Transcriptional analysis of a superoxide dismutase gene of Borrelia burgdorferi (original) (raw)
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Characterization of secA-sod operon in Borrellia burgdorferi
Borrelia burgdorferi, the causative agent of Lyme disease, has been characterized as a microaerophilic spirochete. O2 consumption and utilization potentially yield reactive oxygen intermediates, such as superoxide, hydroxyl radicals, and hydrogen peroxide. This study investigated the expression of the sod gene, which encodes the only, identified oxidative defense mechanism in B. burgdorferi. Using primer extension analysis and RT-PCR, it was found that sod and secA are organized as a single transcriptional unit under the control of σ 70-like promoter upstream of the secA open reading frame. Generally, gene expression decreases with increased distance from the promoter; however, secA expression was observed to be relatively lower amounts than sod. Both genes were expressed in all phases of growth, with greatest expression observed in stationary phase. Because transcription of most sod genes is tightly regulated by iron concentration, the secA-sod gene expression was analyzed in borrelial cells grown in varying amounts of metal. Ironrestriction did not significantly affect growth nor did it affect transcription of secA or sod. Likewise, no major differences in transcription were observed with restricting or supplementing media with manganese. To test the possibility that the borrelial SOD may function with either iron or manganese as a cofactor as in cambialistic SODs, SOD activity was assayed and the highest activity was observed in increased manganese concentrations. Protein expression profiles of Sh-2-82 cultured in metal-defined media were investigated by one-and two-dimensional gel electrophoresis. Approximately 15 proteins were differentially expressed in response to metal concentration. iv ACKNOWLEDGMENTS There are many individuals who have played pivotal roles in helping me become the scientist and the person that I am today. As a graduate student at the University of Louisville, I was most influenced by my major professor, Dr. Faye Austin. I thank her for the opportunity to work in her laboratory on B. burgdorferi. Under her guidance, I received a good foundation in the difficult study of RNA genetics. From her, I have learned the value of attention to detail in experimental design, experimentation, and record-keeping, which will follow me throughout my scientific career. Dr. Uldis Streips has also been a key player in my success. I appreciate the time he has devoted to my project and me. I have good memories of our trips to WindRiver where scientific thoughts and ideas were exchanged in an informal, enjoyable manner. I also thank Dr. Robert Stout, Dr. Thomas Geoghegan, and Dr. Ronald Doyle, for their helpful suggestions during this project and their participation as committee members. Additionally, I thank Joan Gagel for her coordination efforts in the preparation of this dissertation. For her kindness and assistance, I would like to thank Jan Powars. There were many others who befriended me and supported me throughout my graduate study, and I am thankful for all of my friends at the University of Louisville. Most of all, I am grateful to my husband, Greg, and our two sons, Matthew and Joshua, for their love, support and understanding. Through a family effort, we have accomplished this goal together. v TABLE OF CONTENTS ABSTRACT….……………………………………………………………………………. iii ACKNOWLEDGMENTS…..……………………………………………………………... iv TABLE OF CONTENTS………………………………………………………………….. LIST OF TABLES….……………………………………………………………………… v viii LIST OF FIGURES………………………………………………………………………... ix LIST OF ABBREVIATIONS…………………………….…………………...…………... xi INTRODUCTION….……………………………………………………………………… I. Lyme disease….…………………………………………………………………. A. Epidemiology……………………………………………….…………….. B. Clinical features…………………………………………………………... C. Immunopathogenesis……………………………………………………… D. Treatment……………………………………………….………………… E. Vaccine……………………………………………………………………. II. Borrelia burgdorferi….…………….…………………………………………… A. Taxonomy ……………………………………………………………… B. Genetic organization……………………………………………………… C. Morphological features…………………………………………………… D. Metabolism…..…………………………………………………………… E. Virulence Factors….……………………………………………………… III. Transcriptional control in bacteria...…………………………………………… A. Organization of bacterial promoters……………………………………… B. Organization of borrelial promoters……………………………………… C. Processing of bacterial mRNA…..……………………………………….. IV. Bacterial responses to stress and environmental signals….…………………… A. Stationary phase….……………………………………………………….. B. Heat shock response….…………………………………………………… C. Borrelial heat shock response…………………….……………………….. D. Oxidative stress response….……………………………………………… E. Borrelial oxidative stress response……………….………………………. F. Metalloregulation of oxidative stress…..………………………………… V. SecA-dependent protein secretion……………………………………………… A. Overview of secretion mechanisms….…………………………………… B. Transcriptional organization and regulation of SecA…..………………… C. Secretory genes in B. burgdorferi…….…………………………………...
Characterization of a Conditional bosR Mutant in Borrelia burgdorferi
Infection and Immunity, 2010
Borrelia burgdorferi, the etiological agent of Lyme disease, adapts to unique host environments as a consequence of its complex life cycle that spans both arthropod and mammalian species. In this regard, B. burgdorferi must adapt to various environmental signals, pHs, temperatures, and O 2 and CO 2 levels to establish infectious foci. We hypothesize that the BosR protein functions as a global regulator that is required for both borrelial oxidative homeostasis and pathogenesis. To assess the role of BosR in B. burgdorferi, we constructed an IPTG (isopropyl--D-thiogalactopyranoside)-regulated bosR strain. The selective decrease of bosR resulted in a change in growth when cells were cultured either anaerobically or microaerobically; however, a distinct growth defect was observed for anaerobically grown B. burgdorferi relative to the growth attenuation observed for microaerobically grown B. burgdorferi. B. burgdorferi cells in which BosR levels were reduced were more sensitive to hydrogen peroxide and produced lower levels of NapA (Dps) and SodA, proteins involved in the oxidative stress response. In addition, the levels of OspC and DbpA were also induced coincident with increased BosR levels, suggesting that BosR interfaces with the RpoS regulatory cascade, which is known to modulate virulence gene expression in B. burgdorferi. Taken together, these results indicate that BosR is involved in the resistance of B. burgdorferi to oxidative stressors and affects the expression of genes, either directly or indirectly, whose products are important in borrelial pathogenesis.
Molecular Microbiology, 2004
Borrelia burgdorferi , the aetiologic agent of Lyme disease, modulates gene expression in response to changes imposed by its arthropod vector and mammalian hosts. As reactive oxygen species (ROS) are known to vary in these environments, we asked how B. burgdorferi responds to oxidative stress. The B. burgdorferi genome encodes a PerR homologue (recently designated BosR) that represses the oxidative stress response in other bacteria, suggesting a similar function in B. burgdorferi . When we tested the sensitivity of B. burgdorferi to ROS, one clonal non-infectious B. burgdorferi isolate exhibited hypersensitivity to t -butyl hydroperoxide when compared with infectious B. burgdorferi and other non-infectious isolates. Sequence analysis indicated that the hypersensitive non-infectious isolates bosR allele contained a single nucleotide substitution, converting an arginine to a lysine ( bosRR39K ). Mutants in bosRR39K exhibited an increase in resistance to oxidative stressors when compared with the parental non-infectious strain, suggesting that BosRR39K functioned as a repressor. Complementation with bosRR39K and bosR resulted in differential sensitivity to t -butyl hydroperoxide, indicating that these alleles are functionally distinct. In contrast to BosR, BosRR39K did not activate transcription of a napA promoter-lacZ reporter in Escherichia coli nor bind the napA promoter/operator domain. However, we found that both BosR and BosRR39K bound to the putative promoter/operator region of superoxide dismutase ( sodA ). In addition, we determined that cells lacking BosRR39K synthesized fourfold greater levels of the decorin binding adhesin DbpA suggesting that BosRR39K regulates genes unrelated to oxidative stress. Based on these data, we propose that the single amino acid substitution, R39K, dramatically alters the activity of BosR by altering its ability to bind DNA at target regulatory sequences.
Microbiology (Reading, England), 2006
Borrelia burgdorferi regulates gene expression in response to environmental conditions, including temperature, pH, redox potential and host factors. B. burgdorferi encodes a PerR homologue designated BosR, which presumably serves as a global regulator of genes involved in the oxidative stress response. Infectious B. burgdorferi strain B31 is resistant to oxidative stressors in vitro, whereas the non-infectious isolate was sensitive due, in part, to a point mutation that converts an arginine to a lysine at residue 39 of BosR. Subsequent insertional inactivation of this bosRR39K allele (bosRR39K : : kan(R)) restored resistance to oxidative stressors. These observations suggest that the B. burgdorferi non-infectious bosRR39K : : kan(R) strain may transcribe genes that are also expressed in infectious B. burgdorferi cells, but are repressed in the bosRR39K background, thus explaining the different oxidative stress phenotypes observed between these isolates. To test this hypothesis, macr...
Molecular Microbiology, 2011
In a microarray analysis of the RpoS regulon in mammalian host-adapted Borrelia burgdorferi, bb0728 (cdr) was found to be dually-transcribed by the sigma factors σ 70 and RpoS. The cdr gene encodes a coenzyme A disulfide reductase (CoADR) that reduces CoA-disulfides to CoA in an NADH-dependent manner. Based on the abundance of CoA in B. burgdorferi and the biochemistry of the enzyme, CoADR has been proposed to play a role in the spirochete's response to reactive oxygen species (ROS). To better understand the physiologic function(s) of Bb CoADR, we generated a B. burgdorferi mutant in which the cdr gene was disrupted. RT-PCR and 5′-RACE analysis revealed that cdr and bb0729 are co-transcribed from a single transcriptional start site upstream of the bb0729 coding sequence; a shuttle vector containing the bb0729-cdr operon and upstream promoter element was used to complement the cdr mutant. Although the mutant was no more sensitive to hydrogen peroxide than its parent, it did exhibit increased sensitivity to high concentrations of t-butyl-hydroperoxide, an oxidizing compound that damages spirochetal membranes. Characterization of the mutant during standard (15% oxygen, 6% CO 2) and anaerobic (<1% O 2 , 9-13% CO 2) cultivation at 37°C revealed a growth defect under both conditions that was particularly striking during anaerobiosis. The mutant was avirulent by needle inoculation and showed decreased survival in feeding nymphs, but displayed no survival defect in unfed flat nymphs. Based on these results, we propose that Bb CoADR is necessary to maintain optimal redox ratios for CoA/CoA-disulfide and NAD + /NADH during periods of rapid replication throughout the enzootic cycle, to support thiol-disulfide homeostasis, and to indirectly protect the spirochete against peroxide-mediated membrane damage; one or more of these functions are essential for infection of the mammalian host by B. burgdorferi.
BosR (BB0647) governs virulence expression in Borrelia burgdorferi
Molecular Microbiology, 2000
Borrelia burgdorferi (Bb), the Lyme disease spirochaete, encodes a potential ferric uptake regulator (Fur) homologue, BosR (BB0647). Thus far, a role for BosR in Bb metabolism, gene regulation or pathogenesis has not been determined, largely due to the heretofore inability to inactivate bosR in low-passage, infectious Bb isolates. Herein, we report the generation of the first bosR-deficient mutant in a virulent strain of Bb. Whereas the bosR mutant persisted normally in ticks, the mutant was unable to infect mice, indicating that BosR is essential for Bb infection of a mammalian host. Moreover, transcriptional profiling of the bosR mutant showed that a number of genes were either positively or negatively influenced by BosR deficiency, suggesting that BosR may function both as a global repressor and activator in Bb. Strikingly, our study showed that BosR controls the expression of two major virulence-associated Bb lipoproteins, OspC and DbpA, likely via an influence on the alternative sigma factor, RpoS. This study thus not only has elucidated another key virulence gene of Bb, but also provides new insights into a previously unknown layer of gene regulation governing RpoS in Bb.
Infection and Immunity, 2005
Borrelia burgdorferi, the etiologic agent of Lyme disease, is genetically heterogeneous. Previous studies have shown a significant association between the frequency of hematogenous dissemination in Lyme disease patients and the genotype of the infecting B. burgdorferi strain. Comparative transcriptional profiling of two representative clinical isolates with distinct genotypes (BL206 and B356) was undertaken. A total of 78 open reading frames (ORFs) had expression levels that differed significantly between the two isolates. A number of genes with potential involvement in nutrient uptake (BB0603, BBA74, BB0329, BB0330, and BBB29) have significantly higher expression levels in isolate B356. Moreover, nearly 25% of the differentially expressed genes are predicted to be localized on the cell surface, implying that these two isolates have cell surface properties that differ considerably. One of these genes, BBA74, encodes a protein of 257 amino acid residues that has been shown to possess porin activity. BBA74 transcript level was >20-fold higher in B356 than in BL206, and strain B356 contained three-to fivefold more BBA74 protein. BBA74 was disrupted by the insertion of a kanamycin resistance cassette into the coding region. The growth rates of both wild-type and mutant strains were essentially identical, and cultures reached the same final cell densities. However, the mutant strains consistently showed prolonged lags of 2 to 5 days prior to the induction of log-phase growth compared to wild-type strains. It is tempting to speculate that the absence of BBA74 interferes with the enhanced nutrient uptake that may be required for the entry of cells into log-phase growth. These studies demonstrate the value of comparative transcriptional profiling for identifying differences in the transcriptomes of B. burgdorferi clinical isolates that may provide clues to pathogenesis. The 78 ORFs identified here are a good starting point for the investigation of factors involved in the hematogenous dissemination of B. burgdorferi.
Infection and Immunity, 2008
Borrelia burgdorferi, the causative agent of Lyme disease in the United States, regulates numerous genes encoding lipoproteins on linear plasmid 54 in response to environmental cues. We analyzed a subset of these genes/proteins that were historically categorized as paralogous gene family 54 (BBA64, BBA65, BBA66, BBA68, BBA69, BBA70, BBA71, and BBA73) and found that the expression of several genes was influenced by the N -S regulatory cascade at the level of transcription and protein synthesis. Moreover, we established in this and a previous study that BBA65, BBA66, BBA69, BBA71, and BBA73 are temporally expressed during persistent infection of immunocompetent mice, as determined by quantitative real time-PCR of ear tissue, by enzyme-linked immunosorbent assay, and by immunoblotting. Correspondingly, BBA65, BBA66, BBA71, and BBA73 proteins were detectable in infectious B. burgdorferi B31 isolates but undetectable in noninfectious isolates. BBA65, BBA66, BBA71, and BBA73 proteins were also found to partition into the Triton X-114 detergent phase and were sensitive to protease treatment of intact cells, indicating that they are membrane associated and surface localized. Lastly, Southern blotting and PCR with specific gene primer/probes for BBA64, BBA65, BBA66, BBA71, and BBA73 suggest that many of these genes are conserved among the B. burgdorferi sensu lato isolates and the relapsing-fever Borrelia species. Together, the data presented suggest that these genes may play a part in Borrelia infection and/or pathogenicity that could extend beyond the sensu lato group.