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PubMed2001

Do conventionally and biologically cultivated soils differ in bacterial diversity and community structure?

The effects of a long-term herbicide treatment on the microbial community of an agricultural soil were investigated. Therefor, molecular techniques were used to evaluate the structure and diversity of the soil microbial community. Eubacterial and group-specific primers for methanotrophs type I and II were used to amplify 16S rRNA gene fragments from total soil DNA. These fragments were subsequently separated by denaturing gradient gel electrophoresis (DGGE). The structure of the methanotrophic community was affected by the herbicides as the patterns obtained from a herbicide treated soil (conventional) clustered separately from the control soil (biological). It seems that group-specific PCR followed by DGGE is a very powerful and sensitive technique to differentiate fields, which have received a herbicide treatment from those who did not. The diversity of the methanotrophic community was quantified by calculating the Shannon-Weaver index of biodiversity. The type I methanotrophs showed a significant decreased biodiversity due to the herbicide treatment but the diversity of the methanotrophs type II was slightly higher in the herbicide treated soil.

Bacteria↗
PubMed1998

Quinone profiling of bacterial communities in natural and synthetic sewage activated sludge for enhanced phosphate removal.

Respiratory quinones were used as biomarkers to study bacterial community structures in activated sludge reactors used for enhanced biological phosphate removal (EBPR). We compared the quinone profiles of EBPR sludges and standard sludges, of natural sewage and synthetic sewage, and of plant scale and laboratory scale systems. Ubiquinone (Q) and menaquinone (MK) components were detected in all sludges tested at molar MK/Q ratios of 0.455 to 0.981. The differences in MK/Q ratios were much larger when we compared different wastewater sludges (i.e., raw sewage and synthetic sewage) than when we compared sludges from the EBPR and standard processes or plant scale and laboratory scale systems. In all sludges tested a Q with eight isoprene units (Q-8) was the most abundant quinone. In the MK fraction, either tetrahydrogenated MK-8 or MK-7 was the predominant type, and there was also a significant proportion of MK-6 to MK-8 in most cases. A numerical cluster analysis of the profiles showed that the sludges tested fell into two major clusters; one included all raw sewage sludges, and the other consisted of all synthetic sewage sludges, independent of the operational mode and scale of the reactors and the phosphate accumulation. These data suggested that Q-8-containing species belonging to the class Proteobacteria (i.e., species belonging to the beta subclass) were the major constituents of the bacterial populations in the EBPR sludge, as well as in standard activated sludge. Members of the class Actinobacteria (gram-positive bacteria with high DNA G+C contents) were the second most abundant group in both types of sludge. The bacterial community structures in activated sludge processes may be affected more by the nature of the influent wastewater than by the introduction of an anaerobic stage into the process or by the scale of the reactors.

Journal Article↗
PubMed1986

[Mass-spectrometric determination of the structure of glycopeptides from the bacterial cell wall (illustrated by Lactobacillus bulgaricus)].

Mass spectrometry has been applied to the structural analysis of one of the glycopeptides from blastolysin, antitumor bacterial preparation isolated from the Lactobacillus bulgaricus cell wall. The glycopeptide (MW 10,000) was subjected to partial acid hydrolysis (6 N HCl, 100 degrees C) and the resulting products were dansylated or trifluoroacetylated and methylated or deuteromethylated. The mixture of these derivatives was examined by high-performance liquid chromatography or gas chromatography followed by mass spectrometry using electron impact and ammonia chemical ionization techniques.

Amino Acids↗
PubMed1975

Halococcus morrhuae: a sulfated heteropolysaccharide as the structural component of the bacterial cell wall.

The qualitative and quantitative composition of purifed cell wall of Halococcus morrhuae CCM 859 was determined. Glucose, mannose, galactose; glucuronic and galacturonic acids; glucosamine, galactosamine, gulosaminuronic acid; acetate, glycine and sulfate are found as major constituents. The amino sugars are N-acetylated. It was not possible to fractionate the cell wall in chemically different polymers. Evidence is presented that the major cell wall polymer of this strain is a complex heterolgycan which seems, like the peptidoglycan of most bacteria, to be responsible for the rigidity and stability of the cell wall. In addition it could be proved that this heteroglycan is sulfated and therefore differs considerably from previously described bacterial cell wall polymers.

Amino Sugars↗
PubMed2001

Cell compartmentalisation in planctomycetes: novel types of structural organisation for the bacterial cell.

The organisation of cells of the planctomycete species Pirellula marina, Isosphaera pallida, Gemmata obscuriglobus, Planctomyces maris and "Candidatus Brocadia anammoxidans" was investigated based on ultrastructure derived from thin-sections of cryosubstituted cells, freeze-fracture replicas, and in the case of Gemmata obscuriglobus and Pirellula marina, computer-aided 3-D reconstructions from serial sections of cryosubstituted cells. All planctomycete cells display a peripheral ribosome-free region, termed here the paryphoplasm, surrounding the perimeter of the cell, and an interior region including any nucleoid regions as well as ribosome-like particles, bounded by a single intracytoplasmic membrane (ICM), and termed the pirellulosome in Pirellula species. Immunogold labelling and RNase-gold cytochemistry indicates that in planctomycetes all the cell DNA is contained wholly within the interior region bounded by the ICM, and the paryphoplasm contains no DNA but at least some of the cell's RNA. The ICM in Isosphaera pallida and Planctomyces maris is invaginated such that the paryphoplasm forms a major portion of the cell interior in sections, but in other planctomycetes it remains as a peripheral zone. In the anaerobic ammonium-oxidising ("anammox" process) chemoautotroph "Candidatus Brocadia anammoxidans" the interior region bounded by ICM contains a further internal single-membrane-bounded region, the anammoxosome. In Gemmata obscuriglobus, the interior ICM-bounded region contains the nuclear body, a double-membrane-bounded region containing the cell's nucleoid and all genomic DNA in addition to some RNA. Shared features of cell compartmentalisation in different planctomycetes are consistent with the monophyletic nature of the planctomycetes as a distinct division of the Bacteria. The shared organisational plan for the planctomycete cell constitutes a new type not known in cells of other bacteria.

Bacteria↗
PubMed2006

Community structure of actively growing bacterial populations in plant pathogen suppressive soil.

The bacterial community in soil was screened by using various molecular approaches for bacterial populations that were activated upon addition of different supplements. Plasmodiophora brassicae spores, chitin, sodium acetate, and cabbage plants were added to activate specific bacterial populations as an aid in screening for novel antagonists to plant pathogens. DNA from growing bacteria was specifically extracted from the soil by bromodeoxyuridine immunocapture. The captured DNA was fingerprinted by terminal restriction fragment length polymorphism (T-RFLP). The composition of the dominant bacterial community was also analyzed directly by T-RFLP and by denaturing gradient gel electrophoresis (DGGE). After chitin addition to the soil, some bacterial populations increased dramatically and became dominant both in the total and in the actively growing community. Some of the emerging bands on DGGE gels from chitin-amended soil were sequenced and found to be similar to known chitin-degrading genera such as Oerskovia, Kitasatospora, and Streptomyces species. Some of these sequences could be matched to specific terminal restriction fragments on the T-RFLP output. After addition of Plasmodiophora spores, an increase in specific Pseudomonads could be observed with Pseudomonas-specific primers for DGGE. These results demonstrate the utility of microbiomics, or a combination of molecular approaches, for investigating the composition of complex microbial communities in soil.

Bacteria↗
PubMed1995

Aspartyl-tRNA synthetase of the hyperthermophilic archaeon Pyrococcus sp. KOD1 has a chimerical structure of eukaryotic and bacterial enzymes.

The aspartyl-tRNA synthetase (AspRS)-encoding gene from the archaeon, Pyrococcus sp. KOD1 (KOD1), was cloned and sequenced, and expressed in Escherichia coli. The purified AspRS possessed an aminoacyation activity for tRNA extracted from KOD1. Analysis of the deduced amino-acid sequence (438 aa, 50,893 Da) revealed that the AspRS of KOD1 is a chimerical protein of bacteria and eukarya. Regional analysis showed high sequence similarity to higher eukaryotic enzymes in the central and C-terminal regions which are important for catalytic activity of the enzyme. In contrast, the N-terminal portion exhibits bacterial features and does not possess the higher eukaryotic sequence which is involved in high molecular weight (HMW) complex formation. These results suggest that archaeon AspRS has a eukaryotic-type catalytic mechanism without forming the HMW complex. This is the first example which shows that an archaeal protein possesses eukaryotic and bacterial features.

Amino Acid Sequence↗
PubMed2005

Cloning, bacterial expression, purification and structural characterization of N-terminal-repetitive domain of gamma-Gliadin.

The gene encoding the repetitive domain located in the N-terminal half of gamma-Gliadin from wheat endosperm has been subcloned into a thioredoxin expression system (pET102/D-Topo). It was over-expressed as fusion protein with thioredoxin in Escherichia coli. Thioredoxin was removed by enterokinase cleavage or by acid cleavage at the respective engineered recognition sites. The soluble N-terminal half of gamma-Gliadin was purified by affinity and reverse-phase chromatography. While, the enterokinase cleavage leaded to only one species detectable by mass spectroscopy, the acid cleavage resulted in a three different length polypeptides, due to the presence of the same number of acid cleavage sites. The secondary structure of the purified protein domain was analysed by circular dichroism, showing an spectral shape common to a Poly(Pro) II conformation. The spectrum is dominated by a large negative peak centred around 201 nm and a broad shoulder centred around 225 nm. Also, the temperature denaturation process was studied. The differences observed in the spectra show two main tendencies, the increment of the shoulder intensity, and the drop of the intensity of the peak around 201. When the sample was cooled down, the change on intensity of the shoulder around 225 was completely reversible and that around the 201 nm peak reached a reversibility of 90%. Such structure and thermal behaviour are characteristic of the repetitive domains of the wheat prolamins.

Chromatography, High Pressure Liquid↗
PubMed2002

The effects of endometrial scarification on uterine steroid receptors, bacterial flora and histological structure in the bitch.

Following laparotomy, the endometrium of six nulliparous Beagle bitches was scarified at the base of one uterine horn during early metoestrus, when the peripheral plasma P(4) concentration was >10 ng/ml; intrauterine swabs were taken at the same time for bacteriological culture. Twenty-one days later, a bilateral ovariohysterectomy was performed and segments of the scarified and non-scarified parts of the tubular genital tract removed; at the same time, swabs were taken from the uterine lumen. Tissue samples were collected and examined for histopathological structure, and the presence of nuclear oestrogen (ER) and progesterone (PR) receptors using an immunocytochemical method. The immunoreactivity was scored semiquantitatively, incorporating both the intensity and distribution of specific staining of the receptors using a simplified histoscore (H-score). All uterine swabs were sterile, and in three of the six bitches there were noticeable changes with distension of the uterine lumen with secretions and debris and distension of the endometrial gland ducts of the scarified uterine segment. There were no statistically significant differences in the H-scores of ER or PR between scarified and non-scarified segments, except for PR H-scores in the glandular epithelium where the values for the scarified were significantly higher than for the non-scarified endometrium (mean+/-S.E.M. is 129.9+/-22.8 versus 59.5+/-12.6; P<0.05). Thus, trauma can modify the structure of the endometrium and the characteristics of the PR. Whether changes in PR expression are involved in the pathogenesis of CEH/pyometra in the bitch could not be ascertained from this study.

Animals↗
PubMed1991

Primary donor structure and interactions in bacterial reaction centers from near-infrared Fourier transform resonance Raman spectroscopy.

Preresonance Raman and resonance Raman spectra of the primary donor (P) from reaction centers of the Rhodobacter (Rb.) sphaeroides R26 carotenoidless strain in the P and P+ states, respectively, were obtained at room temperature with 1064-nm excitation and a Fourier transform spectrometer. These spectra clearly indicate that the chromophore modes are observable over those of the protein with no signs of interference below 1800 cm-1. The chromophore modes are dominated by those of the bacteriochlorophylls (BChl a), and it is estimated that, in the P state, ca. 65% of the Raman intensity of the BChl a modes arises from the primary donor. This permits the direct observation of a vibrational spectrum of the primary donor at preresonance with the excitonic 865-nm band. The Raman spectrum of oxidized reaction centers in the presence of ferricyanide clearly exhibits bands arising from a BChl a+ species. The magnitude of the frequency shift of a keto carbonyl of neutral P from 1691 to 1717 cm-1 upon P+ formation strongly suggests that one BChl molecule in P+ carries nearly the full +1 charge. Our results indicate that the unpaired electron in P.+ does not share a molecular orbital common to the two components of the dimer on the time scale of the resonance Raman effect (ca. 10(-13) s).

Fourier Analysis↗