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.     

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.     

Effects of wounds on the terpene content of twigs of maritime pine (Pinus pinaster Ait.)

Summary The effect of wounds on the volatile terpene composition of the living bark of Pinus pinaster Ait. (maritime pine) twigs was investigated with respect to the processes of mono- and sesquiterpene hydrocarbon biosynthesis. The large increase in the amounts of - and -pinene is a characteristic feature after a mechanical injury, whereas the quantities of the other terpenes are only slightly increased. This is due to the reactivation of the resin duct secretory cells of primary origin located in cortical tissues. The effect of wounding is observed over a long period and the terpene profiles are very different at the end of the experiments as compared with the initial profiles of the same tissues. The traumatic essential oil (obtained after mechanical traumatism) resembles an oleoresin extracted from tissues of secondary origin. Statistical analysis underlines the effects of the between-tree variations and of the dates of application of the wound.     

Functional regions of the mouse thrombopoietin receptor cytoplasmic domain: evidence for a critical region which is involved in differentiation and can be complemented by erythropoietin.

Thrombopoietin (TPO) is the major regulator of growth and differentiation of megakaryocytes. To identify functionally important regions in the cytoplasmic domain of the TPO receptor, mpl, we introduced wild-type mpl and deletion mutants of murine mpl into the granulocyte-macrophage colony-stimulating factor (GM-CSF)- or erythropoietin (EPO)-dependent human cell line UT7. TPO induced differentiation of UT7-Wtmpl cells, not parental UT7 cells, along the megakaryocytic lineage, as evidenced by decreased proliferation, changes in cell morphology, and increased surface expression and mRNA levels of megakaryocytic markers CD41, CD61, and CD42b. When UT7-mpl cells were cultured long-term in EPO instead of GM-CSF, the TPO effect was dominant over that of EPO. Moreover, the differentiation induced by TPO was more pronounced for cells shifted from EPO to TPO than for cells shifted from GM-CSF to TPO, as shown by the appearance of polyploid cells. Mutational analysis of the cytoplasmic domain of mpl showed that proliferation and maturation functions of mpl can be uncoupled. Two functional regions were identified: (i) the first 69 amino acids comprising the cytokine receptor motifs, box I and box 2, which are necessary for both TPO-induced mitogenesis and maturation; and (ii) amino acids 71 to 94, which are dispensable for proliferation but required for differentiation. Surprisingly, however, EPO could complement this latter domain for TPO-induced differentiation, suggesting a close relationship between EPO and TPO signaling.     

Suppressors of thermosensitive mutations in the DNA polymerase δ gene of Saccharomyces cerevisiae

DNA polymerases (Pol) α, δ and ε are necessary for replication of nuclear DNA. Po1δ interacts permanently or transiently with numerous accessory proteins whose identification may shed light on the function(s) of Po18. In vitro mutagenesis was used to induce thermosensitive (ts) mutations in the DNA polymerase δ gene (POL3). We have attempted to clone two recessive extragenic suppressors of such is mutants (sdp1 for mutation pol3-14 and sdp5-1 for mutation pol3-11) by transforming thermoresistant haploid strains pol3-14 sdpl and pol3-11 sdp5-1 with wild-type genomic libraries in singlecopy or multicopy vectors. None of the thermosensitive transformants so obtained was identified as being sdp1 or sdp5-1. Instead, three genes were cloned whose products interfere with the activity of suppressors. One of them is the type 1 protein phosphatase gene, D1S2. Another is a novel gene, ASM4, whose gene product is rich in asparagine and glutamine residues.     

Constitutive activation of a variant of the env-mpl oncogene product by disulfide-linked homodimerization.

The myeloproliferative leukemia retrovirus (MPLV) has the v-mpl cellular sequences transduced in frame with the deleted and rearranged Friend murine leukemia virus env gene. The resulting env-mpl fusion oncogene is responsible for an acute myeloproliferative disorder induced in mice by MPLV. v-mpl is a truncated form of the c-mpl gene which encodes the receptor for thrombopoietin. We investigated the contribution of the Env-Mpl extracellular domain in the constitutive activation of this truncated cytokine receptor and found that the rearrangement of the env sequences in the env-mpl fusion gene was not required for oncogenicity. A pathogenic variant, DEL3MPLV, was generated, which differs from MPLV by the deletions of 22 amino acids of the Env signal peptide, all of the mature Env sequences, and 18 N-terminal amino acids of the v-Mpl extracellular domain. The resulting del3-mpl oncogene product conserves in its extracellular region the first 12 amino acids of the Env signal sequence including a cysteine residue, and 25 amino acids of the v-Mpl. We show here that a mutation converting this cysteine to a glycine completely abolishes del3-mpl oncogenicity and that the del3-mpl oncogene product is constitutively activated by disulfide-linked homodimerization.     

Carbonic anhydrase,carbondioxide levels and growth of Nitrosomonas

The ammonia oxidizing bacterium Nitrosomonas europaea was grown either (a) with added bicarbonate in the absence of added CO₂ (bubbled through the culture), (b) with added bicarbonate plus low added CO₂ (0.03% v/v), or (c) without added bicarbonate with high added CO₂ (1% v/v). Cell doubling times of 12 h were observed in 1% cultures; doubling times of 2 to 3-fold longer wre found with 0.03% CO₂ and/or bicarbonate grown cultures. The specific activity of carbonic anhydrase was 40–80% lower in cultures grown on 1% CO₂. These results are compared with those in heterotrophic and photosynthetic microorganisms.Scientific Contribution Number 1241 from the New Hampshire Agricultural Experiment Station     

Morphology and dynamics of protruding spirochete periplasmic flagella.

We recently characterized the three-dimensional shape of Treponema phagedenis periplasmic flagella (PFs). In the course of these studies, we observed protrusions on swimming cells that resembled PFs. Here we present a detailed characterization of the shape, structure, and motion of these protrusions. Although protrusion formation occurred primarily in wild-type cells during the stationary phase, a large fraction of exponential-phase cells of cell cylinder helicity mutants (greater than 90% of mutant T-52) had protrusions. These results suggest that cells bearing protrusions can still participate in cell division. T. phagedenis protrusions had the identical helix handedness, pitch, and diameter to those of purified PFs. Protrusions were not present on mutants unable to synthesize PFs, but were present in all motile revertants which regained PFs. These results, taken together with electron microscope observations, suggest that protrusions consist of PFs surrounded by an outer membrane sheath. To analyze protrusion movements, we held cells against a coverglass surface with optical tweezers and observed the motion of protrusions by video-enhanced differential interference contrast light microscopy. Protrusions were found to gyrate in both clockwise and counterclockwise directions, and direct evidence was obtained that protrusions rotate. Protrusions were also observed on Treponema denticola and Borrelia burgdorferi. These were also left-handed and had the same helix handedness, pitch, and diameter as purified PFs from their respective species. The PFs from T. denticola had a helix diameter of 0.26 microns and a helix pitch of 0.78 micron; PFs from B. burgdorferi had a helix diameter of 0.28 micron and a helix pitch of 1.48 microns. Protrusions from these spirochete species had similar structures and motion to those of T. phagedenis. Our results present direct evidence that PFs rotate and support previously proposed models of spirochete motility.