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PubMed2006

Protein extraction and fingerprinting optimization of bacterial communities in natural environment.

Recent development in molecular approaches allows access to genetic structure and diversity of indigenous microbial communities. In contrast, the functional analysis of microorganisms in their environment is still hampered by methodological limitations. Analysis of total proteins expressed at the whole community level (metaproteome) has been proposed to characterize the functional structure of microbial communities in their environment. However, developments are still required to perform such analysis. Our aim was to optimize methods to extract and characterize metaproteome of indigenous microbial communities. Experiments were first conducted in monoxenic bacterial cultures, and various methods were examined to define a procedure of protein extraction ensuring an efficient recovery regardless of the taxonomic affiliation of the cells. These developments were next applied to characterize the metaproteome from indigenous bacterial communities in freshwater samples. Bacterial cells were recovered from water using a high-speed density gradient centrifugation method before protein extraction and fingerprinting. The reactivity and sensitivity of this metaproteomic approach were tested by analyzing the variations of protein fingerprints according to perturbations (cadmium or mercury contamination). The genetic structure of the corresponding communities was also characterized by automated ribosomal spacer analysis (ARISA) DNA fingerprinting. Both protein and DNA fingerprints were statistically analyzed. Results obtained showed that the method developed for protein recovery and fingerprinting was efficient, sensitive, and reproducible. Both the functional and genetic structures of the freshwater bacterial community were complex and varied with perturbations. These variations occurred at both population and protein expression levels and were specific to the perturbation applied.

Bacteria↗

PubMed2005

Proteogenomic approaches for the molecular characterization of natural microbial communities.

At the present time we know little about how microbial communities function in their natural habitats. For example, how do microorganisms interact with each other and their physical and chemical surroundings and respond to environmental perturbations? We might begin to answer these questions if we could monitor the ways in which metabolic roles are partitioned amongst members as microbial communities assemble, determine how resources such as carbon, nitrogen, and energy are allocated into metabolic pathways, and understand the mechanisms by which organisms and communities respond to changes in their surroundings. Because many organisms cannot be cultivated, and given that the metabolisms of those growing in monoculture are likely to differ from those of organisms growing as part of consortia, it is vital to develop methods to study microbial communities in situ. Chemoautotrophic biofilms growing in mine tunnels hundreds of meters underground drive pyrite (FeS(2)) dissolution and acid and metal release, creating habitats that select for a small number of organism types. The geochemical and microbial simplicity of these systems, the significant biomass, and clearly defined biological-inorganic feedbacks make these ecosystem microcosms ideal for development of methods for the study of uncultivated microbial consortia. Our approach begins with the acquisition of genomic data from biofilms that are sampled over time and in different growth conditions. We have demonstrated that it is possible to assemble shotgun sequence data to reveal the gene complement of the dominant community members and to use these data to confidently identify a significant fraction of proteins from the dominant organisms by mass spectrometry (MS)-based proteomics. However, there are technical obstacles currently restricting this type of "proteogenomic" analysis. Composite genomic sequences assembled from environmental data from natural microbial communities do not capture the full range of genetic potential of the associated populations. Thus, it is necessary to develop bioinformatics approaches to generate relatively comprehensive gene inventories for each organism type. These inventories are critical for expression and functional analyses. In proteomic studies, for example, peptides that differ from those predicted from gene sequences can be measured, but they generally cannot be identified by database matching, even if the difference is only a single amino acid residue. Furthermore, many of the identified proteins have no known function. We propose that these challenges can be addressed by development of proteogenomic, biochemical, and geochemical methods that will be initially deployed in a simple, natural model ecosystem. The resulting approach should be broadly applicable and will enhance the utility and significance of genomic data from isolates and consortia for study of organisms in many habitats. Solutions draining pyrite-rich deposits are referred to as acid mine drainage (AMD). AMD is a very prevalent, international environmental problem associated with energy and metal resources. The biological-mineralogical interactions that define these systems can be harnessed for energy-efficient metal recovery and removal of sulfur from coal. The detailed understanding of microbial ecology and ecosystem dynamics resulting from the proposed work will provide a scientific foundation for dealing with the environmental challenges and technological opportunities, and yield new methods for analysis of more complex natural communities.

Ecosystem↗

PubMed2025

A deep metagenomic atlas of Qinghai-Xizang Plateau lakes reveals their microbial diversity and salinity adaptation mechanisms.

The Qinghai-Xizang Plateau (QXP), harboring the planet's highest density of plateau lakes, offers an exceptional biogeographic environment for studying extremophilic microbial communities and their adaptation to salinity. Through deep metagenomic sequencing, we construct the Qinghai-Xizang Lake Sediment Genome (QXLSG) catalog, a high-resolution genomic catalog comprising 5,866 metagenome-assembled genomes (MAGs), 58.16 million non-redundant protein encoding genes, and 19,008 biosynthetic gene clusters. Notably, 80.78% of the 2,742 species-level MAGs represent undescribed taxa, significantly expanding the known microbial diversity. Salinity emerges as the primary environmental factor influencing microbial community. Functional annotation highlights that the "salt-out" strategy, particularly the uptake of glycine betaine, is the main mechanism for salinity tolerance. This strategy is prevalent in both hypersaline lake communities and the dominant microbial phyla. Overall, this study provides a crucial genetic resource for future bioprospecting and deepens our understanding of the fundamental mechanisms of microbial adaptation to extreme saline environments.

Lakes↗

PubMed2006

mRNA-based parallel detection of active methanotroph populations by use of a diagnostic microarray.

A method was developed for the mRNA-based application of microbial diagnostic microarrays to detect active microbial populations. DNA- and mRNA-based analyses of environmental samples were compared and confirmed via quantitative PCR. Results indicated that mRNA-based microarray analyses may provide additional information on the composition and functioning of microbial communities.

Ecosystem↗

PubMed1996

Econutrition and health maintenance--a new concept to prevent GI inflammation, ulceration and sepsis.

The nutritional needs of the gastrointestinal mucosa per se has until today been largely neglected. It is a rather novel finding, that the lower part of the digestive tract is most dependent on luminal nutrition for maintaining its integrity, structure and function. Even the most complete parenteral nutrition (PN) regimen cannot, in the absence of adequate enteral nutrition (EN), fully prevent the development of mucosal atrophy in the lower part of the digestive tract, especially the colon. Nor can PN prevent the downregulation of the colon's many important functions. Increased microbial translocation and a predisposition to sepsis are consequences of inadequate luminal nutrition. Such developments can only be prevented by oral feeding and the local 'manufacturing' of essential nutrients in the colon. Probiotic bacteria are also important, especially with respect to the function of the colonic mucosa, which is the focus of this review.

Journal Article↗

PubMed2005

Epithelial cells and their neighbors. IV. Bacterial contributions to intestinal epithelial barrier integrity.

Mammalian intestinal surfaces are in constant and intimate contact with a vast consortium of indigenous commensal bacteria. As a result, gut epithelia have evolved an array of strategies for limiting bacterial invasion into deeper tissues, helping to preserve the mutually beneficial nature of intestinal host-microbial relationships. In this review, we discuss a growing body of evidence indicating that commensal bacteria are actively involved in shaping the very barriers that confine them to the gut lumen. By modulating epithelial inflammatory responses, antimicrobial protein expression, and tissue repair functions, indigenous microbial populations are essential for the maintenance of healthy mucosal surfaces.

Animals↗

PubMed2006

Microbial activities in soils of a former sawmill area.

To find out microbial metabolic functioning and toxicity in a former sawmill area, carbon dioxide evolution, methane oxidation potential, 10 hydrolytic enzyme activities, Vibrio fischeri test, fluorescein diacetate hydrolysis activity (FDA), soil pH, carbon, nitrogen and pentachlorophenol (PCP) content were measured at four sites. The area is contaminated with aged chlorophenols. Chlorophenol content of soil was analyzed with a novel HPLC-MS technique, which allowed to measure chlorophenols without derivatization. The sites had a pollution gradient from 0.5 to 15 microg PCP g dw of soil(-1). Endogenous carbon dioxide evolution, methane oxidation potential, butyrate-esterase, acetate-esterase, sulphatase and aminopeptidase activities were lower at the site 2 than 3, although the site 2 and 3 had similar content of carbon and nitrogen. The soil was toxic in V. fischeri test at the site 2, which had high content of PCP (3.93+/-1.00 microg PCP g dw of soil(-1)). The results indicated that endogenous carbon dioxide evolution, methane oxidation potential, butyrate-esterase, acetate-esterase, sulphatase and aminopeptidase activities were sensitive to PCP in the soil. The results indicated that alpha-glucosidase, beta-glucosidase, beta-xylosidase, beta-cellobiosidase, phosphomonoesterase, N-acetyl-glucosaminidase activity and FDA hydrolysis activity were not sensitive to PCP in the soil. Soil processes involved in the cycling of carbon, nitrogen, sulphur and phosphorus were only slightly vulnerable in the former sawmill area and most sensitive microbial species were probably replaced with more tolerant ones to maintain and recover functioning of the former sawmill soils.

Acetylesterase↗

PubMed1995

Do de-N-glycosylation enzymes have an important role in plant cells?

In this review de-N-glycosylation was defined as the removal of the glycan(s) from a N-glycosylprotein, by means of enzymes acting on the di-N-acetylchitobiosyl part of the invariant pentasaccharide inner-core of N-glycosylproteins. Peptide-N4-(N-acetyl-beta-D-glucosaminyl) asparagine amidases (PNGase) and endo-N-acetyl-beta-D-glucosaminidases (ENGase) were both considered as de-N-glycosylation enzymes. A detailed description of the characterization and the function of plant PNGases and ENGases is presented, together with a brief presentation on the occurrence and the current knowledge on the function of microbial and animal enzymes. De-N-glycosylation of plant glycoproteins was proposed as a possible mechanism for the release of oligosaccharides displaying biological activities and the removal of N-glycans could also explain the regulation of protein activity. Each enzyme seems to have a specific function during germination and post-germinative development. All the arguments concur that de-N-glycosylation enzymes have an important role in plant cells and confirm that the N-glycosylation/de-N-glycosylation system should occur more commonly than presently recognized in living organisms.

Amidohydrolases↗

PubMed2002

Statistical analysis of denaturing gel electrophoresis (DGE) fingerprinting patterns.

Technical developments in molecular biology have found extensive applications in the field of microbial ecology. Among these techniques, fingerprinting methods such as denaturing gel electrophoresis (DGE, including the three options: DGGE, TGGE and TTGE) has been applied to environmental samples over this last decade. Microbial ecologists took advantage of this technique, originally developed for the detection of single mutations, for the analysis of whole bacterial communities. However, until recently, the results of these high quality fingerprinting patterns were restricted to a visual interpretation, neglecting the analytical potential of the method in terms of statistical significance and ecological interpretation. A brief recall is presented here about the principles and limitations of DGE fingerprinting analysis, with an emphasis on the need of standardization of the whole analytical process. The main content focuses on statistical strategies for analysing the gel patterns, from single band examination to the analysis of whole fingerprinting profiles. Applying statistical method make the DGE fingerprinting technique a promising tool. Numerous samples can be analysed simultaneously, permitting the monitoring of microbial communities or simply bacterial groups for which occurrence and relative frequency are affected by any environmental parameter. As previously applied in the fields of plant and animal ecology, the use of statistics provides a significant advantage for the non-ambiguous interpretation of the spatial and temporal functioning of microbial communities.

Bacteria↗

PubMed2001

In search of natural substrates and inhibitors of MDR pumps.

The function of microbial MDRs remains a hotly debated subject. Given the very broad substrate specificities of some MDRs, like the RND pumps that can extrude all classes of amphipathic compounds (cationic, neutral, and anionic), it seems difficult to develop a rationale for pinpointing possible natural substrates of these translocases. At the same time, several clues can be used to guide our search for natural MDR substrates. One is the fact that amphipathic cations appear to be the preferred substrates of MDRs. These substances are extruded by MDRs of all 5 known families and are the almost exclusive substrates of SMR and MF family MDRs. The universal nature of amphipathic cations as MDR substrates suggests that these were the substances that fueled the evolution of MDR pumps. Two factors apparently favored this particular class of molecules for the role of original MDR substrates--need and opportunity. Unlike other substances, amphipathic cations accumulate in the cell driven by the membrane potential, which makes cations potentially the most dangerous toxins. At the same time, amphipathic cations are highly hydrated and do not permeate the membrane as readily as neutral compounds, making it feasible to design a defense based on an efflux pump. The paucity of known cationic (non-basic) antimicrobials might be a result of using MDR-expressing microbial cells for antibiotic discovery. Plant amphipathic cations, the berberine alkaloids, are good MDR substrates. The Berberis plants produce 5'-methoxyhydnocarpin-D, an MDR inhibitor that potentiates the action of berberine. It is suggested that the further evolution of MDR pumps was determined largely by the barrier function of the membrane they reside in. Thus Gram negative bacteria have an outer membrane barrier that slows the penetration of virtually all amphipathic molecules, and transenvelope MDRs of the RND and EmrAB-type extrude their substrates across this barrier. A low permeability of the cytoplasmic membrane of yeast similarly allows for the operation of broad-specificity ABC and MF MDRs. The presence of MDR sensors that regulate the expression of some MDR pumps strongly suggests that defense against external toxins is the function of these MDRs. The BmrR transcriptional activator of the MerR family induces expression of the Bmr pump in B. subtilis and is a sensor specifically designed to recognize amphipathic cations. Similarly, the OacR repressor binds chemically unrelated cations, which leads to the expression of the QacA pump in S. aureus. In E. coli, the EmrR sensor of the MarR repressor family binds unrelated neutral molecules, allowing for expression of the transenvelope EmrAB pump.

Anti-Bacterial Agents↗

PubMed2002

Microbial behavior in a heterogeneous world.

Most microorganisms are motile during at least part of their life cycle, because they need to find optimal conditions in a patchy world. The sheer volume of microorganisms in the biosphere means that their motile sensory behavior also contributes to the global transformation and cycling of matter. How microorganisms move and how they orient themselves using environmental cues are integral to understanding the complex structure and function of microbial communities, but although motility in response to external stimuli was first described more than 120 years ago, understanding of the cellular and molecular mechanisms involved has only been achieved more recently.

Anaerobiosis↗

PubMed1995

Effect of gamma-irradiation on the effector function of T lymphocytes in microbial control.

A chlamydial-specific T cell clone, capable of inhibiting the growth of infectious Chlamydia in vivo and in vitro, was employed to investigate the effect of gamma-irradiation on the ability of effector T cells to control infections. Clone 2.14-0 (CD4+), specific for the Chlamydia trachomatis biovar agent of mouse pneumonitis (MoPn), was irradiated with varying doses (0, 2.5, 5.0, 10.0, 20.0 and 40.0 Gy) and its biological functions and ability to inhibit the intraepithelial growth of MoPn were assessed. The results revealed that although gamma-irradiation drastically reduced the proliferative response of the clone to antigen, it did not affect the phenotype and the effector function of inhibiting chlamydial growth. The preservation of anti-chlamydial function after gamma-irradiation correlated with the retention of IFN-gamma and TNF-alpha secretion in response to antigenic stimulation. We conclude that the biologic functions of T cells requiring proliferation and differentiation are more likely to be adversely affected by gamma-irradiation on the short-term, but the effector functions, possibly associated with cytokines and cytolysis, may be preserved among persisting effector T cells in an irradiated individual.

Animals↗

PubMed2005

Molecular characterization of PeNhaD1: the first member of the NhaD Na+/H+ antiporter family of plant origin.

PeNhaD1 encodes a putative Na+/H+ antiporter from the salt-resistant tree Populus euphratica. It is the first characterization of a member of the NhaD type ion transporter family of plant origin. Homology searches revealed its close relation to functionally characterized microbial Na+/H+ antiporters VpNhaD and VcNhaD. Na+/H+ antiporters have proven to play a key role in salt resistance, both in plants and bacteria. Under salt stress transcript levels of PeNhaD1 were maintained only in the salt-resistant P. euphratica, but collapsed in Populus x canescens, a salt-sensitive species. To address the function of PeNhaD1, complementation studies with the salt-sensitive Escherichia coli EP432 mutant strain, lacking activity of the two Na+/H+ antiporters EcNhaA and EcNhaB were carried out. PeNhaD1 was able to restore growth of EP432 under stress imposed by up to 400 mM NaCl demonstrating its protective function. Growth rates of EP432 were always highest at pH 5.5 while growth was suppressed under salt stress at pH 7.0 and pH 8.0 suggesting that the antiporter activity is strongly pH dependent. Element analyses of EP432 cells complemented with PeNhaD1 growing under salt stress showed that salt resistance was correlated with a significant reduction in sodium accumulation. These results suggest that PeNhaD1 might play a role in the salt resistance of P. euphratica.

Adaptation, Physiological↗

PubMed2000

Microbiology of flooded rice paddies.

Flooded rice paddies are one of the major biogenic sources of atmospheric methane. Apart from this contribution to the 'greenhouse' effect, rice paddy soil represents a suitable model system to study fundamental aspects of microbial ecology, such as diversity, structure, and dynamics of microbial communities as well as structure-function relationships between microbial groups. Flooded rice paddy soil can be considered as a system with three compartments (oxic surface soil, anoxic bulk soil, and rhizosphere) characterized by different physio-chemical conditions. After flooding, oxygen is rapidly depleted in the bulk soil. Anaerobic microorganisms, such as fermentative bacteria and methanogenic archaea, predominate within the microbial community, and thus methane is the final product of anaerobic degradation of organic matter. In the surface soil and the rhizosphere well-defined microscale chemical gradients can be measured. The oxygen profile seems to govern gradients of other electron acceptors (e.g., nitrate, iron(III), and sulfate) and reduced compounds (e.g., ammonium, iron(II), and sulfide). These gradients provide information about the activity and spatial distribution of functional groups of microorganisms. This review presents the current knowledge about the highly complex microbiology of flooded rice paddies. In Section 2 we describe the predominant microbial groups and their function with particular regard to bacterial populations utilizing polysaccharides and simple sugars, and to the methanogenic archaea. Section 3 describes the spatial and temporal development of microscale chemical gradients measured in experimentally defined model systems, including gradients of oxygen and dissolved and solid-phase iron(III) and iron(II). In Section 4, the results of measurements of microscale gradients of oxygen, pH, nitrate-nitrite, and methane in natural rice fields and natural rice soil cores taken to the laboratory will be presented. Finally, perspectives of future research are discussed (Section 5).

Archaea↗

PubMed2006

Control of microbial communities by the macrofauna: a sensitive interaction in the context of extreme summer temperatures?

Climate models predict an increasing frequency of extremely hot summer events in the northern hemisphere for the near future. We hypothesised that microbial grazing by the metazoan macrofauna is an interaction that becomes unbalanced at high temperatures due to the different development of the grazing rates of the metazoans and the growth rates of the microbial community with increasing temperature. In order to test this hypothesis, we performed grazing experiments in which we measured the impact of increasing temperatures on the development of the grazing rates of riverine mussels in relation to the growth rates of a unicellular prey community (a natural heterotrophic flagellate community from a large river). In a first experimental series using Corbicula fluminea as a grazer and under the addition of a carbon source (yeast extract), the increase of the prey's growth rates was considerably stronger than that of the predator's grazing rates when temperatures were increased from 19 to over 25 degrees C. This was also the outcome when the mussels had been acclimatized to warm temperatures. Hereafter, specific experiments with natural river water at temperatures of 25 and 30 degrees C were performed. Again, a strong decrease of the mussels' grazing rates in relation to the flagellate growth rates with increasing temperature occurred for two mussel species (C. fluminea and Dreissena polymorpha). When performing the same experiment using a benthic microbial predator community (biofilms dominated by ciliates) instead of the benthic mussels, an increase of the grazing rates relative to the growth rates with temperature could be observed. Our data suggest that predator-prey interactions (between metazoans and microbes) that are balanced at moderate temperatures could become unbalanced at high temperatures. This could have significant effects on the structure and function of microbial communities in light of the predicted increasing frequency of summer heat waves.

Animals↗

PubMedDate not supplied

Microbial genomics: from sequence to function.

The era of genomics (the study of genes and their function) began a scant dozen years ago with a suggestion by James Watson that the complete DNA sequence of the human genome be determined. Since that time, the human genome project has attracted a great deal of attention in the scientific world and the general media; the scope of the sequencing effort, and the extraordinary value that it will provide, has served to mask the enormous progress in sequencing other genomes. Microbial genome sequencing, of particular interest to the community studying emerging infectious diseases, prompted the series of articles presented in the following pages. These articles review technological and scientific advances that have occurred since publication of the Haemophilus influenzae genome sequence in July 1995; that was the first demonstration that an entire genome sequence could be deciphered by a "shotgun" approach, i.e., the sequencing and assembly of random fragments of the genome. This is now the method of choice for sequencing of most other genomes, including human (as performed by Celera Genomics).

Genome, Bacterial↗

PubMed2024

Effects of initial corncob particle size on the short-term composting for preparation of cultivation substrates for Pleurotus ostreatus.

The short-term composting based on corncob for preparing Pleurotus ostreatus cultivation medium originated from agricultural production practices and so lacked systematic investigation. In this study, the influences of a Dafen (15 mm, DFT) and Xiaofen (5 mm, XFT) initial particle size (IPS) of corncob on the microbial succession and compost quality were examined. Results demonstrated that XFT compost was better suited for mushroom cultivation due to its high biological efficiency of 70 % and the absence of contamination. The composting microbes differed significantly between the DFT and XFT composts. During composting, the genera of Bacillus, Acinetobacter, Lactobacillus, Streptomyces, and Paenibacillus were majorly found in the DFT compost, while Acinetobacter, Lactobacillus, Puccinia, Bacteroides, and Bacillus genera dominated the XFT compost. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that throughout the thermophilic phase, XFT compost had much greater relative abundances of sequences relevant to energy, carbohydrate, and amino acid metabolism than DFT compost. Analysis of network correlations and Mantel tests indicated that IPS reduction could increase microbial interactions. Overall, adjusting the IPS of corncob to 5 mm increased microbial interactions, improved compost quality, and thereby boosted the P. ostreatus yield. These findings will be pertinent in optimizing the composting process of cultivation medium for P. ostreatus.

Composting↗

PubMed2002

Collision frequencies of microbial aggregates with small particles by differential sedimentation.

Collision and coagulation rates between microbial aggregates and small particles were measured for individual aggregates (1.0-2.5 mm) that settled through a suspension of fluorescent yellow-green (YG) particles (2.83 microm) placed in a settling column. The microbial aggregates, with an average fractal dimension of 2.26, were generated in a lab-scale sequencing batch reactor (SBR) and also collected from a full-scale activated sludge (AS) treatment system. As calculated from comparisons between the settling velocities observed and those predicted by Stokes' law for impermeable particles, the average fluid collection efficiencies were 0.08 for the SBR aggregates and 0.14 for the AS flocs, which were much lower than those previously reported for nonbiological aggregates of latex microspheres. The collision frequency functions between microbial aggregates and small YG particles were 2 orders of magnitude lower than predicted by the rectilinear model but 1 order of magnitude greater than predicted by a curvilinear model. The overall scavenging efficiencies of suspended particles by the falling microbial aggregates compared well with those observed for the nonbiological aggregates, while the particle removal efficiencies from the flow internal to the microbial aggregates were 1 order of magnitude higher than those of the nonbiological aggregates. It is argued that the permeability of microbial aggregates could be reduced by exopolymeric material clogging the pores within the aggregates. The internal permeation through a bioaggregate thus may not be significant enough to be included in the calculation of its settling velocity; however, the intra-aggregate flow cannot be simply neglected where coagulation is concerned. Streamlines still can penetrate the interior of microbial aggregates, allowing greater collision frequencies with other particles than predicted by the curvilinear model. The narrow and convoluted internal flow passages resulting from the collection of extracellular polymeric substances may also contribute to the higher interior particle removal efficiencies of microbial aggregates than those of more permeable, nonbiological aggregates.

Geologic Sediments↗