Genome-Wide Transposon Mutagenesis of
            <i>Borrelia burgdorferi</i>
            for Identification of Phenotypic Mutants

Stewart, Philip E.; Hoff, Jessica; Fischer, Elizabeth R.; Krum, Jonathan G.; Rosa, Patricia A.

doi:10.1128/aem.70.10.5973-5979.2004

Cited by 87 publications

(102 citation statements)

References 26 publications

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“…We have also demonstrated that it is possible to generate and identify large numbers of L. biflexa mutants using transposon mutagenesis. Similarly to a recent study on the spirochete B. burgdorferi (30), we showed that this mutagenesis approach yields a randomly distributed set of insertion mutations throughout the genome, which can be screened for phenotypes affecting diverse aspects of metabolism and physiology.…”

Section: Discussionmentioning

confidence: 99%

Isolation and Characterization of FecA- and FeoB-Mediated Iron Acquisition Systems of the SpirocheteLeptospira biflexaby Random Insertional Mutagenesis

2005

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The specific mechanisms by which Leptospira spp. acquire iron from their ecological niches are unknown. A major factor contributing to our ignorance of spirochetal biology is the lack of methods for genetic analysis of these organisms. In this study, we have developed a system for random transposon mutagenesis of Leptospira biflexa using a mariner transposon, Himar1. To demonstrate the validity of Himar1 in vivo transposon mutagenesis in L. biflexa, a screen of mutants for clones impaired in amino acid biosynthesis was first performed, enabling the identification of tryptophan and glutamate auxotrophs. To investigate iron transporters, 2,000 L. biflexa transposon mutants were screened onto media with and without hemin, thus allowing the identification of five hemin-requiring mutants, and the putative genes responsible for this phenotype were identified. Three mutants had distinct insertions in a gene encoding a protein which shares homology with the TonB-dependent receptor FecA, involved in ferric citrate transport. We also identified two mutants with a Himar1 insertion into a feoB-like gene, the product of which is required for ferrous iron uptake in many bacterial organisms. Interestingly, the growth inhibition exhibited by the fecA and feoB mutants was relieved by deferoxamine, suggesting the presence of a ferric hydroxamate transporter. These results confirm the importance of iron for the growth of Leptospira and its ability to use multiple iron sources.

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Section: Discussionmentioning

confidence: 99%

Isolation and Characterization of FecA- and FeoB-Mediated Iron Acquisition Systems of the SpirocheteLeptospira biflexaby Random Insertional Mutagenesis

2005

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“…A subsequent study demonstrated that CpG methylation of foreign plasmid DNA enhanced transformation of B. burgdorferi carrying lp56, consistent with an R-M system encoded by bbq67 (13). High-throughput genetic transformation techniques, like transposon mutagenesis, have been effective only in B31 strains that lack lp25 and lp56 (6,54).…”

Section: Discussionmentioning

confidence: 99%

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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“…Approximately fivefold more colonies, i.e., 500 transformants per g of DNA, were obtained with plasmids pMKL and pMSL than with plasmid pSC189 expressing transposase from its native promoter. In the spirochete Borrelia burgdorferi, a recent study demonstrated that a high number of mutants could only be obtained when the Himar1 transposase was expressed from this flgB promoter (17). It has to be noted that due to the presence of a hyperactive transposase in plasmid pSC189 and derivatives, these plasmids may not be stable in E. coli (4).…”

mentioning

confidence: 99%

“…The promoterless hyperactive transposase C9 was linked to the spirochetal promoter of B. burgdorferi flgB as previously described (17). The plasmids are derivatives of pSC189 (4) bearing the native C9 transposase.…”

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confidence: 99%

Random Insertional Mutagenesis ofLeptospira interrogans, the Agent of Leptospirosis, Using amarinerTransposon

et al. 2005

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The recent availability of the complete genome sequences of Leptospira interrogans, the agent of leptospirosis, has allowed the identification of several putative virulence factors. However, to our knowledge, attempts to carry out gene transfer in pathogenic Leptospira spp. have failed so far. In this study, we show that the Himar1 mariner transposon permits random mutagenesis in the pathogen L. interrogans. We have identified genes that have been interrupted by Himar1 insertion in 35 L. interrogans mutants. This approach of transposon mutagenesis will be useful for understanding the spirochetal physiology and the pathogenic mechanisms of Leptospira, which remain largely unknown.

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Genome-Wide Transposon Mutagenesis of Borrelia burgdorferi for Identification of Phenotypic Mutants

Cited by 87 publications

References 26 publications

Isolation and Characterization of FecA- and FeoB-Mediated Iron Acquisition Systems of the SpirocheteLeptospira biflexaby Random Insertional Mutagenesis

Isolation and Characterization of FecA- and FeoB-Mediated Iron Acquisition Systems of the SpirocheteLeptospira biflexaby Random Insertional Mutagenesis

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

Random Insertional Mutagenesis ofLeptospira interrogans, the Agent of Leptospirosis, Using amarinerTransposon

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