Cellulose as extracellular polysaccharide of hot spring sulfur-turf bacterial mat

The carbohydrate fraction of a hot spring sulfur-turf bacterial mat was shown to contain cellulose by the examination of neutral sugar composition, methylation analysis, and the identification of free oligosacchrides obtained from an acetolyzate of the desulfurized sulfur-turf mat. This suggested that the sulfur-oxidizing bacteria composing the sulfur-turf were producers of cellulose.     

Phylogenetic Evidence for the Existence of Novel Thermophilic Bacteria in Hot Spring Sulfur-Turf Microbial Mats in Japan

So-called sulfur-turf microbial mats, which are macroscopic white filaments or bundles consisting of large sausage-shaped bacteria and elemental sulfur particles, occur in sulfide-containing hot springs in Japan. However, no thermophiles from sulfur-turf mats have yet been isolated as cultivable strains. This study was undertaken to determine the phylogenetic positions of the sausage-shaped bacteria in sulfur-turf mats by direct cloning and sequencing of 16S rRNA genes amplified from the bulk DNAs of the mats. Common clones with 16S rDNA sequences with similarity levels of 94.8 to 99% were isolated from sulfur-turf mat samples from two geographically remote hot springs. Phylogenetic analysis showed that the phylotypes of the common clones formed a major cluster with members of the Aquifex-Hydrogenobacter complex, which represents the most deeply branching lineage of the domain bacteria. Furthermore, the bacteria of the sulfur-turf mat phylotypes formed a clade distinguishable from that of other members of the Aquifex-Hydrogenobacter complex at the order or subclass level. In situ hybridization with clone-specific probes for 16S rRNA revealed that the common phylotype of sulfur-turf mat bacteria is that of the predominant sausage-shaped bacteria.Microbial mats develop in a wide variety of aquatic environments, including geothermal hot springs and hydrothermal vents. There are several types of thermophilic microbial mats, e.g., those of cyanobacteria, anoxygenic phototrophic bacteria, and chemotrophic sulfur bacteria, which differ according to the physical and chemical conditions they favor and other environmental factors (10, 38). These microbial mats in thermal habitats have been studied extensively as a peculiar microbial community of the ecosystem, in relation to the phylogeny and evolution of thermophilic prokaryotes, or as a source of new functional enzymes.So-called sulfur-turf microbial mats are macroscopic bundles of white filaments consisting of colorless sulfur bacteria and elemental sulfur particles that form in shallow streams of sulfide-containing high-temperature hot springs. Since first reported by Miyoshi in 1897 (33), this kind of microbial mat has been recorded for several geographically remote hot springs in Japan, although there have been only scattered reports of sulfur-turf microbial mats or chemotrophic sulfur streamers in geothermal springs in other countries (9, 13, 14). The sulfur-turf mats generally develop within a temperature range of 45 to 73°C, within a pH range of 6 to 9, and at discrete sulfide-oxygen interfaces in geothermal springs. These characteristics suggest that the major constituents of the sulfur-turf prokaryotic community are (hyper)thermophilic, neutrophilic, microaerophilic, and chemolithotrophic bacteria. Early studies of these sulfur-turf mats distinguished microscopically three morphotypes of bacteria, two of which were tentatively named Thiovibrio miyoshi and Thiothrix miyoshi (15). Moreover, in situ ecophysiological and microscopic studies have shown that one of these bacteria, the large sausage-shaped “Thiovibrio miyoshi,” predominates in sulfur-turf mats and oxidizes environmental sulfide to elemental sulfur and then to sulfate via thiosulfate (27–31). So far, however, it has not been possible to isolate and cultivate any thermophilic prokaryotes from the sulfur-turf mats predominated by these sausage-shaped bacteria with artificial media, and no attempt has been made to clarify their taxonomic and phylogenetic positions.Determination of 16S rRNA genes is a useful research strategy for identifying uncultivated prokaryotes and is now commonly performed in ecological studies. This technique, involving PCR amplification of 16S rRNA genes or synthesis of cDNAs from bulk 16S rRNAs of natural mixed microbial populations, has been used successfully for the phylogenetic characterization of prokaryotes in hydrothermal environments (6, 7, 34, 40, 41, 47, 48). In the present study, this approach was applied to characterize the sausage-shaped bacteria in sulfur-turf mats without isolating and cultivating them. Here we report that sulfur-turf mats contain novel thermophilic bacteria belonging to the earliest-branching lineage of the domain bacteria.     

Composites of bacterial cellulose and paper made with a rotating disk bioreactor

Suspended solids in the nutrient medium for Acetobacter xylinium in a rotating disk bioreactor become incorporated into the gelatinous mat of bacterial cellulose as it forms. Embedding fibers of ordinary cellulose creates composites with enhanced strength and the toughness of bacterial cellulose. Purified cellulose and elongated fibers from paper are incorporated differently than are spherical particles such as silica gel. About 90% of the final cellulose can come from scrap paper, and dried composite sheets were much stronger than plain bacterial cellulose per unit area.     

Preparation of ultrafine oxidized cellulose mats via electrospinning

Ultrafine oxidized cellulose (OC) mats were prepared by oxidation of ultrafine cellulose mats produced by electrospinning and subsequent deacetylation of cellulose acetate for potential applications in nonwoven adhesion barriers. When ultrafine cellulose mats were oxidized with a mixture of HNO3/H3PO4 - NaNO2 (2/1/1.4 v/v/wt %), their ultrafine mat structure remained unchanged. The yield and carboxyl content of OC mats were 86.7% and 16.8%, respectively. OC showed lower crystallinity than cellulose because the oxidation of cellulose proceeded via disruption of hydrogen bonds between cellulose chains. The swelling behaviors of ultrafine OC mats were dependent on the type of swelling solution. In a physiological salt solution, their degree of swelling was approximately 230%.     

Changes in Quinone Profiles of Hot Spring Microbial Mats with a Thermal Gradient

The respiratory and photosynthetic quinones of microbial mats which occurred in Japanese sulfide-containing neutral-pH hot springs at different temperatures were analyzed by spectrochromatography and mass spectrometry. All of the microbial mats that developed at high temperatures (temperatures above 68°C) were so-called sulfur-turf bacterial mats and produced methionaquinones (MTKs) as the major quinones. A 78°C hot spring sediment had a similar quinone profile. Chloroflexus-mixed mats occurred at temperatures of 61 to 65°C and contained menaquinone 10 (MK-10) as the major component together with significant amounts of either MTKs or plastoquinone 9 (PQ-9). The sunlight-exposed biomats growing at temperatures of 45 to 56°C were all cyanobacterial mats, in which the photosynthetic quinones (PQ-9 and phylloquinone) predominated and MK-10 was the next most abundant component in most cases. Ubiquinones (UQs) were not found or were detected in only small amounts in the biomats growing at temperatures of 50°C and above, whereas the majority of the quinones of a purple photosynthetic mat growing at 34°C were UQs. A numerical analysis of the quinone profiles was performed by using the following three parameters: dissimilarity index (D), microbial divergence index (MD_q), and bioenergetic divergence index (BD_q). A D matrix tree analysis showed that the hot spring mats consisting of the sulfur-turf bacteria, Chloroflexus spp., cyanobacteria, and purple phototrophic bacteria formed distinct clusters. Analyses of MD_q and BD_q values indicated that the microbial diversity of hot spring mats decreased as the temperature of the environment increased. The changes in quinone profiles and physiological types of microbial mats in hot springs with thermal gradients are discussed from evolutionary viewpoints.     

Microbial Mat Boundaries between Chemolithotrophs and Phototrophs in Geothermal Hot Spring Effluents

Among the various microbial mats that develop in geothermal hot springs in solfataric fields, colorless sulfur-turf (ST)—macroscopic white bundles consisting of large sickle-shaped bacteria belonging to Aquificales and elemental sulfur particles–develops in a limited environment of geothermal effluent containing hydrogen sulfide with neutral pH and low in oxygen. Photosynthetic cyanobacterial mat (CY) often grow just downstream of chemolithotrophic ST, or they coexist with ST where the temperature is slightly lower. Knowledge of the environmental regimes of these microbial mats will lead to better understanding of the distribution of thermophilic microorganisms on the Earth and provide clues about evolutionary processes in the microbial ecosystems of the Precambrian era. We studied the environmental parameters of the boundary zone and examined the distribution of these types of mats and measured the in situ growth rates of the microorganisms composing them. In situ examination revealed that temperature and Eh constrain the development of the microbial mats. At the boundary between ST and CY, temperature and Eh ranged between 51.1°C and 63.2°C and between ?112 mV and ?25 mV, respectively. These environmental parameters were not significantly different among Japanese, Yellowstone (North American), and Icelandic hot spring effluents with genetically similar thermal sulfur oxidizers. Sickle-shaped bacteria rarely coexist with cyanobacteria, although they can potentially grow in some CY environments. This suggests that the boundary between ST and CY might be partly determined by exclusive ecological competition.     

Roles of Cellulose and Xyloglucan in Determining the Mechanical Properties of Primary Plant Cell Walls

The primary cell walls of growing and fleshy plant tissue mostly share a common set of molecular components, cellulose, xyloglucan (XyG), and pectin, that are required for both inherent strength and the ability to respond to cell expansion during growth. To probe molecular mechanisms underlying material properties, cell walls and analog composites from Acetobacter xylinus have been measured under small deformation and uniaxial extension conditions as a function of molecular composition. Small deformation oscillatory rheology shows a common frequency response for homogenized native cell walls, their sequential extraction residues, and bacterial cellulose alone. This behavior is characteristic of structuring via entanglement of cellulosic rods and is more important than cross-linking with XyG in determining shear moduli. Compared with cellulose alone, composites with XyG have lower stiffness and greater extensibility in uniaxial tension, despite being highly cross-linked at the molecular level. It is proposed that this is due to domains of cross-linked cellulose behaving as mechanical elements, whereas cellulose alone behaves as a mat of individual fibrils. The implication from this work is that XyG/cellulose networks provide a balance of extensibility and strength required by primary cell walls, which is not achievable with cellulose alone.     

Cellulolytic, fermentative, and methanogenic guilds in benthic periphyton mats from the Florida Everglades

Phosphorus enrichment caused by runoff from agricultural areas has resulted in ecosystem-level changes in the northern Florida Everglades, including a loss of periphyton mats from nutrient-impacted areas. The potential for methanogenesis resulting from the anaerobic decomposition of cellulose and fermentation products, and the microorganisms responsible for these processes, were studied in mats from a region not impacted by nutrient enrichment. Methane was produced from periphyton incubated with cellulose, propionate, butyrate, and formate, with an accumulation of fatty acids in incubations. The accumulation of fatty acids may have been caused by the inhibition of syntrophic oxidation, a potentially significant route for methane production in soils. Sequence analysis of 16S rRNA genes characteristic of Clostridium, the primary genus responsible for anaerobic decomposition and fermentation in soils of the area, indicated that Clostridium Cluster I assemblages present in the mat differed from those in the soils of the area. Significantly, sequences characteristic of the Clostridium group that dominates the soils of the area, group XIV, were not detected in the mat. These results indicate that benthic periphyton is probably a significant source of methane in the Everglades, and the responsible microorganisms differ significantly from those in the soils of the area.     

Superhydrophobicity of cellulose triacetate fibrous mats produced by electrospinning and plasma treatment

Nano/micro-fibrous cellulose triacetate (CTA) mats were prepared by electrospinning a fixed concentration of CTA with different methylene chloride (MC)/ethanol (EtOH) ratios and with various concentrations of CTA at a fixed MC/EtOH 80/20 (v/v) ratio. All of the electrospun CTA mats had a high water contact angle (WCA) compared to the CTA cast film. At a solvent composition of 80/20 (v/v) and 5 wt.% CTA concentration, the CTA mat without plasma treatment had good surface roughness and electrospinning processability, and its WCA was 142°. To further improve its hydrophobicity, the CTA fibrous mat electrospun from the 5 wt.% solution of CTA was treated with a CF₄ plasma for various times. Superhydrophobicity could be obtained after the CF₄ plasma treatment. The WCA of the CTA mat reached as high as 153° after plasma treatment for 60 s.     

Spirochaeta caldaria sp. nov., a thermophilic bacterium that enhances cellulose degradation by Clostridium thermocellum

Two strains of obligately anaerobic, thermophilic spirochetes were isolated from cyanobacterial mat samples collected at freshwater hot springs in Oregon and Utah, USA. The isolates grew optimally between 48° and 52°C, and did not grow at 25° or 60°C. Both strains fermented various pentoses, hexoses, and disaccharides. Amino acids or cellulose did not serve as fermentable substrates for growth. H₂, CO₂, acetate, and lactate were end products of d-glucose fermentation. On the basis of physiological characteristics, guanine + cytosine content of DNA, and comparisons of 16S ribosomal RNA sequences, it was concluded that the two isolates were representatives of a novel species of Spirochaeta for which the name Spirochaeta caldaria is proposed. One of the two strains was grown in coculture with a thermophilic cellulolytic bacterium (Clostridium thermocellum) in a medium containing cellulose as the only fermentable substrate. In the coculture cellulose was broken down at a faster rate than in the clostridial monoculture. The results are consistent with the suggestion that interactions between cellulolytic bacteria and non-cellulolytic spirochetes enhance cellulose breakdown in natural environments in which cellulose-containing plant material is biodegraded.     

Ultrastructure and adhesion properties of Ruminococcus albus.

Morphological studies have shown that cells of the anaerobic rumen bacterium Ruminococcus albus have electron-translucent granules of reserve carbohydrate in their cytoplasm, and that they have a polysaccharide "coat" layer external to their gram-negative cell wall. This coat layer, which stains specifically with ruthenium red, forms a compact mat of fibers adjacent to the cell, and fibrous elements also project as much as 0.6 mum from the cells. These radial fibers are clearly visualized by freeze-etching, and can be seen to extend throughout the extensive intercullular space in centrifuged pellets of these bacteria. Cells of R. albus adhere to cellulose fibers added to the culture medium, and the coat material is seen to mediate this adhesion in addition to its function in the general protection of these cells.     

Characterization of mat A-2, mat A-3 and deltamatA mating-type mutants of Neurospora crassa.

The mating-type locus of Neurospora crassa regulates mating identity and entry into the sexual cycle. The mat A idiomorph encodes three genes, mat A-1, mat A-2, and mat A-3. Mutations in mat A-1 result in strains that have lost mating identity and vegetative incompatibility with mat a strains. A strain containing mutations in both mat A-2 and mat A-3 is able to mate, but forms few ascospores. In this study, we describe the isolation and characterization of a mutant deleted for mat (deltamatA), as well as mutants in either mat A-2 or mat A-3. The deltamatA strain is morphologically wild type during vegetative growth, but it is sterile and heterokaryon compatible with both mat A and mat a strains. The mat A-2 and mat A-3 mutants are also normal during vegetative growth, mate as a mat A strain, and produce abundant biparental asci in crosses with mat a, and are thus indistinguishable from a wild-type mat A strain. These data and the fact that the mat A-2 mat A-3 double mutant makes few asci with ascospores indicate that MAT A-2 and MAT A-3 are redundant and may function in the same pathway. Analysis of the expression of two genes (sdv-1 and sdv-4) in the various mat mutants suggests that the mat A polypeptides function in concert to regulate the expression of some sexual development genes.     

Effect of adhesive on the morphology and mechanical properties of electrospun fibrous mat of cellulose acetate

Ultrafine fibers of cellulose acetate/poly(butyl acrylate) (CA/PBA) composite in which PBA acted as an adhesive and CA acted as a matrix, were successfully prepared as fibrous mat via electrospinning. The morphology observation from the electrospun CA/PBA composite fibers, after treatment with heat hardener, revealed that the fibers were cylindrical and had point-bonded structures. SEM, FT-IR spectra, Raman spectra, TGA analysis, and mechanical properties measurement were used to study the different properties of hybrid mats. The tensile strength of blend fibrous electrospun mats was found to be effectively increased. This resultant enhancement of the mechanical properties of polymer fibrous mats, caused by generating the point-bonded structures (due to adhesive), could increase the number of potential applications of mechanically weak electrospun CA fibers.     

The fission yeast homologue of CENP-B, Abp1, regulates directionality of mating-type switching

In fission yeast, mating-type switching involves replacing genetic information contained at the expressed mat1 locus by that of either the mat2P or mat3M donor loci. Donor selection is nonrandom, as mat1P cells preferentially use mat3M for switching, whereas mat1M cells use mat2P. Switching directionality is determined by the cell-type-specific distribution of the Swi2-Swi5 complex that, in mat1P cells, localises to mat3M and, only in mat1M cells, spreads to mat2P in a heterochromatin-dependent manner. Mechanisms regulating spreading of Swi2-Swi5 across heterochromatin are not fully understood. Here, we show that the fission yeast homologue of CENP-B, Abp1, binds to the silent domain of the mating-type locus and regulates directionality of switching. Deletion of abp1 prevents utilisation of mat2P, as when heterochromatin is disrupted and spreading of Swi2-Swi5 is impaired. Our results show that, indeed, deletion of abp1 abolishes spreading of Swi2-Swi5 to mat2P. However, in abp1Delta cells, heterochromatin organisation at the mating-type locus is preserved, indicating that Abp1 is actually required for efficient spreading of Swi2-Swi5 through heterochromatin. Cbh1 and Cbh2, which are also homologous to CENP-B, have only a minor contribution to the regulation of directionality of switching, which is in contrast with the strong effects observed for Abp1.     

Cloning the Mating Types of the Heterothallic Fungus Podospora Anserina: Developmental Features of Haploid Transformants Carrying Both Mating Types

DNAs that encode the mating-type functions (mat+ and mat-) of the filamentous fungus Podospora anserina were cloned with the use of the mating-type A probe from Neurospora crassa. Cloning the full mat information was ascertained through gene replacement experiments. Molecular and functional analyses of haploid transformants carrying both mating types lead to several striking conclusions. Mat+ mat- strains are dual maters. However, the resident mat information is dominant to the mat information added by transformation with respect to fruiting body development and ascus production. Moreover, when dual mating mat+ mat- strains are crossed to mat+ or mat- testers, there is strong selection, after fertilization, that leads to the loss from the mat+ mat- nucleus of the mat information that matches that of the tester. Finally, the mat locus contains at least two domains, one sufficient for fertilization, the other necessary for sporulation.     

Initiation of meiotic recombination by double-strand DNA breaks in S. pombe

Mitotic gene conversion and reciprocal recombination have recently been shown to be efficiently initiated by double-strand DNA breaks (DSBs) in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. We tested whether DSBs could also initiate meiotic recombination at the mat1 locus in S. pombe. The mat1 switching-mechanism-generated DSB found in mitotically growing cells can be repaired without mat1 switching, since strains deleted for both donor loci (mat2-P and mat3-M) have the break but do not produce inviable cells. A (mat1-P X mat1-M) cross produced a high frequency (20%) of 3:1 gene conversions of mat1 in meiotic tetrads. Gene conversion events were associated with the recombination of flanking markers. Strains lacking the DSB failed to convert. Thus, the DSB at mat1 promotes efficient meiotic recombination in fission yeast.     

Swi6, a Gene Required for Mating-Type Switching, Prohibits Meiotic Recombination in the Mat2-Mat3 ``cold Spot'''' of Fission Yeast

Mitotic interconversion of the mating-type locus (mat1) of the fission yeast Schizosaccharomyces pombe is initiated by a double-strand break at mat1. The mat2 and mat3 loci act as nonrandom donors of genetic information for mat1 switching such that switches occur primarily (or only) to the opposite mat1 allele. Location of the mat1 "hot spot" for transposition should be contrasted with the "cold spot" of meiotic recombination located within the adjoining mat2-mat3 interval. That is, meiotic interchromosomal recombination in mat2, mat3 and the intervening 15-kilobase region does not occur at all. swi2 and swi6 switching-deficient mutants possess the normal level of double-strand break at mat1, yet they fail to switch efficiently. By testing for meiotic recombination in the cold spot, we found the usual lack of recombination in a swi2 mutant but a significant level of recombination in a swi6 mutant. Therefore, the swi6 gene function is required to keep the donor loci inert for interchromosomal recombination. This finding, combined with the additional result that switching primarily occurs intrachromosomally, suggests that the donor loci are made accessible for switching by folding them onto mat1, thus causing the cold spot of recombination.