[Chemical structure of bacterial cell wall. Determination of molecular structure of peptides-glycan in cell wall].
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Amino Sugars↗
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High level bacterial expression of uteroglobin, a dimeric eukaryotic protein with two interchain disulfide bridges, in its natural quaternary structure.
Bacterial expression of eukaryotic proteins is a tool of ever-increasing importance in biochemistry and molecular biology. However, the majority of the recombinant eukaryotic proteins that have been expressed in bacteria are produced as fusion proteins and not in their native conformation. In particular, correct formation of quaternary structures by recombinant proteins in bacterial hosts has been reported very rarely. To our knowledge, correct intracellular formation of multimeric structures containing more than one interchain disulfide bridge has not been reported so far. We have constructed three plasmids which are able to direct expression of recombinant rabbit uteroglobin, a homodimeric protein with two interchain disulfide bridges, in Escherichia coli. Among these, the plasmid pLE103-1, in which the expression of recombinant uteroglobin is controlled by a bacteriophage T7 late promoter, is by far the most efficient. With pLE103-1, recombinant uteroglobin production reached about 10% of total bacterial soluble proteins. This protein accumulated in bacterial cells in dimeric form, as it is naturally found in the rabbit uterus. Recombinant uteroglobin was purified to near-homogeneity and its NH2-terminal amino acid sequence was confirmed to be identical to that of its natural counterpart, except for 2 Ala residues the codons for which were added during the plasmid construction. This protein was found to be as active a phospholipase A2 inhibitor as natural uteroglobin on a molar basis. To our knowledge, this is the first report of high level bacterial expression of a full length eukaryotic homodimeric protein with two interchain disulfide bridges in its natural, biologically active form. The plasmid pLE103-1 may be useful to explore structure-function relationships of rabbit uteroglobin. In addition, this plasmid may be useful in obtaining high level bacterial expression of other eukaryotic proteins with quaternary structure, as well as for other general applications requiring efficient bacterial expression of cDNAs.
Animals↗
[Chemical structure of bacterial cell wall. 14. Determination of molecular structure of peptide-glycan of the cell wall].
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Amino Acids↗
Characterization of bacterial community structure on a weathered pegmatitic granite.
This study exploited the contrasting major element chemistry of a pegmatitic granite to investigate mineralogical influences on bacterial community structure. Intact crystals of variably weathered muscovite, plagioclase, K-feldspar, and quartz were extracted, together with whole-rock granite. Environmental scanning electron microscopy revealed a diversity of bacterial structures, with rods and cocci clearly visible on surfaces of all mineral types. Bacterial automated ribosomal intergenic spacer analysis was used to generate a ribotype profile for each mineral. A randomization test revealed that community fingerprints differed between different mineral types, whereas canonical correspondence analysis (CCA) showed that mineral chemistry affected individual bacterial ribotypes. CCA also revealed that Al, Si, and Ca had a significant impact on bacterial community structure within the system, which contrasts with the finding within fungal communities that although Al and Si also had a significant impact, K rather than Ca was important. The bacterial populations associated with different minerals were different. Members of each of these populations were found almost exclusively on a single mineral type, as was previously reported for fungal populations. These results show that bacterial community structure was driven by the chemical composition of minerals, indicating selective pressure by individual chemical elements on bacterial populations in situ.
Bacteria↗
[Methods in study of chemical structure of bacterial cell wall. (12). Determination of molecular structure of cell wall peptide-glycan].
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Cell Wall↗
[Chemical structure of bacterial cell wall. 8. Determination of molecular structure of peptide-glycan in the cell wall].
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Acids↗
Bacterial community structures in MBRs treating municipal wastewater: relationship between community stability and reactor performance.
Bacterial community structures in pilot-scale conventional membrane bioreactors (CMBRs) and hybrid MBRs (HMBRs) which were combined with pre-coagulation/sedimentation were analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and fluorescence in situ hybridization (FISH) techniques. The results were compared with the community structure in a full-scale activated sludge (AS) process treating the same municipal wastewater. The Dice index (Cs) of similarity analysis of DGGE banding patterns demonstrated that the microbial community in AS was more similar to those in CMBR1 and CMBR2 than HMBR1 and HMBR2. This suggested that influent wastewater composition had a larger impact on bacterial community structures. Long-term community structure changes in the HMBRs and CMBRs were monitored and analyzed over 240 days by Non-metric multidimensional scaling (NMDS) analysis of DGGE banding patterns. The NMDS analysis revealed that both HMBRs and CMBRs had marked changes in community structures during the first about 100 days. Thereafter the perpetual fluctuations of bacterial community structures were observed in both HMBRs and CMBRs, even though the stable MBR performances (the performance was measured as membrane permeability and removal of dissolved organic carbon, DOC) were achieved. These results suggest that not only the stability, but also the adequate dynamics ("flexibility") of the bacterial community structure are important for the stable performance of the MBRs treating complex municipal wastewater.
Bacteria↗
A new bacterial flagellar structure found in campylobacters.
A previously unrecognized bacterial flagellar structure present in Campylobacter jejuni and Campylobacter fetus is described. The structure is located just below the insertion of the flagellar apparatus through the bacterial wall. It is disc shaped, approximately 100 nm in diameter and has eleven spokes radiating from a central hole. The function of this new structure and its possible importance in the taxonomy of campylobacters and other spiral micro-organisms are discussed.
Campylobacter fetus↗
Bacterial community structure and diversity in a century-old manure-treated agroecosystem.
Changes in soil microbial community structure and diversity may reflect environmental impact. We examined 16S rRNA gene fingerprints of bacterial communities in six agroecosystems by PCR amplification and denaturing gradient gel electrophoresis (PCR-DGGE) separation. These soils were treated with manure for over a century or different fertilizers for over 70 years. Bacterial community structure and diversity were affected by soil management practices, as evidenced by changes in the PCR-DGGE banding patterns. Bacterial community structure in the manure-treated soil was more closely related to the structure in the untreated soil than that in soils treated with inorganic fertilizers. Lime treatment had little effect on bacterial community structure. Soils treated with P and N-P had bacterial community structures more closely related to each other than to those of soils given other treatments. Among the soils tested, a significantly higher number of bacterial ribotypes and a more even distribution of the bacterial community existed in the manure-treated soil. Of the 99 clones obtained from the soil treated with manure for over a century, two (both Pseudomonas spp.) exhibited 100% similarity to sequences in the GenBank database. Two of the clones were possible chimeras. Based on similarity matching, the remaining 97 clones formed six major clusters. Fifty-six out of 97 were assigned taxonomic units which grouped into five major taxa: alpha-, beta-, and gamma-Proteobacteria (36 clones), Acidobacteria (16 clones), Bacteroidetes (2 clones), Nitrospirae (1 clone), and Firmicutes (1 clone). Forty-one clones remained unclassified. Results from this study suggested that bacterial community structure was closely related to agroecosystem management practices conducted for over 70 years.
Agriculture↗
Structural diversity of bacterial communities in a heavy metal mineralized granite outcrop.
This laboratory study of a variably mineralized and hydrothermally altered granite outcrop investigated the influences of rock-surface chemistry and heavy metal content on resident bacterial populations. Results indicated that elevated heavy metal concentrations had a profound impact on bacterial community structure, with strong relationships found between certain ribotypes and particular chemical/heavy metal elements. Automated ribosomal intergenic sequence analysis (ARISA) was used to assess the nature and extent of bacterial diversity, and this was combined with chemical analysis and multivariate statistics to identify the main geochemical factors influencing bacterial community structure. A randomization test revealed significant changes in bacterial structure between samples, while canonical correspondence analysis (CCA) related each individual ARISA profile to linear combinations of the chemical variables (mineralogy, major element and heavy metal concentrations) revealing the geochemical factors that correlated with changes in the ARISA data. anova was performed to further explore interactions between individual ribotypes and chemical/heavy metal composition, and revealed that a high proportion of ribotypes correlated significantly with heavy metals.
Bacteria↗
Size Selective Feeding in Cyclidium glaucoma (Ciliophora, Scuticociliatida) and Its Effects on Bacterial Community Structure: A Study from a Continuous Cultivation System.
Three aspects of size selective feeding by the scuticociliate Cyclidium glaucoma were studied in continuous cultivation systems. Firstly, grazing-induced changes in abundance, biomass, and size structure of a bacterial community were investigated. Secondly, we studied possible grazing-protection mechanisms of bacteria as a response to permanent presence of the predator. And finally, we were looking for potential feedback mechanisms within this predator-prey relationship, i.e., how the ciliate population reacted to a changed, more grazing-protected bacterial community. The first stage of the cultivation system consisted of the alga Cryptomonas sp. and the accompanying mixed bacterial community. These organisms were transferred to two second stage vessels, a control stage without ciliates and a second one inoculated with C. glaucoma. After the first week, the abundance of bacteria in the latter decreased by 60% and remained stable until the end of the experiment (65 d), whereas bacterial biomass was less affected (393 mg C L-1 during days 0-7, 281 mg C L-1 afterwards). The mean bacterial cell volume doubled from 0.089 mm3 to 0.167 mm3, which was mainly due to increasing cell widths. During the whole investigation period formation of colonies or filaments was not observed, but we found a clear feedback of ciliates on bacterial size. An increase in bacterial cell volume was always followed by a decline of the predator population, resulting in a yet undescribed type of microbial predator-prey relation. Literature and our own data on the optimal food size range grazed by C. glaucoma showed that bacterial cell width rather than length was responsible for that observed phenomenon. Finally, we suggest that uptake rates of spherical latex beads give only limited information on truly ingestible prey volumes and that prey geometry should be considered in future studies on size selective feeding of protists.
Journal Article↗
Ranking the magnitude of crop and farming system effects on soil microbial biomass and genetic structure of bacterial communities.
Biological soil characteristics such as microbial biomass, community structures, activities, and functions may provide important information on environmental and anthropogenic influences on agricultural soils. Diagnostic tools and detailed statistical approaches need to be developed for a reliable evaluation of these parameters, in order to allow classification and quantification of the magnitude of such effects. The DOK long-term agricultural field experiment was initiated in 1978 in Switzerland for the evaluation of organic and conventional farming practices. It includes three representative Swiss farming systems with biodynamic, bio-organic and conventional fertilization and plant protection schemes along with minerally fertilized and unfertilized controls. Effects on microbial soil characteristics induced by the long-term management at two different stages in the crop rotation, i.e. winter wheat after potato or corn, were investigated by analyzing soil bacterial community structures using analysis of PCR-amplified rRNA genes by terminal restriction fragment length polymorphism and ribosomal intergenic spacer analysis. Application of farmyard manure consistently revealed the strongest influence on bacterial community structures and biomass contents. Effects of management and plant protection regimes occurred on an intermediate level, while the two stages in the crop rotation had a marginal influence that was not significant.
Agriculture↗
[A method of preparing specimens for the electron microscopic study of bacterial fimbrial structures].
A method for making bacterial preparations for studies of fimbrial structures by electron microscopy has been developed, making use of impressions of 'young' colonies in agar microscopy. The suggested method involves three steps: cultivation of the examined bacteria in lamellar media till they attain the 'young' age; agar microscopy of the grown culture and making of impressions from the selected sites; contrasting of the objects and drying of the preparations. The new method is simple, economic, highly reliable and yields good results. It maximally rules out the factors conducive to rupture and injury of fimbria, observed in traditional method of making the preparations.
Bacteriological Techniques↗
[Chemical structure of bacterial cell walls. 9. Determination of the molecular structure of peptide-glycan in the cell wall].
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Amino Acids↗
The SAXS solution structure of RF1 differs from its crystal structure and is similar to its ribosome bound cryo-EM structure.
Bacterial class I release factors (RFs) are seen by cryo-electron microscopy (cryo-EM) to span the distance between the ribosomal decoding and peptidyl transferase centers during translation termination. The compact conformation of bacterial RF1 and RF2 observed in crystal structures will not span this distance, and large structural rearrangements of RFs have been suggested to play an important role in termination. We have collected small-angle X-ray scattering (SAXS) data from E. coli RF1 and from a functionally active truncated RF1 derivative. Theoretical scattering curves, calculated from crystal and cryo-EM structures, were compared with the experimental data, and extensive analyses of alternative conformations were made. Low-resolution models were constructed ab initio, and by rigid-body refinement using RF1 domains. The SAXS data were compatible with the open cryo-EM conformation of ribosome bound RFs and incompatible with the crystal conformation. These conclusions obviate the need for assuming large conformational changes in RFs during termination.
Cryoelectron Microscopy↗
Application of the use of high-throughput technologies to the determination of protein structures of bacterial and viral pathogens.
The Structural Proteomics In Europe (SPINE) programme is aimed at the development and implementation of high-throughput technologies for the efficient structure determination of proteins of biomedical importance, such as those of bacterial and viral pathogens linked to human health. Despite the challenging nature of some of these targets, 175 novel pathogen protein structures (approximately 220 including complexes) have been determined to date. Here the impact of several technologies on the structural determination of proteins from human pathogens is illustrated with selected examples, including the parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens.
Animals↗
Effect of sheep urine deposition on the bacterial community structure in an acidic upland grassland soil.
The effect of the addition of synthetic sheep urine (SSU) and plant species on the bacterial community composition of upland acidic grasslands was studied using a microcosm approach. Low, medium, and high concentrations of SSU were applied to pots containing plant species typical of both unimproved (Agrostis capillaris) and agriculturally improved (Lolium perenne) grasslands, and harvests were carried out 10 days and 50 days after the addition of SSU. SSU application significantly increased both soil pH (P < 0.005), with pH values ranging from pH 5.4 (zero SSU) to pH 6.4 (high SSU), and microbial activity (P < 0.005), with treatment with medium and high levels of SSU displaying significantly higher microbial activity (triphenylformazan dehydrogenase activity) than treatment of soil with zero or low concentrations of SSU. Microbial biomass, however, was not significantly altered by any of the SSU applications. Plant species alone had no effect on microbial biomass or activity. Bacterial community structure was profiled using bacterial automated ribosomal intergenic spacer analysis. Multidimensional scaling plots indicated that applications of high concentrations of SSU significantly altered the bacterial community composition in the presence of plant species but at different times: 10 days after application of high concentrations of SSU, the bacterial community composition of L. perenne-planted soils differed significantly from those of any other soils, whereas in the case of A. capillaris-planted soils, the bacterial community composition was different 50 days after treatment with high concentrations of SSU. Canonical correspondence analysis also highlighted the importance of interactions between SSU addition, plant species, and time in the bacterial community structure. This study has shown that the response of plants and bacterial communities to sheep urine deposition in grasslands is dependent on both the grass species present and the concentration of SSU applied, which may have important ecological consequences for agricultural grasslands.
Agrostis↗
Bacterial community structure analyses to assess pollution of water and sediments in the Lake Shkodra/Skadar, Balkan Peninsula.
GOAL, SCOPE AND BACKGROUND: Lake Skadar is the largest lake of the Balkan Peninsula, located along the Montenegro-Albanian border. The unique features of the lake and wide range of endemic, and rare or endangered plant and animal species, resulted in the classification of the Skadar as a wetland site of international significance. In spite of its importance, the lake is influenced by inflowing waters from the river Moraca and other regional rivers contaminated by the industry, municipal and agricultural activities in the area. Therefore, the lake has been the subject of various physical, chemical, biological and toxicological examinations. However, community-level analyses are most relevant to assess the effect of stressors on aquatic ecosystems. In the present study, bacterial community structure among differently polluted sites of the lake was compared using a genetic fingerprinting technique. METHODS: Water and sediment samples were collected from five differently polluted sampling sites on the Lake Skadar in spring and autumn of the same year. The bacterial community structure in the samples was characterized and compared by temporal temperature gel electrophoresis (TTGE) analysis of polymerase chain reaction-amplified bacterial 16S rRNA genes. RESULTS AND DISCUSSION: The TTGE analysis resulted in many distinguishable and reproducible band patterns, allowing a reliable comparison of bacterial communities among sampling sites. Results on the bacterial community structure revealed that three of the selected locations can be considered as sites that have not shown any pollution degradation determined by our method, due to similar structure of a bacterial community in the sediment samples. On the other hand, significant shifts in the bacterial community structure in the mouth of the rivers Moraca and Plavnica were shown. Since the results coincide with some of the bioassays and chemical analysis performed previously, the changes in bacterial community structure are explained as an effect of antropogenic pollution on the lake ecosystem by waters of the river Moraca and the stream Plavnica. CONCLUSION: The TTGE has proven to be an efficient and reliable method to monitor bacterial dynamics and community shifts in aquatic environments, especially in sediments. Within the variety of environmental quality assessments, the use of TTGE analyses from bacterial communities is strongly recommended, particularly as an initial investigation. However, in any conclusion on the state of the environment, the TTGE results should be combined to some other biological, chemical and hydrological data. RECOMMENDATION AND OUTLOOK: Since prokaryotes are a crucial group of organisms in the biosphere, the ecosystem function studies are largely based on bacterial communities. Therefore, bacterial community structure analysis should be a part of an integrated weight of evidence approach in pollution assessment. In case of Triad approach, consisting of chemical analyses, bioassays, and community studies in the field, the TTGE bacterial community structure analyses should be placed in the later Triad leg. In comparison to other community studies, based on various biotic indices, the TTGE bacterial community analysis has proven to be very sensitive, reliable and less time consuming.
Albania↗