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Soil microorganisms, such as bacteria and fungi, play central roles in soil fertility and promoting plant health. This review examines and compares the various methods used to study microbial diversity in soil.
The use of molecular phylogenetic approaches in microbial ecology has revolutionized our view of microbial diversity at high temperatures and led to the proposal of a new kingdom within the Archaea, namely, the "Korarchaeota." We report here the occurrence of another member of this archaeal group and a deeply rooted bacterial sequence from a thermal spring in Yellowstone National Park (USA). The DNA of a mixed community growing at 83 degrees C, pH 7.6, was extracted and the small subunit ribosomal RNA gene (16S rDNA) sequences were obtained using the polymerase chain reaction. The products were cloned and five different phylogenetic types ("phylotypes") were identified: four archaeal phylotypes, designated pBA1, pBA2, pBA3, and pBA5, and only one bacterial phylotype, designated pBB. pBA5 is very closely related to the korarchaeotal phylotype, pJP27, from Obsidian Pool in Yellowstone National Park. The pBB phylotype is a lineage within the Aquificales and, based on 16S rRNA sequence, is different enough from the members of the Aquificales to constitute a different genus. In situ hybridization with bacterial-specific and Aquificales-specific fluorescent oligonucleotide probes indicated the bacterial population dominated the community and most likely contributed significantly to biogeochemical cycling within the community.
Biological methods for decontamination promise an improved substitute for ineffective and costly physico-chemical remediation methods, although so far only a fraction of the total microbial diversity (i.e. the culturable fraction with metabolic potential) has been harnessed for this purpose. Exploring and exploiting the "overlooked" genetic resource might ameliorate concerns associated with the degradation of recalcitrant and xenobiotic pollutants that are not degraded or only poorly degraded by known culturable bacteria. Recent advances in the molecular genetics of biodegradation and in knowledge-based methods of rational protein modification provide insight into the development of "designer biocatalysts" for environmental restoration. The application of such genetically engineered microorganisms (GEMs) in the environment has been limited, however, owing to the risks associated with uncontrolled growth and proliferation of the introduced biocatalyst and horizontal gene transfer. Programming rapid death of the biocatalyst soon after the depletion of the pollutant could minimize the risks in developing these technologies for successful bioremediation.
Peptide nucleic acid (PNA)-mediated PCR clamping (H. Orum, P. E. Nielsen, M. Egholm, R. H. Berg, O. Buchardt, and C. Stanley, Nucleic Acids Res. 21:5332-5336, 1993) was introduced as a novel procedure to selectively amplify ribosomal DNAs (rDNAs) which are not frequently found in clone libraries generated by standard PCR from complex microbial consortia. Three different PNA molecules were used; two of these molecules (PNA-ALF and PNA-EUB353) overlapped with one of the amplification primers, whereas PNA-1114F hybridized to the middle of the amplified region. Thus, PCR clamping was achieved either by competitive binding between the PNA molecules and the forward or reverse primers (competitive clamping) or by hindering polymerase readthrough (elongation arrest). Gene libraries generated from mixed rDNA templates by using PCR clamping are enriched for clones that do not contain sequences homologous to the appropriate PNA oligomer. This effect of PCR clamping was exploited in the following two ways: (i) analysis of gene libraries generated by PCR clamping with PNA-ALF together with standard libraries reduced the number of clones which had to be analyzed to detect all of the different sequences present in an artificial rDNA mixture; and (ii) PCR clamping with PNA-EUB353 and PNA-1114F was used to selectively recover rDNA sequences which represented recently described phylogenetic groups (NKB19, TM6, cluster related to green nonsulfur bacteria) from an anaerobic, dechlorinating consortium described previously. We concluded that PCR clamping might be a useful supplement to standard PCR amplification in rDNA-based studies of microbial diversity and could be used to selectively recover members of undescribed phylogenetic clusters from complex microbial communities.
Bacterial biofilm is a common phenomenon in both natural and engineered systems which often becomes a source of contamination and microbially influenced corrosion. It is thought that formation of biofilm in the monoculture of several bacterial species is regulated by acylhomoserine lactone (AHL) quorum-sensing signals. In this study, we investigated the microbial diversity and existence of AHL-producing and AHL-degrading bacterial species in the biofilm samples from a water reclamation system located in a tropical environment. 16S ribosomal DNA sequencing analysis indicated the presence of at least 11 bacterial species, including the frequently encountered bacterial pathogens Pseudomonas aeruginosa and Klebsiella pneumoniae, and several rare pathogens. We showed that only two groups of isolates, belonging to P. aeruginosa and Enterobacter agglomerans, produced AHL signals. We also found that three bacterial isolates, i.e., Agrobacterium tumefaciens XJ01, Bacillus cereus XJ08, and Ralstonia sp. XJ12, expressed AHL degradation enzymes. Furthermore, we showed that P. aeruginosa isolate HL43 was virulent against animal model Caenorhabditis elegans and released 2-6-fold more pyocyanin cytotoxin than P. aeruginosa strains PA01 and PA14, the two commonly used laboratory strains. These data indicate the complexity and importance of biofilm research in water reclamation.
An Aroclor 1260 (polychlorinated biphenyl, PCB)-laden soil and one heavily contaminated with polycyclic aromatic hydrocarbons (PAHs) from a secure, engineered landfill site in Québec were analyzed for microbial diversity using a clone library of the 16S rDNA sequences. Phylogenetic analysis revealed that three phyla and their major subdivisions of the domain Bacteria were highly represented in these samples despite the high pollution, particularly by PAHs. None of the 16S rDNA sequences obtained matched known sequences from cultivated bacterial species or from 16S rDNA sequences amplified directly from other environmental samples.
As the global threat of drug-resistant pathogens continues to rise, new strategies and resources are required to accelerate and advance the drug discovery process. We believe that rapid progress in metagenomics has opened up a new era in the study of marine microbial diversity that enables direct access to the genomes of numerous uncultivable microorganisms. This review outlines recent developments and future trends in metagenomics-based drug discovery in marine microbial communities and their associated chemical prosperity.
The human gut is populated with as many as 100 trillion cells, whose collective genome, the microbiome, is a reflection of evolutionary selection pressures acting at the level of the host and at the level of the microbial cell. The ecological rules that govern the shape of microbial diversity in the gut apply to mutualists and pathogens alike.
We have investigated the molecular phylogeny of cold-seep sediments obtained from the Nankai Trough, at depths of about 600, 2,000, and 3,300 m, and compared the microbial diversity profiles of those sediments samples. The gamma-Proteobacteria that might function as sulfide oxidizers and the symbiotically related delta-Proteobacteria which might function as sulfate reducers were identified amongst the bacteria from all depths of the sediments. However, anoxic methane oxidizing archaea (ANME) and methanogens were only found in the 600 m deep sediments. These results indicated that the cold-seep microbial sulfur circulation system could be functioning in the shallow seep sediment at a depth of 600 m and the microbial activities at these sites might be more dynamic than at other deeper cold-seep sites.
We analyzed the variation with depth in the composition of members of the domain Bacteria in samples from alkaline, hypersaline, and currently meromictic Mono Lake in California. DNA samples were collected from the mixolimnion (2 m), the base of the oxycline (17.5 m), the upper chemocline (23 m), and the monimolimnion (35 m). Composition was assessed by sequencing randomly selected cloned fragments of 16S rRNA genes retrieved from the DNA samples. Most of the 212 sequences retrieved from the samples fell into five major lineages of the domain Bacteria: alpha- and gamma-Proteobacteria (6 and 10%, respectively), Cytophaga-Flexibacter-Bacteroides (19%), high-G+C-content gram-positive organisms (Actinobacteria; 25%), and low-G+C-content gram-positive organisms (Bacillus and Clostridium; 19%). Twelve percent were identified as chloroplasts. The remaining 9% represented beta- and delta-Proteobacteria, Verrucomicrobiales, and candidate divisions. Mixolimnion and oxycline samples had low microbial diversity, with only 9 and 12 distinct phylotypes, respectively, whereas chemocline and monimolimnion samples were more diverse, containing 27 and 25 phylotypes, respectively. The compositions of microbial assemblages from the mixolimnion and oxycline were not significantly different from each other (P = 0.314 and 0.877), but they were significantly different from those of chemocline and monimolimnion assemblages (P < 0.001), and the compositions of chemocline and monimolimnion assemblages were not significantly different from each other (P = 0.006 and 0.124). The populations of sequences retrieved from the mixolimnion and oxycline samples were dominated by sequences related to high-G+C-content gram-positive bacteria (49 and 63%, respectively) distributed in only three distinct phylotypes, while the population of sequences retrieved from the monimolimnion sample was dominated (52%) by sequences related to low-G+C-content gram-positive bacteria distributed in 12 distinct phylotypes. Twelve and 28% of the sequences retrieved from the chemocline sample were also found in the mixolimnion and monimolimnion samples, respectively. None of the sequences retrieved from the monimolimnion sample were found in the mixolimnion or oxycline samples. Elevated diversity in anoxic bottom water samples relative to oxic surface water samples suggests a greater opportunity for niche differentiation in bottom versus surface waters of this lake.
: The Japan Trench land slope at a depth of 6,400 m is the deepest cold-seep environment with Calyptogena communities. Sediment samples from inside and beside the Calyptogena communities were collected, and the microbial diversity in the sediment samples was studied by molecular phylogenetic techniques. From DNA extracted directly from the sediment samples, 16S rDNAs were amplified by the polymerase chain reaction method. The sequences of the amplified 16S rDNAs selected by restriction fragment length polymorphism analysis were determined and compared with sequences in DNA databases. The results showed that 33 different bacterial 16S rDNA sequences from the two samples analyzed fell into similar phylogenetic categories, the alpha-, gamma-, delta-, and varepsilon-subdivisions of Proteobacteria, Cytophaga, and gram-positive bacteria; some of the 16S rDNA sequences were common to both samples. delta- and varepsilon-Proteobacteria-related sequences were abundant in both sediments. These sequences are mostly related to sulfate-reducing or sulfur-reducing bacteria and epibionts, respectively. Eight different archaeal 16S rDNA sequences were cloned from the sediments. The majority of the archaeal 16S rDNA sequences clustered in Crenarchaeota and showed high similarities to marine group I archaeal rDNA. A Methanococcoides burtonii-related sequence obtained from the sediment clustered in the Euryarchaeota indicating that M. burtonii-related strains in the area of Calyptogena communities may contribute to production of methane in this environment. From these results, we propose a possible model of sulfur circulation within the microbial community and that of Calyptogena clams in the cold-seep environment.
This paper compares two control strategies for a nitrification process. The objective is to achieve partial nitrification and thus to accumulate nitrite instead of nitrate. To this end, change in temperature setpoint and active control of oxygen and ammonia concentrations are evaluated in the long term. Evaluation is made on the control performances that are obtained, but also--and more importantly--on the microbial diversity. In particular, it is shown that the combined oxygen and ammonia control strategy is more appropriate since shift in the temperature setpoint strongly affects the composition of the microbial ecosystem present in the reactor whereas active control of oxygen and ammonia does not.
After nearly 10 years of PCR-based analysis of prokaryotic small-subunit ribosomal RNAs for ecological studies it seems necessary to summarize reported pitfalls of this approach which will most likely lead to an erroneous description on the microbial diversity of a given habitat. The following article will cover specific aspects of sample collection, cell lysis, nucleic acid extraction, PCR amplification, separation of amplified DNA, application of nucleic probes and data analysis.
Use of Pseudomonas sp. strain MT5 to prevent and treat Flavobacterium columnare infection was studied in 2 experiments with fingerling rainbow trout Oncorhynchus mykiss. In the first experiment, length heterogeneity analysis of PCR-amplified DNA fragments (LH-PCR) was used to assess the effect of antagonistic baths on the microbial diversity of healthy and experimentally infected fish. In the 148 samples studied, no difference was found between bathed and unbathed fish, and 3 fragment lengths were detected most frequently: 500 (in 75.7% of the samples), 523 (62.2%) and 517 bp (40.5%). The species contributing to these fragment sizes were Pseudomonas sp., Rhodococcus sp. and F. columnare, respectively. A specific PCR for detection of Pseudomonas sp. MT5 was designed, but none of the tissue samples were found to be positive, most likely indicating poor adhesion of the strain during bathing. LH-PCR was found to be a more powerful tool for detecting F. columnare in fish tissue than traditional culture methods (chi2 = 3.9, df = 1, p < 0.05). Antagonistic baths had no effect on the outbreak of infection or on fish mortality. F. columnare was also detected in healthy fish prior to and after experimental infection, indicating that these fish were carriers of the disease. In the second experiment, intensive Pseudomonas sp. MT5 antagonistic baths were given daily to rainbow trout suffering from a natural columnaris infection. Again, the antagonistic bacteria had no effect on fish mortality, which reached 95 % in both control and antagonist-treated groups in 7 d.
Cold, water-saturated soils play important biogeochemical roles, yet almost nothing is known about the identity and habitat of microbes active under such conditions. We investigated the year-round microenvironment of an alpine tundra wet meadow soil in the Colorado Rocky Mountains, focusing on the biogeochemistry and microbial diversity of spring snowmelt--a dynamic time for alpine ecosystems. In situ measurements revealed spring and autumn periods of long-term temperature stability near 0 degrees C, and that deeper soil (30 cm) was more stable than surface soil, with more moderate summers and winters, and longer isothermal phases. The soil was saturated and water availability was limited by freezing rather than drying. Analyses of bioavailable redox species showed a shift from Mn reduction to net Fe reduction at 2-3 cm depth, elevated SO4(2-) and decreased soluble Zn at spring snowmelt. Terminal restriction fragment length polymorphism profiles detected a correlated shift in bacterial community composition at the surface to subsurface transition. Bacterial and archaeal small-subunit rRNA genes were amplified from saturated spring soil DNA pooled along a depth profile. The most remarkable feature of these subsurface-biased libraries was the high relative abundance of novel, uncultivated Chloroflexi-related sequences comprising the third largest bacterial division sampled, and representing seven new Chloroflexi subdivisions, thereby dramatically expanding the known diversity of this bacterial division. We suggest that these novel Chloroflexi are active at near -0 degrees C temperatures, under likely anoxic conditions, and utilize geochemical inputs such as sulfide from upslope weathering.
Litter from the chicken industry can present several environmental challenges, including offensive odors and runoff into waterways leading to eutrophication. An economically viable solution to the disposal of waste from chicken houses is treatment to produce a natural, granulated fertilizer that can be commercially marketed for garden and commercial use. Odor of the final product is important in consumer acceptance, and an earthy odor is desirable. By understanding and manipulating the microbial processes occurring during this process, it may be possible to modify the odors produced. Geosmin and related volatiles produced by soil actinomycetes are responsible for earthy odors, and actinomycetes are likely to be present in the composting manure. Bacterial communities at each stage of the process were analyzed by culturing studies and denaturing gradient gel electrophoresis (DGGE). The processing steps changed the culturable bacterial community, but the total community was shown by DGGE to be stable throughout the process. A local agricultural soil was analyzed in parallel as a potential source of geosmin-producing actinomycetes. This agricultural soil had higher microbial diversity than the compost at both the culturable and the molecular levels. Actinomycete bacteria were isolated and analyzed by AromaTrax, a gas chromatography-olfactometry system. This system enables the odor production of individual isolates to be monitored, allowing for rational selection of strains for augmentation experiments to improve the odor of the final fertilizer product.
Models of evolutionary processes postulate that new alleles appear in populations through random spontaneous mutation. Alleles that confer a competitive advantage in particular environments are selected and populations can be taken over by individuals expressing these advantageous mutations. We have studied the evolutionary process by using Escherichia coli cultures incubated for prolonged periods of time in stationary phase. The populations of surviving cells were shown to be highly dynamic, even after many months of incubation. Evolution proceeded along different paths even when the initial conditions were identical. As cultures aged, the takeovers by fitter mutants were incomplete, resulting in the coexistence of multiple mutant forms and increased microbial diversity. Thus, the study of bacterial populations in stationary phase provides a model system for understanding the evolution of diversity in natural populations.
Lechuguilla Cave is an ancient, deep, oligotrophic subterranean environment that contains an abundance of low-density ferromanganese deposits, the origin of which is uncertain. To assess the possibility that biotic factors may be involved in the production of these deposits and to investigate the nature of the microbial community in these materials, we carried out culture-independent, small subunit ribosomal RNA (SSU rRNA) sequence-based studies from two sites and from manganese and iron enrichment cultures inoculated with ferromanganese deposits from Lechuguilla and Spider Caves. Sequence analysis showed the presence of some organisms whose closest relatives are known iron- and manganese-oxidizing/reducing bacteria, including Hyphomicrobium, Pedomicrobium, Leptospirillum, Stenotrophomonas and Pantoea. The dominant clone types in one site grouped with mesophilic Archaea in both the Crenarchaeota and Euryarchaeota. The second site was dominated almost entirely by lactobacilli. Other clone sequences were most closely related to those of nitrite-oxidizing bacteria, nitrogen-fixing bacteria, actinomycetes and beta- and gamma-Proteobacteria. Geochemical analyses showed a fourfold enrichment of oxidized iron and manganese from bedrock to darkest ferromanganese deposits. These data support our hypothesis that microorganisms may contribute to the formation of manganese and iron oxide-rich deposits and a diverse microbial community is present in these unusual secondary mineral formations.