[show abstract][hide abstract] ABSTRACT: Developing scientific expertise in the classroom involves promoting higher-order cognitive skills as well as content mastery. Effective use of constructivism can facilitate these outcomes. However this is often difficult to accomplish when delivery of content is paramount. Utilizing many of the tenets of constructivist pedagogy, we have designed an Oxford-style debate assignment to be used in an introductory microbiology course. Two teams of students were assigned a debatable topic within microbiology. Over a five-week period students completed an informative web page consisting of three parts: background on the topic, data-based positions for each side of the argument, and a data-based persuasive argument to support their assigned position. This was followed by an in-class presentation and debate. Analysis of student performance on knowledge-based questions shows that students retain debate-derived content acquired primarily outside of lectures significantly better than content delivered during a normal lecture. Importantly, students who performed poorly on the lecture-derived questions did as well on debate-derived questions as other students. Students also performed well on questions requiring higher-order cognitive skills and in synthesizing data-driven arguments in support of a position during the debate. Student perceptions of their knowledge-base in areas covered by the debate and their skills in using scientific databases and analyzing primary literature showed a significant increase in pre- and postassignment comparisons. Our data demonstrate that an Oxford-style debate can be used effectively to deliver relevant content, increase higher-order cognitive skills, and increase self-efficacy in science-specific skills, all contributing to developing expertise in the field.
Journal of microbiology & biology education : JMBE. 01/2013; 14(1):2-11.
[show abstract][hide abstract] ABSTRACT: The spirochetes in the Borrelia burgdorferi sensu lato genospecies group cycle in nature between a tick vector and a vertebrate host. The current assemblage of B. burgdorferi sensu lato, of which three species cause Lyme disease in humans, originated from a rapid species radiation that occurred near the origin of the clade. These bacteria have a unique genome that is highly segmented and predominantly composed of linear replicons. One of the circular plasmids is a prophage that exists as several isoforms in each cell and can be transduced to other cells, likely contributing to an otherwise relatively anemic level of horizontal gene transfer, which nevertheless appears to be adequate to permit strong natural selection and adaptation in populations of B. burgdorferi. Although the molecular genetic toolbox is meager, several antibioticresistant mutants have been isolated, and the resistance alleles, as well as some exogenous genes, have been fashioned into markers to dissect gene function. Genetic studies have probed the role of the outer membrane lipoprotein OspC, which is maintained in nature by niche selection and negative frequency-dependent selection. One of the most intriguing genetic systems in B. burgdorferi is vls recombination, which generates antigenic variation during infection of mammalian hosts. Expected final online publication date for the Annual Review of Genetics Volume 46 is November 02, 2012. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
Annual Review of Genetics 09/2012; · 17.44 Impact Factor
[show abstract][hide abstract] ABSTRACT: While the roles of rpoS(Bb) and RpoS-dependent genes have been studied extensively within the mammal, the contribution of the RpoS regulon to the tick-phase of the Borrelia burgdorferi enzootic cycle has not been examined. Herein, we demonstrate that RpoS-dependent gene expression is prerequisite for the transmission of spirochetes by feeding nymphs. RpoS-deficient organisms are confined to the midgut lumen where they transform into an unusual morphotype (round bodies) during the later stages of the blood meal. We show that round body formation is rapidly reversible, and in vitro appears to be attributable, in part, to reduced levels of Coenzyme A disulfide reductase, which among other functions, provides NAD+ for glycolysis. Our data suggest that spirochetes default to an RpoS-independent program for round body formation upon sensing that the energetics for transmission are unfavorable.
[show abstract][hide abstract] ABSTRACT: The molecular architecture and composition of the outer membrane (OM) of Treponema pallidum (Tp), the noncultivable agent of venereal syphilis, differ considerably from those of typical Gram-negative bacteria. Several years ago we described TP0453, the only lipoprotein associated with the inner leaflet of the Tp OM. Whereas polypeptides of other treponemal lipoproteins are hydrophilic, non-lipidated TP0453 can integrate into membranes, a property attributed to its multiple amphipathic helices (AHs). Furthermore, membrane integration of the TP0453 polypeptide was found to increase membrane permeability, suggesting the molecule functions in a porin-like manner. To better understand the mechanism of membrane integration of TP0453 and its physiological role in Tp OM biogenesis, we solved its crystal structure and used mutagenesis to identify membrane insertion elements. The crystal structure of TP0453 consists of an α/β/α-fold and includes five stably folded AHs. In high concentrations of detergent, TP0453 transitions from a closed to open conformation by lateral movement of two groups of AHs, exposing a large hydrophobic cavity. Triton X-114 phase partitioning, liposome floatation assay, and bis-1-anilino-8-naphthalenesulfonate binding revealed that two adjacent AHs are critical for membrane sensing/integration. Using terbium-dipicolinic acid complex-loaded large unilamellar vesicles, we found that TP0453 increased efflux of fluorophore only at acidic pH. Gel filtration and cross-linking experiments demonstrated that one AH critical for membrane sensing/insertion also forms a dimeric interface. Based on structural dynamics and comparison with Mycobacterium tuberculosis lipoproteins LprG and LppX, we propose that TP0453 functions as a carrier of lipids, glycolipids, and/or derivatives during OM biogenesis.
Journal of Biological Chemistry 09/2011; 286(48):41656-68. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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 disulphide reductase (CoADR) that reduces CoA-disulphides 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 spirochaete's response to reactive oxygen species. To better understand the physiologic function(s) of BbCoADR, 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 BbCoADR is necessary to maintain optimal redox ratios for CoA/CoA-disulphide and NAD(+) /NADH during periods of rapid replication throughout the enzootic cycle, to support thiol-disulphide homeostasis, and to indirectly protect the spirochaete against peroxide-mediated membrane damage; one or more of these functions are essential for infection of the mammalian host by B. burgdorferi.
[show abstract][hide abstract] ABSTRACT: Current thinking emphasizes the primacy of CD14 in facilitating recognition of microbes by certain TLRs to initiate pro-inflammatory signaling events and the importance of p38-MAPK in augmenting such responses. Herein, this paradigm is challenged by demonstrating that recognition of live Borrelia burgdorferi not only triggers an inflammatory response in the absence of CD14, but one that is, in part, a consequence of altered PI3K/AKT/p38-MAPK signaling and impaired negative regulation of TLR2. CD14 deficiency results in increased localization of PI3K to lipid rafts, hyperphosphorylation of AKT, and reduced activation of p38. Such aberrant signaling leads to decreased negative regulation by SOCS1, SOCS3, and CIS, thereby compromising the induction of tolerance in macrophages and engendering more severe and persistent inflammatory responses to B. burgdorferi. Importantly, these altered signaling events and the higher cytokine production observed can be mimicked through shRNA and pharmacological inhibition of p38 activity in CD14-expressing macrophages. Perturbation of this CD14/p38-MAPK-dependent immune regulation may underlie development of infectious chronic inflammatory syndromes.
[show abstract][hide abstract] ABSTRACT: Lyme disease is caused by transmission of the spirochete Borrelia burgdorferi from ticks to humans. Although much is known about B. burgdorferi replication, the routes and mechanisms by which it disseminates within the tick remain unclear. To better understand this process, we imaged live, infectious B. burgdorferi expressing a stably integrated, constitutively expressed GFP reporter. Using isolated tick midguts and salivary glands, we observed B. burgdorferi progress through the feeding tick via what we believe to be a novel, biphasic mode of dissemination. In the first phase, replicating spirochetes, positioned at varying depths throughout the midgut at the onset of feeding, formed networks of nonmotile organisms that advanced toward the basolateral surface of the epithelium while adhering to differentiating, hypertrophying, and detaching epithelial cells. In the second phase of dissemination, the nonmotile spirochetes transitioned into motile organisms that penetrated the basement membrane and entered the hemocoel, then migrated to and entered the salivary glands. We designated the first phase of dissemination "adherence-mediated migration" and provided evidence that it involves the inhibition of spirochete motility by one or more diffusible factors elaborated by the feeding tick midgut. Our studies, which we believe are the first to relate the transmission dynamics of spirochetes to the complex morphological and developmental changes that the midgut and salivary glands undergo during engorgement, challenge the conventional viewpoint that dissemination of Lyme disease-causing spirochetes within ticks is exclusively motility driven.
The Journal of clinical investigation 11/2009; 119(12):3652-65. · 15.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have previously demonstrated that phagocytosed Borrelia burgdorferi induces activation programs in human peripheral blood mononuclear cells that differ qualitatively and quantitatively from those evoked by equivalent lipoprotein-rich lysates. Here we report that ingested B. burgdorferi induces significantly greater transcription of proinflammatory cytokine genes than do lysates and that live B. burgdorferi, but not B. burgdorferi lysate, is avidly internalized by monocytes, where the bacteria are completely degraded within phagolysosomes. In the course of these experiments, we discovered that live B. burgdorferi also induced a dose-dependent decrease in monocytes but not a decrease in dendritic cells or T cells and that the monocyte population displayed morphological and biochemical hallmarks of apoptosis. Particularly noteworthy was the finding that apoptotic changes occurred predominantly in monocytes that had internalized spirochetes. Abrogation of phagocytosis with cytochalasin D prevented the death response. Heat-killed B. burgdorferi, which was internalized as well as live organisms, induced a similar degree of apoptosis of monocytes but markedly less cytokine production. Surprisingly, opsonophagocytosis of Treponema pallidum did not elicit a discernible cell death response. Our combined results demonstrate that B. burgdorferi confined to phagolysosomes is a potent inducer of cytosolic signals that result in (i) production of NF-kappaB-dependent cytokines, (ii) assembly of the inflammasome and activation of caspase-1, and (iii) induction of programmed cell death. We propose that inflammation and apoptosis represent mutually reinforcing components of the immunologic arsenal that the host mobilizes to defend itself against infection with Lyme disease spirochetes.
Infection and immunity 02/2008; 76(1):56-70. · 4.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: Borrelia burgdorferi (Bb) adapts to its arthropod and mammalian hosts by altering its transcriptional and antigenic profiles in response to environmental signals associated with each of these milieus. In studies presented here, we provide evidence to suggest that mammalian host signals are important for modulating and maintaining both the positive and negative aspects of mammalian host adaptation mediated by the alternative sigma factor RpoS in Bb. Although considerable overlap was observed between genes induced by RpoS during growth within the mammalian host and following temperature-shift, comparative microarray analyses demonstrated unequivocally that RpoS-mediated repression requires mammalian host-specific signals. A substantial portion of the in vivo RpoS regulon was uniquely upregulated within dialysis membrane chambers, further underscoring the importance of host-derived environmental stimuli for differential gene expression in Bb. Expression profiling of genes within the RpoS regulon by quantitative reverse transcription polymerase chain reaction (qRT-PCR) revealed a level of complexity to RpoS-dependent gene regulation beyond that observed by microarray, including a broad range of expression levels and the presence of genes whose expression is only partially dependent on RpoS. Analysis of Bb-infected ticks by qRT-PCR established that expression of rpoS is induced during the nymphal blood meal but not within unfed nymphs or engorged larvae. Together, these data have led us to postulate that RpoS acts as a gatekeeper for the reciprocal regulation of genes involved in the establishment of infection within the mammalian host and the maintenance of spirochetes within the arthropod vector.
[show abstract][hide abstract] ABSTRACT: Acquisition of transition metals is central to the struggle between a bacterial pathogen and its mammalian host. Previous studies demonstrated that Treponema pallidum encodes a cluster-9 (C9) ABC transporter (troABCD) whose solute-binding protein component (TroA) ligands Zn(2+) and Mn(2+) with essentially equal affinities. Bioinformatic analysis revealed that T. pallidum encodes an additional C9 transporter (tp0034-36) orthologous to Zn(2+)-uptake (Znu) systems in other bacteria; the binding protein component, ZnuA, contains a His-rich tract characteristic of C9 Zn(2+)-binding proteins. Metal analysis and metal-reconstitution studies demonstrated that ZnuA is a Zn(2+)-binding protein; parallel studies confirmed that TroA binds Zn(2+), Mn(2+) and Fe. Circular dichroism showed that ZnuA, but not TroA, undergoes conformational changes in the presence of Zn(2+). Using isothermal titration calorimetry (ITC), we demonstrated that TroA binds Zn(2+) and Mn(2+) with affinities approximately 100-fold greater than those previously reported. ITC analysis revealed that ZnuA contains multiple Zn(2+)-binding sites, two of which are high-affinity and presumed to be located within the binding pocket and His-rich loop. Quantitative reverse transcription polymerase chain reaction of tro and znu transcripts combined with immunoblot analysis of TroA and ZnuA confirmed that both transporters are simultaneously expressed in T. pallidum and that TroA is expressed at much greater levels than ZnuA. Collectively, our findings indicate that T. pallidum procures transition metals via the concerted utilization of its general metal (Tro) and Zn(2+) (Znu) transporters. Sequestration of periplasmic Zn(2+) by ZnuA may free up TroA binding capacity for the importation of Fe and Mn(2+).
[show abstract][hide abstract] ABSTRACT: We examined the interactions of live and lysed spirochetes with innate immune cells. THP-1 monocytoid cells were activated to comparable extents by live Borrelia burgdorferi and by B. burgdorferi and Treponema pallidum lysates but were poorly activated by live T. pallidum. Because THP-1 cells poorly internalized live spirochetes, we turned to an ex vivo peripheral blood mononuclear cell system that would more closely reflect spirochete-mononuclear phagocyte interactions that occur during actual infection. In this system, B. burgdorferi induced significantly greater monocyte activation and inflammatory cytokine production than did borrelial lysates or T. pallidum, and only B. burgdorferi elicited gamma interferon (IFN-gamma) from NK cells. B. burgdorferi was phagocytosed avidly by monocytes, while T. pallidum was not, suggesting that the enhanced response to live B. burgdorferi was due to phagocytosis of the organism. When cytochalasin D was used to block phagocytosis of live B. burgdorferi, cytokine production decreased to levels comparable to those induced by B. burgdorferi lysates, while the IFN-gamma response was abrogated altogether. In the presence of human syphilitic serum, T. pallidum was efficiently internalized and initiated responses resembling those observed with live B. burgdorferi, including the production of IFN-gamma by NK cells. Depletion of monocytes revealed that they were the primary source of inflammatory cytokines, while dendritic cells (DCs) directed IFN-gamma production from innate lymphocytes. Thus, phagocytosis of live spirochetes initiates cell activation programs in monocytes and DCs that differ qualitatively and quantitatively from those induced at the cell surface by lipoprotein-enriched lysates. The greater stimulatory capacity of B. burgdorferi versus T. pallidum appears to be explained by the successful recognition and phagocytosis of B. burgdorferi by host cells and the ability of T. pallidum to avoid detection and uptake by virtue of its denuded outer membrane rather than by differences in surface lipoprotein expression.
Infection and Immunity 05/2007; 75(4):2046-62. · 4.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: Borrelia burgdorferi survives in an enzootic cycle, and Dps proteins protect DNA against damage during starvation or oxidative stress. The role of a Dps homologue encoded by Borrelia in spirochaete survival was assessed. Dps-deficient spirochaetes were infectious in mice via needle-inoculation at the dose of 10(5) spirochaetes. Larval ticks successfully acquired Dps-deficient spirochaetes via a blood meal on mice. However, after extended periods within unfed nymphs, the Dps-deficient spirochaetes failed to be transmitted to a new host when nymphs fed. Our data suggest that Dps functions to protect the spirochaetes during dormancy in unfed ticks, and in its absence, the spirochaetes become susceptible during tick feeding. dps is differentially expressed in vivo- low in mice and high in ticks - but constitutively expressed in vitro, showing little change during growth or in response to oxidative stress. Borrelia Dps forms a dodecameric complex capable of sequestering iron. The Dps-deficient spirochaetes showed no defect in starvation and oxidative stress assays, perhaps due to the lack of iron in spirochaetes grown in vitro. Dps is critical for spirochaete persistence within ticks, and strategies to interfere with Dps could potentially reduce Borrelia populations in nature and thereby influence the incidence of Lyme disease.
[show abstract][hide abstract] ABSTRACT: Members of the ospE/ospF/elp lipoprotein gene families of Borrelia burgdorferi, the Lyme disease agent, are transcriptionally upregulated in response to the influx of blood into the midgut of an infected tick. We recently have demonstrated that despite the high degree of similarity between the promoters of the ospF (P(ospF)) and ospE (P(ospE)) genes of B. burgdorferi strain 297, the differential expression of ospF is RpoS-dependent, while ospE is controlled by sigma(70). Herein we used wild-type and RpoS-deficient strains of B. burgdorferi and Escherichia coli to analyse transcriptional reporters consisting of a green fluorescent protein (gfp) gene fused to P(ospF), P(ospE), or two hybrid promoters in which the -10 regions of P(ospF) and P(ospE) were switched [P(ospF ) ((E - 10)) and P(ospE) ((F - 10)) respectively]. We found that the P(ospF)-10 region is both necessary and sufficient for RpoS-dependent recognition in B. burgdorferi, while sigma(70) specificity for P(ospE) is dependent on elements outside of the -10 region. In E. coli, sigma factor selectivity for these promoters was much more permissive, with expression of each being primarily due to sigma(70). Alignment of the sequences upstream of each of the ospE/ospF/elp genes from B. burgdorferi strains 297 and B31 revealed that two B31 ospF paralogues [erpK (BBM38) and erpL (BBO39)] have -10 regions virtually identical to that of P(ospF). Correspondingly, expression of gfp reporters based on the erpK and erpL promoters was RpoS-dependent. Thus, the sequence of the P(ospF)-10 region appears to serve as a motif for RpoS recognition, the first described for any B. burgdorferi promoter. Taken together, our data support the notion that B. burgdorferi utilizes sequence differences at the -10 region as one mechanism for maintaining the transcriptional integrity of RpoS-dependent and -independent genes activated at the onset of tick feeding.
[show abstract][hide abstract] ABSTRACT: While numerous positively regulated loci have been characterized during the enzootic cycle of Borrelia burgdorferi, very little is known about the mechanism(s) involved in the repression of borrelial loci either during tick feeding or within the mammalian host. Here, we report that the alternative sigma factor RpoS is required for the in vivo-specific repression of at least two RpoD-dependent B. burgdorferi loci, ospA and lp6.6. The downregulation of ospA and Ip6.6 appears to require either a repressor molecule whose expression is RpoS dependent or an accessory factor which enables RpoS to directly interact with the ospA and Ip6.6 promoter elements, thereby blocking transcription by RpoD. The central role for RpoS during the earliest stages of host adaptation suggests that tick feeding imparts signals to spirochetes that trigger the RpoS-dependent repression, as well as expression, of in vivo-specific virulence factors critical for the tick-to-mammalian host transition.
Journal of Bacteriology 12/2005; 187(22):7845-52. · 3.19 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have isolated in vitro fluoroquinolone-resistant mutants of the Lyme disease agent, Borrelia burgdorferi. Mutations in parC, which encodes a subunit of topoisomerase IV, were associated with loss of susceptibility to sparfloxacin, moxifloxacin, and Bay-Y3118, but not ciprofloxacin. This is the first description of fluoroquinolone resistance in the spirochete phylum.
Antimicrobial Agents and Chemotherapy 11/2005; 49(10):4354-7. · 4.57 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have isolated in vitro fluoroquinolone-resistant mutants of the Lyme disease agent, Borrelia burgdorferi. Mutations in parC, which encodes a subunit of topoisomerase IV, were associated with loss of susceptibility to sparfloxacin, moxifloxacin, and Bay-Y3118, but not ciprofloxacin. This is the first description of fluoroquin-olone resistance in the spirochete phylum. Borrelia burgdorferi, a bacterium in the spirochete phylum, is the causative agent of Lyme disease (27, 33, 34). It has an unusual genome comprised of a small linear chromosome and a large complement of both linear and circular plasmids (1, 11). Both DNA gyrase and topoisomerase IV map to the linear chromosome in B. burgdorferi (11, 15, 19, 31). DNA gyrase and topoisomerase IV are prokaryotic type II topoisomerases, a group of enzymes that alter DNA topology by breaking and resealing both strands of the double helix. DNA gyrase maintains negative supercoiling in the cell, and topoisomerase IV relaxes supercoiled DNA and decatenates daughter DNA after replication (8, 9, 26, 41). Both DNA gyrase and topoisomerase IV are tetramers comprised of two A subunits (GyrA or ParC) and two B subunits (GyrB or ParE). The A subunits are involved in the double-stranded nicking and resealing reactions, while the B subunits are re-sponsible for providing energy through ATP hydrolysis (26). Fluoroquinolones are chemotherapeutic agents that target type II topoisomerases by preventing the resealing step in the topoisomerase mechanism (7, 8, 13, 22, 35). Fluoroquinolone treatment results in ternary DNA-topoisomerase-fluoroquino-lone complexes that cause lethal double-stranded DNA breaks (3, 17, 40) and block transcription and replication (36, 37). Resistance to fluoroquinolones usually maps to fluoroquino-lone resistance-determining regions (QRDRs) that are found in the A subunits of DNA gyrase and topoisomerase IV, en-coded by gyrA and parC, respectively (4, 25, 39). Mapping a first-step mutation to one of these genes indicates that the respective gene encodes the primary target of fluoroquinolo-nes (7, 8, 14, 23). Gram-positive bacteria tend to have topo-isomerase IV as the primary target, while gram-negative bac-teria tend to have a primary target of DNA gyrase; however, the primary target also depends on the particular fluoroquin-olone (3, 10, 18, 24, 39). We have isolated fluoroquinolone-resistant first-step mu-tants of B. burgdorferi by selection in increasing doses of three different fluoroquinolones in vitro. Although B. burgdorferi is not susceptible to many fluoroquinolones (5, 12, 20, 21, 30), recently developed fluoroquinolones demonstrate some ther-apeutic potential (16). This study provides the first description of fluoroquinolone resistance in the spirochete phylum and is only the second report, to our knowledge, in which genomic mutations have been associated with antibiotic resistance in B. burgdorferi (32). Selection of fluoroquinolone-resistant mutants. High-pas-sage B. burgdorferi strain B31 was grown at 34°C in Barbour-Stoenner-Kelly (BSK)-H medium (Sigma). The fluoroquinolo-nes moxifloxacin, ciprofloxacin, sparfloxacin, and Bay-Y3118 were generously provided by Peter Heisig (Abteilung Pharmazeutische Biologie und Mikrobiologie, Institut für Pharmazie, Universität Hamburg). The concentration at which 50% of growth is inhibited (IC 50) of each fluoroquinolone was determined for wild-type strain B31 (Table 1) using suscepti-bility assays as previously described (30). Fluoroquinolones at the IC 50 were added to B31 cultures. Each culture was evalu-ated by dark-field microscopy for growth after 7 days. Non-growing cultures were continually passaged 1:10 into the iden-tical antibiotic concentration until growth was observed by dark-field microscopy, and growing cultures were diluted 10-fold into medium containing twice the concentration of the respective fluoroquinolone. Cultures growing in 16-fold the wild-type IC 50 of a particular fluoroquinolone were plated for isolation in semisolid medium containing 10-fold the IC 50 , and cultures growing in 128-fold the wild-type IC 50 were plated on 100-fold the IC 50 , as previously described (29). Five to 10 colonies were selected from each plate; no mutant was identi-fied from ciprofloxacin-treated cultures despite exhaustive ef-forts. Mutations in parC of fluoroquinolone-resistant B. burgdor-feri. DNA was isolated from fluoroquinolone-resistant B. burg-dorferi as previously described (31). The region of the gyrA gene (BB0435) encoding the QRDR was amplified by PCR with primers gyrB 1885F (5-GTAATTAATCTTGATGTGT AA-3) and gyrA 538R (5-TTCCAACAGCAATTCCAC-3). The region of the parC gene (BB0035) encoding the QRDR of topoisomerase IV was amplified with parC 68F (5-CTATTG CTAGTGTTGTTGATGGG-3) and parC 311R (5-CTAGA AGCAGAAGCAGGATCAC-3).
[show abstract][hide abstract] ABSTRACT: Borrelia burgdorferi, the Lyme disease spirochete, undergoes dramatic changes in antigenic composition as it cycles between its arthropod and mammalian hosts. A growing body of evidence suggests that these changes reflect, at least in part, the need for spirochetes to adapt to the physiological stresses imposed by abrupt changes in environmental conditions and nutrient availability. In many microorganisms, global responses are mediated by master regulators such as alternative sigma factors, with Escherichia coli RpoS (sigmaS) serving as a prototype. The importance of this transcriptional activator in other bacteria, coupled with the report by Hubner et al. (A. Hubner, X. Yang, D. M. Nolen, T. G. Popova, F. C. Cabello, and M. V. Norgard, Proc. Natl. Acad. Sci. USA 98:12724-12729, 2001) demonstrating that the borrelial RpoS ortholog controls expression of OspC and decorin-binding protein A (DbpA), prompted us to examine more closely the roles of RpoS-dependent and -independent differential gene expression in physiological adaptation by the Lyme disease spirochete. We observed that B. burgdorferi rpoS (rpoSBb) was induced following temperature shift and transcript levels were further enhanced by reduced pH (pH 6.8). Using quantitative real-time reverse transcription-PCR (RT-PCR), we demonstrated that, in contrast to its ortholog (rpoSEc) in Escherichia coli, rpoSBb was expressed at significant levels in B. burgdorferi throughout all phases of growth following temperature shift. By comparing a B. burgdorferi strain 297 rpoSBb mutant to its wild-type counterpart, we determined that RpoSBb was not required for survival following exposure to a wide range of environmental stresses (i.e., temperature shift, serum starvation, increased osmolality, reactive oxygen intermediates, and increased or reduced oxygen tension), although the mutant was more sensitive to extremes of pH. While B. burgdorferi strains lacking RpoS were able to survive within intraperitoneal dialysis membrane chambers at a level equivalent to that of the wild type, they were avirulent in mice. Lastly, RT-PCR analysis of the ospE-ospF-elp paralogous lipoprotein families complements earlier findings that many temperature-inducible borrelial loci are controlled in an RpoSBb-independent manner. Together, these data point to fundamental differences between the role(s) of RpoS in B. burgdorferi and that in E. coli. Rather than functioning as a master regulator, RpoSBb appears to serve as a stress-responsive activator of a subset of virulence determinants that, together with the RpoS-independent, differentially expressed regulon, encompass the spirochete's genetic programs required for mammalian host adaptation.
Infection and Immunity 12/2004; 72(11):6433-45. · 4.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: Borrelia burgdorferi, the causative agent of Lyme disease, encodes an RpoS ortholog (RpoS(Bb)) that controls the temperature-inducible differential expression of at least some of the spirochete's lipoprotein genes, including ospC and dbpBA. To begin to dissect the determinants of RpoS(Bb) recognition of, and selectivity for, its dependent promoters, we linked a green fluorescent protein reporter to the promoter regions of several B. burgdorferi genes with well-characterized expression patterns. Consistent with the expression patterns of the native genes/proteins in B. burgdorferi strain 297, we found that expression of the ospC, dbpBA, and ospF reporters in the spirochete was RpoS(Bb) dependent, while the ospE and flaB reporters were RpoS(Bb) independent. To compare promoter recognition by RpoS(Bb) with that of the prototype RpoS (RpoS(Ec)), we also introduced our panel of constructs into Escherichia coli. In this surrogate, maximal expression from the ospC, dbpBA, and ospF promoters clearly required RpoS, although in the absence of RpoS(Ec) the ospF promoter was weakly recognized by another E. coli sigma factor. Furthermore, RpoS(Bb) under the control of an inducible promoter was able to complement an E. coli rpoS mutant, although RpoS(Ec) and RpoS(Bb) each initiated greater activity from their own dependent promoters than they did from those of the heterologous sigma factor. Genetic analysis of the ospC promoter demonstrated that (i) the T(-14) in the presumptive -10 region plays an important role in sigma factor recognition in both organisms but is not as critical for transcriptional initiation by RpoS(Bb) as it is for RpoS(Ec); (ii) the nucleotide at the -15 position determines RpoS or sigma(70) selectivity in E. coli but does not serve the same function in B. burgdorferi; and (iii) the 110-bp region upstream of the core promoter is not required for RpoS(Ec)- or RpoS(Bb)-dependent activity in E. coli but is required for maximal expression from this promoter in B. burgdorferi. Taken together, the results of our studies suggest that the B. burgdorferi and E. coli RpoS proteins are able to catalyze transcription from RpoS-dependent promoters of either organism, but at least some of the nucleotide elements involved in transcriptional initiation and sigma factor selection in B. burgdorferi play a different role than has been described for E. coli.
Journal of Bacteriology 12/2004; 186(21):7390-402. · 3.19 Impact Factor
[show abstract][hide abstract] ABSTRACT: Borrelia burgdorferi, which causes Lyme disease in humans, has an unusual genome composed of a linear chromosome and up to 21 extrachromosomal elements. Experimental data suggest that two of these elements, linear plasmids lp25 and lp28-1, play essential roles for infectivity in mice. In this study, we prove the essential natures of these two plasmids by selectively displacing lp25 or lp28-1 in an infectious wild-type clone with incompatible shuttle vectors derived from the native plasmids, rendering the respective transformants noninfectious to mice. Conversely, restoration of plasmid lp25 or lp28-1 in noninfectious clones that naturally lack the corresponding plasmid reestablished infectivity in mice. This approach establishes the ability to manipulate the plasmid content of strains by eliminating or introducing entire plasmids in B. burgdorferi and will be valuable in assessing the roles of plasmids even in unsequenced B. burgdorferi strains.
Infection and Immunity 11/2004; 72(10):5938-46. · 4.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: To enhance genetic manipulation of the Lyme disease spirochete Borrelia burgdorferi, we assayed the aadA gene for the ability to confer resistance to the antibiotics spectinomycin and streptomycin. Using the previously described pBSV2 as a backbone, a shuttle vector, termed pKFSS1, which carries the aadA open reading frame fused to the B. burgdorferi flgB promoter was constructed. The hybrid flgB promoter-aadA cassette confers resistance to spectinomycin and streptomycin in both B. burgdorferi and Escherichia coli. pKFSS1 has a replication origin derived from the 9-kb circular plasmid and can be comaintained in B. burgdorferi with extant shuttle vector pCE320, which has a replication origin derived from a 32-kb circular plasmid, or pBSV2, despite the fact that pKFSS1 and pBSV2 have the same replication origin. Our results demonstrate the availability of a new selectable marker and shuttle vector for genetically dissecting B. burgdorferi at the molecular level.
Journal of Bacteriology 12/2003; 185(22):6723-7. · 3.19 Impact Factor