Habitat fragmentation is associated to gut microbiota diversity of an endangered primate: implications for conservation.

The expansion of agriculture is shrinking pristine forest areas worldwide, jeopardizing the persistence of their wild inhabitants. The Udzungwa red colobus monkey (Procolobus gordonorum) is among the most threatened primate species in Africa. Primarily arboreal and highly sensitive to hunting and habitat destruction, they provide a critical model to understanding whether anthropogenic disturbance impacts gut microbiota diversity. We sampled seven social groups inhabiting two forests (disturbed vs. undisturbed) in the Udzungwa Mountains of Tanzania. While Ruminococcaceae and Lachnospiraceae dominated in all individuals, reflecting their role in extracting energy from folivorous diets, analysis of genus composition showed a marked diversification across habitats, with gut microbiota α-diversity significantly higher in the undisturbed forest. Functional analysis suggests that such variation may be associated with food plant diversity in natural versus human-modified habitats, requiring metabolic pathways to digest xenobiotics. Thus, the effects of changes in gut microbiota should not be ignored to conserve endangered populations.

Spirochaeta dissipatitropha sp. nov., an alkaliphilic, obligately anaerobic bacterium, and emended description of the genus Spirochaeta Ehrenberg 1835.

A novel obligately anaerobic, mesophilic, alkaliphilic spirochaete, strain ASpC2(T), was isolated from an anaerobic sediment of alkaline, hypersaline Owens Lake in California, USA. The Gram-negative cells are motile, helical in shape and 0.23 x 8.0-18.0 mum. Growth occurs within the following ranges: 13-41 degrees C, with optimal growth at 35 degrees C; 1-3 % (w/v) NaCl, with optimal growth at 2 % (w/v) NaCl; and pH 7.8-10.5, with optimal growth at pH 10.0. The novel isolate is strictly alkaliphilic and requires high concentrations of carbonate ions in the medium. It utilizes some sugars, some organic acids, some amino acids, Casamino acids, yeast extract and peptone. The main end products of glucose fermentation are CO(2) and acetate. Strain ASpC2(T) is resistant to kanamycin and rifampicin, but sensitive to ampicillin, chloramphenicol, gentamicin and tetracycline. The DNA G+C content of the new isolate is 43.8 mol%, its genome size is 6 x 10(8) Da and the melting temperature of its genomic DNA is 71 degrees C. DNA-DNA hybridization experiments demonstrated 46 % similarity with the phylogenetically most closely related species, Spirochaeta asiatica Z-7591(T). On the basis of physiological and molecular properties, the new isolate belongs taxonomically to a novel species within the genus Spirochaeta, for which the name Spirochaeta dissipatitropha sp. nov. is proposed (type strain, ASpC2(T)=ATCC BAA-1083(T)=JCM 12856(T)). S. dissipatitropha ASpC2(T) is the second strain in the genus (after Spirochaeta smaragdinae SEBR 4228(T)) that is able to use proteolysis products as the sole energy source, and additional tests have shown that other halo-alkaliphilic spirochaetes (Spirochaeta americana, Spirochaeta alkalica and Spirochaeta africana) are also able to grow on yeast extract alone; therefore, an emended description for the genus Spirochaeta is given.

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)).

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.

Spirochaeta coccoides sp. nov., a novel coccoid spirochete from the hindgut of the termite Neotermes castaneus.

A novel spirochete strain, SPN1, was isolated from the hindgut contents of the termite Neotermes castaneus. The highest similarities (about 90%) of the strain SPN1 16S rRNA gene sequence are with spirochetes belonging to the genus Spirochaeta, and thus, the isolate could not be assigned to the so-called termite clusters of the treponemes or to a known species of the genus Spirochaeta. Therefore, it represents a novel species, which was named Spirochaeta coccoides. In contrast to all other known validly described spirochete species, strain SPN1 shows a coccoid morphology and is immotile. The isolated strain is obligately anaerobic and ferments different mono-, di-, and oligosaccharides by forming formate, acetate, and ethanol as the main fermentation end products. Furthermore, strain SPN1 is able to grow anaerobically with yeast extract as the sole carbon and energy source. The fastest growth was obtained at 30 degrees C, the temperature at which the termites were also grown. The cells possess different enzymatic activities that are involved in the degradation of lignocellulose in the termite hindgut, such as beta-D-glucosidase, alpha-L-arabinosidase, and beta-D-xylosidase. Therefore, they may play an important role in the digestion of breakdown products from cellulose and hemicellulose in the termite gut.

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.

[The role of oxygen in the regulation of the metabolism of aerotolerant spirochetes, a major component of "Thiodendron" bacterial sulfur mats]. / Rol' kisloroda v reguliatsii metabolizma aérotolerantnykh spirokhet--osnovnogo komponenta bakterial'nykh sernykh matov "Thiodendron".

Two spirochete strains isolated earlier from "Thiodendron" bacterial sulfur mats grew better under microaerobic (0.3-0.5 mg O2/l) than under anaerobic conditions. The microaerobic growth of these strains was accompanied by a twofold increase in the cell yield and the efficiency of glucose utilization, despite an amount of ATP (and hence glucose) was spent in this case for the synthesis of exopolysaccharides. Glucose metabolism under microaerobic conditions gave rise to more oxidized products (acetate and carbon dioxide) than under anaerobic conditions (formate, ethanol, pyruvate, and hydrogen). The paper considers two putative mechanisms implemented by aerotolerant spirochetes: adaptive (the use of a more efficient pathway of glucose catabolism) and protective (an enhanced synthesis of exopolysaccharides and the reduction of hydrogen peroxide by the reduced sulfur compounds thiosulfate and sulfide, yielding elemental sulfur). The formation of "Thiodendron" bacterial sulfur mats in saltwater environments is also discussed.

Characterization of a novel spirochete associated with the hydrothermal vent polychaete annelid, Alvinella pompejana.

A highly integrated, morphologically diverse bacterial community is associated with the dorsal surface of Alvinella pompejana, a polychaetous annelid that inhabits active high-temperature deep-sea hydrothermal vent sites along the East Pacific Rise (EPR). Analysis of a previously prepared bacterial 16S ribosomal DNA (rDNA) library identified a spirochete most closely related to an endosymbiont of the oligochete Olavius loisae. This spirochete phylotype (spirochete A) comprised only 2.2% of the 16S rDNA clone library but appeared to be much more dominant when the same sample was analyzed by denaturing gradient gel electrophoresis (DGGE) and the terminal restriction fragment length polymorphism procedure (12 to 18%). PCR amplification of the community with spirochete-specific primers used in conjunction with DGGE analysis identified two spirochete phylotypes. The first spirochete was identical to spirochete A but was present in only one A. pompejana specimen. The second spirochete (spirochete B) was 84.5% similar to spirochete A and, more interestingly, was present in the epibiont communities of all of the A. pompejana specimens sampled throughout the geographic range of the worm (13 degrees N to 32 degrees S along the EPR). The sequence variation of the spirochete B phylotype was less than 3% for the range of A. pompejana specimens tested, suggesting that a single spirochete species was present in the A. pompejana epibiotic community. Additional analysis of the environments surrounding the worm revealed that spirochetes are a ubiquitous component of high-temperature vents and may play an important role in this unique ecosystem.

Spirochete periplasmic flagella and motility.

Spirochetes have a unique structure, and as a result their motility is different from that of other bacteria. They also have a special attribute: spirochetes can swim in a highly viscous, gel-like medium, such as that found in connective tissue, that inhibits the motility of most other bacteria. In spirochetes, the organelles for motility, the periplasmic flagella, reside inside the cell within the periplasmic space. A given periplasmic flagellum is attached only at one end of the cell, and depending on the species, may or may not overlap in the center of the cell with those attached at the other end. The number of periplasmic flagella varies from species to species. These structures have been shown to be directly involved in spirochete motility, and they function by rotating within the periplasmic space. The mechanics of motility also vary among the spirochetes. In Leptospira, a motility model developed several years ago has been extensively tested, and the evidence supporting this model is convincing. Borrelia burgdorferi swims differently, and a model of its motility has been recently put forward. This model is based on analyzing the motion of swimming cells, high voltage electron microscopy of fixed cells, and mutant analysis. To better understand spirochete motility on a more molecular level, the proteins and genes involved in motility are being analyzed. Spirochete periplasmic flagellar filaments are among the most complex of bacterial flagella. They are composed of the FlaA sheath proteins, and in many species, multiple FlaB core proteins. Allelic exchange mutagenesis of the genes which encode these proteins is beginning to yield important information with respect to periplasmic flagellar structure and function. Although we are at an early stage with respect to analyzing the function, organization, and regulation of many of the genes involved in spirochete motility, unique aspects have already become evident. Future studies on spirochete motility should be exciting, as only recently have complete genome sequences and tools for allelic exchange mutagenesis become available.

Spirochaeta smaragdinae sp. nov., a new mesophilic strictly anaerobic spirochete from an oil field.

An obligately anaerobic spirochete designated strain SEBR 4228T (T = type strain) was isolated from an oil field of Congo, Central Africa. The strain grew optimally with a sodium chloride concentration of 5% (sodium chloride concentration) growth range 1.0-10%) at 37 degrees C (growth temperature range 20-40 degrees C) and pH of 7.0-7.2 (pH growth range pH 5.5-8.0). Strain SEBR 4228T grew on carbohydrates (glucose, fructose, ribose, D-xylose, galactose, mannitol and mannose), glycerol, fumarate, peptides and yeast extract. Yeast extract was required for growth and could not be replaced by vitamins. It reduced thiosulfate and sulfur, to H2S. Glucose was oxidised to lactate, acetate, CO2 and H2S in the presence of thiosulfate but in its absence lactate, ethanol, CO2 and H2 were produced. Fumarate was fermented to acetate and succinate. The G + C content of strain SEBR 4228T was 50%. Strain SEBR 4228T was spiral shaped measuring 5-30 by 0.3-0.5 micron and was motile with a corkscrew-like motion. Electron microscopy revealed the presence of periplasmic flagella in a 1-2-1 arrangement. Strain SEBR 4228T possessed features typical of the members of the genus Spirochaeta. 16S rRNA sequence analysis revealed that it was closely related to Spirochaeta bajacaliforniensis (similarity 98.6%). The lack of DNA homology with S. bajacaliforniensis (38%), together with other phenotypic differences, indicated that strain SEBR 4228T is a new species, which we have designated Spirochaeta smaragdinae. The type strain is SEBR 4228T (= DSM 11293).

Spirochete chemotaxis, motility, and the structure of the spirochetal periplasmic flagella.

Spirochetes have a unique motility system that is characterized by flagellar filaments contained within the outer membrane sheath. Direct evidence using video microscopy has recently been obtained which indicates that these periplasmic flagella (PF) rotate in several spirochetal species. This rotation generates thrust. As shown for one spirochete, Spirochaeta aurantia, motility is driven by a proton motive force. Spirochete chemotaxis has been most thoroughly studied in S. aurantia. This spirochete exhibits three distinct behaviours, runs of smooth swimming, reversals and flexing. These behaviours are modulated by addition of attractants such that S. aurantia swims towards higher concentrations of attractants in a spatial gradient. Unlike the prototypical bacterium, Escherichia coli, chemotaxis in S. aurantia involves fluctuations in membrane potential. The PF of a number of spirochetes have been examined in considerable detail. For most species, the PF filaments are complex, consisting of an assembly of several different polypeptides. There are several antigenically related core polypeptides surrounded by an outer layer consisting of a different polypeptide. Borrelia burgdorferi and Spirochaeta zuelzerae represent exceptions where the filaments are composed of a single major polypeptide species. The genes encoding the filament polypeptides from several spirochete species have been cloned and analysed. Apparently, the outer layer polypeptides of S. aurantia, Treponema pallidum and Serpulina hyodysenteriae are transcribed from sigma-70-like promoters, whereas the core polypeptide genes are transcribed from sigma-28-like promoters. A gene encoding the hook polypeptide in Treponema phagedenis has been cloned and analysed. The product of this gene shows significant similarity to the E. coli hook protein, FlgE, and homologs have been identified in T. pallidum and B. burgdorferi.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemotaxis mutants of Spirochaeta aurantia.

Five Spirochaeta aurantia chemotaxis mutants were isolated. One mutant (the che-101 mutant) never reversed, one (the che-200 mutant) flexed predominantly, two (the che-300 and che-400-1 mutants) exhibited elevated reversal frequencies, and one (the che-400 mutant) exhibited chemotactically unstimulated behavior similar to that of the wild-type strain. The che-101 and che-400 mutants were essentially nonchemotactic, whereas the che-200, che-300, and che-400-1 mutants showed impaired chemotactic responses. Protein methylation in response to attractant addition appeared normal in all of the mutants. Compared with the wild type, all of the mutants exhibited significantly altered membrane potential responses to the attractant xylose.

Motility and chemotaxis of Spirochaeta aurantia: computer-assisted motion analysis.

A computer program has been designed to study behavior in populations of Spirochaeta aurantia cells, and this program has been used to analyze changes in behavior in response to chemoattractants. Three kinds of behavior were distinguished: smooth swimming, flexing, and reversals in direction of swimming after a short pause (120 ms). Cell populations exposed to chemoattractants spent, on average, 66, 33, and 1% of the time in these modes, respectively. After the addition of a chemoattractant, behavior was modified transiently--smooth swimming increased, flexing decreased, and reversals were suppressed. After addition of D-xylose (final concentration, 10 mM), the adaptation time (the time required for the populations to return to the unmodified behavior) for S. aurantia was 1.5 to 2.0 min. A model to explain the behavior of S. aurantia and the response of cells to chemoattractants is described. This model includes a coordinating mechanism for flagellar motor operation and a motor switch synchronizing device.

Early evolution of microtubules and undulipodia.

A critique of both autogeneous and symbiotic hypotheses for the origin of microtubules and cilia and eukaryotic flagella (undulipodia) is presented. It is proposed that spirochetes provided the ancient eukaryotic cell with microtubules twice; cytoplasmic microtubules originated from phagocytosed spirochetes whereas axopodial tubules of undulipodia were transformed from ectosymbiotic spirochetes. A role in transport for microtubules in spirochetes together with a detailed scenario by which free-living spirochetes attached as ectosymbionts and subsequently differentiated into undulipodia is outlined. A mechanism for the continuity of motility in the form of "training" of the novel microtubular axoneme by the ancient spirochete motility apparatus is proposed. Transitional states (missing links) are unlikely to have survived. Constraints regarding the nature of the host cell are discussed. A corresponding flowchart of the early evolution of eukaryotes is presented in which plastids and mitochondria are polyphyletic in their origins.

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.

Inhibition of Spirochaeta aurantia chemotaxis by neurotoxins.

The effects of neurotoxic compounds on the chemotactic response of Spirochaeta aurantia were investigated. In the presence of neurotoxins that affect action potential generation and transmission in excitable eucaryotic cells, D-xylose taxis was inhibited by 69 to 93%. Inhibition of chemotaxis was not due to decreased viability or motility. This study supports the hypothesis that the molecular basis for sensory signal transduction in S. aurantia involves ion fluxes across the cytoplasmic membrane.

A voltage clamp inhibits chemotaxis of Spirochaeta aurantia.

Anaerobic conditions were employed to study the relationship between membrane potential and chemotaxis in Spirochaeta aurantia. When cells were grown anaerobically and suspended in anaerobic potassium phosphate buffer (pH 5.5), membranes did not appear to be polarized. Nevertheless, motility was supported by a transmembrane pH gradient, and the anaerobic cells exhibited D-xylose taxis. Introduction of trace amounts of air into anaerobic cell suspensions resulted in a transient membrane polarization. The addition of valinomycin to cells suspended under anaerobic conditions did not alter the steady-state value of membrane potential appreciably but served to clamp membrane potential at the preset level. Although there was no detectable effect of valinomycin on the motility of anaerobic cells in potassium phosphate buffer, D-xylose taxis was completely inhibited by this treatment. These data indicate the the action of valinomycin as a voltage clamp serves to inhibit the chemotaxis of S. aurantia and provide evidence to support the suggestion that the mechanism of chemotaxis in this organism involves the transduction of sensory signals in the form of membrane potential fluctuations.

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.

Chemotaxis of Spirochaeta aurantia: involvement of membrane potential in chemosensory signal transduction.

The effects of valinomycin and nigericin on sugar chemotaxis in Spirochaeta aurantia were investigated by using a quantitative capillary assay, and the fluorescent cation, 3,3'-dipropyl-2,2'-thiodicarbocyanine iodide was used as a probe to study effects of chemoattractants on membrane potential. Addition of a chemoattractant, D-xylose, to cells in either potassium or sodium phosphate buffer resulted in a transient membrane depolarization. In the presence of valinomycin, the membrane potential of cells in potassium phosphate buffer was reduced, and the transient membrane depolarization that resulted from the addition of D-xylose was eliminated. Although there was no detectable effect of valinomycin on motility, D-xylose taxis of cells in potassium phosphate buffer was completely inhibited by valinomycin. In sodium phosphate buffer, valinomycin had little effect on membrane potential or D-xylose taxis. Nigericin is known to dissipate the transmembrane pH gradient of S. aurantia in potassium phosphate buffer. This compound did not dissipate the membrane potential or the transient membrane depolarization observed upon addition of D-xylose to cells in either potassium or sodium phosphate buffer. Nigericin did not inhibit D-xylose taxis in either potassium or sodium phosphate buffer. This study indicates that the membrane potential but not the transmembrane pH gradient of S. aurantia is somehow involved in chemosensory signal transduction.