Predicting prokaryotic ecological niches using genome sequence analysis

Automated DNA sequencing technology is so rapid that analysis has become the rate-limiting step. Hundreds of prokaryotic genome sequences are publicly available, with new genomes uploaded at the rate of approximately 20 per month. As a result, this growing body of genome sequences will include microorganisms not previously identified, isolated, or observed. We hypothesize that evolutionary pressure exerted by an ecological niche selects for a similar genetic repertoire in those prokaryotes that occupy the same niche, and that this is due to both vertical and horizontal transmission. To test this, we have developed a novel method to classify prokaryotes, by calculating their Pfam protein domain distributions and clustering them with all other sequenced prokaryotic species. Clusters of organisms are visualized in two dimensions as 'mountains' on a topological map. When compared to a phylogenetic map constructed using 16S rRNA, this map more accurately clusters prokaryotes according to functional and environmental attributes. We demonstrate the ability of this map, which we term a "niche map", to cluster according to ecological niche both quantitatively and qualitatively, and propose that this method be used to associate uncharacterized prokaryotes with their ecological niche as a means of predicting their functional role directly from their genome sequence.     

Identification and characterization of metagenomic fragments from tidal flat sediment

Phylogenetic surveys based on cultivation-independent methods have revealed that tidal flat sediments are environments with extensive microbial diversity. Since most of prokaryotes in nature cannot be easily cultivated under general laboratory conditions, our knowledge on prokaryotic dwellers in tidal flat sediment is mainly based on the analysis of metagenomes. Microbial community analysis based on the 16S rRNA gene and other phylogenetic markers has been widely used to provide important information on the role of microorganisms, but it is basically an indirect means, compared with direct sequencing of metagenomic DNAs. In this study, we applied a sequence-based metagenomic approach to characterize uncultivated prokaryotes from tidal flat sediment. Two large-insert genomic libraries based on fosmid were constructed from tidal flat metagenomic DNA. A survey based on end-sequencing of selected fosmid clones resulted in the identification of clones containing 274 bacterial and 16 archaeal homologs in which majority were of proteobacterial origins. Two fosmid clones containing large metagenomic DNAs were completely sequenced using the shotgun method. Both DNA inserts contained more than 20 genes encoding putative proteins which implied their ecological roles in tidal flat sediment. Phylogenetic analyses of evolutionary conserved proteins indicate that these clones are not closely related to known prokaryotes whose genome sequence is known, and genes in tidal flat may be subjected to extensive lateral gene transfer, notably between domains Bacteria and Archaea. This is the first report demonstrating that direct sequencing of metagenomic gene library is useful in underpinning the genetic makeup and functional roles of prokaryotes in tidal flat sediments.     

Are viruses driving microbial diversification and diversity?

Viruses can influence the genetic diversity of prokaryotes in various ways. They can affect the community composition of prokaryotes by 'killing the winner' and keeping in check competitive dominants. This may sustain species richness and the amount of information encoded in genomes. Viruses can also transfer (viral and host) genes between species. Such mechanisms have probably influenced the speciation of prokaryotes. Whole-genome sequencing has clearly revealed the importance of (virus-mediated) gene transfer. However, its significance for the ecological performance of aquatic microbial communities is only poorly studied, although the few available reports indicate a large potential. Here, we present data supporting the hypothesis that viral genes and viral activity generate genetic variability of prokaryotes and are a driving force for ecological functioning and evolutionary change.     

A Novel Nutritional Predictor Links Microbial Fastidiousness with Lowered Ubiquity,Growth Rate,and Cooperativeness

Understanding microbial nutritional requirements is a key challenge in microbiology. Here we leverage the recent availability of thousands of automatically generated genome-scale metabolic models to develop a predictor of microbial minimal medium requirements, which we apply to thousands of species to study the relationship between their nutritional requirements and their ecological and genomic traits. We first show that nutritional requirements are more similar among species that co-habit many ecological niches. We then reveal three fundamental characteristics of microbial fastidiousness (i.e., complex and specific nutritional requirements): (1) more fastidious microorganisms tend to be more ecologically limited; (2) fastidiousness is positively associated with smaller genomes and smaller metabolic networks; and (3) more fastidious species grow more slowly and have less ability to cooperate with other species than more metabolically versatile organisms. These associations reflect the adaptation of fastidious microorganisms to unique niches with few cohabitating species. They also explain how non-fastidious species inhabit many ecological niches with high abundance rates. Taken together, these results advance our understanding microbial nutrition on a large scale, by presenting new nutrition-related associations that govern the distribution of microorganisms in nature.     

The environment,microbes and bioremediation: microbial activities modulated by the environment

Microorganisms in nature are largely responsible for the biodegradation and removal of toxic and non-toxic chemicals. Many organisms are also known to have specific ecological niches for proliferation and colonization. The nature of the environment dictates to a large extent the biodegradability of synthetic compounds by modulating the evolutionary processes in microorganisms for new degradative genes. Similarly, environmental factors often determine the extent of microbial gene expression by activating or repressing specific gene or sets of genes through a sensory signal transduction process. Understanding how the environment modulates microbial activity is critical for successful bioremediative applications.     

Non-Darwinian and Darwinian prokaryotic and eukaryotic evolution--an enigma in cell biology conservation

Our theory is embarrassingly simple. What made today's prokaryotes and modern cyanobacteria so robust is the fact that in their origin, back in the Archean (3 billion years ago), selection did not play a central role in evolution, it had only a transitory role. Asexual reproduction, mutation, drift and sampling variance in local demes were more important especially when they were accompanied by population catastrophes, where millions perished. Metazoans are generally macroscopic, sexually reproducing, ecologically specialized organisms whose history is full of extinctions and radiations leading to morphological change. On the other hand, prokaryotes, thanks to their origin, avoid extinction because as a group they have slowly evolved as generalists. Specialization appears to be less important than ecological versatility and metabolic unspecialization. Modern cyanobacteria keep on using that strategy.     

Genomics, environmental genomics and the issue of microbial species

A microbial species concept is crucial for interpreting the variation detected by genomics and environmental genomics among cultivated microorganisms and within natural microbial populations. Comparative genomic analyses of prokaryotic species as they are presently described and named have led to the provocative idea that prokaryotes may not form species as we think about them for plants and animals. There are good reasons to doubt whether presently recognized prokaryotic species are truly species. To achieve a better understanding of microbial species, we believe it is necessary to (i) re-evaluate traditional approaches in light of evolutionary and ecological theory, (ii) consider that different microbial species may have evolved in different ways and (iii) integrate genomic, metagenomic and genome-wide expression approaches with ecological and evolutionary theory. Here, we outline how we are using genomic methods to (i) identify ecologically distinct populations (ecotypes) predicted by theory to be species-like fundamental units of microbial communities, and (ii) test their species-like character through in situ distribution and gene expression studies. By comparing metagenomic sequences obtained from well-studied hot spring cyanobacterial mats with genomic sequences of two cultivated cyanobacterial ecotypes, closely related to predominant native populations, we can conduct in situ population genetics studies that identify putative ecotypes and functional genes that determine the ecotypes' ecological distinctness. If individuals within microbial communities are found to be grouped into ecologically distinct, species-like populations, knowing about such populations should guide us to a better understanding of how genomic variation is linked to community function.     

The question of DNA repair in hyperthermophilic archaea

Hyperthermophilic archaea grow at temperatures that destabilize the primary structure of DNA and in evolutionary terms they are highly divergent from other well studied microorganisms. These prokaryotes should therefore require DNA damage repair to be unusually effective, and could employ novel mechanisms for this repair. Recent genome sequence analyses and biochemical and genetic assays suggest a distribution of DNA repair strategies that raises intriguing questions for future study.     

Examination of Antarctic prokaryotic diversity through molecular comparisons

Prokaryotes perform key functions in Antarctic ecosystems, and knowledge of the taxonomy of Antarctic prokaryotes is a prerequisite for the transfer of information between fields of scientific inquiry. The taxonomy of prokaryotes has been greatly revised and improved due to the refinements afforded by molecular techniques such as 16S rRNA sequencing. Past inventories of Antarctic microbial diversity are difficult to reconcile with the developing, phylogenetically-based taxonomy.Antarctic prokaryotes are considerably diverse and most evolutionary groups are represented, including representatives of both Archaea and Bacteria. The diversity appears unique due to the ease with which new species can be isolated; however, that may be a result of our vastly incomplete knowledge of both Antarctic and non-Antarctic prokaryotic diversity. Use of the 16S rRNA gene as a molecular clock would suggest that the majority of Antarctic prokaryotes diverged from their nearest known non-Antarctic relatives long before a stable ice-sheet developed in Antarctica. The time of colonization (or recolonization) of Antarctic environments by individual species may have been very recent in evolutionary time scales.     

How mutualisms arise in phytoplankton communities: building eco‐evolutionary principles for aquatic microbes

Extensive sampling and metagenomics analyses of plankton communities across all aquatic environments are beginning to provide insights into the ecology of microbial communities. In particular, the importance of metabolic exchanges that provide a foundation for ecological interactions between microorganisms has emerged as a key factor in forging such communities. Here we show how both studies of environmental samples and physiological experimentation in the laboratory with defined microbial co‐cultures are being used to decipher the metabolic and molecular underpinnings of such exchanges. In addition, we explain how metabolic modelling may be used to conduct investigations in reverse, deducing novel molecular exchanges from analysis of large‐scale data sets, which can identify persistently co‐occurring species. Finally, we consider how knowledge of microbial community ecology can be built into evolutionary theories tailored to these species’ unique lifestyles. We propose a novel model for the evolution of metabolic auxotrophy in microorganisms that arises as a result of symbiosis, termed the Foraging‐to‐Farming hypothesis. The model has testable predictions, fits several known examples of mutualism in the aquatic world, and sheds light on how interactions, which cement dependencies within communities of microorganisms, might be initiated.     

Evolution of prokaryotic two-component systems: insights from comparative genomics

Two-component systems (TCSs) are diverse and abundant signal transduction pathways found predominantly in prokaryotes. This review focuses on insights into TCS evolution made possible by the sequencing of whole prokaryotic genomes. Typical TCSs comprise an autophosphorylating protein (a histidine kinase), which transfers a phosphoryl group onto an effector protein (a response regulator), thus modulating its activity. Histidine kinases and response regulators are usually found encoded as pairs of adjacent genes within a genome, with multiple examples in most prokaryotes. Recent studies have shed light on major themes of TCS evolution, including gene duplication, gene gain/loss, gene fusion/fission, domain gain/loss, domain shuffling and the emergence of complexity. Coupled with an understanding of the structural and biophysical properties of many TCS proteins, it has become increasingly possible to draw inferences regarding the functional consequences of such evolutionary changes. In turn, this increase in understanding has the potential to enhance both our ability to rationally engineer TCSs, and also allow us to more powerfully correlate TCS evolution with behavioural phenotypes and ecological niche occupancy.     

Phylodynamic Inference and Model Assessment with Approximate Bayesian Computation: Influenza as a Case Study

A key priority in infectious disease research is to understand the ecological and evolutionary drivers of viral diseases from data on disease incidence as well as viral genetic and antigenic variation. We propose using a simulation-based, Bayesian method known as Approximate Bayesian Computation (ABC) to fit and assess phylodynamic models that simulate pathogen evolution and ecology against summaries of these data. We illustrate the versatility of the method by analyzing two spatial models describing the phylodynamics of interpandemic human influenza virus subtype A(H3N2). The first model captures antigenic drift phenomenologically with continuously waning immunity, and the second epochal evolution model describes the replacement of major, relatively long-lived antigenic clusters. Combining features of long-term surveillance data from the Netherlands with features of influenza A (H3N2) hemagglutinin gene sequences sampled in northern Europe, key phylodynamic parameters can be estimated with ABC. Goodness-of-fit analyses reveal that the irregularity in interannual incidence and H3N2''s ladder-like hemagglutinin phylogeny are quantitatively only reproduced under the epochal evolution model within a spatial context. However, the concomitant incidence dynamics result in a very large reproductive number and are not consistent with empirical estimates of H3N2''s population level attack rate. These results demonstrate that the interactions between the evolutionary and ecological processes impose multiple quantitative constraints on the phylodynamic trajectories of influenza A(H3N2), so that sequence and surveillance data can be used synergistically. ABC, one of several data synthesis approaches, can easily interface a broad class of phylodynamic models with various types of data but requires careful calibration of the summaries and tolerance parameters.     

Phylogenetic systematics of microorganisms inhabiting thermal environments

Thermal habitats harbor specialized communities of thermophilic microorganisms, primarily prokaryotes. This review considers modern systematics of prokaryotes and the place of thermophilic archaea and bacteria in it. Among the existing hierarchical classifications of prokaryotes, the bulk of attention is given to the one accepted in the current second edition of "Bergey's Manual of Systematic Bacteriology", which is primarily based on 16S rRNA phylogeny and phenotypic properties of the organisms. Analysis of the genomics data shows that they on the whole agree with the 16S rRNA-based system, although revealing the significance of the evolutionary role of lateral transfer, duplication, and loss of genes. According to the classification elaborated in the current edition of "Bergey's Manual", the prokaryotes currently culturable under laboratory conditions are distributed among 26 phyla, two of which belong to the domain Archaea and 24 to the domain Bacteria. Six phyla contain exclusively thermophiles, and eleven phyla contain thermophiles along with mesophiles, thermophiles being usually separated phylogenetically and representing high-level taxa (classes, orders). In light of the data on the topology of the 16S rRNA-based phylogenetic tree and some other data, this review discusses the probable hyperthermophilic nature of the universal common ancestor.     

On the origin of synonymous codon usage divergence between thermophilic and mesophilic prokaryotes

Synonymous codon usage analysis between thermophilic and mesophilic prokaryotes has gained wide attention in recent years. Although it is known that thermophilic and mesophilic prokaryotes use different subset of synonymous codons, no reason for this difference is known so far. In the present communication, by analyzing a large number of thermophilic and mesophilic prokaryotes, we provide evidence that bias in the selection of synonymous codons between thermophilic and mesophilic prokaryotes is related to differential folding pattern of mRNA secondary structures. Moreover, we observe that error-minimizing property has significant influence in differentiating the synonymous codon usage between thermophilic and mesophilic prokaryotes. Biological implications of these results are discussed.     

Plant-microbe-soil interactions in the rhizosphere: an evolutionary perspective

Soils are the product of the activities of plants, which supply organic matter and play a pivotal role in weathering rocks and minerals. Many plant species have a distinct ecological amplitude that shows restriction to specific soil types. In the numerous interactions between plants and soil, microorganisms also play a key role. Here we review the existing literature on interactions between plants, microorganisms and soils, and include considerations of evolutionary time scales, where possible. Some of these interactions involve intricate systems of communication, which in the case of symbioses such as the arbuscular mycorrhizal symbiosis are several hundreds of millions years old; others involve the release of exudates from roots, and other products of rhizodeposition that are used as substrates for soil microorganisms. The possible reasons for the survival value of this loss of carbon over tens or hundreds of millions of years of evolution of higher plants are discussed, taking a cost-benefit approach. Co-evolution of plants and rhizosphere microorganisms is discussed, in the light of known ecological interactions between various partners in terrestrial ecosystems. Finally, the role of higher plants, especially deep-rooted plants and associated microorganisms in the weathering of rocks and minerals, ultimately contributing to pedogenesis, is addressed. We show that rhizosphere processes in the long run are central to biogeochemical cycles, soil formation and Earth history. Major anticipated discoveries will enhance our basic understanding and allow applications of new knowledge to deal with nutrient deficiencies, pests and diseases, and the challenges of increasing global food production and agroecosystem productivity in an environmentally responsible manner.     

Identification of microorganisms by PCR amplification and sequencing of a universal amplified ribosomal region present in both prokaryotes and eukaryotes

The small ribosomal subunit contains 16S rRNA in prokaryotes and 18S rRNA in eukaryotes. Even though it has been known that some small ribosomal sequences are conserved in 16S rRNA and 18S rRNA molecules, they have been used separately for taxonomic and phylogenetic studies. Here, we report the existence of two highly conserved ribosomal sequences in all organisms that allow the amplification of a zone containing approximately 495 bp in prokaryotes and 508 bp in eukaryotes which we have named the "Universal Amplified Ribosomal Region" (UARR). Amplification and sequencing of this zone is possible using the same two universal primers (U1F and U1R) designed on the basis of two highly conserved ribosomal sequences. The UARR encompasses the V6, V7 and V8 domains from SSU rRNA in both prokaryotes and eukaryotes. The internal sequence of this zone in prokaryotes and eukaryotes is variable and the differences become less marked on descent from phyla to species. Nevertheless, UARR sequence allows species from the same genus to be differentiated. Thus, by UARR sequence analysis the construction of universal phylogenetic trees is possible, including species of prokaryotic and eukaryotic microorganisms together. Single isolates of prokaryotic and eukaryotic microorganisms from different sources can be identified by amplification and sequencing of UARR using the same pair of primers and the same PCR and sequencing conditions.     

Biodegradation of organic pollutants by halophilic bacteria and archaea

Hypersaline environments are important for both surface extension and ecological significance. As all other ecosystems, they are impacted by pollution. However, little information is available on the biodegradation of organic pollutants by halophilic microorganisms in such environments. In addition, it is estimated that 5% of industrial effluents are saline and hypersaline. Conventional nonextremophilic microorganisms are unable to efficiently perform the removal of organic pollutants at high salt concentrations. Halophilic microorganisms are metabolically different and are adapted to extreme salinity; these microorganisms are good candidates for the bioremediation of hypersaline environments and treatment of saline effluents. This literature survey indicates that both the moderately halophilic bacteria and the extremely halophilic archaea have a broader catabolic versatility and capability than previously thought. A diversity of contaminating compounds is susceptible to be degraded by halotolerant and halophile bacteria. Nevertheless, significant research efforts are still necessary in order to estimate the true potential of these microorganisms to be applied in environmental processes and in the remediation of contaminated hypersaline ecosystems. This effort should be also focused on basic research to understand the overall degradation mechanism, to identify the enzymes involved in the degradation process and the metabolism regulation.     

Contribution of SAR11 bacteria to dissolved dimethylsulfoniopropionate and amino acid uptake in the North Atlantic ocean

SAR11 bacteria are abundant in marine environments, often accounting for 35% of total prokaryotes in the surface ocean, but little is known about their involvement in marine biogeochemical cycles. Previous studies reported that SAR11 bacteria are very small and potentially have few ribosomes, indicating that SAR11 bacteria could have low metabolic activities and could play a smaller role in the flux of dissolved organic matter than suggested by their abundance. To determine the ecological activity of SAR11 bacteria, we used a combination of microautoradiography and fluorescence in situ hybridization (Micro-FISH) to measure assimilation of (3)H-amino acids and [(35)S]dimethylsulfoniopropionate (DMSP) by SAR11 bacteria in the coastal North Atlantic Ocean and the Sargasso Sea. We found that SAR11 bacteria were often abundant in surface waters, accounting for 25% of all prokaryotes on average. SAR11 bacteria were typically as large as, if not larger than, other prokaryotes. Additionally, more than half of SAR11 bacteria assimilated dissolved amino acids and DMSP, whereas about 40% of other prokaryotes assimilated these compounds. Due to their high abundance and activity, SAR11 bacteria were responsible for about 50% of amino acid assimilation and 30% of DMSP assimilation in surface waters. The contribution of SAR11 bacteria to amino acid assimilation was greater than would be expected based on their overall abundance, implying that SAR11 bacteria outcompete other prokaryotes for these labile compounds. These data suggest that SAR11 bacteria are highly active and play a significant role in C, N, and S cycling in the ocean.