MicrobiologyBytes

In this article in Microbiology Today Ed Feil describes how we must brace ourselves for the next wave of data as new sequencing techniques become available to determine and compare many sequences at once. The enormous amount of data soon to be generated will bring exciting new insights into how micro-organisms within communities evolve and interact:

Regardless of the species in question, announcements of completed genome sequencing projects in the mainstream media almost invariably make reference to ‘cracking a code’ or ‘deciphering a genetic blueprint’. For bacteria, these over-used analogies spectacularly fail to give a true sense of the fluidity of genome evolution. The doe-eyed assumption in the mid-1990s that a single genome sequence can safely be considered as a prescriptive ‘solution’ for a given bacterial species has been dramatically falsified. By the late 1990s, multiple genome sequences for Escherichia coli revealed extensive differences in gene content between strains, and it rapidly became clear that, for many taxa, an individual genome is most usefully considered as one of many possible combinations of genes drawn from a vast pool known as the pangenome. When faced with such a maelstrom, our natural inclination (as good cladists) is to try and tidy it up, and catalogue strains into pockets of relatedness. Fortunately, phylogenetic analyses are possible, even for very variable species like E. coli, because one can readily identify genes which are universally present in all strains. These essential ‘core’ genes can be thought of as representing the operating system of a given species. In contrast, the specialist software is provided by ‘non-core’ or ‘accessory’ genes which are variably present or absent, are commonly acquired by horizontal transfer, and tend to be restricted to hypervariable regions called genomic islands. These two sets of genes present a fundamental duality in bacterial genomics. Whilst core genes can satisfy our requirements for molecular phylogeny (i.e. what a strain is), accessory genes often play a significant role in adaptation and phenotypic differences (i.e. what a strain does). Conflicts between these two can go a long way to explaining the mystery behind the muddle that is bacterial systematics.

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While single cell studies have focused on bacteria and cyanobacteria, single virions have yet to be isolated and genomically described using similar mechanisms. Viruses are ubiquitous and the most numerous and diverse biological entities on our planet. Nearly all aspects of our lives are influenced by viruses through shaping the environments that surround us, our immune responses and even our genomes. The field of environmental viral metagenomics has gained momentum over the past several years; however, sequencing of individual environmental viral genomes is currently dependent on the establishment of cultivable virus-host systems. With this in mind, if less than one percent of microbial populations can be cultured using standard microbiological techniques due to incongruencies in direct counts versus cultivatable microbes, then only a very small number of viruses have the likelihood of being genomically described. Currently, viral genomic sequences are lacking in public databases, with the exception of human viruses and those of agricultural and industrial significance (e.g. Lactococcal phages). Clearly, a better understanding of virus diversity and evolution will not be achieved until the genomes of a broad range of viruses are available.

This paper introduces an approach for isolating and characterizing the genomes of viruses called “Single Virus Genomics” (SVG). The benefits of SVG will be far-reaching, enabling novel virus discovery in a variety of clinical and environmental settings, altering our understanding of virus evolution, adaptation and ecology and facilitating the interpretation of viral genomic and metagenomic data by providing suitable reference genomes.

Single Virus Genomics: A New Tool for Virus Discovery. 2011 PLoS ONE 6(3): e17722. doi:10.1371/journal.pone.0017722
Whole genome amplification and sequencing of single microbial cells has significantly influenced genomics and microbial ecology by facilitating direct recovery of reference genome data. However, viral genomics continues to suffer due to difficulties related to the isolation and characterization of uncultivated viruses. We report here on a new approach called ‘Single Virus Genomics’, which enabled the isolation and complete genome sequencing of the first single virus particle. A mixed assemblage comprised of two known viruses; E. coli bacteriophages lambda and T4, were sorted using flow cytometric methods and subsequently immobilized in an agarose matrix. Genome amplification was then achieved in situ via multiple displacement amplification (MDA). The complete lambda phage genome was recovered with an average depth of coverage of approximately 437X. The isolation and genome sequencing of uncultivated viruses using Single Virus Genomics approaches will enable researchers to address questions about viral diversity, evolution, adaptation and ecology that were previously unattainable.

A new study examines how bacterial and archaeal genomic repertoires evolve to face new challenges by acquiring genes from other individuals. Microbes live and thrive in incredibly diverse and harsh conditions, from boiling or freezing water to the human immune system. This remarkable adaptability results from their ability to quickly modify their repertoire of protein functions by gaining, losing and modifying their genes. Microbes were known to modify genes to expand their repertoire of protein families in two ways: via duplication processes followed by slow functional specialization, in the same way as large multicellular organisms like us, and by acquiring different genes directly from other microbes. The latter process, known as horizontal gene transfer (HGT), is notoriously conspicuous in the spread of antibiotic resistance, turning some bacteria into drug-resistant ‘superbugs’ such as MRSA (methicillin-resistant Staphylococcus aureus), a serious public health concern.

The researchers examined a large database of microbial genomes, including some of the most virulent human pathogens, to discover whether duplication or HGT was the most common expansion method. They show that gene family expansion can indeed follow both routes, but unlike large multicellular organisms, it predominantly takes place by horizontal transfer. Thus, quick diversification of microbial functions results from the recruitment by microbes of pre-existing adaptations from other microbes. The study concludes with the observation that, since microbes invented the majority of life’s biochemical diversity, from respiration to photosynthesis, we should recognize the predominant role of HGT in the diversification of all protein families.

Horizontal Transfer, Not Duplication, Drives the Expansion of Protein Families in Prokaryotes. (2011) PLoS Genet 7(1): e1001284. doi:10.1371/journal.pgen.1001284
Gene duplication followed by neo- or sub-functionalization deeply impacts the evolution of protein families and is regarded as the main source of adaptive functional novelty in eukaryotes. While there is ample evidence of adaptive gene duplication in prokaryotes, it is not clear whether duplication outweighs the contribution of horizontal gene transfer in the expansion of protein families. We analyzed closely related prokaryote strains or species with small genomes (Helicobacter, Neisseria, Streptococcus, Sulfolobus), average-sized genomes (Bacillus, Enterobacteriaceae), and large genomes (Pseudomonas, Bradyrhizobiaceae) to untangle the effects of duplication and horizontal transfer. After removing the effects of transposable elements and phages, we show that the vast majority of expansions of protein families are due to transfer, even among large genomes. Transferred genes — xenologs — persist longer in prokaryotic lineages possibly due to a higher/longer adaptive role. On the other hand, duplicated genes — paralogs — are expressed more, and, when persistent, they evolve slower. This suggests that gene transfer and gene duplication have very different roles in shaping the evolution of biological systems: transfer allows the acquisition of new functions and duplication leads to higher gene dosage. Accordingly, we show that paralogs share most protein–protein interactions and genetic regulators, whereas xenologs share very few of them. Prokaryotes invented most of life’s biochemical diversity. Therefore, the study of the evolution of biology systems should explicitly account for the predominant role of horizontal gene transfer in the diversification of protein families.

Related:

• Barriers to Horizontal Gene Transfer
• Evolution of Clostridium difficile

We know of a large number of diseases or medical conditions that affect humans more severely than non-human primates. Humans are more sensitive than chimpanzees to the severe effects of certain virus infections, such as progression of HIV to AIDS or severe complications from hepatitis B. These differences likely arise from different immune responses to infection among species. However, due to the lack of comparative functional data across species, it remains unclear how the immune system of humans and other primates differ.

This paper present the first genome-wide characterization of functional differences in innate immune responses between humans and our closest evolutionary relatives. The results indicate that “core” immune responses, those that are critical to fight any invading pathogen, are the most conserved across primates and that much of the divergence in immune responses is observed in genes that are involved in response to specific microbial and viral agents. Human-specific immune responses are enriched for genes involved in apoptosis and cancer biology, as well as with genes previously associated with susceptibility to infectious diseases or immune-related disorders. Finally, it shows that chimpanzee-specific immune signaling pathways are enriched for HIV–interacting genes. These observations may help explain known inter-species differences in susceptibility to infectious diseases. Though detailed species-specific gene expression patterns were identified in this study, more experiments will be required to assess the phenotypic impact of those unique immune responses. Future studies will also test the immune response of each species to specific infectious agents. This is only the first step in characterizing inter-species differences in immune response.

Functional Comparison of Innate Immune Signaling Pathways in Primates. (2010) PLoS Genet 6(12): e1001249. doi:10.1371/journal.pgen.1001249
Humans respond differently than other primates to a large number of infections. Differences in susceptibility to infectious agents between humans and other primates are probably due to inter-species differences in immune response to infection. Consistent with that notion, genes involved in immunity-related processes are strongly enriched among recent targets of positive selection in primates, suggesting that immune responses evolve rapidly, yet providing only indirect evidence for possible inter-species functional differences. To directly compare immune responses among primates, we stimulated primary monocytes from humans, chimpanzees, and rhesus macaques with lipopolysaccharide (LPS) and studied the ensuing time-course regulatory responses. We find that, while the universal Toll-like receptor response is mostly conserved across primates, the regulatory response associated with viral infections is often lineage-specific, probably reflecting rapid host–virus mutual adaptation cycles. Additionally, human-specific immune responses are enriched for genes involved in apoptosis, as well as for genes associated with cancer and with susceptibility to infectious diseases or immune-related disorders. Finally, we find that chimpanzee-specific immune signaling pathways are enriched for HIV–interacting genes. Put together, our observations lend strong support to the notion that lineage-specific immune responses may help explain known inter-species differences in susceptibility to infectious diseases.

Related:

• Genomic fossils in lemurs shed light on HIV
• You Are Not What You Eat

The PLoS ONE Prokaryotic Genome Collection is an attempt to present and highlight a number of important articles that describe whole genome sequence and/or comparative genomics of important prokaryotic organisms:

A Flood of Microbial Genomes – Do We Need More?
Complete genome sequences of important bacterial pathogens and industrial organisms hold significant consequences and opportunities for human health, industry and the environment. Addressing biological and clinical problems through genome sequence based approaches offers many commercial opportunities. The aftermath of whole genome sequencing has revealed new insights into evolution of bacterial lifestyles including strategies for adaptation to new niches and overcoming competitors. Whole genome sequences representing more than 1500 prokaryotic organisms combined with the dozens (to hundreds) of strain re-sequencing projects are posing mind boggling problems on the optimal utilization of the resultant ‘omic’ datasets. Consequently, microbiologists are confronted with the challenge to translate these data into better human and animal healthcare solutions and pursue basic research approaches to interpret the data in ecological and evolutionary perspectives. New informatic approaches towards optimal utilization, holistic integration and meaningful interpretation of the genome sequence data are extremely necessary.

Related:

• Global functional atlas of Escherichia coli proteins

In 2002, the publication of the genome of Plasmodium falciparum, the most malignant agent of malaria, generated hope in the fight against this deadly disease by the opportunities it offered to discover new drug targets. Since then results have not lived up to the expectations. The development of comparative genomics to further understanding of P. falciparum has indeed been hindered by a lack of knowledge of closely related species’ genomes. Only one species, P. reichenowi, infecting chimpanzees, was previously known as a sister lineage of P. falciparum.

Researchers based in Gabon and France have now reported the discovery of a new malaria agent infecting chimpanzees in Central Africa. To investigate the diversity of Plasmodium parasites circulating in chimpanzees in Africa, the team collected blood from 19 wild-borne animals kept as pets by villagers in Gabon. Two were found infected by a Plasmodium parasite. This new species, named Plasmodium gaboni, is a close relative of the most virulent human malaria agent, P. falciparum. Based on its whole mitochondrial genome, they demonstrate that this new species is a close relative of P. falciparum and P. reichenowi. The analysis of its genome should thus offer the opportunity to explore P. falciparum specific adaptations to humans. These results suggest that malaria may have been present in early hominoids and may have experienced a radiation along with that of its hosts. This discovery highlights the paucity of our knowledge on the richness of Plasmodium species infecting primates and suggests more research in this area is urgently needed.

A New Malaria Agent in African Hominids. 2009 PLoS Pathog 5(5): e1000446
Plasmodium falciparum is the major human malaria agent responsible for 200 to 300 million infections and one to three million deaths annually, mainly among African infants. The origin and evolution of this pathogen within the human lineage is still unresolved. A single species, P. reichenowi, which infects chimpanzees, is known to be a close sister lineage of P. falciparum. Here we report the discovery of a new Plasmodium species infecting Hominids. This new species has been isolated in two chimpanzees (Pan troglodytes) kept as pets by villagers in Gabon (Africa). Analysis of its complete mitochondrial genome (5529 nucleotides including Cyt b, Cox I and Cox III genes) reveals an older divergence of this lineage from the clade that includes P. falciparum and P. reichenowi (21+/-9 Myrs ago using Bayesian methods and considering that the divergence between P. falciparum and P. reichenowi occurred 4 to 7 million years ago as generally considered in the literature). This time frame would be congruent with the radiation of hominoids, suggesting that this Plasmodium lineage might have been present in early hominoids and that they may both have experienced a simultaneous diversification. Investigation of the nuclear genome of this new species will further the understanding of the genetic adaptations of P. falciparum to humans. The risk of transfer and emergence of this new species in humans must be now seriously considered given that it was found in two chimpanzees living in contact with humans and its close relatedness to the most virulent agent of malaria.

Related:

• Malaria: now you see me, now you don’t
• New ways to beat malaria
• Tracing resistance to antimalarial drugs across Africa

Because of its central position in the microbial research community, the Gram-negative bacterium Escherichia coli plays a leading role in investigations of the fundamental molecular biology of bacteria. This experimentally tractable microbe is a workhorse in basic and applied research aimed at elucidating the mechanistic basis of prokaryotic processes and traits, including those of pathogens. The ever-expanding availability of genomic resources makes E. coli particularly well-suited to systematic investigations of microbial protein components and functional relationships on a global scale. These include a genome-wide collection of single-gene deletion strains along with extensive knowledge of regulatory circuits and metabolic pathways. Yet despite being the most highly studied model bacterium, a recent comprehensive community annotation effort for the fully sequenced reference K-12 laboratory strains indicated that only half (54%) of the protein-coding gene products of E. coli currently have experimental evidence indicative of a biological role. The remaining genes have either only generic, homology-derived functional attributes (e.g. “predicted DNA-binding”) or no discernable physiological significance. Some of these functional “orphans” may have eluded characterization in part because they exhibit mild mutant phenotypes, are expressed at low or undetectable levels, or have limited homology to annotated genes.

One goal of modern biology is to chart groups of proteins that act together to perform biological processes via direct and indirect interactions. Such groupings are sometimes called functional modules. The types of protein interaction within modules include physical interactions that generate protein complexes and biochemical associations that make up metabolic pathways. Researchers have combined proteomic and bioinformatic tools and used them to decipher a large number of protein interactions, complexes and functional modules with high confidence. In addition, exploring the topology of the resulting interaction networks, they successfully predicted specific biological roles for a number of proteins with previously unknown functions, and identified some potential drug targets. Although their work is focused on E. coli, their phylogenetic projections suggest that a considerable fraction of observations and predictions can be extrapolated to many other bacterial taxa. As all the data derived from this study are publicly available (at eNet), others may build on this work for further hypothesis-driven studies of gene function discovery.

Global functional atlas of Escherichia coli encompassing previously uncharacterized proteins. PLoS Biol 7(4): e1000096
One-third of the 4,225 protein-coding genes of Escherichia coli K-12 remain functionally unannotated (orphans). Many map to distant clades such as Archaea, suggesting involvement in basic prokaryotic traits, whereas others appear restricted to E. coli, including pathogenic strains. To elucidate the orphans’ biological roles, we performed an extensive proteomic survey using affinity-tagged E. coli strains and generated comprehensive genomic context inferences to derive a high-confidence compendium for virtually the entire proteome consisting of 5,993 putative physical interactions and 74,776 putative functional associations, most of which are novel. Clustering of the respective probabilistic networks revealed putative orphan membership in discrete multiprotein complexes and functional modules together with annotated gene products, whereas a machine-learning strategy based on network integration implicated the orphans in specific biological processes. We provide additional experimental evidence supporting orphan participation in protein synthesis, amino acid metabolism, biofilm formation, motility, and assembly of the bacterial cell envelope. This resource provides a ‘‘systems-wide’’ functional blueprint of a model microbe, with insights into the biological and evolutionary significance of previously uncharacterized proteins.

Related:

• An introduction to genomics (video)
• Digitizing Life (video)
• The end of the world? Dr Franken-Venter? Nope