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 cheA1::kan cheA2::ermC. 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.spirochete ͉ periplasmic flagella ͉ Lyme disease ͉ chemotaxis ͉ motility

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.Bacteria move toward or away from environments that are favorable or unfavorable, respectively, to enhance their survival (reviewed in references 5, 63, and 65). When this movement is in response to chemicals, the process is termed chemotaxis. Flagella or periplasmic flagella, depending upon their location in a cell, are responsible for locomotion in many species of bacteria. Regulation of flagellar rotation and chemotaxis has been studied most extensively in Escherichia coli and Salmonella enterica serovar Typhimurium, and phosphorylation of the response regulator CheY plays an important role in regulating the swimming pattern of cells (reviewed in references 5, 8, 12, 60, 63, and 65). The concentration of phosphorylated CheY (CheY-P) determines whether a cell runs or tumbles. In the absence of attractants, the concentration of CheY-P is relatively high, and CheY-P diffuses to and binds the flagellar switch protein FliM, switching flagellar rotation from a default counterclockwise (CCW) state to a clockwise (CW) rotation. CW rotation of one or more flagella disrupts flagellar bundles, causing cells to tumble and reorient direction during the next run (37, 64). Although CheY-P autodephosphorylates, E. coli CheZ is required for efficient CheY-P dephosphorylation, allowing rapid responses to the environment (53). Thus, functionally reducing CheY-P in null mutants of cheA (encoding the protein that transfers phosphate to CheY) or cheY resu...

The toxicity of homologous series of organic solvents has been investigated for the gram-positive bacteria, Arthrobacter sp. and Nocardia sp., and the gram-negative bacteria, Acinetobacter sp. and Pseudomonas sp. The hydrophobicity of the solvent, expressed by its logP(octanol), proves to be a good measure for the toxicity of solvents in a two-phase system. The transition from toxic to nontoxic solvents occurs between logP(octanol) 3 and 5 and depends on the homologous series. No correlation has been found between the hydrophobicity of the substituent on the alkyl backbone of the solvent and the location of the transition point in toxicity. The logP(octanol), above which all solvents are nontoxic, is used to express the solvent tolerance of the bacteria. In general, the solvent tolerance of gram-negative bacteria is found to be slightly higher than that of gram-positive bacteria, but this does not hold for all homologous series of organic solvents investigated.Because the toxicity effects of organic solvents in a two-phase system can be ascribed to molecular as well as phase toxicity effects, molecular toxicity effects were investigated separately in a one-phase system with subsaturating amounts of organic solvent. The solvent concentration in the aqueous phase, at which 50% of the metabolic activity of the bacteria is lost, is used to express solvent toxicity. This concentration is found to be similar for the gram-positive Arthrobacter and the gram-negative Acinetobacter. Assuming the critical membrane concentration theory (G. J. Osborne et al. Enzyme Microb. Technol. 1990, 12: 281-291) to be valid, it can be concluded that differences in solvent tolerance between these two bacteria, cannot be ascribed to differences in response to molecular toxicity. Prediction of the toxicity of any solvent, using the critical membrane theory, appears to be possible in the case of alkanols or alkyl acetates. However, prediction of the toxicity of ethers appears to be impossible.

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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.Lyme disease is caused by the motile spirochete Borrelia burgdorferi. This disease is the most prevalent arthropod-borne infection in the United States, with 19,804 cases reported in 2004 (11). The clinical course of B. burgdorferi infections includes symmetrical spread of the spirochetes through the dermis resulting in a rash referred to as erythema migrans and invasion of the blood and deep organs (6). Disease manifestations also include arthritis, cardiac abnormalities, and neuropathies. The life cycle of B. burgdorferi involves transmission from a tick vector to mammals or birds and back to the tick over the course of several seasons (54).A robust motility-and-chemotaxis system is likely to be vital for B. burgdorferi in its overall life cycle. Many of the motility and chemotaxis genes are expressed in both the tick and the mammalian host, and several are upregulated in the laboratory that mimic in vivo conditions (8,16,21,45,48,57). Furthermore, approximately 6% of its chromosomal genome encodes putative chemotaxis and motility genes (18) and between 10 and 14% of the total cellular protein is composed of flagellar filament proteins (42). Thus, this system is both evidentl...

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SummaryAn enzyme-linked immunosorbent assay (ELISA) to measure Streptobacillus moniliformis antibodies in mice and rats was developed. Twelve S. moniliformis strains originating from cases of rat-bite fever and Haverhill fever in man and from various rodent species, showed considerable serological relationship.The ELISA appeared specific since antibodies to S. moniliformis were absorbed by autologous and homologous antigen, but not by heterologous bacterial antigens.Acholeplasma laidlaw;; showed partial serological relationship with S. moniliformis.The ELISA was validated using experimental infections in mice and rats. These studies and observations in naturally infected feral rats, confirmed that S. moniliformis is difficult to grow on primary isolation, and that the ELISA for S. moniliformis antibodies revealed more contaminated animals than culture. Keywords: Streptobacillus moniliformis;Serology; ELISA; Mouse; Rat Streptobacillus monilijormis has been associated with rat-bite fever and Haverhill fever in humans, following a bite and contamination of food by rat urine respectively. The rat has been identified as the main reservoir of S. monilijormis and the bacterium is considered a commensal inhabitant of the nasopharynx in this species of animal. S. moni/iformis has been reported to

SummaryStreptobacillus moniliformis was isolated from the middle ear of 9 of 16 rats used for otological studies. Examination of rat sera for the presence of anti-Streptobacillus moniliformis antibodies using an ELISA technique resulted in 15 seropositive animals. The source of the S. moniliformis infection was not determined.