This chapter focuses on how metagenomic data are applied to examine the genomic heterogeneity of natural microbial populations. It highlights the opportunities and challenges inherent to the approach and describes recently developed methods to maximally leverage the potential of these datasets while tackling some of the challenges. We describe how performing population genomic analyses using metagenomic data allows (1) resolution of ecologically and genetically cohesive populations in the environment, (2) tracking of evolutionary processes within them, and (3) application of metatranscriptomic and metaproteomic analyses to determine the in situ physiology of distinct populations. While challenges remain that are inherent to the approach, the current wave of new bioinformatic tools is starting to realize the theoretical potential of metagenomics to peer into the spatiotemporal dynamics of the genetic structure of natural populations.

Since the first two complete bacterial genome sequences were published in 1995, the science of bacteria has dramatically changed. Using third-generation DNA sequencing, it is possible to completely sequence a bacterial genome in a few hours and identify some types of methylation sites along the genome as well. Sequencing of bacterial genome sequences is now a standard procedure, and the information from tens of thousands of bacterial genomes has had a major impact on our views of the bacterial world. In this review, we explore a series of questions to highlight some insights that comparative genomics has produced. To date, there are genome sequences available from 50 different bacterial phyla and 11 different archaeal phyla. However, the distribution is quite skewed towards a few phyla that contain model organisms. But the breadth is continuing to improve, with projects dedicated to filling in less characterized taxonomic groups. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system provides bacteria with immunity against viruses, which outnumber bacteria by tenfold. How fast can we go? Second-generation sequencing has produced a large number of draft genomes (close to 90 % of bacterial genomes in GenBank are currently not complete); third-generation sequencing can potentially produce a finished genome in a few hours, and at the same time provide methlylation sites along the entire chromosome. The diversity of bacterial communities is extensive as is evident from the genome sequences available from 50 different bacterial phyla and 11 different archaeal phyla. Genome sequencing can help in classifying an organism, and in the case where multiple genomes of the same species are available, it is possible to calculate the pan- and core genomes; comparison of more than 2000 Escherichia coli genomes finds an E. coli core genome of about 3100 gene families and a total of about 89,000 different gene families. Why do we care about bacterial genome sequencing? There are many practical applications, such as genome-scale metabolic modeling, biosurveillance, bioforensics, and infectious disease epidemiology. In the near future, high-throughput sequencing of patient metagenomic samples could revolutionize medicine in terms of speed and accuracy of finding pathogens and knowing how to treat them.

Background and aims

Microbial communities are the primary drivers of organic matter decomposition in peatlands. However, limited knowledge is available regarding depth-dependent microbial community structure and function in East Asian peatlands, using cultivation independent approaches.

Methods

We investigated the vertical stratification of prokaryote and fungal communities in a moderately rich fen in northeast China (Hani) and a rich fen in southwest China (Riganqiao).

Results

Fungal and prokaryotic operational taxonomic unit (OTU) composition exhibited strong site and/or depth responses. Prokaryotic OTUs exhibited the greatest alpha diversity at the mesotelm ‘hot spot’, whereas the predicted metagenomic metabolic functions did not align with the pattern of prokaryote alpha diversity. The large cover of shrubs contributed to a greater relative abundance of ericoid- and ecto-mycorrhizal fungi at Hani, whereas Riganqiao showed more arbuscular mycorrhizal fungi. Soil pH and water table depth were among the predominant abiotic factors associated with microbial community composition.

Conclusions

Projected shifts in hydrology and/or vegetation with global change may cause substantial impacts on peatland microorganisms and thus the associated biogeochemistry. It is critical to better understand the mechanism of the discrepancy between microbial community structure and the functions at the mesotelm ‘hot spot’ when evaluating the ecosystem functions in peatlands.

Background

Genomes of Methanosarcina spp. are among the largest archaeal genomes. One suggested reason for that is massive horizontal gene transfer (HGT) from bacteria. Genes of bacterial origin may be involved in the central metabolism and solute transport, in particular sugar synthesis, sulfur metabolism, phosphate metabolism, DNA repair, transport of small molecules etc. Horizontally transferred (HT) genes are considered to play the key role in the ability of Methanosarcina spp. to inhabit diverse environments. At the moment, genomes of three Methanosarcina spp. have been sequenced, and while these genomes vary in length and number of protein-coding genes, they all have been shown to accumulate HT genes. However, previous estimates had been made when fewer archaeal genomes were known. Moreover, several Methanosarcinaceae genomes from other genera have been sequenced recently. Here, we revise the census of genes of bacterial origin in Methanosarcinaceae.

Results

About 5 % of Methanosarcina genes have been shown to be horizontally transferred from various bacterial groups to the last common ancestor either of Methanosarcinaceae, or Methanosarcina, or later in the evolution. Simulation of the composition of the NCBI protein non-redundant database for different years demonstrates that the estimates of the HGT rate have decreased drastically since 2002, the year of publication of the first Methanosarcina genome.

The phylogenetic distribution of HT gene donors is non-uniform. Most HT genes were transferred from Firmicutes and Proteobacteria, while no HGT events from Actinobacteria to the common ancestor of Methanosarcinaceae were found. About 50 % of HT genes are involved in metabolism. Horizontal transfer of transcription factors is not common, while 46 % of horizontally transferred genes have demonstrated differential expression in a variety of conditions. HGT of complete operons is relatively infrequent and half of HT genes do not belong to operons.

Conclusions

While genes of bacterial origin are still more frequent in Methanosarcinaceae than in other Archaea, most HGT events described earlier as Methanosarcina-specific seem to have occurred before the divergence of Methanosarcinaceae. Genes horizontally transferred from bacteria to archaea neither tend to be transferred with their regulators, nor in long operons.

Microbial taxonomy should provide adequate descriptions of bacterial, archaeal, and eukaryotic microbial diversity in ecological, clinical, and industrial environments. Its cornerstone, the prokaryote species has been re-evaluated twice. It is time to revisit polyphasic taxonomy, its principles, and its practice, including its underlying pragmatic species concept. Ultimately, we will be able to realize an old dream of our predecessor taxonomists and build a genomic-based microbial taxonomy, using standardized and automated curation of high-quality complete genome sequences as the new gold standard.

The bacterial tree of life has recently undergone significant expansion, chiefly from candidate phyla retrieved through genome-resolved metagenomics. Bypassing the need for genome availability, we present a snapshot of bacterial phylogenetic diversity based on the recovery of high-quality SSU rRNA gene sequences extracted from nearly 7000 metagenomes and all available reference genomes. We illuminate taxonomic richness within established bacterial phyla together with environmental distribution patterns, providing a revised framework for future phylogeny-driven sequencing efforts.

Sponge-microbe symbiotic interactions are important features of modern marine ecosystems. It is likely that these ancient partnerships are as old as the phylum Porifera. Powerful new tools have exposed remarkable microbial diversity within sponge tissues. We are now able to study the composition and structure of the microbial communities at unprecedented levels of resolution. We also recognize that the partnership cannot be disaggregated and should be considered as an integrated holobiont. New hypotheses (e.g., the sponge loop hypothesis) have opened exciting avenues for future experimental work that link holobiont performance from micro- to macro-perspectives. This type of research has taken on added significance given that our planet is experiencing accelerating rates of ocean warming and ocean acidification. It is essential that we examine how sponges respond to environmental stressors that are increasing in intensity and frequency. This review focuses on the molecular and functional ecology of sponge-based microbial symbioses. We discuss the coevolutionary processes that operate to generate partner specificity or to maintain promiscuous partnerships and consider reciprocal selective forces that shape the material exchanges that occur between the partners. We focus attention on the functional ecological role the holobiont plays in marine habitats. The role that the symbionts play in host physiology, and ultimately in the function of sponges on marine ecosystems, is also discussed. We stand to gain important basic information about symbiotic interactions through the detailed study of sponge-microbe interactions, but important practical lessons will be afforded to resource managers who are looking for strategies to protect aquatic habitats worldwide.

Background

Renewable energy production is currently a major issue worldwide. Biogas is a promising renewable energy carrier as the technology of its production combines the elimination of organic waste with the formation of a versatile energy carrier, methane. In consequence of the complexity of the microbial communities and metabolic pathways involved the biotechnology of the microbiological process leading to biogas production is poorly understood. Metagenomic approaches are suitable means of addressing related questions. In the present work a novel high-throughput technique was tested for its benefits in resolving the functional and taxonomical complexity of such microbial consortia.

Results

It was demonstrated that the extremely parallel SOLiD™ short-read DNA sequencing platform is capable of providing sufficient useful information to decipher the systematic and functional contexts within a biogas-producing community. Although this technology has not been employed to address such problems previously, the data obtained compare well with those from similar high-throughput approaches such as 454-pyrosequencing GS FLX or Titanium. The predominant microbes contributing to the decomposition of organic matter include members of the Eubacteria, class Clostridia, order Clostridiales, family Clostridiaceae. Bacteria belonging in other systematic groups contribute to the diversity of the microbial consortium. Archaea comprise a remarkably small minority in this community, given their crucial role in biogas production. Among the Archaea, the predominant order is the Methanomicrobiales and the most abundant species is Methanoculleus marisnigri. The Methanomicrobiales are hydrogenotrophic methanogens. Besides corroborating earlier findings on the significance of the contribution of the Clostridia to organic substrate decomposition, the results demonstrate the importance of the metabolism of hydrogen within the biogas producing microbial community.

Conclusions

Both microbiological diversity and the regulatory role of the hydrogen metabolism appear to be the driving forces optimizing biogas-producing microbial communities. The findings may allow a rational design of these communities to promote greater efficacy in large-scale practical systems. The composition of an optimal biogas-producing consortium can be determined through the use of this approach, and this systematic methodology allows the design of the optimal microbial community structure for any biogas plant. In this way, metagenomic studies can contribute to significant progress in the efficacy and economic improvement of biogas production.

Isolation and cultivation of microorganisms enables in combination with cultivation-independent methods comprehensive research on ecology and function of microbes. The availability of cultures provides opportunities for ecophysiological experiments, enables high-quality genome research and represents a mandatory prerequisite for the description of new taxa of prokaryotes. Unfortunately, access to microorganism by cultivation methods is still quite limited, and the majority of the microbial diversity remains uncultured so far. This chapter discusses the potential reasons for this lack in cultivability and reviews advances in cultivation methods for prokaryotes. Detailed analysis of the media and methods used by taxonomists for isolation of bacterial strains used for description of >1000 new species revealed that the description of new taxa in the ranks of species and genera is currently not methodically limited. On the other hand, isolation of strains enabling the description of taxa representing higher ranks is obviously strongly limited by the currently applied methods. Consequently, different cultivation strategies are required according to the scientific goals of the respective research. While taxonomists interested in the description of new species affiliated with any genus or family can isolate new strains suitable for this task by using standard cultivation methods and media, ecologists interested in cultivation of model organisms appearing in natural systems with high cell numbers, as well as taxonomists interested in isolation of strains representing new higher ranking taxa like orders, classes and phyla, should rather use high-throughput non-standard methods in combination with optimized screening protocols.

Interactions with microorganisms might enable house dust mites (HDMs) to derive nutrients from difficult-to-digest structural proteins and to flourish in human houses. We tested this hypothesis by investigating the effects of changes in the mite culture growth and population of two HDM species on HDM microbiome composition and fitness. Growing cultures of laboratory and industrial allergen-producing populations of Dermatophagoides farinae (DFL and DFT, respectively) and Dermatophagoides pteronyssinus (DPL and DPT, respectively) were sampled at four time points. The symbiotic microorganisms of the mites were characterized by DNA barcode sequencing and quantified by qPCR using universal/specific primers. The population growth of mites and nutrient contents of mite bodies were measured and correlated with the changes in bacteria in the HDM microbiome. The results showed that both the population and culture age significantly influenced the microbiome profiles. Cardinium formed 93% and 32% of the total sequences of the DFL and DFT bacterial microbiomes, respectively, but this bacterial species was less abundant in the DPL and DPT microbiomes. Staphylococcus abundance was positively correlated with increased glycogen contents in the bodies of mites, and increased abundances of Aspergillus, Candida, and Kocuria were correlated with increased lipid contents in the bodies of mites. The xerophilic fungus Wallemia accounted for 39% of the fungal sequences in the DPL microbiome, but its abundance was low in the DPT, DFL, and DFT microbiomes. With respect to the mite culture age, we made three important observations: the mite population growth from young cultures was 5–8-fold higher than that from old cultures; specimens from old cultures had greater abundances of fungi and bacteria in their bodies; and yeasts predominated in the gut contents of specimens from young cultures, whereas filamentous mycelium prevailed in specimens from old cultures. Our results are consistent with the hypothesis that mites derive nutrients through associations with microorganisms.