bases. In addition to genetic engineering, oligoinduced mutagenesis may have evolutionary implications as a mechanism to incorporate divergent DNA sequences with only short regions of homology.

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Section: Resultssupporting

confidence: 91%

Oligonucleotides stimulate genomic alterations of Legionella pneumophila

Bryan

Swanson²

2011

Molecular Microbiology

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“…The lagging strand bias in E. coli , Pseudomonas syringae and in Mycobacterium tuberculosis is approximately 3 to 10-fold, 10-fold and 1,000 to 10,000-fold, respectively. 12 , 21 , 23 One hypothesis for this lagging strand bias is that more single-stranded DNA is exposed during replication on the lagging strand because of the formation of the different Okazaki fragments. At present it is unknown why the magnitude of strand bias in recombineering in Mycobacterium tuberculosis is different from E. coli .…”

Section: Resultsmentioning

confidence: 99%

Exploring optimization parameters to increase ssDNA recombineering inLactococcus lactisandLactobacillus reuteri

et al. 2012

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Single-stranded DNA (ssDNA) recombineering is a technology which is used to make subtle changes in the chromosome of several bacterial genera. Cells which express a single-stranded DNA binding protein (RecT or Bet) are transformed with an oligonucleotide which is incorporated via an annealing and replication-dependent mechanism. By in silico analysis we identified ssDNA binding protein homologs in the genus Lactobacillus and Lactococcus lactis. To assess whether we could further improve the recombineering efficiency in Lactobacillus reuteri ATCC PTA 6475 we expressed several RecT homologs in this strain. RecT derived from Enterococcus faecalis CRMEN 19 yielded comparable efficiencies compared with a native RecT protein, but none of the other proteins further increased the recombineering efficiency. We successfully improved recombineering efficiency 10-fold in L. lactis by increasing oligonucleotide concentration combined with the use of oligonucleotides containing phosphorothioate-linkages (PTOs). Surprisingly, neither increased oligonucleotide concentration nor PTO linkages enhanced recombineering in L. reuteri 6475. To emphasize the utility of this technology in improving probiotic features we modified six bases in a transcriptional regulatory element region of the pdu-operon of L. reuteri 6475, yielding a 3-fold increase in the production of the antimicrobial compound reuterin. Directed genetic modification of lactic acid bacteria through ssDNA recombineering will simplify strain improvement in a way that, when mutating a single base, is genetically indistinguishable from strains obtained through directed evolution.

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“…While attempting and optimizing the recombineering technology in S. oneidensis , Corts et al found that in the control reactions with no recombinase present, the ssDNA oligos can site-specifically recombine in the chromosome of S. oneidensis , albeit at a much lower frequency compared to the W3Beta-mediated recombination; ∼10 –4 recombinants/viable cells (a total of ∼10 4 recombinants in 10 8 total cells) . This finding resembled the results from Swingle et al , who also obtained recombinase-independent mutants in Pseudomonas syringae while attempting to use the λ-Red system at a similar frequency. , This strategy, however, resulted in an ∼800-fold decrease in recombinants and, thus, a strong selection or highly efficient screening method is needed. The advantage of this approach is that no protein expression is required and, thus, no plasmids or cloning are necessary, which makes it a straightforward technique convenient for those other Shewanella species with no basic bioengineering tools yet available.…”

Section: Genetic Tools For Control Of Electron Transfer Pathways In N...mentioning

confidence: 63%

Engineering Wired Life: Synthetic Biology for Electroactive Bacteria

et al. 2021

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Electroactive bacteria produce or consume electrical current by moving electrons to and from extracellular acceptors and donors. This specialized process, known as extracellular electron transfer, relies on pathways composed of redox active proteins and biomolecules and has enabled technologies ranging from harvesting energy on the sea floor, to chemical sensing, to carbon capture. Harnessing and controlling extracellular electron transfer pathways using bioengineering and synthetic biology promises to heighten the limits of established technologies and open doors to new possibilities. In this review, we provide an overview of recent advancements in genetic tools for manipulating native electroactive bacteria to control extracellular electron transfer. After reviewing electron transfer pathways in natively electroactive organisms, we examine lessons learned from the introduction of extracellular electron transfer pathways into Escherichia coli. We conclude by presenting challenges to future efforts and give examples of opportunities to bioengineer microbes for electrochemical applications.

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Oligonucleotide recombination: A hidden treasure

Cited by 8 publications

References 26 publications

Oligonucleotides stimulate genomic alterations of Legionella pneumophila

Oligonucleotides stimulate genomic alterations of Legionella pneumophila

Exploring optimization parameters to increase ssDNA recombineering inLactococcus lactisandLactobacillus reuteri

Engineering Wired Life: Synthetic Biology for Electroactive Bacteria

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