The chimeric genome of Sphaerochaeta: nonspiral spirochetes that break with the prevalent dogma in spirochete biology.

UNLABELLED: Spirochaetes is one of a few bacterial phyla that are characterized by a unifying diagnostic feature, namely, the helical morphology and motility conferred by axial periplasmic flagella. Their unique morphology and mode of propulsion also represent major pathogenicity factors of clinical spirochetes. Here we describe the genome sequences of two coccoid isolates of the recently described genus Sphaerochaeta which are members of the phylum Spirochaetes based on 16S rRNA gene and whole-genome phylogenies. Interestingly, the Sphaerochaeta genomes completely lack the motility and associated signal transduction genes present in all sequenced spirochete genomes. Additional analyses revealed that the lack of flagella is associated with a unique, nonrigid cell wall structure hallmarked by a lack of transpeptidase and transglycosylase genes, which is also unprecedented in spirochetes. The Sphaerochaeta genomes are highly enriched in fermentation and carbohydrate metabolism genes relative to other spirochetes, indicating a fermentative lifestyle. Remarkably, most of the enriched genes appear to have been acquired from nonspirochetes, particularly clostridia, in several massive horizontal gene transfer events (>40% of the total number of genes in each genome). Such a high level of direct interphylum genetic exchange is extremely rare among mesophilic organisms and has important implications for the assembly of the prokaryotic tree of life. IMPORTANCE: Spiral shape and motility historically have been the unifying hallmarks of the phylum Spirochaetes. These features also represent important virulence factors of highly invasive pathogenic spirochetes such as the causative agents of syphilis and Lyme disease. Through the integration of genome sequencing, microscopy, and physiological studies, we conclusively show that the spiral morphology and motility of spirochetes are not universal morphological properties. In particular, we found that the genomes of the members of the recently described genus Sphaerochaeta lack the genes encoding the characteristic flagellar apparatus and, in contrast to most other spirochetes, have acquired many metabolic and fermentation genes from clostridia. These findings have major implications for the isolation and study of spirochetes, the diagnosis of spirochete-caused diseases, and the reconstruction of the evolutionary history of this important bacterial phylum. The Sphaerochaeta sp. genomes offer new avenues to link ecophysiology with the functionality and evolution of the spirochete flagellar apparatus.

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)

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The impact of cleaner processing on nutrient availability in the bleached kraft industry.

The pulp and paper industry has invested heavily over recent years in cleaner processing, to reduce losses and minimise its impact on the environment. Over the past fifteen years, a New Zealand integrated bleached kraft mill has undergone a comprehensive programme of upgrades to increase production, reduce water consumption and streamline its biological treatment process. Whilst the overall discharge of contaminants from the site decreased, the treatment system performance did not show a concurrent improvement as may have been expected. Reduced BOD removal, low dissolved oxygen levels, and poor solids settlability were symptomatic of phosphorus limitation in the aerated lagoon treatment system. The wastewater entering the system was found to be phosphorus limited at a BOD:P ratio of 100:0.2. Mono-ammonium-phosphate was supplemented, at approximately 30 kg P/d, to raise the phosphorus levels to a BOD:P ratio of 100:0.3. Treatment efficiencies improved very quickly after phosphorus dosage, with a 50% reduction in BOD and TSS discharge, a significant increase in dissolved oxygen levels, and improved BOD removal (85% to 93%). This case study demonstrates that whilst more closed operation can result in reduced discharge of organic loads, there may be negative impacts on the availability of nutrients for balanced biological growth.

Spirochaeta aurantia has diacetyl chloramphenicol esterase activity.

The free-living spirochete Spirochaeta aurantia was nearly as susceptible to diacetyl chloramphenicol, the product of chloramphenicol acetyltransferase, as it was to chloramphenicol itself. This unexpected susceptibility to diacetyl chloramphenicol was wholly or partly the consequence of intrinsic carboxylesterase activity, as indicated by high-performance liquid chromatography, thin-layer chromatography, and microbiological assays. The esterase converted the diacetate to chloramphenicol, thus inhibiting spirochete growth. The esterase activity was cell associated, reduced by proteinase K, eliminated by boiling, and independent of the presence of either chloramphenicol or diacetyl chloramphenicol. S. aurantia extracts also hydrolyzed other esterase substrates, and two of these, alpha-napthyl acetate and 4-methylumbelliferyl acetate, identified an esterase of approximately 75 kDa in a nondenaturing gel. Carboxylesterases occur in Streptomyces species, but in this study their activity was weaker than that of S. aurantia. The S. aurantia esterase could reduce the effectiveness of cat as either a selectable marker or a reporter gene in this species.

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).

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.

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.

Syphilitic lymphadenitis: immunofluorescent identification of spirochetes from imprints.

We are reporting two cases of early syphilis with inguinal buboes which were surgically excised. Th clinical impression was lymphoma in one case and inguinal hernia in the other. The correct and specific diagnosis was quickly established by the application of fluorescent antibody technique to imprints of the enlarged lymph node.

Morphology and physiology of Spirochaeta aurantia strains isolated from aquatic habitats.

1. Seven strains of Spirochaeta aurantia were isolated from pond and swamp water by means of a selective technique which utilized the ability of these organisms to move through bacterial filters and to diffuse through agar media. Although most of the isolations were accomplished when enrichment media low in carbohydrates were used, all seven strains were found to be exclusively saccharolytic. 2. The isolates could be divided into two groups on the basis of cell morphology: a loosely coiled group, and a tightly coiled group with markedly smaller wave length and wave apmlitude. Spirochetes of the latter group possessed a slightly lower GC content in their DNA. The isolates were facultative anaerobes, synthesized carotenoid pigments which conferred an orange color to aerobic colonies, and utilized a variety of carbohydrates--but not amino acids--as energy sources. Exogenous thiamine was required by six isolates tested, riboflavin by four, and biotin by one. The major products of glucose fermentation were acetate, ethanol, CO2 and H2. Growth of the isolates was inhibited by a variety of antibiotics. Determinations of GC contents of DNA showed that strains of S. aurantia are phylogenetically distant from spirochetes classified in the genera Treponema and Leptospira. 3. S. aurantia populations inoculated in the center of agar medium plates migrated in the form of growth rings toward the periphery of the plates. The rate of migration of glucose-utilizing rings was greatest at low glucose concentrations (e.g., 0.02 g/100 ml). It was concluded that migration of cells present in these rings was mainly due to a chemotactic response to glucose which served both as the attractant and the substrate. Chemotaxis of S. aurantia toward glucose may be used as a selective factor in isolating this bacterium from natural environments. 4. The subspecific epithet stricta is proposed to recognize, taxonomically, the tightly coiled strains of S. aurantia.

Peptidoglycan of free-living anaerobic spirochetes.

Electron microscope examination of negatively stained or thin-sectioned cells of Spirochaeta stenostrepta treated with penicillin or lysozyme showed that the peptidoglycan was present as a thin, electron-dense layer adjacent and external to the cytoplasmic membrane. The peptidoglycan was isolated from cells of S. stenostrepta and Spirochaeta litoralis by a procedure including treatments with sodium lauryl sulfate and Pronase. Hydrolysates of the isolated S. stenostrepta and S. litoralis peptidoglycans contained glucosamine, muramic acid, glutamic acid, l-ornithine, and alanine in molar ratios of 0.90:0.85:1.00:1.00:1.40 and of 0.63:0.63:0.99:1.00:1.41, respectively. Determination of N-terminal residues suggested that nearly 50% of the ornithine in S. stenostrepta and S. litoralis peptidoglycans was involved in peptide cross-linkage. The peptidoglycan layer of S. stenostrepta was sensitive to lysozyme and myxobacter AL-1 protease.

Shape of Treponema pallidum.

Treponema pallidum was found to be not helical, but a flat wave twisted into one to five different planes per cell.