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Microbial diversity and activity along the forefields of two receding glaciers.

Forefields of two receding glaciers were sampled along either a 150 or 200 m long transect at identical spatial intervals for assessment of soil microbial activity and community diversity trends. The forefields belonged to the Dammaglacier (forefield area is 157 ha, 2000 m above sea level) and Rotfirnglacier (100 ha, 2200 m) and at the time of sampling were receding at an estimated rate of 8 and 10 m yr(-1) over the past 5 years, respectively. Direct counting of bacteria (DAPI staining), assessment of dehydrogenase activity (DH), and fluorescein diacetate hydrolysis activity (FDA) were performed to estimate bacteria number and soil microbial activity. Along the Dammaglacier forefield (from youngest to oldest soil), bacteria number (8.21 x 10(7) to 1.49 x 10(9) cells g(-1) soil), DH activity (0 to 61 mg TTC reduced g(-1) soil h(-1)), and FDA activity (0 to 100 mg fluorescein produced g-1 soil h-1) increased, suggesting the development of microbial populations increasing in number and activity. The Rotfirn forefield exhibited similar trends per gram of soil in bacteria number (1.13 x 10(8) to 5.93 x 10(9) cells), DH activity (0 to 36 mg TTC reduced), and FDA activity (2 to 70 mg fluorescein produced), but with more variability among samples than the Damma forefield samples. Molecular assessment of bacterial diversity included denaturing gradient gel electrophoresis (DGGE) and ribosomal intergenic spacer analysis (RISA) of soil DNA. DGGE and RISA revealed that the composition and succession of bacterial populations were different in both forefields. Comparison of Shannon diversity index values indicated that all populations sampled from the Damma forefield were significantly different (p < 0.05). Conversely, similar populations existed in the Rotfirn forefield succession. Overall, the results indicate that diverse bacterial assemblages increasing in number and activity characterize these glacier forefield soils with both forefield successions exhibiting differing modes of bacterial community establishment.

Bacteria↗

Microbial diversity of hydrothermal sediments in the Guaymas Basin: evidence for anaerobic methanotrophic communities.

Microbial communities in hydrothermally active sediments of the Guaymas Basin (Gulf of California, Mexico) were studied by using 16S rRNA sequencing and carbon isotopic analysis of archaeal and bacterial lipids. The Guaymas sediments harbored uncultured euryarchaeota of two distinct phylogenetic lineages within the anaerobic methane oxidation 1 (ANME-1) group, ANME-1a and ANME-1b, and of the ANME-2c lineage within the Methanosarcinales, both previously assigned to the methanotrophic archaea. The archaeal lipids in the Guaymas Basin sediments included archaeol, diagnostic for nonthermophilic euryarchaeota, and sn-2-hydroxyarchaeol, with the latter compound being particularly abundant in cultured members of the Methanosarcinales. The concentrations of these compounds were among the highest observed so far in studies of methane seep environments. The delta-(13)C values of these lipids (delta-(13)C = -89 to -58 per thousand) indicate an origin from anaerobic methanotrophic archaea. This molecular-isotopic signature was found not only in samples that yielded predominantly ANME-2 clones but also in samples that yielded exclusively ANME-1 clones. ANME-1 archaea therefore remain strong candidates for mediation of the anaerobic oxidation of methane. Based on 16S rRNA data, the Guaymas sediments harbor phylogenetically diverse bacterial populations, which show considerable overlap with bacterial populations of geothermal habitats and natural or anthropogenic hydrocarbon-rich sites. Consistent with earlier observations, our combined evidence from bacterial phylogeny and molecular-isotopic data indicates an important role of some novel deeply branching bacteria in anaerobic methanotrophy. Anaerobic methane oxidation likely represents a significant and widely occurring process in the trophic ecology of methane-rich hydrothermal vents. This study stresses a high diversity among communities capable of anaerobic oxidation of methane.

Anaerobiosis↗

A two-species test of the hypothesis that spatial isolation influences microbial diversity in soil.

The hypothesis that spatial isolation is a key determinant of microbial community structure in soils was evaluated by examining the competitive dynamics of two species growing on a single resource in a uniform sand matrix under varied moisture content. One species dominated the community under highly connected, saturated treatments, suggesting that these conditions allow competitive interactions to structure the community. As moisture content decreased, however, the less competitive species became established in the community. This effect was most pronounced at a matric water potential of -0.14 MPa where estimates of final population density and species fitness were equal. A second but more closely related species pair exhibited a similar response to decreasing moisture, suggesting that the effects of spatial isolation we observed are not simply a species-pair-specific phenomenon. These findings indicate that spatial isolation, created by low moisture content, plays an important role in structuring soil microbial communities.

Cupriavidus necator↗

Recombinant environmental libraries provide access to microbial diversity for drug discovery from natural products.

To further explore possible avenues for accessing microbial biodiversity for drug discovery from natural products, we constructed and screened a 5,000-clone "shotgun" environmental DNA library by using an Escherichia coli-Streptomyces lividans shuttle cosmid vector and DNA inserts from microbes derived directly (without cultivation) from soil. The library was analyzed by several means to assess diversity, genetic content, and expression of heterologous genes in both expression hosts. We found that the phylogenetic content of the DNA library was extremely diverse, representing mostly microorganisms that have not been described previously. The library was screened by PCR for sequences similar to parts of type I polyketide synthase genes and tested for the expression of new molecules by screening of live colonies and cell extracts. The results revealed new polyketide synthase genes in at least eight clones. In addition, at least five additional clones were confirmed by high-pressure liquid chromatography analysis and/or biological activity to produce heterologous molecules. These data reinforce the idea that exploiting previously unknown or uncultivated microorganisms for the discovery of novel natural products has potential value and, most importantly, suggest a strategy for developing this technology into a realistic and effective drug discovery tool.

Amino Acid Sequence↗

Molecular microbial diversity of a spacecraft assembly facility.

In ongoing investigations to map and archive the microbial footprints in various components of the spacecraft and its accessories, we have examined the microbial populations of the Jet Propulsion Laboratory's Spacecraft Assembly Facility (JPL-SAF). Witness plates made up of spacecraft materials, some painted with spacecraft qualified paints, were exposed for approximately 7 to 9 months at JPL-SAF and examined the particulate materials collected for the incidence of total cultivable aerobic heterotrophs and heat-tolerant (80 degrees C for 15-min.) spore-formers. The results showed that the witness plates coated with spacecraft qualified paints attracted more dust particles than the non-coated stainless steel witness plates. Among the four paints tested, witness plates coated with NS43G accumulated the highest number of particles, and hence attracted more cultivable microbes. The conventional microbiological examination revealed that the JPL-SAF harbors mainly Gram-positive microbes and mostly spore-forming Bacillus species. Most of the isolated microbes were heat resistant to 80 degrees C and proliferate at 60 degrees C. The phylogenetic relationships among 23 cultivable heat-tolerant microbes were examined using a battery of morphological, physiological, molecular and chemotaxonomic characterizations. By 16S rDNA sequence analysis, the isolates fell into seven clades: Bacillus licheniformis, B. pumilus, B. cereus, B. circulans, Staphylococcus capitis, Planococcus sp. and Micrococcus lylae. In contrast to the cultivable approach, direct DNA isolation, cloning and 16S rDNA sequencing analysis revealed equal representation of both Gram-positive and Gram-negative microorganisms.

Bacillus↗

[Effect of ecological organic fertilizer on tomato bacterial wilt and soil microbial diversities].

The effects of ecological organic fertilizer (EOF) on tomato bacterial wilt and soil microbial community were studied through field trial and fatty acid methyl esters (FAMEs) analysis. The results showed that the incidence rate of tomato bacterial wilt was 100% in continuous cropped soil, and significantly reduced to 39% and 50% when applying EOF at 1.0 kg.m-2 and 0.5 kg.m-2, respectively. There was no significant difference between two fertilization treatments. The fatty acid fingerprints of soil microbial community were greatly changed, and the relative contents of cyc17:0 cited as biomarker for GN bacteria, c18:2 w6 as biomarker for fungi and c16:1 11c as biomarker for arbuscular mycorrhizal (AM) fungi all increased when applying EOF. The minimum distance of cluster analysis was 1.08 between control and treatments of applying ecological organic fertilizer, and 0.70 between different fertilization treatments. Applying EOF could adjust soil microbial community structure, benefit for soil beneficial microbes, and develop stability and disease suppression of soil ecological system, and hence, improve soil quality.

Ecology↗

Microbial diversity in coastal subsurface sediments: a cultivation approach using various electron acceptors and substrate gradients.

Microbial communities in coastal subsurface sediments are scarcely investigated and have escaped attention so far. But since they are likely to play an important role in biogeochemical cycles, knowledge of their composition and ecological adaptations is important. Microbial communities in tidal sediments were investigated along the geochemical gradients from the surface down to a depth of 5.5 m. Most-probable-number (MPN) series were prepared with a variety of different carbon substrates, each at a low concentration, in combination with different electron acceptors such as iron and manganese oxides. These achieved remarkably high cultivation efficiencies (up to 23% of the total cell counts) along the upper 200 cm. In the deeper sediment layers, MPN counts dropped significantly. Parallel to the liquid enrichment cultures in the MPN series, gradient cultures with embedded sediment subcores were prepared as an additional enrichment approach. In total, 112 pure cultures were isolated; they could be grouped into 53 different operational taxonomic units (OTU). The isolates belonged to the Proteobacteria, "Bacteroidetes," "Fusobacteria," Actinobacteria, and "Firmicutes." Each cultivation approach yielded a specific set of isolates that in general were restricted to this single isolation procedure. Analysis of the enrichment cultures by PCR and denaturing gradient gel electrophoresis revealed an even higher diversity in the primary enrichments that was only partially reflected by the culture collection. The majority of the isolates grew well under anoxic conditions, by fermentation, or by anaerobic respiration with nitrate, sulfate, ferrihydrite, or manganese oxides as electron acceptors.

Actinobacteria↗

Microbial diversity in ikaite tufa columns: an alkaline, cold ecological niche in Greenland.

Ikaite tufa columns from the Ikka Fjord in south-western Greenland constitute a natural, stable environment at low temperature and with a pH ranging from neutral at the exterior to very alkaline (pH 10.4) at the interior of the column. Phylogenetic analysis of culturable organisms revealed ten different isolates representing three of the major bacterial divisions. Nine of the isolates showed 94-99% similarity to known sequences, whereas one isolate displayed a low degree of similarity (less than 90%) to a Cyclobacterium species. Seven of the isolates were shown to be cold active alkaliphiles, whereas three isolates showed optimal growth at neutral pH. Phylogenetic analysis of DNA isolated directly from the ikaite material demonstrated the presence of a microbial flora more diverse than the culturable isolates. Whereas approximately half of the phylotypes showed 90-99% similarity to known meso- or thermophilic alkaliphiles, the rest of the sequences displayed less than 90% similarity when compared to known 16S rRNA gene sequences in databases. Thus, in the present paper, we demonstrate that ikaite columns that host a specialized macroscopic flora and fauna also contain a unique, cold active, alkaliphilic microflora.

Alkalies↗

Marine microbial diversity: can it be determined?

Estimates of the order of magnitude for the total number of microbial species on Earth range from 10(3) to 10(9). Despite global dispersal of microorganisms, this number is probably rather large. The total biodiversity of an ecosystem is composed of two elements: first, a set of abundant taxa that carry out most ecosystem functions, grow actively and suffer intense losses through predation and viral lysis. These taxa are retrievable with molecular techniques but are difficult to grow in culture. Second, there is a seed bank of many rare taxa that are not growing or grow extremely slowly, do not experience viral lysis and predation is reduced. Such taxa are seldom retrieved by molecular techniques but many can be grown in culture, which explains the dictum 'everything is everywhere'.

Animals↗

Microbial diversity in ultra-high-pressure rocks and fluids from the Chinese Continental Scientific Drilling Project in China.

Microbial communities in ultra-high-pressure (UHP) rocks and drilling fluids from the Chinese Continental Scientific Drilling Project were characterized. The rocks had a porosity of 1 to 3.5% and a permeability of approximately 0.5 mDarcy. Abundant fluid and gas inclusions were present in the minerals. The rocks contained significant amounts of Fe2O3, FeO, P2O5, and nitrate (3 to 16 ppm). Acridine orange direct counting and phospholipid fatty acid analysis indicated that the total counts in the rocks and the fluids were 5.2 x 10(3) to 2.4 x 10(4) cells/g and 3.5 x 10(8) to 4.2 x 10(9) cells/g, respectively. Enrichment assays resulted in successful growth of thermophilic and alkaliphilic bacteria from the fluids, and some of these bacteria reduced Fe(III) to magnetite. 16S rRNA gene analyses indicated that the rocks were dominated by sequences similar to sequences of Proteobacteria and that most organisms were related to nitrate reducers from a saline, alkaline, cold habitat; however, some phylotypes were either members of a novel lineage or closely related to uncultured clones. The bacterial communities in the fluids were more diverse and included Proteobacteria, Bacteroidetes, gram-positive bacteria, Planctomycetes, and Candidatus taxa. The archaeal diversity was lower, and most sequences were not related to any known cultivated species. Some archaeal sequences were 90 to 95% similar to sequences recovered from ocean sediments or other subsurface environments. Some archaeal sequences from the drilling fluids were >93% similar to sequences of Sulfolobus solfataricus, and the thermophilic nature was consistent with the in situ temperature. We inferred that the microbes in the UHP rocks reside in fluid and gas inclusions, whereas those in the drilling fluids may be derived from subsurface fluids.

Atmospheric Pressure↗

Microbial diversity of cold-seep sediments in Sagami Bay, Japan, as determined by 16S rRNA gene and lipid analyses.

Microbial communities in Calyptogena sediment and microbial mats of Sagami Bay, Japan, were characterized using 16S rRNA gene sequencing and lipid biomarker analysis. Characterization of 16S rRNA gene isolated from these samples suggested a predominance of bacterial phylotypes related to Gammaproteobacteria (57-64%) and Deltaproteobacteria (27-29%). The Epsilonproteobacteria commonly found in cold seeps and hydrothermal vents were only detected in the microbial mat sample. Significantly different archaeal phylotypes were found in Calyptogena sediment and microbial mats; the former contained only Crenarchaeota clones (100% of the total archaeal clones) and the latter exclusively Euryarchaeota clones, including the anaerobic oxidation of methane archaeal groups ANME-2a and ANME-2c. Many of these lineages are as yet uncultured and undescribed groups of bacteria and archaea. Phospholipid fatty acid analysis suggested the presence of sulphate-reducing and sulphur-oxidizing bacteria. Results of intact glyceryl dialkyl glyceryl tetraether lipid analysis indicated the presence of nonthermophilic marine planktonic archaea. These results suggest that the microbial community in the Sagami Bay seep site is distinct from previously characterized cold-seep environments.

Bacteroides↗

Microbial diversity in the deep sea and the underexplored "rare biosphere".

The evolution of marine microbes over billions of years predicts that the composition of microbial communities should be much greater than the published estimates of a few thousand distinct kinds of microbes per liter of seawater. By adopting a massively parallel tag sequencing strategy, we show that bacterial communities of deep water masses of the North Atlantic and diffuse flow hydrothermal vents are one to two orders of magnitude more complex than previously reported for any microbial environment. A relatively small number of different populations dominate all samples, but thousands of low-abundance populations account for most of the observed phylogenetic diversity. This "rare biosphere" is very ancient and may represent a nearly inexhaustible source of genomic innovation. Members of the rare biosphere are highly divergent from each other and, at different times in earth's history, may have had a profound impact on shaping planetary processes.

Biodiversity↗

Monitoring of microbial diversity by fluorescence in situ hybridization and fluorescence spectrometry.

The goal of the research was the development of a simple method to quantify microbial groups in environmental samples. Fluorescence intensity was measured in the sample before and after whole cell fluorescence in situ hybridization with rRNA-targeted, fluorochrome-labeled oligonucleotide probes. To determine specific and non-specific binding of different oligonucleotide probes the following approaches have been used: (1) incubation of the sample with probes at two different temperatures; (2) hybridization of labeled probe in the presence of unlabeled probe; (3) incubation of the sample with labeled specific probe or labeled nonsense probe. Specific binding (hybridization) of the probe was calculated as the difference between total binding and non-specific binding of the probe. Specific binding was 40-50% of total binding in the environmental samples tested. The ratio of the specific binding of different probes may be used to quantify the ratio of different microbial groups in the environmental samples. This quantification is suitable for the microbiological monitoring of microbial aggregates because it is a simple technique and the results can be measured by a portable fluorometer.

Bacteria↗

Phylogenetic Analysis of Microbial Diversity in the Rhizoplane of Oilseed Rape (Brassica napus cv. Westar) Employing Cultivation-Dependent and Cultivation-Independent Approaches.

The structure of the microbial rhizoplane community of the important crop plant oilseed rape was studied by using a culture-dependent as well as a culture-independent approach based on 16S rDNA amplification. After isolation of the microbial community from the rhizoplane of oilseed rape (Brassica napus cv. Westar), the collected suspension was divided into two parts. One part was used for cultivation of bacteria onto three different growth media to establish a culture collection. From the other part of the rhizoplane suspension, genomic DNA was isolated and purified. Thereafter, 16S rDNA was amplified by PCR and cloned to obtain a library of 16S rDNA genes representative for the bacterial communities of this habitat. Phylogenetic 16S rDNA sequence analysis of 103 clones of this library revealed considerable differences from the corresponding nucleotide sequences of 111 cultured bacteria. Whereas the 16S rDNA clone library was dominated by a-Proteobacteria and bacteria of the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum (51% and 30%, respectively), less than 17% of the cultured bacteria belonged to these two groups. More than 64% of the cultivated isolates were allocated to the b- and g-subclasses of the Proteobacteria, which were present in the clone library at about 14%. Most of the clones of the a-Proteobacteria of the library showed highest similarity to Bradyrhizobium sp. No such bacteria were found in the culture collection. Similarly, the second dominant group of the clone library comprising members of the CFB phylum was represented in the culture collection by a single isolate. The phylogenetic analysis of isolates of the culture collection clearly emphasized the need to use different growth media for recovery of rhizoplane bacteria. Whereas most of the a-Proteobacteria were recovered on complex medium, most of the b-Proteobacteria were isolated onto minimal media. Our results demonstrate that the combined approach pursued in this paper is necessary to explore the biodiversity of bacterial rhizoplane communities.

Journal Article↗

A shallow BTEX and MTBE contaminated aquifer supports a diverse microbial community.

Microbial communities in subsurface environments are poorly characterized and the impacts of anthropogenic contamination on their structure and function have not been adequately addressed. The release of contaminant(s) to a previously unexposed environment is often hypothesized to decrease the diversity of the affected community. We characterized the structure of microbial communities along a gradient of benzene, toluene, ethylbenzene, and xylene (BTEX) and methyl-tert-butyl-ether (MTBE) contamination, resulting from a petroleum spill, within a shallow sandy aquifer at Vandenberg Air Force Base (VAFB) in Lompoc, CA. Differences in microbial community composition along the contaminant plume were assessed via a combinatorial approach utilizing denaturing gradient gel electrophoresis (DGGE), cloning and sequencing, intergenic transcribed spacer analysis (ITS), and comparative phylogenetic analysis of partial 16S rDNA sequences. Substantial bacterial sequence diversity, similar levels of species richness, and similar phylo-groups (including the Cytophaga-Flavobacterium-Bacteroidetes group and numerous members of the alpha-, beta-, gamma-, delta-, and epsilon-groups of the proteobacteria) were observed in both uncontaminated and contaminated regions of the aquifer. High-resolution measures (ITS fingerprinting and phylogenetic inference) readily separated communities impacted by the original petroleum spill (in source zone) from those in other parts of the aquifer and indicated that communities exposed to MTBE only were similar to communities in uncontaminated regions. Collectively, these data suggest that petroleum contamination alters microbial community structure at the species and subspecies level. Further study is required to determine whether these changes have an impact on the functioning of this subsurface ecosystem.

Bacteria↗