Characterization of the cell surface glycolipid from Spirochaeta aurantia.

Spirochaeta aurantia is a free-living saprophytic spirochete that grows easily in simple laboratory media, and thus can be used as a model for the investigation of surface carbohydrate structures in spirochetae, which are normally not available in sufficient amounts. Freeze-substitution electron microscopy indicated the presence of a capsule-like material projecting from the surface of S. aurantia. Extraction of cells gave two major glycolipids, the one with a higher molecular mass glycolipid was designated large glycolipid A (LGLA). LGLA contained small amount of branched and unsaturated O-linked fatty acids, L: -rhamnose, L: -fucose, D: -xylose, D: -mannose, D: -glucosamine, D: -glycero-D: -gluco-heptose (DDglcHep), D: -glycero-D: -manno-heptose (DDHep), and a novel branched tetradeoxydecose monosaccharide, which we proposed to call aurantose (Aur). The carbohydrate structure of LGLA was extremely complex and consisted of the repeating units built of 11 monosaccharides, arrangement of nine of them was determined as: - [- 3 - beta - DDglcHep - 3 - beta - D - GlcNAc - 2 - beta - D - Man - ] - which wasdeduced from the NMR and chemical data on the LGLA and its fragments, obtained by various degradations. Tentative position of two remaining sugars is proposed. LGLA was negative for gelation of Limulus amebocyte lysate, did not contain lipid A, and was unable to activate any known Toll-like receptors.

Spirochaeta cellobiosiphila sp. nov., a facultatively anaerobic, marine spirochaete.

A facultatively anaerobic, marine spirochaete, designated strain SIP1(T), was isolated from interstitial water from a cyanobacteria-containing microbial mat. Cells of strain SIP1(T) were 0.3-0.4x10-12 mum in size, helical with a body pitch of approximately 1.4 mum and motile by means of two to four periplasmic flagella (one, or occasionally two, being inserted near each end of the cell). Cells were catalase-negative and used a variety of monosaccharides and disaccharides and pectin as energy sources, growing especially well on cellobiose. Neither organic acids nor amino acids were utilized as energy sources. One or more amino acids in tryptone and one or more components of yeast extract were required for growth. Growth was observed at 9-37 degrees C (optimally at or near 37 degrees C), at initial pH 5-8 (optimally at initial pH 7.5) and in media prepared with 20-100 % (v/v) seawater (optimally at 60-80 %) or 0.10-1.00 M NaCl (optimally at 0.30-0.40 M). The products of cellobiose fermentation were acetate, ethanol, CO(2), H(2) and small amounts of formate. Aerated cultures performed incomplete oxidation of cellobiose to acetate (and, presumably, CO(2)) plus small amounts of ethanol and formate, but exhibited a Y(cellobiose) that was only slightly greater than that of cellobiose-fermenting anoxic cultures. The G+C content of the genomic DNA of strain SIP1(T) was 41.4 mol%, the lowest among known spirochaetas. On the basis of its 16S rRNA gene sequence, strain SIP1(T) was grouped among other members of the genus Spirochaeta, but it bore only 89 % similarity with respect to its closest known relatives, Spirochaeta litoralis and Spirochaeta isovalerica, two marine obligate anaerobes. On the basis of its phenotypic properties and phylogenetic position, strain SIP1(T) represents a novel species of the genus Spirochaeta, for which the name Spirochaeta cellobiosiphila sp. nov. is proposed. The type strain is SIP1(T) (=ATCC BAA-1285(T) =DSM 17781(T)).

Phylogenetic diversity and temporal variation in the Spirochaeta populations from two Mediterranean microbial mats

Spirochetes are among the bacterial groups often observed in hydrogen-sulfide-rich layers of coastal microbial mats. However, relatively few spirochetes from these microbial mats have been described and characterized. We used 16S rDNA phylogenetic analysis to investigate the spirochetal diversity of microbial mats from two locations in the western Mediterranean (Ebro Delta, Spain, and Camargue, France). Samples from each location were monitored in the spring and winter over a period of 1 to 2 years. In the sequence analysis of 332 clones derived from samples of both locations, 42 novel phylotypes of not-yet-cultivated spirochetes belonging to the genus Spirochaeta were detected. None of the phylotypes were identified as known culturable species of Spirochaeta or previously identified phylotypes cloned from other hypersaline microbial mat such as Guerrero Negro, Mexico. Eight of the phylotypes were common to Ebro and Camargue mats, and two (IF058 and LL066) were present both in spring and winter. Some phylotypes appeared to show seasonal variation, i.e., they were found only in the spring, but not in the winter. Ebro and Camargue phylotypes, like phylotypes from Guerrero Negro, grouped according to the vertical gradient of oxygen and sulfide in the mat. Some phylotypes, such as LH073, IE028, LH042, or LG013 were harbored in low H2S or H2S-O2 interface zone. In contrast, major phylotypes were detected in deeper layers and they were likely strict anaerobes and high tolerant to H2S. The presence of spirochetes in differently located microbial mats suggests that they constitute very diverse and stable populations involved in a well-integrated metabolic symbiosis (i.e., permanent physiological cooperation) with other guild populations in the mats, where they maintain a coordinated functional and stable community (AU)

No disponible

Enigmatic dual symbiosis in the excretory organ of Nautilus macromphalus (Cephalopoda: Nautiloidea).

Symbiosis is an important driving force in metazoan evolution and the study of ancient lineages can provide an insight into the influence of symbiotic associations on morphological and physiological adaptations. In the 'living fossil' Nautilus, bacterial associations are found in the highly specialized pericardial appendage. This organ is responsible for most of the excretory processes (ultrafiltration, reabsorption and secretion) and secretes an acidic ammonia-rich excretory fluid. In this study, we show that Nautilus macromphalus pericardial appendages harbour a high density of a beta-proteobacterium and a coccoid spirochaete using transmission electron microscopy, comparative 16S rRNA sequence analysis and fluorescence in situ hybridization (FISH). These two bacterial phylotypes are phylogenetically distant from any known bacteria, with ammonia-oxidizing bacteria as the closest relatives of the beta-proteobacterium (above or equal to 87.5% sequence similarity) and marine Spirochaeta species as the closest relatives of the spirochaete (above or equal to 89.8% sequence similarity), and appear to be specific to Nautilus. FISH analyses showed that the symbionts occur in the baso-medial region of the pericardial villi where ultrafiltration and reabsorption processes take place, suggesting a symbiotic contribution to the excretory metabolism.

The periplasmic flagellum of spirochetes.

Although spirochete periplasmic flagella have many features similar to typical bacterial flagella, they are unique in their structure and internal periplasmic location. This location provides advantages for pathogenic spirochetes to enter and to adapt in the appropriate host, and to penetrate through matrices that inhibit the motility of most other bacteria. These flagella are complex, and they dynamically interact with the spirochete cell cylinder in novel ways. Electron microscopy, tomography and three-dimensional reconstructions have provided new insights into flagellar structure and its relationship to the spirochetal cell cylinder. Recent advances in genetic methods have begun to shed light on the composition of the spirochete flagellum, and on the regulation of its synthesis. Because spirochetes have a high length to width ratio, their cells provide an opportunity to study two important features. These include the polarity or distribution of flagellar synthesis as well as the mechanisms required for coordination of the movement of the cell ends, to enable it to move in the forward or reverse direction.

Appendiceal spirochetosis.

Appendiceal spirochetosis is rarely described in the literature. We assessed the incidence of spirochetosis in a series of 109 appendectomies (76 adult and 33 pediatric cases) done for either suspected acute appendicitis or as a concurrent procedure. Appendiceal spirochetosis was identified on hematoxylin-eosin sections and confirmed by Steiner and Steiner stain and ultrastructural study in two women and two men. One of these four patients was found to have HIV infection. The HIV status of the other three patients was unknown. We observed appendiceal spirochetosis to be an uncommon phenomenon that affects both sexes, occurring with or without symptoms of acute appendicitis. None of our four positive cases occurred in the pediatric age group.

Spirochaeta alkalica sp. nov., Spirochaeta africana sp. nov., and Spirochaeta asiatica sp. nov., alkaliphilic anaerobes from the Continental Soda Lakes in Central Asia and the East African Rift.

During a study of microbial communities in athalassic bodies of water, three new species within the genus Spirochaeta were described. These are alkaliphilic Spirochaeta alkalica sp. nov. Z-7491 (DSM 8900) and halophilic S. africana sp. nov. Z-7692 (DSM 8902) from the soda-depositing Lake Magadi in Central Africa and haloalkaliphilic S. asiatica sp. nov. Z-7591 (DSM 8901) from Lake Khatyn, Central Asia. These mesophilic spirochetes develop at pHs of > 9 as anaerobic saccharolytic dissipotrophs. The DNA base compositions (moles percent G+C) of the strains were as follows: S. alkalica Z-7491, 57.1; S. africana Z-7692, 56.1; and S. asiatica Z-7591, 49.2. The optimum growth parameters (temperature, pH, and NaCl concentration [percent, wt/vol], respectively) were as follows: for S. alkalica Z-7491, 35 degrees C, 9.2, and 5 to 7%; for S. africana Z-7692, 35 degrees C, 9.3, and 5 to 7%; and for S. asiatica Z-7591, 35 degrees C, 8.9, and 3 to 6%. The products of glucose fermentation were acetate, hydrogen, ethanol, and lactate, in different proportions, for S. alkalica and S. africana; for S. asiatica, they were acetate, ethanol, and lactate. S. asiatica is strictly anaerobic, while S. alkalica and S. africana are rather aerotolerant. All three species group within the radiation of the majority of the species of the genus Spirochaeta. Studies of the genes encoding 16S rRNA indicate a possible fanning out of the phylogenetic tree of spirochetes.

A monoclonal antibody reacting with the cell envelope of spirochaetes isolated from cases of intestinal spirochaetosis in pigs and humans.

A monoclonal antibody (mAb) designed BJL/AC1 was prepared against the cell envelope of an intestinal spirochaete (strain 3295) that was isolated from a pig with intestinal spirochaetosis. The mAb reacted with a band of approximately 29 kDa in cell envelope preparations from 13 porcine and 11 human spirochaetes isolated from cases of intestinal spirochaetosis, but did not react with preparations made from a range of other intestinal spirochaetes. Immunogold labelling demonstrated that the reactive epitope was located on the cell envelope of the strains causing intestinal spirochaetosis. The mAb was used in an indirect immunofluorescence test to detect spirochaetes in the faeces of pigs with experimentally induced intestinal spirochaetosis. The mAb should prove to be a useful reagent for detection and identification of spirochaetes that are specifically associated with intestinal spirochaetosis.

[Relationship between Melkersson-Rosenthal syndrome and spirochetes infection].

By using Warthin-Starry spirochete special stain method and a transmission electron microscope for the first time, we detected spirochetes separately in lesions of 23 cases of MRS and 5 cases of MRS. Their shapes and distributive places were described. 11 cases were treated by high dose of penicillin, and 10 were responsive. This result provided further basis for a conjecture that the attack of MRS may be related to the infection of spirochetes.

N-terminal amino acid sequences and amino acid compositions of the Spirochaeta aurantia flagellar filament polypeptides.

The amino-terminal sequences and amino acid compositions of the three major and two minor polypeptides constituting the filaments of Spirochaeta aurantia periplasmic flagella were determined. The amino-terminal sequence of the major 37.5-kDa outer layer polypeptide is identical to the sequence downstream of the proposed signal peptide of the protein encoded by the S. aurantia flaA gene. However, the amino acid composition of the 37.5-kDa polypeptide is not in agreement with that inferred from the sequence of flaA. The 34- and 31.5-kDa major filament core polypeptides and the 33- and 32-kDa minor core polypeptides show a striking similarity to each other, and the amino-terminal sequences of these core polypeptides show extensive identity with homologous proteins from members of other genera of spirochetes. An additional 36-kDa minor polypeptide that occurs occasionally in preparations of S. aurantia periplasmic flagella appears to be mixed with the 37.5-kDa outer layer polypeptide or a degradation product of this polypeptide.

Biochemical and cytological analysis of the complex periplasmic flagella from Spirochaeta aurantia.

The periplasmic flagella of Spirochaeta aurantia were isolated and were found to be ultrastructurally and biochemically complex. Generally, flagellar filaments were 18 to 20 nm in diameter and appeared to consist of an 11 to 13-nm-wide inner region and an outer layer. The hook-basal body region consisted of two closely apposed disks connected to a hook by a rod. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified flagella together with a Western blot analysis of a motility mutant that produces hooks and basal bodies but not flagellar filaments revealed that the filaments were composed of three major polypeptides of 37,500, 34,000, and 31,500 apparent molecular weight (37.5K, 34K, and 31.5K polypeptides) and three minor polypeptides of 36,000, 33,000, and 32,000 apparent molecular weight (36K, 33K, and 32K polypeptides). Purified hook-basal body preparations were greatly enriched in three polypeptides in the range of 62,000 to 66,000 apparent molecular weight. Immunogold labeling experiments with a monoclonal antibody specific for the 37.5K flagellin and one that reacts with an epitope common to the 36K, 34K, 33K, 32K, and 31.5K flagellins revealed that the 37.5K major polypeptide was a component of the outer layer, whereas one or more of the other polypeptides constituted the core.

Tubulinlike protein from Spirochaeta bajacaliforniensis.

Tubulin proteins are the fundamental subunits of all polymeric microtubule-based eukaryotic structures. Long, hollow structures each composed of 13 protofilaments as revealed by electron microscopy, microtubules (240 angstroms in diameter) are nearly ubiquitous in eukaryotes. These proteins have been the subject of intense biochemical and biophyiscal interest since the early 1970s and are of evolutionary interest as well. If tubulin-based structures (i.e., neurotubules, mitotic spindle tubules, centrioles, kinetosomes, axonemes, etc.) evolved from spirochetes by way of motility symbioses, tubulin homologies with spirochete proteins should be detectable. Tubulin proteins are widely thought to be limited to eukaryotes. Yet both azotobacters and spirochetes have shown immunological cross-reactivity with antitubulin antibodies. In neither of these studies was tubulin isolated nor any specific antigen identified as responsible for the immunoreactivity. Furthermore, although far less uniform in structure than eukaryotic microtubules, various cytoplasmic fibers and tubules (as seen by electron microscopy) have been reported in several types of prokaryotes (e.g., Spirochaeta; large termite spirochetes; treponemes; cyanobacteria; and Azotobacter. This work forms a part of our long-range study of the possible prokaryotic origin of tubulin and microtubules. Spirochetes are helically shaped gram-negative motile prokaryotes. They differ from all other bacterial in that the position of their flagella is periplasmic: their flagella lie between the inner and outer membranes of the gram-negative cell wall. Some of the largest spirochetes have longitudinally aligned 240 angstrom microtubules. Unfortunately, in spite of many attempts, all of the larger spirochetes (family Pillotaceae) with well-defined cytoplasmic tubules and antitubulin immunoreactivity are not cultivable. However, a newly described spirochete species (Spirochaeta bajacaliforniensis) possessing cytoplasmic fibers displays antitubulin immunoreactivity in whole-cell preparations. Since preliminary observations suggested that Spirochaeta bajacaliforniensis proteins may be related to eukaryotic tubulins, their characterization was undertaken. Brain tubulin can be purified by utilizing its ability to polymerize at warm temperatures and to depolymerize in the cold. After several cycles of sedimentation and redissolution the microtubule fraction is comprised of 75% tubulin and 20% high molecular mass microtubule-associated proteins (MAPs). In this paper we report that components of cell lysates, prepared from a spirochete that contains cytoplasmic fibers (Spirochaeta bajacaliforniensis), also exhibit the property of temperature-dependent cyclical sedimentation. Additionally we report the identification and characterization of the polypeptide responsible for cross-reactivity with antitubulin antiserum.

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.

Congenital spirochetosis in calves: association with epizootic bovine abortion.

Congenital spirochetosis was encountered as a newly recognized infection of cattle. The spirochete was seen in blood of fetuses with lesions of epizootic bovine abortion. A spirochete with morphologic features similar to those found in the fetuses was detected in Ornithodoros coriaceus ticks. Ticks collected from rangelands were allowed to feed on cows that then produced epizootic bovine abortion-affected fetuses, and the fetuses had spirochetosis. Inapparent spirochetosis also was found in fetuses in clinically normal cattle sent to slaughter. Only a few lesions were seen in abattoir-collected fetuses. Fetal spirochetosis was common in the bovine population studied, and it appeared that infection may be limited only by the availability of the tick vector.

Spirochaetes in the equine caecum.

Two morphological types of spirochaete were found in the horse caecum measuring 4 to 6 micron by 0.3 to 0.4 micron and 6 to 8 micron by 0.1 to 0.2 micron. Attempts were made to culture the organisms but none survived subculture beyond the primary isolate. Electron microscopy revealed that many of the organisms were infected by bacteriophages.

Spirochaeta bajacaliforniensis sp. n. from a microbial mat community at Laguna Figueroa, Baja California Norte, Mexico.

A new anaerobic spirochete was isolated from anaerobic muds beneath the laminated sediment in the evaporite flat at Laguna Figueroa, Baja California Norte, Mexico. The organism is a member of the stratified microbial community involved in the deposition of the laminated sediments in the lagoon. The size of the spirochete is 0.3 by 30 micrometers, with a wave amplitude of 0.5 micrometer and a wavelength of 1.25 micrometers. The periplasmic flagella have a 1-2-1 arrangement. The outer membrane of the modified Gram-negative cell wall (the sheath) is irregularly crenulated and has a sillon. The growth medium contained yeast extract, trypticase, cellobiose, sodium thioglycolate and at least 20% natural seawater. Chemically defined artificial seawater media did not support growth. Optimal growth occurred with a seawater concentration of 80% at 36 degrees C and a pH of 7.5. Glucose was fermented to acetate, ethanol, carbon dioxide and hydrogen. The guanine + cytosine content of the DNA was 50 mol %. The spirochete body reacts positively to antibodies raised against eukaryotic brain tubulin protein. On the basis of its free-living anaerobic habitat, its unique morphological and physiological characteristics and G+C ratio, it is proposed that this isolated be considered a new species and names Spirochaeta bajacaliforniensis.

Physiological diversity of rumen spirochetes.

Bovine rumen fluid contained relatively large numbers of spirochetes capable of fermenting polymers commonly present in plant materials. Polymers such as xylan, pectin, and arabinogalactan served as fermentable substrates for the spirochetes, whereas cellulose did not. Furthermore, spirochetes cultured from rumen fluid utilized as growth substrates hydrolysis products of plant polymers (e.g., D-xylose, L-arabinose, D-galacturonic acid, D-glucuronic acid, cellobiose), but did not ferment amino acids. Viable cell counts of spirochetes capable of fermenting individual plant polymers or their hydrolysis products yielded minimum values ranging from 0.2 X 10(6) to 4 X 10(6) cells per ml of rumen fluid. Thirteen strains of rumen spirochetes were characterized in terms of their fermentation products from glucose, the guanine plus cytosine content of their DNA, their ultrastructure, and their ability to ferment pectin, starch, or arabinogalactan. Of the 13 strains, 6 fermented glucose mainly to formate, acetate, and succinate, whereas the remaining 7 strains did not produce succinate, but instead formed ethanol, in addition to formate and acetate. The succinate-forming strains had two periplasmic (axial) fibrils per cell, measured 0.2 to 0.3 by 5 to 8 micrograms, had a guanine plus cytosine content of the DNA ranging from 36 to 38 mol%, and lacked the ability to ferment pectin, starch, or arabinogalactan. The ethanol-forming strains had from 8 to more than 32 periplasmic fibrils per cell, tended to be larger in cell size than the succinate-forming strains, and had a guanine plus cytosine content of the DNA ranging from 41 to 54 mol%. Some of the ethanol-forming strains fermented pectin, starch, or arabinogalactan. The results of this study indicate that the bovine rumen is inhabited by a physiologically and morphologically diverse population of spirochetes. It is likely that these spirochetes contribute significantly to the degradation of plant materials ingested by the ruminants.