Verification and dissection of the ospC operator by using flaB promoter as a reporter in Borrelia burgdorferi

Xu, Qilong; McShan, Kristy; Liang, Fang Ting

doi:10.1016/j.micpath.2008.03.002

Cited by 20 publications

(27 citation statements)

References 48 publications

(84 reference statements)

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“…However, the OspC protein ultimately elicits a robust humoral immune response, and repression of ospC expression is vital for B. burgdorferi persistence in the mouse (56,62,64). A duplicated palindromic sequence consisting of 20-bp inverted repeats is located in the intergenic region between the ospC and guaA open reading frames (28,62,63). This DNA sequence, termed the ospC operator, has been shown to be important for repression of ospC expression (62,63); however, the effect of this region, if any, on guaAB expression is unknown.…”

Section: Resultsmentioning

confidence: 99%

“…A duplicated palindromic sequence consisting of 20-bp inverted repeats is located in the intergenic region between the ospC and guaA open reading frames (28,62,63). This DNA sequence, termed the ospC operator, has been shown to be important for repression of ospC expression (62,63); however, the effect of this region, if any, on guaAB expression is unknown.…”

Section: Resultsmentioning

confidence: 99%

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GuaA and GuaB Are Essential forBorrelia burgdorferiSurvival in the Tick-Mouse Infection Cycle

et al. 2009

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Pathogens lacking the enzymatic pathways for de novo purine biosynthesis are required to salvage purines and pyrimidines from the host environment for synthesis of DNA and RNA. Two key enzymes in purine salvage pathways are IMP dehydrogenase (GuaB) and GMP synthase (GuaA), encoded by the guaB and guaA genes, respectively. While these genes are typically found on the chromosome in most bacterial pathogens, the guaAB operon of Borrelia burgdorferi is present on plasmid cp26, which also harbors a number of genes critical for B. burgdorferi viability. Using molecular genetics and an experimental model of the tick-mouse infection cycle, we demonstrate that the enzymatic activities encoded by the guaAB operon are essential for B. burgdorferi mouse infectivity and provide a growth advantage to spirochetes in the tick. These data indicate that the GuaA and GuaB proteins are critical for the survival of B. burgdorferi in the infection cycle and highlight a potential difference in the requirements for purine salvage in the disparate mammalian and tick environments.Purine metabolism is critical for the growth and virulence in mammals of many bacterial pathogens (11,26,29,33,51). Borrelia burgdorferi, the infectious agent of Lyme borreliosis, lacks the genes encoding the enzymes required for de novo nucleotide synthesis (8, 12) and therefore must rely on salvage of purines and pyrimidines from its hosts for nucleic acid biosynthesis (21, 35). Furthermore, B. burgdorferi lacks the genes encoding key enzymes required for a classic purine salvage pathway, including hpt (hypoxanthine-guanine phosphoribosyltransferase), purA (adenylosuccinate synthase), purB (adenylosuccinate lyase), and the locus encoding a ribonucleotide reductase (4,8,12,35,66). Despite the absence of a ribonucleotide reductase, an enzyme critical for the generation of deoxynucleotides through enzymatic reduction of ribonucleotides (32), a novel purine salvage pathway that involves salvage of deoxynucleosides from the host and interconversion of purine bases to deoxynucleosides by BB0426, a deoxyribosyl transferase, has recently been demonstrated for B. burgdorferi (23) (Fig. 1).In its infection cycle, B. burgdorferi passages between two disparate environments with potentially distinct purine availabilities, the tick vector and a mammalian host. Hypoxanthine is the most abundant purine in mammalian blood (17), and it is available for salvage by B. burgdorferi during the blood meal of an infected tick and during the spirochete's transient presence in the mammalian bloodstream. Despite the absence of the hpt gene, we and others have shown that B. burgdorferi is able to transport and incorporate low levels of hypoxanthine (23,35). During mammalian infection B. burgdorferi resides in various tissues, including the skin, heart, bladder, and joints. Adenine has been shown to be ubiquitous in mammalian tissues (61) and therefore is available for salvage by B. burgdorferi. Guanine is present at low levels in mammalian blood and tissues (17, 61); however, the amount m...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

GuaA and GuaB Are Essential forBorrelia burgdorferiSurvival in the Tick-Mouse Infection Cycle

et al. 2009

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“…Typically, transient induction of RpoS by shifting the temperature, pH, or growth phase of cultures is needed to induce ospC expression in infectious B31 clones carrying lp17 (Fig. 4), indicating that in a wt background, RpoS is made only under certain culture conditions and that the repressor is not made (56,58). However, a constitutively activated RpoN-RpoS pathway has also been observed in some ospAB mutants (28).…”

Section: Discussionmentioning

confidence: 99%

“…Xu and colleagues recently described a palindromic sequence immediately upstream of the ospC promoter that represents a potential operator site to which a repressor could bind (55,56). Although B. burgdorferi mutants lacking this palindromic sequence can initiate mammalian infection, ospC expression is not downregulated in these mutants, and thus they are subsequently recognized and cleared by neutralizing antibodies of the acquired immune response (55).…”

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confidence: 99%

Regulation of the Virulence Determinant OspC by bbd18 on Linear Plasmid lp17 of Borrelia burgdorferi

et al. 2011

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Persistent infection of a mammalian host by Borrelia burgdorferi, the spirochete that causes Lyme disease, requires specific downregulation of an immunogenic outer surface protein, OspC. Although OspC is an essential virulence factor needed by the spirochete to establish infection in the mammal, it represents a potent target for the host acquired immune response, and constitutive expression of OspC results in spirochete clearance. In this study, we demonstrate that a factor encoded on a linear plasmid of B. burgdorferi, lp17, can negatively regulate ospC transcription from the endogenous gene on the circular plasmid cp26 and from an ospC promoter-lacZ fusion on a shuttle vector. Furthermore, we have identified bbd18 as the gene on lp17 that is responsible for this effect. These data identify a novel component of ospC regulation and provide the basis for determining the molecular mechanisms of ospC repression in vivo.

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“…1B). The intergenic region between guaA and ospC, which includes the ospC promoter and regulatory operator sequences up to the ospC start codon (12,20,21,(37)(38)(39), was amplified from B. burgdorferi genomic DNA with primers 13 and 14 (Table 2), cloned into pCR2.1, sequenced, and digested with BamHI and XhoI for ligation into pBH-lacZBb, creating pBHospCp-lacZBb. Constructs were transformed into electrocompetent B. burgdorferi (27) and ⌬lac E. coli cells.…”

Section: Methodsmentioning

confidence: 99%

lacZ Reporter System for Use in Borrelia burgdorferi

Hayes

Jewett

Rosa

2010

Appl Environ Microbiol

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Regulation of gene expression is critical for the ability of Borrelia burgdorferi to adapt to different environments during its natural infectious cycle. Reporter genes have been used successfully to study gene regulation in multiple organisms. We have introduced a lacZ gene into B. burgdorferi, and we show that B. burgdorferi produces a protein with detectable ␤-galactosidase activity in both liquid and solid media when lacZ is expressed from a constitutive promoter. Furthermore, when lacZ is expressed from the ospC promoter, ␤-galactosidase activity is detected only in B. burgdorferi clones that express ospC, and it accurately monitors endogenous gene expression. The addition of lacZ to the repertoire of genetic tools available for use in B. burgdorferi should contribute to a better understanding of how B. burgdorferi gene expression is regulated during the infectious cycle.

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Verification and dissection of the ospC operator by using flaB promoter as a reporter in Borrelia burgdorferi

Cited by 20 publications

References 48 publications

GuaA and GuaB Are Essential forBorrelia burgdorferiSurvival in the Tick-Mouse Infection Cycle

GuaA and GuaB Are Essential forBorrelia burgdorferiSurvival in the Tick-Mouse Infection Cycle

Regulation of the Virulence Determinant OspC by bbd18 on Linear Plasmid lp17 of Borrelia burgdorferi

lacZ Reporter System for Use in Borrelia burgdorferi

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