Microbial diversity and function in soil: from genes to ecosystems

240 Microbial diversity and function in soil: from genes to ecosystems Vigdis Torsvik* and Lise Øvreås† Soils sustain an immense diversity of microbes, which, to a Figure 1 large extent, remains unexplored. A range of novel methods, most of which are based on rRNA and rDNA analyses, have uncovered part of the soil microbial diversity. The next step in the era of microbial ecology is to extract genomic, evolutionary and functional information from bacterial artificial chromosome libraries of the soil community genomes (the metagenome). Sophisticated analyses that apply molecular phylogenetics, DNA microarrays, functional genomics and in situ activity measurements will provide huge amounts of new data, potentially increasing our understanding of the structure and function of soil microbial ecosystems, and the interactions that occur within them. This review summarizes the recent progress in studies of soil microbial communities with focus on novel methods and approaches that provide new insight into the relationship between phylogenetic and functional diversity. Current Opinion in Microbiology Addresses Department of Microbiology, University of Bergen, Post Box 7800, Epifluorescence micrography of soil microorganisms stained with Jahnebakken 5, N-5020 Bergen, Norway 4′,6-diamidino-2-phenylindole (DAPI). The total bacterial count was *e-mail: vigdis.torsvik@im.uib.no 4.2 × 1010 cells gram–1 soil (dry weight) by fluorescent microscopy, † e-mail: lise.ovreas@im.uib.no and 4.2 × 106 colony-forming units gram–1 soil (dry weight) by plating. Current Opinion in Microbiology 2002, 5:240–245 1369-5274/02/$ — see front matter distribution of information, which is directly applicable to © 2002 Elsevier Science Ltd. All rights reserved. the total genetic diversity or complexity in a community. Published online 13 May 2002 The genetic complexity or genome size of microbial Abbreviations community genomes can be assessed by re-association of BAC bacterial artificial chromosome community DNA. Such broad-scale analysis has revealed BrdU 5-bromo-2′-deoxyuridine [4,5] that the community genome size equals the size of DGGE denaturant gradient gel electrophoresis FISH fluorescent in situ hybridization 6000–10 000 Escherichia coli genomes in unperturbed GC guanine + cytosine organic soils, and 350–1500 genomes in arable or heavy- PCR polymerase chain reaction metal-polluted soils. These values may be conservative, as PLFA phospholipid fatty acid genomes representing rare and unrecovered microorgan- isms are probably not included in the analysis. In contrast, Introduction the genomic complexity recovered by culturing methods Microbial diversity in soil ecosystems exceeds, by far, that was less than 40 genomes. The total genomic complexity of eukaryotic organisms. One gram of soil may harbor denotes the confines of diversity in terms of genetic infor- up to 10 billion microorganisms of possibly thousands mation present and provides information about the overall of different species [1•]. As less than 1% of the micro- (potential) taxonomic and functional variability at the com- organisms observed under the microscope (Figure 1) is munity level. Because this represents an average diversity cultivated and characterized, soil ecosystems are, to a large value, no detailed information about taxon and functional extent, uncharted. Microbial diversity describes complexity diversity at lower levels of biological organization is and variability at different levels of biological organization. obtained. A number of methods with higher resolving It encompasses genetic variability within taxons (species), power have been developed for characterization of micro- and the number (richness) and relative abundance bial communities that includes both cultured and (evenness) of taxons and functional groups (guilds) in com- uncultured microorganisms. Most of them are based on munities. Important aspects of diversity at the ecosystem analyses of ribosomal RNA genes (rDNA). They have level are the range of processes, complexity of interactions, uncovered part of the microbial diversity in soil, yielding and number of trophic levels. Thus, measures of microbial sequences from many novel phylogenetic lineages. diversity should include multiple methods integrating Measures of microbial patterns (fingerprinting) and holistic measures at the total community level and partial taxonomic variability have been coupled with analysis of approaches targeting structural or functional subsets [2,3]. functional genes and activity measurements. Such investi- Diversity may also be considered to be the amount and gations aim to reveal and understand the relationship  Microbial diversity and function in soil: from genes to ecosystems Torsvik and Øvreås 241 between structural and functional diversity in soil microbial cell, the study of the collective genomes in a microbial ecosystems. community — the ‘metagenome’ — promises to unlock the secrets of community life. Approximately 60 microbial In this review, we summarize the recent progress in studies genomes have been completely sequenced, and hundreds of soil microbial communities. We present an overview of more are in the process of being sequenced. Complete novel molecular methods for studying all the microorganisms microbial genome sequencing has made it possible to in soil, including those uncultured, and approaches for identify and characterize all genes present in a species. obtaining information from community genome analysis. This means that we get information about novel metabolic The review highlights some recent studies that link phylo- pathways, gene regulatory elements, genes of unknown genetic groups to function and may contribute to exciting function, and genes for pathogenesis, virulence and drug new insight into the relationship between community resistance [9]. This information also provides insights into composition and function. Finally, we discuss the effect of the evolution of genes and species. Furthermore, the soil physical and chemical conditions on microbial diversity understanding of species diversity based on comparative and the importance of functional diversity for soil ecosystem genomics has led to a new epoch for biological investiga- stability and resilience. tions, using techniques such as microarray analysis [10]. The next step in the era of microbial genomics is to extract Methods to describe diversity functional and evolutionary information from these large The indications of the vast diversity of uncultured life in soil datasets and to apply genomics technology to relevant have stimulated development of methods for culture- questions in microbial ecology [11••]. Thus, new technology independent study of microbial communities. These methods will have an important impact on the understanding of soil have employed a combination of nucleic acid characterization microbial ecology. Recently, soil community genomes have and microscopy. Over the past two decades, molecular methods, been cloned using a bacterial artificial chromosome (BAC) especially 16S rRNA gene sequencing, have become very vector. Together, all the genomes of soil microbes can be popular to help identify unknown bacteria [6,7]. In turn, this considered to be one large soil microbial community has led to community analysis using total community DNA genome — the metagenome [12••]. Combined compara- extracted from the environment. Broad-scale analysis of com- tive analyses of core housekeeping genes like rDNA and munity DNA, using techniques such as DNA re-association, functional genes may provide information on both phylo- provides information about the total genetic diversity of a genetic diversity and the potential functional diversity of given bacterial community [4]. A shift in guanine + cytosine microbial communities. There are several ways in which (GC) content can be used to detect changes in microbial environmental microbiology will benefit from microbial community structure, but does not tell us anything about the genomics. Comparative genomic analysis and microarray other diversity parameters, which are richness, evenness and technology may be used to determine patterns of gene composition. Polymerase chain reaction (PCR)-based finger- expression, and to detect novel metabolic pathways. This printing techniques give a higher resolution and provide offers a quick method to access functional information for information about changes in the whole community structure. genes of unknown function. Such information is very useful These fingerprinting techniques, such as phospholipid fatty in functional diversity studies to track highly expressed acid (PLFA) analysis, denaturant gradient gel electrophoresis genes and genes critical in biogeochemical pathways. (DGGE), amplified rDNA restriction analysis (ARDRA), ter- It is also important to understand how microbial cells are minal restriction fragment length polymorphism (T-RFLP) regulated under varying conditions such as carbon supply, and ribosomal intergenic spacer analysis (RISA), provide energy source and electron acceptor availability, thereby information on the species composition, and can be used to obtaining information on microbial community response to compare common species present in samples. However, there environmental changes. The new tools may also increase are some problems and biases in the PCR amplification step our understanding of the process of genome evolution and and, therefore, these methods cannot be used as definite the factors that regulate diversity. indicators of species richness. Despite the PCR problems, a combination of the techniques mentioned above can reveal a Novel methods linking phylogenetic groups to great deal about the microbial community diversity. Recently, their activities and function a method based on integron-targeting PCR assays has been Perhaps the greatest challenge facing microbiology today is developed to recover genes from environmental DNA the problem of linking phylogeny and function. The methods without the necessity to know their sequences [8••]. A based on 16S rRNA analysis provide extensive information comprehensive description and discussion of the potential about the taxa present in an environment, although they and limitations of the methods are given in the overviews of provide little insight into the functional role of each Kozdrój and van Elsas [2] and Johnsen et al. [3]. phylogenetic group. Metagenomic analysis provides some functional information through genomic sequence and Single genome versus community genome expression of traits, but other methods are required to link analysis specific functions with the group responsible for them. Although the complete sequences of more than 60 microbial The concomitant quantitative and comparative analyses genomes have provided critical insights into the individual of expressed rRNA genes and genes for key enzymes in 242 Ecology and industrial microbiology relation to environmental factors can be used to obtain Yin et al. [24•] recently used this technique to determine information about the phylogeny and ecology of functional the extent of functional redundancy along a soil reclamation bacterial groups responsible for processes like denitrification, gradient in a highly contaminated mine spoil. Different nitrification and methane oxidation. carbon amendments were used and significant differences were detected in the microbial populations that had Environmental functional gene arrays could be constructed incorporated BrdU in their DNA, indicating a bacterial using oligonucleotid probes to target gene expression or functional redundancy within this microbial community. A genes coding for key enzymes in all biogeochemical cycles. problem using this technique is that selective stimulation These can be used for specific detection of gene expres- of bacteria that are not actually active before substrate sion in the environment. Investigations in which attempts amendment may occur. have been made to associate specific microorganism or microbial groups with their ecological function have been The effect of soil structure and environmental performed on functional groups of bacteria, using genes for conditions on microbial diversity key enzymes such as nitrate reductase [13], ammonia Soil is a very complex system that comprises a variety of monooxygenase [14] and methane monooxygenase [15]. microhabitats with different physicochemical gradients and Another strategy to look for specific functional groups is to discontinuous environmental conditions. Microorganisms use microsensor measurements of chemical profiles in adapt to microhabitats and live together in consortia with relation to the distribution of different bacterial taxa to more or less sharp boundaries, interacting with each other identify environmental conditions favored by particular and with other parts of the soil biota. A number of investi- bacteria [16,17•]. It remains, however, a major challenge in gations emphasize the impact of soil structure and spatial soil microbial ecology to ascribe microbial processes to isolation on microbial diversity and community structure specific microorganisms. Before the microorganisms are [11••,25,26]. Analysis of the spatial distribution of bacteria at cultivated, their ecological functions must be elucidated microhabitat levels showed that, in soils subjected to differ- through culture-independent characterization that links ent fertilization treatments, more than 80% of the bacteria phylogenetic and functional genetic analyses to metabolic were located in micropores of stable soil micro-aggregates activities in soil. (2–20 µm) [25]. Such microhabitats offer the most favorable conditions for microbial growth with respect to water and Microradioautography, using the uptake of specific radio- substrate availability, gas diffusion and protection against labeled substrates by individual cells, can be used to predation. Particle size had a higher impact on microbial detect and quantify the active populations utilizing this diversity and community structure than did factors like bulk substrate. In order to link the uptake of a specific substrate pH and the type and amount of organic compound input with the phylogenetic identity of a specific bacterial cell, [26]. Results showed that the microbial diversity in fractions microautoradiography has been used in combination with with small soil particles was higher than that in fractions fluorescent in situ hybridization (FISH) of microbial cells with large soil particles, and that most of the soil microbial using fluorescent, group-specific phylogenetic probes community was particle-specific. A high diversity of bacteria (targeting rRNA) and fluorescence microscopy. Studies in belonging to the Holophaga/Acidobacterium division and which microautoradiography and FISH are combined in Prosthecobacter were present in small particles (silt and clay). natural and engineered environments are rapidly increasing Large particles (sand) harbored few members of the [14,16,18,19]. The application of stable isotope probing Holophaga/Acidobacterium division, and were dominated by (SIP) and phospholipid fatty acid (PLFA) labeling to bacteria belonging to the α-proteobacteria. Other investiga- determine functionally active components of microbial tions indicate that the type and amount of available organic communities is also becoming increasingly used in micro- substrates strongly influence the abundance of microbial bial ecology studies [20,21••]. In SIP, 13C-DNA produced groups and their functional diversity in soil ecosystems during the growth of metabolically distinct microbial [27,28]. Smit et al. [29•] used their own data and data from groups on a 13C-enriched carbon source can be resolved recent literature on the distribution of 16S rDNA sequences from 12C-DNA by density gradient centrifugation. The among five main bacterial divisions to search for relation- DNA can then be fractionated, and each fraction can be ships between the abundance of microbial groups and soil further analyzed taxonomically and functionally by gene nutritional status. Their results suggested that soil with a probing and functional analysis. This method, therefore, high content of readily available nutrients showed positive offers a powerful technique for identifying microorganisms selection for α- and γ-proteobacteria, this being indicative of that are actively involved in specific metabolic processes. r-selection, which is selection for bacteria with potentially Another method in which activity can be linked to phylo- high growth rates. In low-nutrient soil or soil with a high genetic information is the incorporation of 5-bromo- content of recalcitrant substrates, the percentage of 2′-deoxyuridine (BrdU) into DNA to detect metabolically Acidobacterium increased, this being indicative of k-selection, active community members in response to substrate or which is selection for bacteria with lower growth potential other stimuli. BrdU can be added to a culture of microbial but higher capability to compete for substrates. The ratio cells, and metabolically active members of a community between the number of proteobacteria and Acidobacterium will then incorporate BrdU into their DNA [22,23]. was suggested to be indicative of the nutritional status of  Microbial diversity and function in soil: from genes to ecosystems Torsvik and Øvreås 243 soils. The ratio was low in oligotrophic soil, medium in high diversity of organic substrate is likely to have a agricultural soil with low organic input, and high in positive effect on the function. The relationship between agricultural soil with high organic input [29•,30]. microbial diversity and soil processes may not be linear because many processes are carried out by a consortium of Competitive interactions are thought to be a key factor microorganisms. In interacting consortia, small linear controlling microbial community structure and diversity changes in microbial diversity may result in non-linear [11••]. Soil structure and water regime influence changes in process. competitive interactions by causing spatial isolation within communities. Soil with high spatial isolation showed high Functional diversity of soil microorganisms microbial diversity, whereas soil with lower spatial isolation Functional diversity is an aspect of the overall microbial showed much lower diversity and was dominated by a few diversity in soil, and encompasses a range of activities. The microorganisms. The high diversity in soil with high spatial relationship between microbial diversity and function in soil isolation may also have been caused by a higher hetero- is largely unknown, but biodiversity has been assumed to geneity of carbon resources in this soil and, consequently, influence ecosystem stability, productivity and resilience a higher niche variation. Soil bacteria are subjected to towards stress and disturbance. The catabolic response considerable seasonal fluctuations in environmental profile (CRP) [33], which is a measure of short-term sub- conditions such as temperature, water content and strate-induced respiration, has been used to calculate the nutrient availability. An important issue to elucidate is how diversity (range and evenness) of catabolic functions environmental changes and seasonal variations influence expressed in situ. Catabolic diversity has been used to inves- qualitative variation in community composition. Smit et al. tigate the effect of stress and disturbance on the diversity [29•] found that bacterial biomass did not change signifi- and resilience of soil microbial communities. When match- cantly during the seasons, but that both culturing and ing soils from different long-term-managed environments molecular fingerprinting techniques demonstrate signifi- (crop and pasture) were subjected to stress and disturbance, cant variations in community composition. Culturing it was demonstrated that microbial communities with low techniques show that the proportion of fast-growing catabolic evenness (crop fields) were less resistant to stress bacteria was lowest in winter and highest in summer, and and disturbance than were microbial communities with high that the highest species richness was found in spring and catabolic evenness (pasture). Other soil properties might autumn. This seemed to correlate with enhanced micro- also have contributed to the resistance. After a major bial activities and nutrient input from fertilization (spring) disturbance (such as landslips, volcanic eruptions or retreat and plant debris after harvest (autumn). Molecular finger- of glaciers), marked changes in functional diversity printing indicated that the community, to a large extent, (catabolic evenness) in developing soil ecosystems have been consisted of stable dominant populations, but that less- demonstrated [34]. The functional diversity was initially low abundant populations, as revealed by low-intensity bands in non-vegetated (underdeveloped) sites, but as vegetation on a DGGE gel, showed distinct seasonal differences. was established, the diversity rapidly increased and, in older Culture-dependent methods and molecular methods successions, the catabolic evenness declined. These diversity reveal strikingly different microbial populations in soil. It patterns broadly paralleled patterns in plant and fungal has been demonstrated that Gram-positive bacteria with diversity. The relationships between microbial community high GC content [29•] and Gram-positive bacteria with low composition and physiological capacity [35] were elucidated GC content [31] were abundant among isolates, but had using PLFA profiles and two functional measures, namely very low abundance or were nearly absent among clones. substrate utilization capacities (BIOLOG) and enzyme activities. Broad-scale community composition (PFLA The microbial diversity of soil and the interactions profiles) correlated well with specific enzyme activities, between different trophic levels were elucidated in a simple especially enzymes responsible for initial degradation of ecosystem model in which primary producers (plants) and macromolecules such as lignocellulose. The lack of correla- decomposers (microbes) were linked through cycling of a tion between BIOLOG and PLFA assays may be due to limiting nutrient factor [32] for the primary producers. The the fact that BIOLOG selects for a minor part of the micro- model shows that the efficiency of nutrient recycling from bial community utilizing readily available simple substrates, organic compounds to decomposers is a key parameter that whereas PLFA includes the total community. Macro- controls ecosystem processes (productivity and biomass of molecule degradation and demineralization enzyme activities the functional groups). The model predicts that microbial may measure functions that immediately respond to litter diversity has a positive effect on nutrient cycling efficiency, input and are more clearly related to changes in the active and contributes to increased ecosystem processes. One part of microbial communities. major effect that microbial diversity can have on ecosystem processes is to ensure that all organic compounds are Conclusions and future directions recycled. Organic compound diversity may have a negative Recent advances in soil community analysis using molecular or neutral effect on a stable ecosystem. Most soils are methods agree with the earlier data on total genetic exposed to fluctuating environmental conditions and, in diversity by indicating an enormous microbial diversity in fluctuating ecosystems and a long-term perspective, the soil. Soil diversity exceeds that of aquatic environments, 244 Ecology and industrial microbiology and is a great resource for biotechnological exploration of designed to target recombination sites that flank gene cassettes associated with integrons. This PCR strategy denotes a unique opportunity for exploita- novel organisms, products and processes. Novel methods tion of novel genes of biotechnological importance by culture-independent and approaches enable us to explore this vast diversity. means. This paper explains that rapid access to a significant genetic resource, independently of prior gene sequence knowledge, enables Studies of sequence information from organisms in soil recovery of the actual gene in a form ready for direct analysis. microhabitats and their gene expression under different 9. MacNeil IA, Tiong CL, Minor C, August PR, Grossman TH, conditions will provide guidelines for designing new and Loiacono KA, Lynch BA, Phillips T, Narula S, Sundaramoorthi R et al.: improved culturing methods that resemble their natural Expression and isolation of antimicrobial small molecules from soil DNA libraries. J Mol Microbiol Biotech 2001, 3:301-308. niches. New tools in bioinformatics and statistical analysis 10. Cho JC, Tiedje JM: Bacterial species determination from enable us to handle the huge amount of data obtained DNA–DNA hybridization by using genome fragments and DNA through multidimensional studies that combine growth- microarrays. Appl Environ Microbiol 2001, 67:3677-3682. independent molecular analyses with analyses of microbial 11. Tiedje JM, Cho JC, Murray A, Treves D, Xia B, Zhou J: Soil teeming growth, activity and physiology, and integrate measures •• with life: new frontiers for soil science. In Sustainable Management of Soil Organic Matter. Edited by Rees RM, Ball BC, Campbell CD, of environmental parameters. Such polyphasic studies Watson CA. CAB International; 2001:393-412. integrate different aspects of microbial diversity and This paper emphasizes the importance of soil biology knowledge to the understanding of the complexity of soil microbial communities. It also provide a more complete picture of microbial diversity and includes an introduction to the application of DNA microarray technology in a deeper understanding of the interactions in soil microbial analysis of environmental microbial communities and for detection of selected ecosystems. Studies of microbial sequences, comparative genes in the environment. genomics and microarray technology will improve our 12. Rondon MR, August PR, Bettermann AD, Brady SF, Grossman TH, •• Liles MR, Loiacono KA, Lynch BA, MacNeil IA, Minor C et al.: Cloning understanding of the structure/function relationships and the soil metagenome: a strategy for accessing the genetic and the effects of abiotic and biotic factors on soil microbial functional diversity of uncultured microorganisms. Appl Environ Microbiol 2000, 66:2541-2547. communities. It is conceivable that the research field For a particular soil microbial community, the authors successfully constructed dealing with the interaction of genomes with the environ- BAC libraries with average insert sizes of 27 kb and 44.5 kb. Preparation of ment will be an important topic in the future. soil DNA adequate for cloning in a BAC library is considerably more challenging then that of DNA from aquatic microbial communities. The authors found clones expressing Dnase, antibacterial protein, lipase and Acknowledgements amylase activities, whereas clones expressing cellulase, chitinase, esterase This work was supported by grant from the Norwegian Research Council and keratinase activities were not found. (NFR), project number 140658/720, and the Nordic Academy for Advanced 13. Wu L, Thompson DK, Li G, Hurt RA, Tiedje JM, Zhou J: Development Study (NorFA). and evaluation of functional gene arrays for detection of selected genes in the environment. Appl Environ Microbiol 2001, 67:5780-5790. References and recommended reading Papers of particular interest, published within the annual period of review, 14. Daims H, Purkhold U, Bjerrum L, Arnold E, Wilderer PA, Wagner M: have been highlighted as: Nitrification in sequencing biofilm batch reactors: lessons from molecular approaches. Water Sci Technol 2001, 43:9-18. • of special interest •• of outstanding interest 15. 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