Development of a Single-Plasmid-Based Regulatable Gene Expression System for
            <i>Borrelia burgdorferi</i>

Whetstine, Christine R.; Slusser, Joyce G.; Zückert, Wolfram R.

doi:10.1128/aem.02825-08

Cited by 32 publications

(40 citation statements)

References 36 publications

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“…pBLS705 contains the lp56 bpaB (allele bpaB56) under the control of a TetR-repressible promoter, post (30,60). Transcription from the post promoter was induced in earlyexponential-phase cultures (approximately 10 5 bacteria/ml) by addition of anhydrotetracycline (ATc) at a final concentration of 0.5 g/ml.…”

Section: Methodsmentioning

confidence: 99%

Borrelia burgdorferi cp32 BpaB Modulates Expression of the Prophage NucP Nuclease and SsbP Single-Stranded DNA-Binding Protein

Chenail¹,

Jutras²,

Adams³

et al. 2012

Journal of Bacteriology

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Section: Methodsmentioning

confidence: 99%

Borrelia burgdorferi cp32 BpaB Modulates Expression of the Prophage NucP Nuclease and SsbP Single-Stranded DNA-Binding Protein

Chenail¹,

Jutras²,

Adams³

et al. 2012

Journal of Bacteriology

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“…The previously described pCRW53 replicates autonomously in both B. burgdorferi and E. coli and contains both a constitutively expressed tetR gene and a TetR-repressible promoter, Post, which drives transcription of gfp (71). By use of overlap extension PCR (36), the pCRW53 multiple cloning sites were deleted, and then the gfp gene was removed and replaced with single BamHI and PstI recognition sequences, producing pSZW53-4.…”

Section: Methodsmentioning

confidence: 99%

“…However, the recent development of an inducible expression system for B. burgdorferi allowed us to take an alternative approach to examine the roles of erp operatorbinding proteins in B. burgdorferi. The previously constructed pCRW53 contains tetR, encoding the tet repressor, and a tetracycline-inducible promoter, Post, that functions in B. burgdorferi (71). This plasmid was modified to place a bpaB or ebfC gene such that it is under the transcriptional control of Post.…”

Section: Bpab and Ebfc Bind To Erp Operators In Vivomentioning

confidence: 99%

BpaB and EbfC DNA-Binding Proteins Regulate Production of the Lyme Disease Spirochete's Infection-Associated Erp Surface Proteins

et al. 2012

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Successful infection of a host requires that the invading pathogen control its production of virulence determinants. The infectious agent must sense its environment and respond by increasing production of appropriate factors and repressing production of unnecessary ones. These features are especially critical for vector-borne pathogens, which must not only efficiently infect two extremely different host types but also be transmitted back and forth between hosts. Deciphering the regulatory pathways used by pathogens to control production of infection-associated proteins provides significant insight into the infectious nature of those organisms. Moreover, regulatory factors are attractive candidates for development of novel preventative and curative therapies.The spirochetal bacterium Borrelia burgdorferi, the agent of Lyme disease, is an excellent model organism for the study of gene regulation by a vector-borne pathogen. B. burgdorferi is genetically tractable, and its natural mammal-tick infectious cycle can be replicated in the laboratory. In addition, infection by B. burgdorferi is a significant cause of human morbidity, being the most commonly reported vector-borne disease in the United States and many other parts of the world (51, 55, 56).B. burgdorferi Erp lipoproteins are produced throughout mammalian infection but are largely repressed during colonization of vector ticks (10,31,48,49). Erp synthesis is greatly enhanced when B. burgdorferi is transmitted from a feeding tick into a warm-blooded host. Regulation of Erp protein production is controlled at the level of transcription (6). Erp proteins are located in the bacterial outer membrane and are exposed to the external environment (25,32,41). Known functions of Erp proteins include binding of host plasmin(ogen), laminin, and the complement regulators factor H and factor H-related proteins 1, 2, and 5 (2,3,11,12,34,37,40,45,59). These functions indicate roles for Erp proteins in host adherence, dissemination, and resistance to the alternative pathway of complement-mediated killing. Borrelial erp genes are located in mono-or bicistronic operons on extrachromosomal cp32 prophages, most of which replicate autonomously as circular episomes (24,60,63,64,72). Individual Lyme spirochetes naturally contain numerous different cp32 elements, each with a unique erp locus, and therefore produce multiple, distinct Erp surface proteins. A bacterium simultaneously expresses its entire repertoire of Erp proteins (26).A highly conserved DNA region immediately 5= of all erp promoters, the erp operator, is required for regulation of erp transcription (see Fig. 1) (6,10,64). Two erp operator-binding proteins have been identified, and their binding sites have been characterized: BpaB (borrelial plasmid ParB analogue) and EbfC (erp-binding factor, chromosomal) (4, 13, 52). BpaB binds with high affinity to a 5-bp sequence within the erp operator (13; C. A. Adams, unpublished). Binding of one BpaB protein to that sequence then facilitates binding of additional BpaB molecules al...

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“…All plasmids generated and used in this study are derivatives of pBSV2 (56). The expression of OspA-CaM fusion proteins was driven either by the constitutive B. burgdorferi flagellin (flaB) promoter (P flaB ) or by the TetR-regulated hybrid P ost promoter (57). Gene fusion constructs were generated by sequence overlap extension PCR (SOE-PCR) (26) using Phusion (New England BioLabs/Finnzymes) high-fidelity DNA polymerase.…”

Section: Methodsmentioning

confidence: 99%

Probing the Borrelia burgdorferi Surface Lipoprotein Secretion Pathway Using a Conditionally Folding Protein Domain

Shi-yong¹,

Zückert²

2011

Journal of Bacteriology

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Surface lipoproteins of Borrelia spirochetes are important virulence determinants in the transmission and pathogenesis of Lyme disease and relapsing fever. To further define the conformational secretion requirements and to identify potential lipoprotein translocation intermediates associated with the bacterial outer membrane (OM), we generated constructs in which Borrelia burgdorferi outer surface lipoprotein A (OspA) was fused to calmodulin (CaM), a conserved eukaryotic protein undergoing calcium-dependent folding. Protein localization assays showed that constructs in which CaM was fused to full-length wild-type (wt) OspA or to an intact OspA N-terminal "tether" peptide retained their competence for OM translocation even in the presence of calcium. In contrast, constructs in which CaM was fused to truncated or mutant OspA N-terminal tether peptides were targeted to the periplasmic leaflet of the OM in the presence of calcium but could be flipped to the bacterial surface upon calcium chelation. This indicated that in the absence of an intact tether peptide, unfolding of the CaM moiety was required in order to facilitate OM traversal. Together, these data further support a periplasmic tether peptide-mediated mechanism to prevent premature folding of B. burgdorferi surface lipoproteins. The specific shift in the OM topology of sequence-identical lipopeptides due to a single-variable change in environmental conditions also indicates that surface-bound Borrelia lipoproteins can localize transiently to the periplasmic leaflet of the OM.

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Development of a Single-Plasmid-Based Regulatable Gene Expression System for Borrelia burgdorferi

Cited by 32 publications

References 36 publications

Borrelia burgdorferi cp32 BpaB Modulates Expression of the Prophage NucP Nuclease and SsbP Single-Stranded DNA-Binding Protein

Borrelia burgdorferi cp32 BpaB Modulates Expression of the Prophage NucP Nuclease and SsbP Single-Stranded DNA-Binding Protein

BpaB and EbfC DNA-Binding Proteins Regulate Production of the Lyme Disease Spirochete's Infection-Associated Erp Surface Proteins

Probing the Borrelia burgdorferi Surface Lipoprotein Secretion Pathway Using a Conditionally Folding Protein Domain

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