Summary 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 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‐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 .
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.
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.
Introducing foreign DNA into the spirochete Borrelia burgdorferi has been almost exclusively accomplished by transformation using electroporation. This process has notably lower efficiencies in the Lyme disease spirochete relative to other, better-characterized Gram-negative bacteria. The rate of success of transformation is highly dependent upon having concentrated amounts of high-quality DNA from specific backgrounds and is subject to significant strain-to-strain variability. Alternative means for introducing foreign DNA (i.e., shuttle vectors, fluorescent reporters, and antibiotic-resistance markers) into B. burgdorferi could be an important addition to the armamentarium of useful tools for the genetic manipulation of the Lyme disease spirochete. Bacteriophage have been well-recognized as natural mechanisms for the movement of DNA among bacteria in a process called transduction. In this study, a method has been developed for using the ubiquitous borrelial phage φBB-1 to transduce DNA between B. burgdorferi cells of both the same and different genetic backgrounds. The transduced DNA includes both borrelial DNA and heterologous DNA in the form of small shuttle vectors. This demonstration suggests a potential use of phage-mediated transduction as a complement to electroporation for the genetic manipulation of the Lyme disease spirochete. This report describes methods for the induction and purification of phage φBB-1 from B. burgdorferi, the use of this phage in transduction assays, and the selection and screening of potential transductants.
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.
Summary 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+ .
All members of the Borrelia genus that have been examined harbour a linear chromosome that is about 900 kbp in length, as well as a plethora of both linear and circular plasmids in the 5-220 kbp size range.Genome sequences for 27 Lyme disease Borrelia isolates have been determined since the elucidation of the B. burgdorferi B31 genome sequence in 1997.The chromosomes, which carry the vast majority of the housekeeping genes, appear to be very constant in gene content and organization across all Lyme disease Borrelia species.The content of the plasmids, which carry most of the genes that encode the differentially expressed surface proteins that interact with the spirochete's arthropod and vertebrate hosts, is much more variable.Lyme disease Borrelia isolates carry between 7-21 different plasmids, ranging in size from 5-84 kbp.All strains analyzed to date harbor three plasmids, cp26, lp54 and lp17.The plasmids are unusual, as compared to most bacterial plasmids, in that they contain many paralogous sequences, a large number of pseudogenes, and, in some cases, essential genes.In addition, a number of the plasmids have features indicating that they are prophages.Numerous methods have been developed for Lyme disease Borrelia strain typing.These have proven valuable for clinical and epidemiological studies, as well as phylogenomic and population genetic analyses.Increasingly, these approaches have been displaced by whole genome sequencing techniques.Some correlations between genome content and pathogenicity have been deduced, and comparative whole genome analyses promise future progress in this arena.
Summary The 32 kb circular plasmid (cp32) family of Borrelia burgdorferi has been the subject of intensive investigation because its members encode numerous differentially expressed lipoproteins. As many as nine different cp32s appear to be capable of stable replication within a single spirochaete. Here, we show that a construct (pCE310) containing a 4 kb fragment from the putative maintenance region of a B. burgdorferi CA‐11.2A cp32 was capable of autonomous replication in both high‐passage B. burgdorferi B31 and virulent B. burgdorferi 297. Deletion analysis revealed that only the member of paralogous family 57 and the adjacent non‐coding segment were essential for replication. The PF32 ParA orthologue encoded by the pCE310 insert was almost identical to the PF32 orthologues encoded on the B31 and 297 cp32‐3 plasmids. The finding that cp32‐3 was selectively deleted in both B31 and 297 transformants carrying pCE310 demonstrated the importance of the PF32 protein for cp32 compatibility and confirmed the prediction that cp32 plasmids expressing identical PF32 paralogues are incompatible. A shuttle vector containing the CA‐11.2A cp32 plasmid maintenance region was used to introduce green, yellow and cyan fluorescent protein reporters into B. burgdorferi . Flow cytometry revealed that the green fluorescent protein was well expressed by almost 90% of both avirulent and infectious transformants. In addition to enhancing our understanding of B. burgdorferi plasmid biology, our results further the development of genetic systems for dissecting pathogenic mechanisms in Lyme disease.
