Characterization of the xylan-degrading microbial community from human faeces

In humans, plant cell wall polysaccharides represent an important source of dietary fibres that are digested by gut microorganisms. Despite the extensive degradation of xylan in the colon, the population structure and the taxonomy of the predominant bacteria involved in degradation of this polysaccharide have not been extensively explored. The objective of our study was to characterize the xylanolytic microbial community from human faeces, using xylan from different botanic origins. The xylanolytic population was enumerated at high level in all faecal samples studied. The predominant xylanolytic organisms further isolated (20 strains) were assigned to Roseburia and Bacteroides species. Some Bacteroides isolates corresponded to the two newly described species Bacteroides intestinalis and Bacteroides dorei. Other isolates were closely related to Bacteroides sp. nov., a cellulolytic bacterium recently isolated from human faeces. The remaining Bacteroides strains could be considered to belong to a new species of this genus. Roseburia isolates could be assigned to the species Roseburia intestinalis. The xylanase activity of the Bacteroides and Roseburia isolates was found to be higher than that of other gut xylanolytic species previously identified. Our results provide new insights to the diversity and activity of the human gut xylanolytic community. Four new xylan-degrading Bacteroides species were identified and the xylanolytic capacity of R. intestinalis was further shown.     

Characterization of an unusual lipoprotein similar to human lipoprotein a isolated from the baboon, Papio sp

An unusual lipoprotein was detected and purified from the blood of some members of a large colony of baboons, Papio sp. This lipoprotein was found to be similar to human lipoprotein a in all respects and is therefore termed lipoprotein a. Baboon lipoprotein a had a density of 1.052 g/ml and was located between low- and high-density lipoproteins in a density gradient ultracentrifugation. However, despite its greater density, baboon lipoprotein a was larger than low-density lipoprotein, based on gradient gel electrophoresis and gel filtration. The lipoprotein contained a very large apolipoprotein (apolipoprotein-lipoprotein a) which was found to consist of an apolipoprotein B linked to another protein called apolipoprotein a by a disulfide bridge(s). In all these characteristics, baboon lipoprotein a was similar to human lipoprotein a.     

Characterization and cloning of plasmids from the iron-oxidizing bacteriumThiobacillus ferrooxidans

More than 100 independent strains ofThiobacillus ferrooxidans were isolated from six different domestic mining sites. Although there was some variation according to sampling site, about 73% of all strains carried more than one plasmid ranging in size from about 2.0 to 30 kilobase-pairs(kb). Among these, four plasmids of 2.4, 4.7, 5.1, and 8.9 kb, designated pTNA33, pTSY91, pTSB121, and pTSB122, respectively, were cloned intoEscherichia coli plasmids. pTSB121 and pTSB122, originated from the sameT. ferrooxidans strain, showed weak homology by Southern blotting, whereas pTSB121 showed high homology with pTSY91 from a different strain. It seems that the occurrence of the plasmid homologous to pTSB121 or pTSB122 is more ubiquitous inThiobacillus. On the other hand, pTNA33 is a unique plasmid because it showed no significant homology with other plasmids.     

Methanogenic sarcina from an anaerobic microbial community degrading p-toluene sulfonate

The methanogenic strain MM isolated from an anaerobic microbial community degrading p-toluene sulfonate showed optimal values of temperature and pH for growth equal to 37 degrees C and 6.3-6.9, respectively. The doubling times of the isolate grown on methanol, acetate, and methylamines under the optimal conditions were 8.8, 19.1, and 10.3-28.1 h, respectively. The growth of strain MM was observed only when the cultivation medium contained casamino acids or p-toluene sulfonate. The G + C content of the DNA of the isolate was 40.3 mol%. This, together with DNA-DNA hybridization data, allowed the new isolate to be identified as a strain of the species Methanosarcina mazei. The new isolate differed from the known representatives of this species in that it was resistant to alkylbenzene sulfonates and able to demethylate p-toluene sulfonate when grown on acetate.     

Characterization of Symbiobacterium toebii, an obligate commensal thermophile isolated from compost

A symbiotic thermophilic bacterium, strain SC-1, was isolated from hay compost (toebi) in Korea. The new isolate exhibited an obligate commensal interaction with a thermophilic Geobacillus strain and required crude extracts and/or culture supernatant from Geobacillus sp. SK-1 for axenic growth. The growth factors from Geobacillus sp. SK-1 were irreversibly inactivated by phenol or protease treatment, suggesting that they might be proteins. The cells of strain SC-1 were non-spore forming, nonmotile rods that were stained Gram-negatively. The isolate was a microaerophilic heterotroph. Growth was observed between 45 degrees and 70 degrees C (optimum: 60 degrees C; 2.4-h doubling time) and pH 6.0 and 9.0 (optimum: pH 7.5). The G+C content of the genomic DNA was 65 mol%, and the major quinones were MK-6 and MK-7. A phylogenetic analysis of its 16S rDNA sequence indicated that strain SC-1 is closely related to Symbiobacterium thermophilum and so was named Symbiobacterium toebii on the basis of its physiological and molecular properties.     

Characterization of an anaerobic marine microbial community exposed to combined fluxes of perchlorate and salinity

Applied Microbiology and Biotechnology - The recent recognition of the environmental prevalence of perchlorate and its discovery on Mars, Earth’s moon, and in meteorites, in addition to its...     

Description of an unusual gram-negative anaerobic rod isolated from periodontal pockets

A Gram-negative rod which grew with an unusual colonial "water-drop" form was isolated from periodontal pocket samples from 12 patients. Six strains were characterized by biochemical tests, cell wall analyses, malate dehydrogenase mobilities, protein profiles, and serology. By these criteria, the organisms formed a group of similar strains which were anaerobic, nonmotile, nonsporing, Gram-negative rods resembling Bacteroides. Comparison of the isolates to American Type Culture Collection strains of Bacteroides showed that they represented a closely related group, distinct from the described species of oral Bacteroides. Initial results on the DNA of the isolates suggested a base ratio of 54-57% G + C. Despite the DNA G + C base ratios currently accepted for the Bacteroides (28-61 mol% G + C), many species fall into a narrower range of 40-52 mol% G + C. This range would exclude the organisms described here and suggests that placing them into the genus Bacteroides may be inappropriate.     

Identification of biofilm matrix-associated proteins from an acid mine drainage microbial community

In microbial communities, extracellular polymeric substances (EPS), also called the extracellular matrix, provide the spatial organization and structural stability during biofilm development. One of the major components of EPS is protein, but it is not clear what specific functions these proteins contribute to the extracellular matrix or to microbial physiology. To investigate this in biofilms from an extremely acidic environment, we used shotgun proteomics analyses to identify proteins associated with EPS in biofilms at two developmental stages, designated DS1 and DS2. The proteome composition of the EPS was significantly different from that of the cell fraction, with more than 80% of the cellular proteins underrepresented or undetectable in EPS. In contrast, predicted periplasmic, outer membrane, and extracellular proteins were overrepresented by 3- to 7-fold in EPS. Also, EPS proteins were more basic by ～2 pH units on average and about half the length. When categorized by predicted function, proteins involved in motility, defense, cell envelope, and unknown functions were enriched in EPS. Chaperones, such as histone-like DNA binding protein and cold shock protein, were overrepresented in EPS. Enzymes, such as protein peptidases, disulfide-isomerases, and those associated with cell wall and polysaccharide metabolism, were also detected. Two of these enzymes, identified as β-N-acetylhexosaminidase and cellulase, were confirmed in the EPS fraction by enzymatic activity assays. Compared to the differences between EPS and cellular fractions, the relative differences in the EPS proteomes between DS1 and DS2 were smaller and consistent with expected physiological changes during biofilm development.     

Taxonomic composition and gene content of a methane-producing microbial community isolated from a biogas reactor

A total community DNA sample from an agricultural biogas reactor continuously fed with maize silage, green rye, and small proportions of chicken manure has recently been sequenced using massively parallel pyrosequencing. In this study, the sample was computationally characterized without a prior assembly step, providing quantitative insights into the taxonomic composition and gene content of the underlying microbial community. Clostridiales from the phylum Firmicutes is the most prevalent phylogenetic order, Methanomicrobiales are dominant among methanogenic archaea. An analysis of Operational Taxonomic Units (OTUs) revealed that the entire microbial community is only partially covered by the sequenced sample, despite that estimates suggest only a moderate overall diversity of the community. Furthermore, the results strongly indicate that archaea related to the genus Methanoculleus, using CO(2) as electron acceptor and H(2) as electron donor, are the main producers of methane in the analyzed biogas reactor sample. A phylogenetic analysis of glycosyl hydrolase protein families suggests that Clostridia play an important role in the digestion of polysaccharides and oligosaccharides. Finally, the results unveiled that most of the organisms constituting the sample are still unexplored.     

Specific characteristics of the strains isolated from a thermoacidophilic microbial community oxidizing antimony sulfide ore

Investigation of the phenotypic properties of three mixotrophic bacteria, strains Sb-K, Sb-F, and Sb-S, isolated from an aboriginal thermoacidophilic microbial community participating in biooxidation of ore with high antimony content (26%) and ore concentrates from the Olympiadinskoe deposit under semicontinuous cultivation conditions at 46 ± 1°C, revealed the differentiating characteristics of these strains. The isolated cultures grew lithotrophically through different numbers of transfers: strains Sb-F and Sb-K grew through seven and eight transfers, respectively, and strain Sb-S grew through two or three transfers. Strains Sb-K and Sb-S utilized a wide range of organic substrates for active organotrophic growth during nine or ten transfers, while strain Sb-F was less tolerant to organic compounds. Strain Sb-K grew on a medium with the ore and sulfide ore concentrates in the pH range of 1.0–3.0. Growth of strains Sb-F and Sb-S occurred in the pH ranges of 1.0–2.5 and 1.5–5.5 on media with Fe²⁺ and S⁰, respectively.. The optimal initial pH values of the media, corresponding to the maximum specific growth rates, were 1.6–1.7, 1.9, and 2.0–3.0 for strains Sb-K, Sb-F, and Sb-S, respectively. All three strains were able to grow within a broad temperature range, 20–65°C, with an optimum at 46°C (Sb-K), 40–46°C (Sb-F), and 48–50°C (Sb-S). According to the results of DNA-DNA hybridization and phylogenetic analysis, as well as their phenotypic characteristics, the isolates can be classified as novel strains of species of the genus Sulfobacillus. Strains Sb-K, Sb-F, and Sb-S, isolated as predominant cultures on the media with sulfide compounds, iron, or sulfur, respectively, were affiliated to the species S. thermotolerans, S. sibiricus, and S. thermosulfidooxidans.     

Structure of an unusual complex-type oligosaccharide isolated from Chinese hamster ovary cells

An asparagine-linked oligosaccharide with an unusual structure has been isolated from Pronase digests of Chinese hamster ovary cell glycoproteins using gel filtration, ion exchange chromatography, and affinity chromatography on lectin-Sepharose columns. The oligosaccharide contained approximately 7.5% of the total cellular glycopeptide galactose and 1.3% of the mannose. The structure of the oligosaccharide was determined by the combination of methylation analysis and digestion with exo- and endo-glycosidases. The oligosaccharide has predominantly a triantennary structure consisting of repeating [Galβ1 → 4GlcNAcβ1 → 3] units in the outer branches linked to a trimannosyl-di-N,N′-acetyl-chitobiose core. It resembles oligosaccharides present on human erythrocyte glycoproteins and corneal keratan sulfate.     

Asthma and climatic conditions: experience from Bermuda,an isolated island community.

A retrospective study of patients attending the emergency department with acute asthma was performed in Bermuda. Climatic data (barometric pressure, rainfall, humidity, and wind strength and direction) were obtained and compared with frequency of exacerbations of asthma. Three factors--namely, relative humidity, average daily temperature, and northeasterly winds--were found to be related to worsening asthma. Owing to Bermuda''s lack of pollution and aeroallergens it was thought that these weather parameters had a direct effect on the asthmatic population.     

Characterization of a new dihemic c(4)-type cytochrome isolated from Thiobacillus ferrooxidans

A new soluble c-type cytochrome has been purified to homogeneity from the acidophilic proteobacterium Thiobacillus ferrooxidans BRGM. It is characterized by an alpha-peak wavelength of 552 nm, a molecular mass of 26 567 Da (as determined by mass spectroscopy) and a pI value of 8. Optical redox titrations at pH 4.0 revealed the presence of two distinguishable redox species with an E(m) of 510 mV and an E(m) of 430 +/- 20 mV. EPR spectra recorded for this heme protein demonstrated the presence of stoichiometric amounts of two low-spin hemes with a g(z)() of 3.08 (510 mV species) and a g(z)() of 3.22 (430 mV species). Modifications of the physicochemical properties of the cytochrome were observed on complex formation with the blue copper protein rusticyanin, another soluble electron carrier in the genus Thiobacillus. N-Terminal sequencing yielded the polypeptide sequence up to the 50th residue. The determined sequence was found to be present (at 100% amino acid identity) in the (unfinished) genome of T. ferrooxidans ATCC 23270, and the corresponding full-length protein turned out to be surprisingly similar (34.5% amino acid identity) to another c(4)-type diheme protein from T. ferrooxidans BRGM [Cavazza, C., et al. (1996) Eur. J. Biochem. 242, 308-314], the gene of which is also present (at 97% amino acid identity) in the T. ferrooxidans ATCC 23270 genome. The physicochemical properties and sequence characteristics of both c(4) cytochromes present in the same bacteria are compared, and the functional role of this new diheme protein in the iron(II)-oxidizing electron transport chain in the genus Thiobacillus is discussed.     

Characterization by EPR spectroscopy of cytochrome b-562 isolated from the cytochrome b-c1 complex of Rhodopseudomonas sphaeroides R-26

The EPR spectra of cytochrome b-562 isolated from the cytochrome b-c1 complex of Rhodopseudomonas sphaeroides were measured at liquid helium temperature. The purified cytochrome b-562 gives a high spin signal at g = 6.0. Anaerobic titration of this signal confirmed the presence of two redox components with Em = 40 and -110 mV at pH 7.5. These values are consistent with the published ones, Em = 55 and -100 mV at pH 7.0, that were optically estimated for the same type of preparation (Iba et al. (1985) FEBS Lett. 183, 151-154). The power saturation behavior of the g = 6.0 signal at different redox potentials indicated a direct spin-spin interaction between these two redox centers.     

Characterization of an unusual sex steroid binding component from the chicken oviduct

In addition to the classical estrogen receptor, chick oviduct cytosol contains a sex steroid binding component (SSB) with specificity for steroidal estrogens, androgens and progestins. We have optimized the measurement of SSB and have further characterized this protein. It was possible to quantitate [3H]estradiol binding to SSB by performing the measurements in the presence of excess diethylstilbestrol, which saturates the estrogen receptor and does not bind to SSB, and by using excess progesterone to determine nonspecific binding. Since SSB appears to be quite unstable with rapid hormone dissociation kinetics, we determined that short incubation times (usually 2 h) at 0 degrees C with 20-30% glycerol in the buffer gave optimal SSB measurements. The affinity of SSB for estradiol (Kd = 20 nM) is about 5% that of the estrogen receptor. In addition to estradiol, several androgens and progestins bind to SSB. However, the nonsteroidal antiestrogen, H1285 does not bind to SSB even though it binds well to the avian estrogen receptor. The tissue content of SSB is about 15-fold greater than for estrogen receptor and is stimulated by estrogen treatment. Whereas labeled SSB cannot be readily resolved by ion-exchange chromatography due to rapid dissociation of hormone from SSB, post-labeling experiments yield binding activity eluting with 0.2 M KCl indicating that SSB is an acidic protein having a chromatography behavior similar to that of estrogen receptor. SSB binding was dramatically reduced by the chaotropic salt, NaSCN, whereas binding to the estrogen receptor was not disrupted. SSB is stabilized by sodium molybdate, a property which is characteristic of steroid receptors. Although the role of SSB in the chick oviduct is yet to be determined, an understanding of its properties is essential for accurate determinations of the estrogen receptor.     

Cellular localization of tolyporphins,unusual tetrapyrroles,in a microbial photosynthetic community determined using hyperspectral confocal fluorescence microscopy

Photosynthesis Research - The cyanobacterial culture HT-58-2, composed of a filamentous cyanobacterium and accompanying community bacteria, produces chlorophyll a as well as the tetrapyrrole...     

Purification and characterization of sulfide:quinone oxidoreductase from an acidophilic iron-oxidizing bacterium, Acidithiobacillus ferrooxidans

Sulfide:quinone oxidoreductase (SQR) was purified from membrane of acidophilic chemolithotrophic bacterium Acidithiobacillus ferrooxidans NASF-1 cells grown on sulfur medium. It was composed of a single polypeptide with an apparent molecular mass of 47 kDa. The apparent K(m) values for sulfide and ubiquinone were 42 and 14 muM respectively. The apparent optimum pH for the SQR activity was about 7.0. A gene encoding a putative SQR of A. ferrooxidans NASF-1 was cloned and sequenced. The gene was expressed in Escherichia coli as a thioredoxin-fusion protein in inclusion bodies in an inactive form. A polyclonal antibody prepared against the recombinant protein reacted immunologically with the purified SQR. Western blotting analysis using the antibody revealed an increased level of SQR synthesis in sulfur-grown A. ferrooxidans NASF-1 cells, implying the involvement of SQR in elemental sulfur oxidation in sulfur-grown A. ferrooxidans NASF-1 cells.     

Characterization of cytochrome c-550 from cyanobacteria

Cytochrome c-550 has been purified from several cyanobacteria. It is a low-potential, auto-oxidizable cytochrome. This cytochrome should not be confused with a degradation product of cytochrome ? which may be formed during the isolation of the latter protein. Cytochromes c-550 are distinctive in size, amino-acid composition and N-terminal amino-acid sequence.