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N W Charon

Publications and source records attributed to N W Charon.

At least 37 records · Page 2Linked to original sources
PubMed1991

Treponema phagedenis encodes and expresses homologs of the Treponema pallidum TmpA and TmpB proteins.

We cloned and sequenced the genes from Treponema phagedenis Kazan 5 encoding proteins homologous to the TmpA and TmpB proteins of Treponema pallidum subsp. pallidum Nichols (hereafter referred to as T. pallidum). Although previous reports suggested that the TmpA and TmpB proteins were specific for T. pallidum, we found that homologs for both were expressed in T. phagedenis Kazan 5 and Reiter. The TmpA protein from T. phagedenis contained the consensus sequence that bacterial lipoproteins require for posttranslational modification and subsequent proteolytic cleavage by signal peptidase II and showed 42% amino acid sequence identity with the TmpA protein from T. pallidum. The TmpB proteins of T. phagedenis and T. pallidum had similar amino acid sequences at their amino- and carboxy-terminal ends. The central portions of both of these proteins contained four repeats of the amino acid sequence EAARKAAE. The TmpB protein from T. phagedenis had an additional amino acid sequence repeat (consensus sequence KAAKE/D) that was not found in the TmpB protein from T. pallidum; this repeat was most remarkable, as it occurred 17 times in succession. These repeated amino acid sequences probably created an extensive alpha-helix region within the TmpB proteins. As with T. pallidum, the stop codon of the T. phagedenis tmpA gene overlapped the start codon of its tmpB gene. Northern blot analysis showed that the T. phagedenis tmpA and tmpB genes were probably transcribed into a single 2.5-kb mRNA molecule. Western blot (immunoblot) analysis demonstrated that both proteins were expressed by T. phagedenis. The high degree of amino acid sequence conservation seen with the TmpA and TmpB proteins from two different Treponema species suggests that they may play crucial roles in the biology of these organisms.

Amino Acid Sequence↗
PubMed1991

The bent-end morphology of Treponema phagedenis is associated with short, left-handed, periplasmic flagella.

Treponema phagedenis Kazan 5 is a spirochete with multiple periplasmic flagella attached near each end of the cell cylinder. Dark-field microscopy revealed that most of the cell is right-handed (helix diameter, 0.23 micron; helix pitch, 1.74 microns), and the ends appear bent. These ends could move and gyrate while the central part of the cell remained stationary. The present study examines the basis for the bent-end characteristic. Motility mutants deficient in periplasmic flagella were found to lack the bent ends, and spontaneous revertants to motility regained the periplasmic flagella and bent-end characteristic. The length of the bent ends (2.40 microns) was found to be similar to the length of the periplasmic flagella as determined by electron microscopy (2.50 microns). The helix diameter of the bent ends was 0.57 micron, and the helix pitch of the bent ends was 1.85 microns. The periplasmic flagella were short relative to the length of the cells (15 microns) and, in contrast to the reports of others, did not overlap in the center of the cell. Similar results were found with T. phagedenis Reiter. The results taken together indicate that there is a causal relationship between the bent-end morphology and the presence of short periplasmic flagella. We report the first three-dimensional description of spirochete periplasmic flagella. Dark-field microscopy of purified periplasmic flagella revealed that these organelles were left-handed (helix diameter, 0.36 microns; helix pitch, 1.26 microns) and only 1 to 2 wavelengths long. Because of a right-handed cell cylinder and left-handed periplasmic flagella along with bent ends having helix diameters greater than those of either the cell cylinder or periplasmic flagella, we conclude that there is a complex interaction of the periplasmic flagella and the cell cylinder to form the bent ends. The results are discussed with respect to a possible mechanism of T. phagedenis motility.

Cell Movement↗
PubMed1990

Multiple-exposure photographic analysis of a motile spirochete.

The Leptospiraceae are thin spirochetes with a unique mode of motility. These spiral-shaped bacteria have internal periplasmic flagella that propel the cells in low-viscosity and gel-like high-viscosity media. A model of Leptospiraceae motility has been previously proposed that states that the subterminally attached periplasmic flagella rotate between the outer sheath and the helical protoplasmic cylinder. The shape of the cell ends and the direction of gyration of these ends are determined by the direction of rotation of the internal periplasmic flagella. Rotation of the periplasmic flagella in one direction causes that end to be spiral-shaped, and rotation in the other direction causes that end to be hook-shaped. One prediction of the model is that these right-handed spirochetes roll clockwise when swimming away from an observer. For maximum swimming efficiency, the model predicts that the sense of the spiral-shaped end is left-handed and gyrates counterclockwise. The present study presents direct evidence that the cell rolls clockwise (protoplasmic cylinder helix diameter = 0.24 micron; pitch = 0.69 micron), the ends gyrate counterclockwise, and the spiral-shaped end is left-handed (helix diameter = 0.6 micron; pitch = 2.7 microns)--as predicted by the model. The hook-shaped end appears approximately planar. The approach used was to illuminate stroboscopically cells slowed by Ficoll and analyze the resultant multiple-exposure photographs focused above and below the axis of the cell. The methodology used should be helpful in analyzing the motility of the larger and more complex spirochetes.

Cell Movement↗
PubMed1988

Motility of the spirochete Leptospira.

Spirochetes are a group of bacteria with a unique ultrastructure and a fascinating swimming behavior. This article reviews the hydrodynamics of spirochete motility, and examines the motility of the spirochete Leptospira in detail. Models of Leptospira motility are discussed, and future experiments are proposed. The outermost structure of Leptospira is a membrane sheath, and within this sheath are a helically shaped cell cylinder and two periplasmic flagella. One periplasmic flagellum is attached subterminally at either end of the cell cylinder and extends partway down the length of the cell. In swimming cells, each end of the cell may assume either a spiral or a hook shape. Translational cells have the anterior end spiral shaped, and the posterior end hook shaped. In the model of Berg et al., the periplasmic flagella are believed to rotate between the sheath and the cell cylinder. Rotation of the anterior periplasmic flagellum causes the generation of a gyrating spiral-shaped wave. This wave is believed sufficient to propel the cells forward in a low-viscosity medium. The cell cylinder concomitantly rolls around the periplasmic flagella in the opposite direction--which allows the cell to literally screw through a gel-like viscous medium without slippage. This model is presented, and it is contrasted to previous models of Leptospira motility.

Cell Movement↗
PubMed1988

Sensitivity of pathogenic and free-living Leptospira spp. to UV radiation and mitomycin C.

The habitats for the two major Leptospira spp. differ. The main habitat of L. biflexa is soil and water, whereas L. interrogans primarily resides in the renal tubules of animals. We investigated whether these two species, along with L. illini (species incertae sedis), differ with respect to their sensitivity to UV radiation. The doses of UV resulting in 37, 10, and 1% survival were determined for representative serovars from each species. L. interrogans serovar pomona was 3.0 to 4.8 times more sensitive to UV than the other Leptospira species under the 37, 10, and 1% survival parameters. In comparison to other bacteria, L. interrogans serovar pomona is among the most sensitive to UV. In a qualitative UV sensitivity assay, L. interrogans serovars were found to be in general more sensitive than L. biflexa serovars. All three species were found to have a photoreactivation DNA repair mechanism. Since organisms that are resistant to UV are often resistant to the DNA cross-linking agent mitomycin C, we tested the relative sensitivity of several Leptospira serovars to this compound. With few exceptions, L. biflexa and L. illini serovars were considerably more resistant to mitomycin C than the L. interrogans serovars. The mitomycin C sensitivity assay could be a useful addition to current characterization tests used to differentiate the Leptospira species.

Dose-Response Relationship, Radiation↗
PubMed1988

Nucleotide sequence analysis of a gene cloned from Leptospira biflexa serovar patoc which complements an argE defect in Escherichia coli.

The genus Leptospira, as a member of the order Spirochaetales, forms one of the most ancient evolutionary branches of the eubacteria. These spirochetes are morphologically and physiologically different from most eubacteria, and little is known about Leptospira genetics. In this communication, we report the first nucleotide sequence of a Leptospira gene. A gene which complements an argE mutation in Escherichia coli was isolated from a plasmid-based genomic library composed of Leptospira biflexa serovar patoc DNA. The functional region for the complementing activity was localized by transposon mutagenesis and restriction enzyme mapping and by subcloning. Nucleotide sequence analysis indicated a single open reading frame within the region containing argE complementing activity. The size of the predicted protein, 31,071 daltons, was in excellent agreement with data obtained from coupled transcription-translation reactions primed with cloned L. biflexa DNA. One surprising result was that the predicted amino acid sequence of this protein closely resembles portions of the beta' subunits of RNA polymerases from bacteria and chloroplasts.

Amino Acid Sequence↗
PubMed1988

Antigenic relatedness and N-terminal sequence homology define two classes of periplasmic flagellar proteins of Treponema pallidum subsp. pallidum and Treponema phagedenis.

The periplasmic flagella of many spirochetes contain multiple proteins. In this study, two-dimensional electrophoresis, Western blotting (immunoblotting), immunoperoxidase staining, and N-terminal amino acid sequence analysis were used to characterize the individual periplasmic flagellar proteins of Treponema pallidum subsp. pallidum (Nichols strain) and T. phagedenis Kazan 5. Purified T. pallidum periplasmic flagella contained six proteins (Mrs = 37,000, 34,500, 33,000, 30,000, 29,000, and 27,000), whereas T. phagedenis periplasmic flagella contained a major 39,000-Mr protein and a group of two major and two minor 33,000- to 34,000-Mr polypeptide species; 37,000- and 30,000-Mr proteins were also present in some T. phagedenis preparations. Immunoblotting with monospecific antisera and monoclonal antibodies and N-terminal sequence analysis indicated that the major periplasmic flagellar proteins were divided into two distinct classes, designated class A and class B. Class A proteins consisted of the 37-kilodalton (kDa) protein of T. pallidum and the 39-kDa polypeptide of T. phagedenis; class B included the T. pallidum 34.5-, 33-, and 30-kDa proteins and the four 33- and 34-kDa polypeptide species of T. phagedenis. The proteins within each class were immunologically cross-reactive and possessed similar N-terminal sequences (67 to 95% homology); no cross-reactivity or sequence homology was evident between the two classes. Anti-class A or anti-class B antibodies did not react with the 29- or 27-kDa polypeptides of T. pallidum or the 37- and 30-kDa T. phagedenis proteins, indicating that these proteins are antigenically unrelated to the class A and class B proteins. The lack of complete N-terminal sequence homology among the major periplasmic flagellar proteins of each organism indicates that they are most likely encoded by separate structural genes. Furthermore, the N-terminal sequences of T. phagedenis and T. pallidum periplasmic flagellar proteins are highly conserved, despite the genetic dissimilarity of these two species.

Amino Acid Sequence↗
PubMed1986

Treponema phagedenis has at least two proteins residing together on its periplasmic flagella.

Treponema phagedenis is an anaerobic, motile spirochete with several periplasmic flagella (PFs) at each cell end. This study provides the first genetic evidence that multiple protein species are associated with the PFs. In addition, these proteins were found to reside together on a given PF. Nonmotile mutants which lacked the PFs were isolated, and spontaneous revertants to motility regained the PFs. These results suggest that the PFs are involved in the motility of T. phagedenis. Isolated PFs had two major protein bands with molecular weights of 33,000 and 39,800, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blots with monoclonal and polyclonal antibodies indicated that both proteins were absent in the PF mutants but present in the revertants. Immunoelectron microscopy revealed that the 39,800-molecular-weight protein was distributed along the entire PF. Immunoprecipitation analysis suggested that the 39,800- and 33,000-molecular-weight proteins were closely associated in situ.

Bacterial Proteins↗
PubMed1986

Antiserum to the 33,000-dalton periplasmic-flagellum protein of "Treponema phagedenis" reacts with other treponemes and Spirochaeta aurantia.

"Treponema phagedenis" periplasmic flagella (PF) have two major protein bands at molecular weights of 33,000 and 39,800 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (R. J. Limberger and N. W. Charon, J. Bacteriol. 166:105-112, 1986). By use of Western blotting and a polyclonal antiserum directed toward the 33,000-molecular-weight PF protein, cell lysates of 12 species of spirochetes were surveyed for reactivity. Eight species of Treponema as well as Spirochaeta aurantia were positive. The results suggest that epitopes residing on the 33,000-molecular-weight PF protein of "T. phagedenis" are evolutionarily well conserved among the spirochetes.

Bacterial Proteins↗
PubMed1984

Cloning of a gene required for tryptophan biosynthesis from Leptospira biflexa serovar patoc into Escherichia coli.

A clone bank, consisting of approx. 8100 colonies, has been created for the spirochete Leptospira biflexa serovar patoc in Escherichia coli using pBR322 as the vector. One of these clones contains the genetic information needed to complement a defect in the trpE gene of E. coli. The information resides on a 20.5-kb plasmid designated pYC1, which carries a 16-kb insert consisting of three HindIII fragments. It does not complement defects in other genes needed for the biosynthesis of tryptophan in E. coli.

Chromosome Mapping↗
PubMed1984

Microcinematographic analysis of tethered Leptospira illini.

A model of Leptospira motility was recently proposed. One element of the model states that in translating cells the anterior spiral-shaped end gyrates counterclockwise and the posterior hook-shaped end gyrates clockwise. We tested these predictions by analyzing cells tethered to a glass surface. Leptospira illini was incubated with antibody-coated latex beads (Ab-beads). These beads adhered to the cells, and subsequently some cells became attached to either the slide or the cover glass via the Ab-beads. As previously reported, these cells rapidly moved back and forth across the surface of the beads. In addition, a general trend was observed: cells tethered to the cover glass rotated clockwise around the Ab-bead; cells tethered to the slide rotated counterclockwise around the Ab-bead. A computer-aided microcinematographic analysis of tethered cells indicated that the direction of rotation of cells around the Ab-bead was a function of both the surface of attachment and the shape of the cell ends. The results can best be explained by assuming that the gyrating ends interact with the glass surface to cause rotation around the Ab-beads. The analysis obtained indicates that the hook- and spiral-shaped ends rotate in the directions predicted by the model. In addition, the tethered cell assay permitted detection of rapid, coordinated reversals of the cell ends, e.g., cells rapidly switched from a hook-spiral configuration to a spiral-hook configuration. These results suggest the existance of a mechanism which coordinates the shape of the cell ends of L. illini.

Cell Movement↗
PubMed1983

Multiple pathways for isoleucine biosynthesis in the spirochete Leptospira.

Spirochetes of the genus Leptospira have previously been shown to use an unusual pathway to synthesize isoleucine. For reasons of convenience, we assume that only one unusual pathway is found in the genus, and we refer to it as the pyruvate pathway. We determined the distribution of this pyruvate pathway in representatives of the seven Leptospira DNA hybridization groups. Our method included labeling the representative strains with radioactive carbon dioxide and other radioactive precursors, fractionating the cells, and determining the specific activities (counts detected per nanomole) of the amino acids found in the protein fractions. On the basis of isoleucine biosynthesis, we found that the genus can be classified as follows: class I primarily, if not exclusively, uses the well-known threonine pathway; class II uses mostly the pyruvate pathway, with a minor amount of isoleucine being synthesized via the threonine pathway; and class III uses the pyruvate pathway exclusively. No relationship appears to exist between the degree of DNA hybridization and the classes of isoleucine biosynthesis. Although the precise intermediates on the pyruvate pathway are unknown, the origin of the carbon skeleton of isoleucine synthesized by this pathway is consistent with a borrowing of the leucine biosynthetic enzymes. However, we found that the pyruvate pathway is not controlled by leucine and that the two isoleucine pathways are independently regulated. Finding major and highly evolved multiple biosynthetic pathways of a specific amino acid within one genus is unique, and, conceivably, represents phylogenetic diversity within Leptospira.

Isoleucine↗
PubMed1981

Movement of antibody-coated latex beads attached to the spirochete Leptospira interrogans.

Antibody-coated latex beads (Ab-beads) were attached to Leptospira interrogans serovars illini 3055 and icterohaemorrhagiae SC1157. The movement of the Ab-beads relative to the motion of the cells was observed by direct darkfield microscopy or was recorded on videotape. When the Ab-beads were attached to the front end of motile cells, the Ab-beads were displaced towards the back end of the cells. When the cells reversed direction, the Ab-beads also reversed direction. A number of hypotheses were proposed and tested to account for this Ab-bead displacement. The one best supported by the evidence states that the Ab-beads are attached to antigens of the outer membrane sheath. These antigens are dragged laterally through the sheath due to the forward motion of the cells and the retarding forces of the medium acting on the beads. The results obtained provide information on the nature of the outer membrane sheath of L. interrogans, the basis for certain movements of spirochetes, and insight on how spirochetes attach to eukaryotic cells and tissues. In addition, the results indicate that antigens can move laterally through membranes as rapidly as 11 micrometers/sec.

Antibodies↗
PubMed1980

Isolation of lambda transducing phage with the bio genes inserted between lambda genes P and Q.

Plaque-forming, biotin-transducing phages were constructed with the bio genes inserted between lambda genes P and Q. These phages were isolated for the eventual aim of fusing the lambda Q gene to the bio operon. The following steps were used to construct these phages: A defective temperature-sensitive lysogen was constructed with the bio genes adjacent to and to the left of lambda genes beta NcI857OPQSRA. Heat-resistant survivors were screened for deletions with endpoints in the bio operon and to the right of lambda P and to the left of lambda A. Five of approximately 1,600 heat-resistant survivors had these properties. Two had the gene order bioAB .... lambda QSRA. When these two strains were lysogenized with lambda cI857b221 and heat induced, the desired transducing phages were obtained. We characterized these phages and studied one in detail. Two-thirds of the plaque-forming transducing phages isolated carried the entire bioB gene and only part of the bioA gene, and one-third carried the entire bioA and bioB genes. The phages isolated lost the bio genes upon propagation, indicating that they contain a partial duplication of phage genes. The duplication was shown not to involve the entire lambda Q gene in one of these phages, lambda bioq1b221. A recombinant of this phage, lambda Nam7am53c17b221, failed to form plaques under biotin-derepression conditions. We conclude that if the lambda Q gene was fused to the bio operon in this phage, not enough lambda Q gene product was made to allow phage propagation.

Bacteriophage lambda↗
PubMed1979

Axial filament involvement in the motility of Leptospira interrogans.

Motility mutants of Leptospira interrogans serovar illini were isolated and analyzed by dark-field and electron microscopy. Mutants were obtained by screening for small colonies after nitrosoguanidine treatment. One class of mutants did not have hook- or spiral-shaped ends. In addition, the axial filaments from these mutants were not coiled. An analysis of revertants of two of the mutants in this class indicated that the mutations were pleiotropic with respect to motility, hook- and spiral-shaped ends, and axial filament coiling. We conclude that the axial filaments and the hook- and spiral-shaped ends are involved in L. interrogans motility.

Cell Wall↗
PubMed1979

Helix handedness of Leptospira interrogans as determined by scanning electron microscopy.

Representative serovars and strains of the seven genetic groups of Leptospira interrogans, and two previously studied serovars, were all found to form exclusively right-handed helices as determined by scanning electron microscopy. No change in handedness occurred in cells grown in a minimal medium (Tween-80 albumin) compared to cells grown in a rich medium (rabbit serum). The right-handedness of the organisms was related to the evolution, cell wall structure, and the mechanism of motility of L. interrogans.

Culture Media↗