Actinobacteria within the acI lineage are often numerically dominating in freshwater ecosystems, where they can account for >50% of total bacteria in the surface water. However, they remain uncultured to date. We thus set out to use single-cell genomics to gain insights into their genetic make-up, with the aim of learning about their physiology and ecological niche. A representative from the highly abundant acI-B1 group was selected for shotgun genomic sequencing. We obtained a draft genomic sequence in 75 larger contigs (sum=1.16 Mb), with an unusually low genomic G+C mol% (∼42%). Actinobacteria core gene analysis suggests an almost complete genome recovery. We found that the acI-B1 cell had a small genome, with a rather low percentage of genes having no predicted functions (∼15%) as compared with other cultured and genome-sequenced microbial species. Our metabolic reconstruction hints at a facultative aerobe microorganism with many transporters and enzymes for pentoses utilization (for example, xylose). We also found an actinorhodopsin gene that may contribute to energy conservation under unfavorable conditions. This project reveals the metabolic potential of a member of the global abundant freshwater Actinobacteria.

This chapter describes biochemical pathways operating in aerobic methylotrophic bacteria. We first define aerobic methylotrophy as a specific metabolic capability and describe the phylogenetic diversity of these bacteria. We then describe enzymes involved in primary oxidation of different single carbon substrates, resulting in formaldehyde or methyl or methylene radical, and describe the variety of pathways used for their assimilation and dissimilation. We also give a brief account of genetic manipulation tools in methylotrophic bacteria and examples of systems approaches for studying their metabolism, including availability of whole genome sequence information.

The honeybee haplodiploid sex determination system depends on genetic variation at the complementary sex-determiner (csd) locus. In closed populations of honeybees, especially those undergoing selective breeding, the number of csd alleles can drop such that brood viability is affected. Here we present two polymerase chain reaction tests that allow the discrimination of csd alleles. Such tests should find utility in bee breeding programmes allowing the tracking and maintenance of csd alleles through successive generations.

Many marine sponges are hosts to dense and phylogenetically diverse microbial communities that are located in the extracellular matrix of the animal. The candidate phylum Poribacteria is a predominant member of the sponge microbiome and its representatives are nearly exclusively found in sponges. Here we used single-cell genomics to obtain comprehensive insights into the metabolic potential of individual poribacterial cells representing three distinct phylogenetic groups within Poribacteria. Genome sizes were up to 5.4 Mbp and genome coverage was as high as 98.5%. Common features of the poribacterial genomes indicated that heterotrophy is likely to be of importance for this bacterial candidate phylum. Carbohydrate-active enzyme database screening and further detailed analysis of carbohydrate metabolism suggested the ability to degrade diverse carbohydrate sources likely originating from seawater and from the host itself. The presence of uronic acid degradation pathways as well as several specific sulfatases provides strong support that Poribacteria degrade glycosaminoglycan chains of proteoglycans, which are important components of the sponge host matrix. Dominant glycoside hydrolase families further suggest degradation of other glycoproteins in the host matrix. We therefore propose that Poribacteria are well adapted to an existence in the sponge extracellular matrix. Poribacteria may be viewed as efficient scavengers and recyclers of a particular suite of carbon compounds that are unique to sponges as microbial ecosystems.

Background

Sediments are massive reservoirs of carbon compounds and host a large fraction of microbial life. Microorganisms within terrestrial aquifer sediments control buried organic carbon turnover, degrade organic contaminants, and impact drinking water quality. Recent 16S rRNA gene profiling indicates that members of the bacterial phylum Chloroflexi are common in sediment. Only the role of the class Dehalococcoidia, which degrade halogenated solvents, is well understood. Genomic sampling is available for only six of the approximate 30 Chloroflexi classes, so little is known about the phylogenetic distribution of reductive dehalogenation or about the broader metabolic characteristics of Chloroflexi in sediment.

Results

We used metagenomics to directly evaluate the metabolic potential and diversity of Chloroflexi in aquifer sediments. We sampled genomic sequence from 86 Chloroflexi representing 15 distinct lineages, including members of eight classes previously characterized only by 16S rRNA sequences. Unlike in the Dehalococcoidia, genes for organohalide respiration are rare within the Chloroflexi genomes sampled here. Near-complete genomes were reconstructed for three Chloroflexi. One, a member of an unsequenced lineage in the Anaerolinea, is an aerobe with the potential for respiring diverse carbon compounds. The others represent two genomically unsampled classes sibling to the Dehalococcoidia, and are anaerobes likely involved in sugar and plant-derived-compound degradation to acetate. Both fix CO₂ via the Wood-Ljungdahl pathway, a pathway not previously documented in Chloroflexi. The genomes each encode unique traits apparently acquired from Archaea, including mechanisms of motility and ATP synthesis.

Conclusions

Chloroflexi in the aquifer sediments are abundant and highly diverse. Genomic analyses provide new evolutionary boundaries for obligate organohalide respiration. We expand the potential roles of Chloroflexi in sediment carbon cycling beyond organohalide respiration to include respiration of sugars, fermentation, CO₂ fixation, and acetogenesis with ATP formation by substrate-level phosphorylation.

The development of culture-independent strategies to study microbial diversity and function has led to a revolution in microbial ecology, enabling us to address fundamental questions about the distribution of microbes and their influence on Earth’s biogeochemical cycles. This article discusses some of the progress that scientists have made with the use of so-called “omic” techniques (metagenomics, metatranscriptomics, and metaproteomics) and the limitations and major challenges these approaches are currently facing. These ‘omic methods have been used to describe the taxonomic structure of microbial communities in different environments and to discover new genes and enzymes of industrial and medical interest. However, microbial community structure varies in different spatial and temporal scales and none of the ‘omic techniques are individually able to elucidate the complex aspects of microbial communities and ecosystems. In this article we highlight the importance of a spatiotemporal sampling design, together with a multilevel ‘omic approach and a community analysis strategy (association networks and modeling) to examine and predict interacting microbial communities and their impact on the environment.

The beginning of the twenty-first century marks the genomic era in microbiology, where many genomes started being completely sequenced. The rate at which this kind of feat is now possible has increased drastically with the development of new sequencing technologies. In this post-genomic scenario, intraspecies genome variation has been examined and the concept of pan-genome, the sum of all genes present in members of one taxonomic group, was proposed. Practical application of this concept is found in the search for universal antigens for vaccine development. On a more fundamental level, these genome comparisons have increased the debate on what is a bacterial species and have shown that lateral gene transfer is more important than previously thought in bacterial genome evolution. Sequencing of the genomes from entire microbial communities has shown that the diversity of microorganisms is much greater than previously thought. Available post-genome technology has allowed the development of successful commercial biotechnology products without resorting to the cultivation of the microorganism of interest. On the opposite side of the spectrum, single cell genomics has allowed sequencing of the genome from individual cells. Accessing these genomes will give a perspective of genome differences between individuals and will facilitate the cultivation of these organisms. Central to all these developments in genome and post-genome technologies is bioinformatics. Some of the strategies used for genome and metagenome data storage and analysis are discussed. Although there are a number of successful examples, the challenge now is to transform the knowledge about genomes and post-genome technologies into commercial products at a greater pace.

Background

Single cell genomics has revolutionized microbial sequencing, but complete coverage of genomes in complex microbiomes is imperfect due to enormous variation in organismal abundance and amplification bias. Empirical methods that complement rapidly improving bioinformatic tools will improve characterization of microbiomes and facilitate better genome coverage for low abundance microbes.

Methods

We describe a new approach to sequencing individual species from microbiomes that combines antibody phage display against intact bacteria with fluorescence activated cell sorting (FACS). Single chain (scFv) antibodies are selected using phage display against a bacteria or microbial community, resulting in species-specific antibodies that can be used in FACS for relative quantification of an organism in a community, as well as enrichment or depletion prior to genome sequencing.

Results

We selected antibodies against Lactobacillus acidophilus and demonstrate a FACS-based approach for identification and enrichment of the organism from both laboratory-cultured and commercially derived bacterial mixtures. The ability to selectively enrich for L. acidophilus when it is present at a very low abundance (<0.2%) leads to complete (>99.8%) de novo genome coverage whereas the standard single-cell sequencing approach is incomplete (<68%). We show that specific antibodies can be selected against L. acidophilus when the monoculture is used as antigen as well as when a community of 10 closely related species is used demonstrating that in principal antibodies can be generated against individual organisms within microbial communities.

Conclusions

The approach presented here demonstrates that phage-selected antibodies against bacteria enable identification, enrichment of rare species, and depletion of abundant organisms making it tractable to virtually any microbe or microbial community. Combining antibody specificity with FACS provides a new approach for characterizing and manipulating microbial communities prior to genome sequencing.

Reproduction in species of eusocial insects is monopolized by one or a few individuals, while the remaining colony tasks are performed by the worker caste. This reproductive division of labor is exemplified by honey bees (Apis mellifera L.), in which a single, polyandrous queen is the sole colony member that lays fertilized eggs. Previous work has revealed that the developmental fate of honey bee queens is highly plastic, with queens raised from younger worker larvae exhibiting higher measures in several aspects of reproductive potential compared to queens raised from older worker larvae. Here, we investigated the effects of queen reproductive potential (“quality”) on the growth and winter survival of newly established honey bee colonies. We did so by comparing the growth of colonies headed by “high-quality” queens (i.e., those raised from young worker larvae, which are more queen-like morphologically) to those headed by “low-quality” queens (i.e., those raised from older worker larvae, which are more worker-like morphologically). We confirmed that queens reared from young worker larvae were significantly larger in size than queens reared from old worker larvae. We also found a significant positive effect of queen grafting age on a colony’s production of worker comb, drone comb, and stored food (honey and pollen), although we did not find a statistically significant difference in the production of worker and drone brood, worker population, and colony weight. Our results provide evidence that in honey bees, queen developmental plasticity influences several important measures of colony fitness. Thus, the present study supports the idea that a honey bee colony can be viewed (at least in part) as the expanded phenotype of its queen, and thus selection acting predominantly at the colony level can be congruent with that at the individual level.