Isolation and characterization of chemotaxis mutants of the Lyme disease Spirochete Borrelia burgdorferi using allelic exchange mutagenesis, flow cytometry, and cell tracking.

Constructing mutants by targeted gene inactivation is more difficult in the Lyme disease organism, Borrelia burgdorferi, than in many other species of bacteria. The B. burgdorferi genome is fragmented, with a large linear genome and 21 linear and circular plasmids. Some of these small linear and circular plasmids are often lost during laboratory propagation, and the loss of specific plasmids can have a significant impact on virulence. In addition to the unusual structure of the B. burgdorferi genome, the presence of an active restriction-modification system impedes genetic transformation. Furthermore, B. burgdorferi is relatively slow growing, with a 7- to 12-h generation time, requiring weeks to obtain single colonies. The beginning part of this chapter details the procedure in targeting specific B. burgdorferi genes by allelic exchange mutagenesis. Our laboratory is especially interested in constructing and analyzing B. burgdorferi chemotaxis and motility mutants. Characterization of these mutants with respect to chemotaxis and swimming behavior is more difficult than for many other bacterial species. We have developed swarm plate and modified capillary tube assays for assessing chemotaxis. In the modified capillary tube chemotaxis assay, flow cytometry is used to rapidly enumerate cells that accumulate in the capillary tubes containing attractants. To assess the swimming behavior and velocity of B. burgdorferi wild-type and mutant cells, we use a commercially available cell tracker referred to as "Volocity." The latter part of this chapter presents protocols for performing swarm plate and modified capillary tube assays, as well as cell motion analysis. It should be possible to adapt these procedures to study other spirochete species, as well as other species of bacteria, especially those that have long generation times.

Identification of specific chemoattractants and genetic complementation of a Borrelia burgdorferi chemotaxis mutant: flow cytometry-based capillary tube chemotaxis assay.

Measuring the chemotactic response of Borrelia burgdorferi, the bacterial species that causes Lyme disease, is relatively more difficult than measuring that of other bacteria. Because these spirochetes have long generation times, enumerating cells that swim up a capillary tube containing an attractant by using colony counts is impractical. Furthermore, direct counts with a Petroff-Hausser chamber is problematic, as this method has a low throughput and necessitates a high cell density; the latter can lead to misinterpretation of results when assaying for specific attractants. Only rabbit serum and tick saliva have been reported to be chemoattractants for B. burgdorferi. These complex biological mixtures are limited in their utility for studying chemotaxis on a molecular level. Here we present a modified capillary tube chemotaxis assay for B. burgdorferi that enumerates cells by flow cytometry. Initial studies identified N-acetylglucosamine as a chemoattractant. The assay was then optimized with respect to cell concentration, incubation time, motility buffer composition, and growth phase. Besides N-acetylglucosamine, glucosamine, glucosamine dimers (chitosan), glutamate, and glucose also elicited significant chemoattractant responses, although the response obtained with glucose was weak and variable. Serine and glycine were nonchemotactic. To further validate and to exploit the use of this assay, a previously described nonchemotactic cheA2 mutant was shown to be nonchemotactic by this assay; it also regained the wild-type phenotype when complemented in trans. This is the first report that identifies specific chemical attractants for B. burgdorferi and the use of flow cytometry for spirochete enumeration. The method should also be useful for assaying chemotaxis for other slow-growing prokaryotic species and in specific environments in nature.

CheX is a phosphorylated CheY phosphatase essential for Borrelia burgdorferi chemotaxis.

Motility and chemotaxis are believed to be important in the pathogenesis of Lyme disease caused by the spirochete Borrelia burgdorferi. Controlling the phosphorylation state of CheY, a response regulator protein, is essential for regulating bacterial chemotaxis and motility. Rapid dephosphorylation of phosphorylated CheY (CheY-P) is crucial for cells to respond to environmental changes. CheY-P dephosphorylation is accomplished by one or more phosphatases in different species, including CheZ, CheC, CheX, FliY, and/or FliY/N. Only a cheX phosphatase homolog has been identified in the B. burgdorferi genome. However, a role for cheX in chemotaxis has not been established in any bacterial species. Inactivating B. burgdorferi cheX by inserting a flgB-kan cassette resulted in cells (cheX mutant cells) with a distinct motility phenotype. While wild-type cells ran, paused (stopped or flexed), and reversed, the cheX mutant cells continuously flexed and were not able to run or reverse. Furthermore, swarm plate and capillary tube chemotaxis assays demonstrated that cheX mutant cells were deficient in chemotaxis. Wild-type chemotaxis and motility were restored when cheX mutant cells were complemented with a shuttle vector expressing CheX. Furthermore, CheX dephosphorylated CheY3-P in vitro and eluted as a homodimer in gel filtration chromatography. These findings demonstrated that B. burgdorferi CheX is a CheY-P phosphatase that is essential for chemotaxis and motility, which is consistent with CheX being the only CheY-P phosphatase in the B. burgdorferi chemotaxis signal transduction pathway.

Asymmetrical flagellar rotation in Borrelia burgdorferi nonchemotactic mutants.

The Lyme disease spirochete Borrelia burgdorferi has bundles of periplasmic flagella subpolarly located at each cell end. These bundles rotate in opposite directions during translational motility. When not translating, they rotate in the same direction, and the cells flex. Here, we present evidence that asymmetrical rotation of the bundles during translation does not depend upon the chemotaxis signal transduction system. The histidine kinase CheA is known to be an essential component in the signaling pathway for bacterial chemotaxis. Mutants of cheA in flagellated bacteria continually rotate their flagella in one direction. B. burgdorferi has two copies of cheA designated cheA1 and cheA2. Both genes were found to be expressed in growing cells. We reasoned that if chemotaxis were essential for asymmetrical rotation of the flagellar bundles, and if the flagellar motors at both cell ends were identical, inactivation of the two cheA genes should result in cells that constantly flex. To test this hypothesis, the signaling pathway was completely blocked by constructing the double mutant cheA1kan cheA2ermC. This double mutant was deficient in chemotaxis. Rather than flexing, it failed to reverse, and it continually translated only in one direction. Video microscopy of mutant cells indicated that both bundles actively rotated. The results indicate that asymmetrical rotation of the flagellar bundles of spirochetes does not depend upon the chemotaxis system but rather upon differences between the two flagellar bundles. We propose that certain factors within the spirochete localize at the flagellar motors at one end of the cell to effect this asymmetry.