One even might be able to predict the cell stiffness; for such calculations certain assumptions like: swimming speed is determined mainly by rotation of the helical cell body have to be made about which we feel not sure. Therefore, these theoretical calculations are not included here. We want to stress, however, the fact that the morphotype of spirochetes well can change.
In the case of Treponema denticola with a endoflagella arrangement an irregular twisted morphology bas been observed. Both planar and helical regions of the cells were observed with endoflagella wrapped around planar regions or lying along the cell axis in helical regions.
In addition, however, also some T. All these data point to the fact that only a combination of light microscopy done under physiological conditions to observe motility with electron microscopy—as we have done here—will allow one to define the cell body plan of spirochetes. Interestingly, an increase in viscosity alone—by addition of Ficoll—slowed down, both B.
On the other hand the swimming speed of B. Similar viscoelasticity effects were reported for T. We therefore conclude that S. The normal habitat of S. RW planned the study, took some of the movies and wrote the manuscript. MU took most of the movies. GW made all electron microscopic pictures.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. We thank W. Liebl for his initial help to grow S. Mora for calculations of swimming speeds, A. Bellack for helpful discussions and two reviewers for their helpful critics; the excellent technical support by E. Papst is gratefully acknowledged. Supplemental Movie 1. Supplemental Movie 2. Supplemental Movie 3. Supplemental Movie 4.
Supplemental Movie 5. Aksenova, H. Spirochaeta thermophila sp. Bernecky, C. Nature , — Braun, T. Archaeal flagellin combines a bacterial Type IV pilin domain with an immunoglobulin-like domain. Canale-Parola, E. Motility and chemotaxis of spirochetes. Charon, N. The unique paradigm of Spirochete motility and chemotaxis.
Coltharp, C. Superresolution microscopy for microbiology. Ford, B. The clarity of images from early single-lens microscopes captured on video. Goldstein, S. Structural analysis of the Leptospiraceae and Borrelia burgdorferi by high-voltage electron microscopy.
PubMed Abstract Google Scholar. Borrelia burgdorferi swims with a planar waveform similar to that of eukaryotic flagella. Gupta, R. A phylogenomic and molecular signature based approach for characterization of the phylum Spirochetes and its major clades: proposal for a taxonomic revision of the phylum. Harman, M. Viscous dynamics of lyme disease and syphilis spirochetes reveal flagellar torque and drag. Herzog, B. Swimming behavior of selected species of Archaea. Horn, C. In vivo observation of cell division of anaerobic hyperthermophiles by using a high-intensity dark-field microscope.
This is especially intriguing because the cp32 plasmid prophages could be prone to particularly rapid horizontal transfer [10] , [40] , [] , []. Have all extant B. In addition, strain B31 linear plasmid lp carries two apparently intact PFam32 genes.
It seems quite possible that plasmids of this compatibility type a 28 th type will be found in other strains. We note that the four sequenced isolates are all from a geographically rather restricted region southern New England and New York , and it will be interesting to determine whether B. The organizational variation within each PFam32 type in the four strains studied here suggests that the overall number of B. Nonetheless, the facts that i some pairs of organizationally identical cognate plasmids exist among these four strains, and that ii there are numerous novel sequence joints that are present in more than one strain, suggest that a limited number of such variants exists for each plasmid compatibility type in nature.
Our analyses also indicate that a many of the rearrangements that formed the different organizational types occurred so recently that the sequences involved have diverged substantially less than one percent since the rearrangement, yet the process is not so fast that every B.
Since no such rearrangement has been observed in the laboratory, such events are in fact quite rare, at least under laboratory conditions. Many important unanswered questions regarding Lyme Borrelia genomics and population structure remain, including the following: Do other B.
How many organizational subtypes within each plasmid compatibility type exist in B. Are any plasmids or plasmid subtypes restricted to particular B. What are the relationships among plasmids of different B. Are plasmids transferred between strains as whole entities or as fragments, and what are the rates of transfer in nature?
Are plasmids transferred only within species or between closely related Borrelia species in nature? What gene set constitutes the B. We have recently sequenced nine additional B.
Low passage cultures of B. The bacterial pellet was washed twice with 10 mM Tris pH 7. All three genomes were sequenced to closure. Briefly, small-insert and medium-insert plasmid libraries were generated by random nebulization and cloning of genomic DNA. The following libraries were generated: N40, one 3—4 kb small-insert and one 6—8 kb medium-insert libraries; JD1, one 2—3 kb small-insert, one 3—4 kb small-insert and one 8—12 kb medium-insert libraries; , one 3—4 kb small-insert and one 10—12 kb medium-insert libraries.
In the random sequencing phase, at least a 9-fold coverage across the genome was achieved from the shotgun sequencing libraries generated for each strain.
More specifically, a total of , and Sanger sequencing reads were generated during the random sequencing phase for N40, JD1 and respectively. All sequence and physical gaps were closed by editing the ends of sequence traces, primer walking or transposon-primed sequencing on plasmid clones, and combinatorial PCR followed by sequencing of the PCR product. A number of the termini of bulk-determined sequence contigs were extended by sequencing DNAs from inverse PCR or by direct PCR amplification using outside primers designed from sequence predicted to be orthologous by comparison with plasmids from one of the other strains.
About 8, 19, and 5 Kbp were determined by these directed methods in the N40, JD1 and plasmid sequences. The lengths of the sequence contigs and plasmid sizes determined by pulsed-field electrophoresis and Southern analysis as in Casjens et al. The linear plasmid sequence contigs are often missing from to terminal bp; this was a poorly understood property of the DNA libraries, not sequencing depth. The GenBank accession numbers for the sequences determined in this study were reported in Schutzer et al.
This first set of open reading frames ORFs was then manually curated so that ORFs equal to or less than 50 codons long not counting the stop codon were removed unless they are homologs of a similarly sized gene of known function, and ORFs in the 51— codon range were only included if their reading frame is intact in cognate sequence in all of the strains that carry the sequence.
ORFs that overlapped were inspected visually and, in some cases, removed. Frameshifts and point mutations were detected, checked and corrected where appropriate.
For ease of comparison, the genome of strain B31 was also re-annotated by this pipeline, and this reannotation can be found in the original accession numbers of the B31 replicons Table S1 and [10] , [11].
Some of the plasmids carry full-length, degenerate pseudogene paralogs of other intact plasmid genes. These were not annotated as they were in the original B31 annotation. The automated ORF searches identified some smaller ORFs within these pseudogenes, and since they could theoretically be expressed they were kept in the predicted ORF list. Translation frameshift and in-frame stop differences among the strains sequenced here were compared to homologs in B.
Table S4 lists the locus tag letters with their corresponding plasmids for all the plasmids in the four current genome sequences as well as for our additional unpublished sequences. In the different genomes, the same locus tag numbers in the B31, N40, and JD1 chromosome, cp26 and lp54 indicate orthology of the corresponding genes; however, organizational differences in the other plasmids made this system unworkable, so the same locus tag numbers on these replicons do not indicate orthology.
Within each homolog cluster, orthologs were distinguished from paralogs by visual inspection of gene orders displayed by the authors' unpublished synteny browser and by matrix comparison with DNA Strider []. Protein multiple sequence alignments were constructed using ClustalW 1. Codon alignments were derived from protein alignment templates using PERL scripts. The right end of the B. Burgdorferi JD1 chromosome. Open reading frame maps for plasmids carried by B.
Neighbor-joining trees of proteins encoded by B. B31, JD1, N40 and replicon sizes and accession numbers. Paralogous protein families in four B. Lyme agent Borrelia plasmid letter appellations for locus tags. We thank Drs. Wrote the paper: SRC. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field.
Abstract Lyme disease is the most common tick-borne human illness in North America. Introduction Bacteria in the spirochete genus Borrelia cause arthropod-borne human diseases such as Lyme disease and relapsing fever, as well as a number of diseases of veterinary importance [1] — [6].
Results and Discussion B. Download: PPT. Figure 1. Length variation of B. Plasmid types, occurrence and nomenclature Plasmid content of sequenced genomes. Table 2. Plasmid variation among B. Figure 2. Linear plasmid contents of B. Variation within each plasmid type in different B. Figure 3. Organizational and open reading frame relationships among four lp17 plasmids.
Figure 5. Comparison of lp plasmids and the vls cassette and vlsE loci. Figure 6. Organizational and open reading frame relationships among three lp plasmids. Figure 7. Figure 8. Organizational and open reading frame relationships among four lp36 plasmids. Figure 9. Organizational and open reading frame relationships among four lp38 plasmids.
Plasmid genes and paralog families Paralogous protein families. Novel plasmid-encoded proteins. Plasmid DNA rearrangements Clearly the patchwork of sequences with near identity to other sequences in the B. Non-homologous recombination. Homologous recombination. Mutational decay and pseudogenes The constant regions of the Borrelia chromosomes have very few obvious pseudogenes unlike the plasmids and plasmid-like right end chromosomal extensions. Some examples are as follows: i Mutations in the JD1 lineage.
Conclusions Overall genome relationships. Rates of horizontal exchange? Plasmid types. Future directions. Sequencing and sequence analysis Sequencing, assembly, and gap closure. Sequence annotation. Open reading frame nomenclature. Supporting Information. Figure S1. Figure S2. Figure S3. Vls cassette regions. Figure S4. Ip copy number. Figure S5. Recombination points. Figure S6. Figure S7. Orthologous non-coding DNA in Ip Table S1.
Table S2. Table S3. Major tandem repeat tracts. Table S4. Acknowledgments We thank Drs. References 1. Steere AC Lyme disease. N Engl J Med — View Article Google Scholar 2.
J Clin Invest — View Article Google Scholar 3. Wien Klin Wochenschr — View Article Google Scholar 4. View Article Google Scholar 5. Nat Rev Microbiol 4: — View Article Google Scholar 6. Infect Dis Clin North Am —, viii. View Article Google Scholar 7. Plasmid 1— View Article Google Scholar 8. Infect Dis Clin North Am —, v. View Article Google Scholar 9. Nat Rev Microbiol 87— View Article Google Scholar Mol Microbiol — Nature — Awareness and better understanding of these variations by researchers in the field is thus highly relevant to improvements in their prevention and treatment, and critical for improvement of human health.
This volume focuses on a series of state of the art presentations of the research taking place in the laboratories of the contributors. As such, it may be useful as an introduction to those individuals entering in the burgeoning field of spirochete research. This book could serve as a stimulus for researchers in the field to widen collaborations and exchanges between investigators in the different geographical areas where spirochetal diseases are common since these interactions can only be of benefit to the field.
This type of encounter is valuable because diseases produced by spirochetes, including Lyme borreliosis, syphilis and leptospirosis, are on the rise worldwide, and because the biology of their causative organisms, their epidemiology, and clinical presentation display important variations in different geographical areas. The meeting was organized with oral presentations by major speakers and poster sessions by students and postdoctoral fellows from Eastern Europe.
This volume includes not only the presentations of the major speakers but also several additional presentations by investigators who were invited but were unable to attend. For many reasons including meeting organization and funding limitations , this volume does not intend to represent a comprehensive coverage of all aspects of spirochete biology. It rather focuses on a series of state of the art presentations of the research taking place in the laboratories of the contributors.
As such, we hope that it may be useful as an introduction to those individuals entering in the burgeoning field of spirochete research. We would also like to believe that the meeting and this book will serve as a stimulus for researchers in the field to widen collaborations and exchanges between investigators in the different geographical areas where spirochetal diseases are common since these interactions can only be of benefit to the field.
Finally, we would like to thank the participants who risked the cold weather to attend the meeting, the authors who despite their inability to attend were willing to submit chapters to this book, the funding institutions mentioned above, and in particular, Drs.
Marylin E. Mary C. Marina Cinco University of Trieste , Dr. We would also like to thank Ms. Leonor Delgado for editorial assistance and Ms. Harriett V. Harrison for her outstanding and continuing assistance in organizing the meeting and in preparing the manuscripts that compose this book.
In all of the major human spirochetal infections, the fundamental pathogenetic event underlying the most serious complications of these diseases is documented or presumed hematogenous dissemination of the spirochete from the site of inoculation to distant sites. Lyme borreliosis is attractive for study of spirochetal dissemination for a variety of reasons including: the availability of a large patient base, the ability to identify patients early during the course of infection, and the ability to culture Lyme borrelia readily in vitro.
Tick feeding per se does not directly lead to blood stream invasion by Lyme borrelia. The genotype of the strain of Borrelia burgdorferi introduced by the tick, however, does appear to be an important determinant of dissemination in humans and other mammals.
Recent evidence suggests that host factors affect both the development of infection and subsequent dissemination of this spirochete as well. In one United States study, independent risk factors for hematogenous dissemination of B. In another United States study, infection due to the least invasive genotype of B.
All of the major human spirochetal infections are also characterized by persistence of the spirochete in mammalian hosts, and depending on the specific spirochete and host involved, this phenomenon may be closely linked to the pathogenesis of important clinical manifestations and to communicability to uninfected hosts. In conclusion, spirochetemia and persistence are common and important pathogenetic features of the major human spirochetal infections.
Transposon mutagenesis is a powerful technique for generating random mutations. Previous studies had shown that the mariner transposon Himar1 could be utilized to introduce mutations in high-passage, noninfectious strains of Lyme disease Borrelia; however, transposon mutagenesis has not been reported in low-passage, infectious strains.
In this pilot study, Himar1 transposon mutants were generated by transforming the infectious, highly transformable B. The data provide 'proof in concept' that random transposon mutagenesis can be utilized for the global analysis of genes involved in the pathogenesis of Lyme disease Borrelia.
Borrelia burgdorferi, the causative agent of Lyme disease, is the most prevalent arthropod-borne infectious agent in the United States and, along with infections attributed to other related Borrelia spp. Much of what is known regarding pathogenic mechanisms of B. Recently, advances by a number of investigators have made it possible to isolate and genetically complement isogenic mutants in infectious B.
The approach described here involves a customized transposon that contains an antibiotic resistance marker expressed from a strong borrelial promoter, which is used to mutagenize a borrelial gene target in vitro. The resulting transposon mutagenized candidates are then mapped and sequenced.
The desired constructs, containing the inactivated gene, are transformed into a low passage but non-infectious B. Using this genetic background, several borrelial genes purported to be involved in B. The loss of infectivity associated with the lp25 deficient background can be restored back to wild-type levels by transformation with the borrelial shuttle vector pBBE22, which encodes the lp25 bbe22 locus. The bbe22 pncA gene of this strain encodes a nicotinamidase enzyme required for de novo biosynthesis of NAD only during infection.
Thus, transformation with pBBE22 serves two purposes, as it: i restores infectivity in the parent background, and ii asks whether the resulting mutant exhibits an infectivity deficient phenotype relative to the parent background using the well-established mouse model of infection.
Subsequent genetic complementation with an intact version of the targeted gene in pBBE22 is then carried out to ensure that the mutant phenotype observed can be attributed to the inactivated gene. The strategy presented here could be applied to the genetic analysis of other borrelial genes to determine their significance in the pathogenesis of Lyme borreliosis. Motility and chemotaxis are important in the disease process for many species of bacteria. When infected ticks feed, B.
Motility and chemotaxis are likely to be involved in this traveling of B. Compared with the well-studied Escherichia coli, motility and chemotaxis in B.
Instead of the E. Furthermore, B. Analysis of the B. Rather, studies indicate that motility genes in B. To determine the role s of individual motility genes, we deployed targeted mutagenesis technology and analyzed the resulting cell phenotypes based on the flagellar structure, cell swimming behavior, and effect of specific gene inactivation on other genes and on chemotaxis.
Novel functions of many of these genes of the Lyme disease organism are summarized. The genus Leptospira belongs to the order Spirochaetales and is composed of both saprophytic and pathogenic members, such as Leptospira biflexa and L.
A major factor contributing to our ignorance of spirochetal biology is the lack of methods for genetic analysis of these organisms. We have developed a system for transposon mutagenesis of L. This mutagenesis approach yields a randomly distributed set of insertion mutations throughout the genome, which can be screened for specific phenotypes. An analysis of transposon mutants has allowed the identification of genes required for diverse biological activities such as amino acid biosynthesis and metal transport.
The development of numerous genetic tools for saprophytic species of Leptospira enables the use of these bacteria such as L. Comparative genomics, in combination with gene inactivation, give us significant functional information on iron homeostasis in Leptospira.
Antibiotic resistance is not a clinical problem with Lyme disease, but it has been extensively employed to genetically dissect the causative agent Borrelia burgdorferi. The first selectable marker was a coumermycin A1-resistant gyrB allele, which encodes a subunit of DNA gyrase, a target of several antibiotics. The utility of coumermycin A1 resistance has been compromised by technical and genetic barriers; resistance to other antibiotics has replaced the gyrB marker.
Fluoroquinolones are another class of antibiotics that target DNA gyrase, as well as its homolog topoisomerase IV. Fluoroquinolone resistance in B. A fluoroquinolone-resistant parC allele has been fashioned into a counter-selectable marker, a genetic tool used to select for the loss of DNA. One of the second-generation selectable markers is a heterologous aadA gene that confers resistance to spectinomycin and streptomycin, which target the small subunit of the ribosome.
Selection with spectinomycin failed due to a high frequency of mutants in the population. Eisenbach and M. Balaban Eds. Harwood, C. Jannasch, and E. Hespell, R. Hungate, R. Johnson, P. Jukes, T. Munro Ed. Kropinski, A. Ghiorse, and E. Leschine, S. Magot, M. Fardeau, O. Arnauld, C.
Lanau, B. Ollivier, P. Thomas, and B. Paster, B. Dewhirst, W. Weisburg, L. Tordoff, G. Fraser, R. Hespell, T. Stanton, L. Zablen, L.
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