Treponema zioleckii sp. nov., a novel fructan-utilizing species of rumen treponemes.

During studies on fructan degradation in the rumen, a Treponema-like bacterium able to utilize Timothy grass fructan, commercial inulin and sucrose as the sole carbon source was recovered from sheep rumen. At least two different fructanolytic enzymes were identified in cell-free extracts of the isolated bacterium. Characterization of the strain by a polyphasic approach indicated that it can be regarded as a representative of a new bacterial species within the genus Treponema. Electron microscopy showed that the bacterium exhibited all of the features typical of spirochetes. The helical cells measured 5.4-11.5 microm x 0.42-0.51 microm and possessed up to seven regular coils. The bacterium utilized various plant mono- and disaccharides as fermentable substrates. Formate, acetate and ethanol in a molar ratio of 16 : 10 : 1 were the end products of glucose fermentation. The major cellular fatty acids were C(13:0), C(14:0), C(14:1), C(15:0), C(15:1) and C(16:0). The nearly complete 16S rRNA gene sequence was obtained, and phylogenetic analysis of the 16S rRNA gene showed the highest similarity to rumen Treponema strain CA. We propose the name Treponema zioleckii sp. nov. for this novel rumen spirochete with strain kT as the type strain.

Novel ultrastructures of Treponema primitia and their implications for motility.

Members of the bacterial phylum Spirochaetes are generally helical cells propelled by periplasmic flagella. The spirochete Treponema primitia is interesting because of its mutualistic role in the termite gut, where it is believed to cooperate with protozoa that break down cellulose and produce H(2) as a by-product. Here we report the ultrastructure of T. primitia as obtained by electron cryotomography of intact, frozen-hydrated cells. Several previously unrecognized external structures were revealed, including bowl-like objects decorating the outer membrane, arcades of hook-shaped proteins winding along the exterior and tufts of fibrils extending from the cell tips. Inside the periplasm, cone-like structures were found at each pole. Instead of the single peptidoglycan layer typical of other Gram-negative bacteria, two distinct periplasmic layers were observed. These layers formed a central open space that contained two flagella situated adjacent to each other. In some areas, the inner membrane formed flattened invaginations that protruded into the cytoplasm. High-speed light microscopic images of swimming T. primitia cells showed that cell bodies remained rigid and moved in a helical rather than planar motion. Together, these findings support the 'rolling cylinder' model for T. primitia motility that posits rotation of the protoplasmic cylinder within the outer sheath.

Electron cryotomography reveals novel structures of a recently cultured termite gut spirochete.

Electron cryotromography, a relatively new methodology in the field of microbiology, has been exploited by Murphy et al. (in this issue of Molecular Microbiology) in their analysis of the recently isolated termite gut spirochete Treponema primitia. Unique structures (bowls, arcades of hooks, cones at the cell ends, two layers of wall material) were evident from the analysis of its surface and internal constituents. These results, coupled to video microscopy analysis of swimming cells, allowed the authors to propose a model of cell motility. This highly significant paper highlights the importance of electron cryotomography to the field of microbiology. It also illustrates that newly cultured recalcitrant bacteria from complex environments are likely to possess novel structures not previously seen in other species.

In situ structure of the complete Treponema primitia flagellar motor.

The bacterial flagellar motor is an amazing nanomachine: built from approximately 25 different proteins, it uses an electrochemical ion gradient to drive rotation at speeds of up to 300 Hz (refs 1, 2). The flagellar motor consists of a fixed, membrane-embedded, torque-generating stator and a typically bidirectional, spinning rotor that changes direction in response to chemotactic signals. Most structural analyses so far have targeted the purified rotor, and hence little is known about the stator and its interactions. Here we show, using electron cryotomography of whole cells, the in situ structure of the complete flagellar motor from the spirochaete Treponema primitia at 7 nm resolution. Twenty individual motor particles were computationally extracted from the reconstructions, aligned and then averaged. The stator assembly, revealed for the first time, possessed 16-fold symmetry and was connected directly to the rotor, C ring and a novel P-ring-like structure. The unusually large size of the motor suggested mechanisms for increasing torque and supported models wherein critical interactions occur atop the C ring, where our data suggest that both the carboxy-terminal and middle domains of FliG are found.

Treponema putidum sp. nov., a medium-sized proteolytic spirochaete isolated from lesions of human periodontitis and acute necrotizing ulcerative gingivitis.

So far, little phenotypic heterogeneity has been detected in cultured oral treponemes with trypsin-like proteolytic activity, and all have been assigned to the species Treponema denticola. However, comparisons of protein patterns and antigen expression in our collection of proteolytic oral treponemes occasionally identified isolates with a unique phenotype; e.g. strain OMZ 830 (=ATCC 700768), which qualified as a 'pathogen-related oral spirochaete' due to the presence of a approximately 37 kDa protein reactive with the Treponema pallidum FlaA-specific mAb H9-2. In addition to such single isolates, a homogeneous group of seven independent strains is described that were highly motile, medium-sized, proteolytic but asaccharolytic spirochaetes and were cultured from human gingivitis, periodontitis and acute necrotizing ulcerative gingivitis in medium OMIZ-Pat supplemented with 1% human serum and antibiotics. Growth of these spirochaetes in OMIZ-Pat was not dependent on, but was stimulated by, human or bovine serum. Carbohydrates were neither required nor stimulatory for growth. The protein and antigen patterns of total cell extracts of these organisms separated by SDS-PAGE were distinct from those of all previously cultured spirochaetes, with highest similarity to T. denticola. The novel spirochaete has a 2 : 4 : 2 arrangement of the periplasmic flagella, similar to T. denticola. However, the flagellin pattern as detected by immunostaining or glycan staining of Western blots readily distinguished the novel group from T. denticola. Also, distinct from reference strains of T. denticola, none of the novel isolates displayed sialidase or dentilisin activities, both of which are expressed by most strains of T. denticola. Trypsin-like activity and other enzymes as detected by API ZYM test were similar to those of T. denticola. The status of a novel species is supported by the 16S rRNA gene sequence, with 98.5% similarity to its closest cultured relative, T. denticola. The name Treponema putidum sp. nov. is proposed (type strain OMZ 758T=ATCC 700334T=CIP 108088T).

Role of dentilisin in Treponema denticola epithelial cell layer penetration.

Treponema denticola is an oral anaerobic spirochete implicated in periodontal diseases. The chymotrypsin-like protease, dentilisin (PrtP), has been suggested to be an important virulence factor of T. denticola. In this study, we examined the role of dentilisin in T. denticola epithelial monolayer penetration by comparing the wild type and prtP mutant. Wild-type T. denticola can disrupt transepithelial resistance (TER) and substantially penetrate the HEp-2 cell layer. The prtP mutant altered the monolayer only slightly and penetrated the Hep-2 layer in very low numbers. The membrane fraction of wild-type T. denticola is able to complement the prtP mutant in monolayer penetration, while the comparable fraction from the mutant has no such effect. Immunofluorescence studies suggested that wild-type T. denticola altered the TER by likely degrading the tight junctional proteins such as ZO-1. Cytotoxicity was not a major factor in the disruption of TER. The outer membrane vesicles (OMVs) of wild-type T. denticola also disrupted epithelial barrier function and penetrated the epithelial layers. Taken together, these results suggest that T. denticola penetrates the epithelial cell monolayers by altering cellular tight junctions.

Insertional inactivation of the prtP gene of Treponema denticola confirms dentilisin's disruption of epithelial junctions.

The purified chymotrypsin-like protease of Treponema denticola, designated dentilisin or PrtP (DDBJ accession no. D83264), can disrupt cell-cell junctions and impair the barrier function of epithelial monolayers in vitro. Serine protease inhibitors block these effects. Yet, the protease is apparently less significant in perturbing intracellular signaling pathways and cytoskeletal rearrangement in fibroblasts. The purpose of this study was to use a PrtP-deficient mutant of T. denticola to confirm that the cytopathic effects of whole bacteria and its outer membrane on epithelial cell junctions were primarily accounted for by the activity of this protease. The prtP gene of ATCC 35405 was inactivated by insertion of an erythromycin-resistance cassette, yielding mutant K1. In contrast to wildtype ATCC 35405, mutant K1 grew in tight cell aggregates; the cells had a disrupted outer sheath, as determined by electron microscopy. When compared by silver stained SDS-PAGE of sonicated extracts of whole cells, the extract of mutant K1 was missing a band at approximately 90 kDa that was present in the wildtype ATCC 35405 strain. Whole cells and Triton X-100 outer membrane (OM) extracts of K1 and the wildtype strains were compared 1) for SAAPNA degrading activity by a colorimetric assay, 2) for stress fiber disruption in human gingival fibroblasts (HGF) by fluorescence microscopy of TRITC-phalloidin stained cells, and 3) the OM extracts only for perturbation of HEp-2 epithelial monolayers by electrical cell-substrate impedance sensing (ECIS). Mutant K-1 cells and OM had no SAPPNA degrading activity that is characteristic of dentilisin. K1 cells had HGF stress fiber disrupting activity (86 +/- 4.5% of HGFs affected) equivalent to both 35405 wildtype strains (84 +/- 3.9% and 71 +/- 14.1% of HGF, respectively). Yet, mutant K1 OM had diminished stress fiber disrupting activity (12.9 +/- 4.6% of HGF) compared with its parent 35405's OM (94.6 +/- 2.9%). The major cytopathogenic difference between the K1 mutant and wildtype strains was in their OM's effect on epithelial cell junctions. ATCC 35405 OM completely disrupted epithelial resistance in a concentration - dependent manner; mutant K1 OM had negligible effects. These data confirm that inactivation of the prtP gene completely reverses T. denticola's disruption of epithelial junctions, but there are pleiotropic effects of the mutation that may account for its apparently diminished effects on the cytoskeleton of HGF when the cells were challenged with OM extracts.

Relationship of Treponema denticola periplasmic flagella to irregular cell morphology.

Treponema denticola is an anaerobic, motile, oral spirochete associated with periodontal disease. We found that the periplasmic flagella (PFs), which are located between the outer membrane sheath and cell cylinder, influence its morphology in a unique manner. In addition, the protein composition of the PFs was found to be quite complex and similar to those of other spirochetes. Dark-field microscopy revealed that most wild-type cells had an irregular twisted morphology, with both planar and helical regions, and a minority of cells had a regular right-handed helical shape. High-voltage electron microscopy indicated that the PFs, especially in those regions of the cell which were planar, wrapped around the cell body axis in a right-handed sense. In those regions of the cell which were helical or irregular, the PFs tended to lie along the cell axis. The PFs caused the cell to form the irregular shape, as two nonmotile, PF-deficient mutants (JR1 and HL51) were no longer irregular but were right-handed helices. JR1 was isolated as a spontaneously occurring nonmotile mutant, and HL51 was isolated as a site-directed mutant in the flagellar hook gene flgE. Consistent with these results is the finding that wild-type cells with their outer membrane sheath removed were also right-handed helices similar in shape to JR1 and HL51. Purified PFs were analyzed by two-dimensional gel electrophoresis, and several protein species were identified. Western blot analysis using antisera to Treponema pallidum PF proteins along with N-terminal amino acid sequence analysis indicated T. denticola PFs are composed of one class A sheath protein of 38 kDa (FlaA) and three class B proteins of 35 kDa (FlaB1 and FlaB2) and one of 34 kDa (FlaB3). The N-terminal amino acid sequences of the FlaA and FlaB proteins of T. denticola were most similar to those of T. pallidum and Treponema phagedenis. Because these proteins were present in markedly reduced amounts or were absent in HL51, PF synthesis is likely to be regulated in a hierarchy similar to that found for flagellar. synthesis in other bacteria.

Treponema saccharophilum sp. nov., a large pectinolytic spirochete from the bovine rumen.

A large, obligately anaerobic spirochete (strain PB) was isolated from bovine rumen fluid by a procedure involving rifampin as a selective agent. The helical cells measured 0.6 to 0.7 micron by 12 to 20 micron and possessed approximately 16 periplasmic flagella inserted near each end of the protoplasmic cylinder. The periplasmic flagella were arranged in a bundle wound around the cell body. Strain PB utilized as fermentable substrates various plant polysaccharides (e.g., pectin, arabinogalactan, starch, and inulin) as well as pentoses, hexoses, disaccharides, and uronic acids. Glucose was fermented to acetate, formate, and ethanol, whereas the fermentation of pectin or glucuronic acid yielded only acetate and formate as major end products. Determinations of radioactivity in end products and assays of enzymatic activities indicated that strain PB catabolized glucose via the Embden-Meyerhof pathway. Extracts of cells grown in pectin-containing media possessed relatively high levels of phospho-2-keto-3-deoxygluconate aldolase activity, an enzymatic activity typical of the Entner-Doudoroff pathway. The guanine-plus-cytosine content of the DNA of strain PB (54 mol%) was considerably higher than that of known host-associated anaerobic spirochetes. This study indicates that strain PB represents a new species of Treponema, for which we propose the name Treponema saccharophilum.

Colonial morphology of treponemes observed by electron microscopy.

The colonial morphology of three strains of cultivable, nonpathogenic treponemes including a human oral treponeme was examined by light and electron microscopy. Treponema phagedenis strains Kazan and Reiter produced large white colonies on the surface of solid media composed of sterility test broth, 0.9 to 3.1% agar, rifampin, and 12.5% rabbit or horse serum. A human oral treponeme, strain G7201 , grew as diffused white zones on 0.9 to 3.1% agar plates. Under the cultural conditions employed agar concentrations slightly affected the time of appearance of colonies of the three strains of treponemes. When the colonies of these three strains were viewed by scanning electron microscopy, differences in their colonial morphology were observed. The 11-day-old colonies of human oral strain G7201 were very small, 5 to 15 micron in diameter, and had a slight irregular border. Kazan treponemes developed circular, entire and low convex colonies. Scanning and transmission electron microscopy revealed that the colonies of Reiter treponemes contained spherical forms almost up to 5 micron in diameter, each consisting of an outer membrane and a treponemal main body. They were very similar to the spherical bodies produced by strain G7201 in sucrose-containing broth.

Helical conformation of Treponema pallidum (Nichols strain), Treponema paraluis-cuniculi, Treponema denticola, Borrelia turicatae, and unidentified oral spirochetes.

Borrelia turicatae (mouse virulent) and Treponema denticola, a small oral treponeme, formed right-handed helices as determined by scanning electron microscopy. Treponema pallidum (Nichols strain), Treponema paraluis-cuniculi, and two unidentified oral spirochetes displayed left-handed helices.

Isolation and antigenic characteristics of axial filaments from the Reiter Treponeme.

Axial filaments were isolated and purified from Reiter treponemes after detergent solubilization of the cells" outer envelope. The axial filaments were separated from the spirochetal cells by shearing, purified by density gradient centrifugation, and fragmented by ultrasonication. Acrylamide gel electrophoresis of dissociated filaments revealed two major protein bands. Gel diffusion precipitin tests and immunoelectrophoresis between a purified axial filament suspension and anti-Reiter treponeme serum gave a single precipitin line. Checkerboard complement fixation tests also gave results consistent with a single antigen-antibody system. Tests with immune sera to other cultivable spirochetes were positive with some and negative with others. In addition, strongly positive reactions were obtained in complement fixation and precipitin tests with sera from rabbits and humans with syphilis and other treponematoses. However, both serological tests gave reactions of partial identity between the antigen(s) of Reiter treponeme axial filaments and those of the pathogenic treponemes. It was concluded from these studies that the axial filaments were probably the cellular locus of the so-called "Reiter protein" antigen of syphilis serology.

Treponeme outer cell envelope: solubilization and reaggregation.

Exposure of the avirulent Kazan 5 treponeme to low concentrations of sodium dodecyl sulfate (1.4 mm) results in the solubilization of the outer envelope. Maximal reaggregation of the outer envelope preparation requires cations, and cation concentration affects both the yield and structure of the aggregates. The reaggregated outer envelope preparation can be resolubilized with the chelating agent, ethylenediaminetetraacetic acid, or with sodium dodecyl sulfate.