In Vitro CpG Methylation Increases the Transformation Efficiency of<i>Borrelia burgdorferi</i>Strains Harboring the Endogenous Linear Plasmid lp56

Chen, Qiang; Fischer, Joshua R.; Benoit, Vivian M.; Dufour, Nicholas; Youderian, Philip; Leong, John M.

doi:10.1128/jb.00324-08

Cited by 42 publications

(50 citation statements)

References 40 publications

(58 reference statements)

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“…Figure 2 shows data from a single experiment/competence preparation for each clone transformed in parallel with all three shuttle vectors. These data indicate that the bbe02 and bbq67 loci of B. burgdorferi represent barriers to shuttle vector transformation, confirming and extending previous findings (13,27,34).…”

Section: Resultssupporting

confidence: 80%

“…In this case, pBSV2 transformants retaining lp25 (bbe02) were obtained only when plasmid DNA was prepared from E. coli dam mutant (data not shown). This outcome was not observed with pKFSS1 or pBSV2G shuttle vectors and was not previously reported for pBSV2 by Chen et al (13). If confirmed, this result could indicate the presence of a unique restriction site on pBSV2 whose cleavage by BBE02 is blocked by in vitro M.SssI methylation unless this modification is itself blocked by overlapping dam methylation.…”

Section: Resultssupporting

confidence: 50%

“…We conclude that dam, dcm, and hsd modification of shuttle vector DNA by E. coli does not form the basis for recognition of foreign DNA by the bbe02 and bbq67 gene products in B. burgdorferi. In separate experiments using in vitro-methylated DNA as described by Chen et al (13), we noticed a potential influence of dam methylation when transforming B. burgdorferi strains carrying both bbe02 and bbq67 with the shuttle vector pBSV2. In this case, pBSV2 transformants retaining lp25 (bbe02) were obtained only when plasmid DNA was prepared from E. coli dam mutant (data not shown).…”

Section: Resultsmentioning

confidence: 99%

“…Several steps were taken to obtain shuttle vector transformants in B. burgdorferi genetic backgrounds that contained bbe02 and bbq67 to serve as a source of modified plasmid DNA from Borrelia. We incorporated in vitro methylation of E. coli-derived plasmid DNA with the methyltransferase M.SssI, as previously described by Chen and colleagues (13), to introduce the shuttle vectors into B. burgdorferi clones that retained linear plasmids lp25 and lp56. These B. burgdorferi transformants were used in turn as a source of Borrelia-modified shuttle vector DNA with which to transform naïve recipient B. burgdorferi.…”

Section: Resultsmentioning

confidence: 99%

“…As described previously by Chen et al (13), between 50 and 100 g of E. coli plasmid DNA was methylated, following the manufacturer's recommendation, with the CpG methyltransferase M.SssI (New England BioLabs or Zymo Research, Irvine, CA). Methylation was assessed by digestion of plasmid DNA with the restriction enzyme BstUI, which is blocked by CpG methylation, before and after in vitro methylation.…”

mentioning

confidence: 99%

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Defining the Plasmid-Borne Restriction-Modification Systems of the Lyme Disease SpirocheteBorrelia burgdorferi

2011

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The restriction-modification (R-M) systems of many bacteria present a barrier to the stable introduction of foreign DNA. The Lyme disease spirochete Borrelia burgdorferi has two plasmid-borne putative R-M genes, bbe02 and bbq67, whose presence limits transformation by shuttle vector DNA from Escherichia coli. We show that both the bbe02 and bbq67 loci in recipient B. burgdorferi limit transformation with shuttle vector DNA from E. coli, irrespective of its dam, dcm, or hsd methylation status. However, plasmid DNA purified from B. burgdorferi transformed naïve B. burgdorferi much more efficiently than plasmid DNA from E. coli, particularly when the bbe02 and bbq67 genotypes of the B. burgdorferi DNA source matched those of the recipient. We detected adenine methylation of plasmid DNA prepared from B. burgdorferi that carried bbe02 and bbq67. These results indicate that the bbe02 and bbq67 loci of B. burgdorferi encode distinct R-M enzymes that methylate endogenous DNA and cleave foreign DNA lacking the same sequence-specific modification. Our findings have basic implications for horizontal gene transfer among B. burgdorferi strains with distinct plasmid contents. Further characterization and identification of the nucleotide sequences recognized by BBE02 and BBQ67 will facilitate efficient genetic manipulation of this pathogenic spirochete.Borrelia burgdorferi sensu lato is a zoonotic pathogen whose natural infectious cycle alternates between a tick vector and rodent or bird reservoir hosts (1,7,8,14,32,33,36). Transmission of B. burgdorferi to humans occurs through the bite of an infected tick and can lead to Lyme disease, which is a major public health concern in areas of North America and Europe where B. burgdorferi is endemic (8, 53).The genomic structure of the spirochete B. burgdorferi is unique, consisting of a linear chromosome of approximately 900 kb and more than 20 linear (lp) and circular (cp) plasmids, ranging in size from ϳ5 kb to 56 kb, in the type strain B31 (9,10,11,19,42). The plasmids of B. burgdorferi are present at unit copy number relative to the chromosome (22), and some are relatively unstable during in vitro propagation (52, 57). The loss of linear plasmids lp25, lp28-1, and lp36 by strain B31 was found to correlate with the loss of infectivity in mice (20,31,45,56), leading to the identification of genes carried on these plasmids that are dispensable in vitro but required in vivo during an experimental infectious cycle (21,26,35,44,47). The loss of two linear plasmids, lp25 and lp56, was shown to correlate with enhanced shuttle vector transformation, suggesting that specific lp25 and lp56 gene products present a barrier to stable introduction of foreign DNA (34). Further studies linked the transformation phenotype of B. burgdorferi strain B31 with the bbe02 and bbq67 genes on lp25 and lp56, respectively, and the putative restriction-modification (R-M) enzymes that they encode (11,27,29,34). The recent demonstration by Chen and colleagues of enhanced transformation of B. burgdorferi follow...

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Section: Resultssupporting

confidence: 80%

Section: Resultssupporting

confidence: 50%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Defining the Plasmid-Borne Restriction-Modification Systems of the Lyme Disease SpirocheteBorrelia burgdorferi

2011

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Genetic Transformation of Borrelia burgdorferi

2011

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The development of robust genetic tools to manipulate Borrelia burgdorferi, the etiologic agent of Lyme disease, now allows investigators to assess the role(s) of individual genes in the context of experimental Lyme borreliosis. This unit is devoted to the description of experimental approaches that are available for the molecular genetic analysis of B. burgdorferi with an emphasis on cultivation, electrotransformation, selection of desired mutants, and genetic complementation of acquired mutants. The intent is to provide a consensus protocol that encapsulates the methodologies currently employed by the B. burgdorferi research community and describe pertinent issues that must be accounted for when working with these pathogenic spirochetal bacteria. Curr. Protoc. Microbiol. 20:12C.4.1‐12C.4.17. © 2011 by John Wiley & Sons, Inc.

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Building a genome engineering toolbox in nonmodel prokaryotic microbes

Freed

Fenster

Smolinski

et al. 2018

Biotech & Bioengineering

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The realization of a sustainable bioeconomy requires our ability to understand and engineer complex design principles for the development of platform organisms capable of efficient conversion of cheap and sustainable feedstocks (e.g., sunlight, CO , and nonfood biomass) into biofuels and bioproducts at sufficient titers and costs. For model microbes, such as Escherichia coli, advances in DNA reading and writing technologies are driving the adoption of new paradigms for engineering biological systems. Unfortunately, microbes with properties of interest for the utilization of cheap and renewable feedstocks, such as photosynthesis, autotrophic growth, and cellulose degradation, have very few, if any, genetic tools for metabolic engineering. Therefore, it is important to develop "design rules" for building a genetic toolbox for novel microbes. Here, we present an overview of our current understanding of these rules for the genetic manipulation of prokaryotic microbes and the available genetic tools to expand our ability to genetically engineer nonmodel systems.

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In Vitro CpG Methylation Increases the Transformation Efficiency ofBorrelia burgdorferiStrains Harboring the Endogenous Linear Plasmid lp56

Cited by 42 publications

References 40 publications

Defining the Plasmid-Borne Restriction-Modification Systems of the Lyme Disease SpirocheteBorrelia burgdorferi

Defining the Plasmid-Borne Restriction-Modification Systems of the Lyme Disease SpirocheteBorrelia burgdorferi

Genetic Transformation of Borrelia burgdorferi

Building a genome engineering toolbox in nonmodel prokaryotic microbes

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