MicrobiologyBytes

One of my New Year resolutions this year was to try to lay off the “omics” papers here on MicrobiologyBytes. Most of them are not very good and even those that are get indigestible fairly quickly. I’m making an exception for this one as it’s reasonably easy to read and highly relevant to chronic obstructive pulmonary disease (COPD), a progressive and eventually fatal lung disease that is projected to be responsible for the fifth largest burden of disease worldwide by 2020.

The results show that the lungs of “healthy” smokers contain a bacterial microbiome that is quantitatively significant, diverse (but of limited membership), and quite distinct from that reported for the oral cavity or nasopharynx. The diversity of the lung bacterial microbiome is lower in subjects with decreased lung function, most commonly associated with dominance by Pseudomonas spp. This is the first study to describe that the numerous microanatomic sites within the lung can give rise to significant differences in bacterial community structure. By demonstrating that one person’s lungs can harbor both generalized areas of “healthy” microbiome and a single site containing a “pathogenic” community, the results suggest a mechanism by which the interaction of lung pathogens and host immunity might contribute to localized disease progression, even in the absence of overt symptoms.

Even if you feel OK, stop smoking, stupid!

Analysis of the Lung Microbiome in the “Healthy” Smoker and in COPD. (2011) PLoS ONE 6(2): e16384. doi:10.1371/journal.pone.0016384
Although culture-independent techniques have shown that the lungs are not sterile, little is known about the lung microbiome in chronic obstructive pulmonary disease (COPD). We used pyrosequencing of 16S amplicons to analyze the lung microbiome in two ways: first, using bronchoalveolar lavage (BAL) to sample the distal bronchi and air-spaces; and second, by examining multiple discrete tissue sites in the lungs of six subjects removed at the time of transplantation. We performed BAL on three never-smokers (NS) with normal spirometry, seven smokers with normal spirometry (“heathy smokers”, HS), and four subjects with COPD (CS). Bacterial 16 s sequences were found in all subjects, without significant quantitative differences between groups. Both taxonomy-based and taxonomy-independent approaches disclosed heterogeneity in the bacterial communities between HS subjects that was similar to that seen in healthy NS and two mild COPD patients. The moderate and severe COPD patients had very limited community diversity, which was also noted in 28% of the healthy subjects. Both approaches revealed extensive membership overlap between the bacterial communities of the three study groups. No genera were common within a group but unique across groups. Our data suggests the existence of a core pulmonary bacterial microbiome that includes Pseudomonas, Streptococcus, Prevotella, Fusobacterium, Haemophilus, Veillonella, and Porphyromonas. Most strikingly, there were significant micro-anatomic differences in bacterial communities within the same lung of subjects with advanced COPD. These studies are further demonstration of the pulmonary microbiome and highlight global and micro-anatomic changes in these bacterial communities in severe COPD patients.

Related:

• Human Microbiome Project (HMP)
• Circumcision alters penis microbiome

How do marine microbial ecosystems respond to climate change and pollution? In this article in Microbiology Today, Jack Gilbert explains how treating microbial marine communities as single cells in a metatranscriptomics approach could shed light on this fundamental question:

If the number of known stars in the Milky Way is multiplied by the number of known galaxies in the universe the result is a huge number, a septillion (1×10²⁴). Yet, large as this is, it pales in comparison to the number of microbial cells found in the world oceans, estimated to be 1 nonillion (1×10³⁰). When we start to include soil, air and organism-associated environments, this number becomes unimaginable. Traditional microbiology is our gold standard for understanding how these trillions and trillions of bacteria function. Basically, we grow the bugs in a laboratory, one species at a time, and test how they respond to chemical stimuli. Ultimately, we sequence their genome and try to map their genes to particular functions. To help make this link we can observe the expression of these genes in response to certain stimuli, so called transcriptomics.

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Related:

• PLoS: A Primer on Metagenomics

Amazingly, all the phages on Earth, if placed end to end, would probably extend a distance equivalent to that of the nearest 60 galaxies. In this article in Microbiology Today, Eric Wommack explains how metagenomics is gradually revealing the amazing diversity and abundance of viruses in the biosphere:

Although the throughput and accuracy of methods for viral direct counting has improved since the 1989 report based on transmission electron microscopy, the ‘factor of 10’ ratio of virus to bacterial abundance within aquatic environments has remained a surprisingly common observation. Extrapolating the ‘factor of 10’ rule to the biosphere has lead to estimates that global viral abundance is in the order of 10³¹ individuals. Assuming an average length dimension of 100 nm, Curtis Suttle has proposed that, lined end-to-end, all the phages on earth would extend a distance equivalent to that of the nearest 60 galaxies (10 million light years, 10²⁴m).

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Related:

• New dimensions of the virus world discovered through metagenomics
• Genomes of unknown marine viruses

Normally, I try to steer people away from the controversial journal Medical Hypotheses, with its policy of publishing on editorial whim rather than peer review. Every once in a while they surprise us by publishing something decent. This is one such article:

Albert Einstein once said that “The true value of a human being can be found in the degree to which he has attained liberation from the self”. For years our traditional view of ‘self’ was restricted to our own bodies; composed of eukaryote cells encoded by our genome. However, in the era of omics technologies and systems biology, this view now extends beyond the traditional limitations of our own core being to include our resident microbial communities. These prokaryote cells outnumber our own cells by a factor of ten and contain at least ten times more DNA than our own genome. In exchange for food and shelter, this symbiont provides us, the host, with metabolic functions far beyond the scope of our own physiological capabilities. In this respect the human body can be considered a superorganism; a communal group of human and microbial cells all working for the benefit of the collective – a view which most certainly attains liberation from self.

The human superorganism – of microbes and men. Medical Hypotheses 2010 74(2): 214-215

Related:

• PLoS: A Primer on Metagenomics
• New dimensions of the virus world discovered through metagenomics

Metagenomics is a discipline that enables the genomic study of uncultured microorganisms. Faster, cheaper sequencing technologies and the ability to sequence uncultured microbes sampled directly from their habitats are expanding and transforming our view of the microbial world. Distilling meaningful information from the millions of new genomic sequences presents a serious challenge to bioinformaticians. In cultured microbes, the genomic data come from a single clone, making sequence assembly and annotation tractable. In metagenomics, the data come from heterogeneous microbial communities, sometimes containing more than 10,000 species, with the sequence data being noisy and partial. From sampling, to assembly, to gene calling and function prediction, bioinformatics faces new demands in interpreting voluminous, noisy, and often partial sequence data. Although metagenomics is a relative newcomer to science, the past few years have seen an explosion in computational methods applied to metagenomic-based research. It is therefore not within the scope of this article to provide an exhaustive review. Rather, we provide here a concise yet comprehensive introduction to the current computational requirements presented by metagenomics, and review the recent progress made. We also note whether there is software that implements any of the methods presented here, and briefly review its utility. Nevertheless, it would be useful if readers of this article would avail themselves of the comment section provided by this journal, and relate their own experiences. Finally, the last section of this article provides a few representative studies illustrating different facets of recent scientific discoveries made using metagenomics.

A Primer on Metagenomics. 2010 PLoS Comput Biol 6(2): e1000667. doi:10.1371/journal.pcbi.1000667

Related:

• Vineyardomics: SOS – Save our Shiraz!
• New dimensions of the virus world discovered through metagenomics

Grapevines are an important global crop which are widely planted throughout temperate regions. Viruses are a significant factor in reducing the quality and quantity of the yield and are known to reduce the productive life of vineyards. Grapevines are subject to infection by more than 60 different viruses, the most known for any crop plant. Most important grapevine virus diseases are caused by complexes of viruses, with up to nine different viruses having been identified in a single vine. In South Africa, as in most grape-growing regions of the world, grapevine leafroll is regarded to be the most significant virus disease affecting grapevine, with Shiraz disease and Shiraz decline becoming more prominent as emerging diseases in the industry.

Present disease diagnostics rely on ELISA or RT-PCR and target the viruses that have historically been associated with these diseases. While these tests are highly specific, they may not result in an accurate reflection of the etiological status of the tested plant, or of the particular disease, since none of the current diagnostic techniques address the potential contribution of other known or unknown viruses that may be involved in the etiology of a particular disease. Moreover, the error prone replication of RNA viruses leads to quasispecies, which can further complicate PCR-based detection assays as not all variants of the virus may be detected.

New and powerful technologies which are able to sequence viruses from environmental samples without the need for laborious and costly purification, cloning and screening techniques can result in the generation of sequence information for the complete virome in an unbiased fashion. This paper describes the use of sequencing-by-synthesis technology on the massively parallel Illumina Genome Analyzer II, to sequence an environmental sample composed of 44 randomly selected vines, to determine the virus profile of a severely diseased vineyard.

Deep sequencing analysis of viruses infecting grapevines: Virome of a vineyard. Virology. Feb 19 2010
Double stranded RNA, isolated from 44 pooled randomly selected vines from a diseased South African vineyard, has been used in a deep sequencing analysis to build a census of the viral population. The dsRNA was sequenced in an unbiased manner using the sequencing-by-synthesis technology offered by the Illumina Genome Analyzer II and yielded 837 megabases of metagenomic sequence data. Four known viral pathogens were identified. It was found that Grapevine leafroll-associated virus 3 (GLRaV-3) is the most prevalent species, constituting 59% of the total reads, followed by Grapevine rupestris stem pitting-associated virus and Grapevine virus A. Grapevine virus E, a virus not previously reported in South African vineyards, was identified in the census. Viruses not previously identified in grapevine were also detected. The second most prevalent virus detected was a member of the Chrysoviridae family similar to Penicillium chrysogenum virus. Sequences aligning to two other mycoviruses were also detected.

Related:

• New dimensions of the virus world discovered through metagenomics
• A Glimpse Into the World of Metagenomics
• Genetic and environmental factors affect gene expression in yeast

Over the past two decades, the study of marine viruses using electron and fluorescent microscopy revealed an unexpected abundance of virus particles. At 10⁶ to 10⁹ particles per milliliter of sea water, viruses are the most abundant microbes in the sea and, most likely, in the entire biosphere. Furthermore, they have emerged as crucial geochemical and ecological factors in marine ecosystems. More recently, extensive data on the metagenomics of marine viruses have been reported. Viral metagenomics is either pursued specifically by deep sequencing of environmental samples enriched for virus particles or emerge serendipitously through detection of virus-specific sequences in databases yielded by other metagenomic projects. The latter type of study is mostly limited to known classes of viruses but the former has the potential to discover completely unknown viruses.

The gene repertoires of the putative marine viromes that were derived by sequencing double-stranded DNA (dsDNA) isolated from the fractions enriched for virus-like particles brought several major surprises and potential concerns. In particular, the estimates of the number of unique viral genotypes yielded breathtaking numbers of >10³⁰, making the marine viromes the most genetically diverse biological communities on earth. The main and highly unexpected findings were that a substantial majority of the putative viral sequences were not significantly similar to any sequences in the current databases, and that those sequences that did have detectable homologs represented, primarily, various bacterial genes often having specific roles in central metabolism rather than distinct classes of genes commonly found in known bacteriophages or other viruses. These remarkable findings suggest two possibilities that are not mutually exclusive. First, known viruses might not be representative of actual viromes, with the implication that marine viruses are the principal reservoir of new genes in the ocean. Second, the samples deemed to represent viromes might be, largely, not of viral origin and reflect contamination of the samples with non-viral DNA, which would indicate a serious shortcoming of the current metagenomic protocols.

This paper applies computational approaches to analyze the marine dsDNA viromes and shows that, despite non-negligible contamination with bacterial genes, these sequences represent a collection that is markedly different in its statistical features from both prokaryotic and known viral genomes. Thus, there seems to be a realistic possibility that the actual marine viromes consist predominantly of virus-like particles that are different from well-characterized phages and might resemble gene transfer agents (GTAs). Although still a young field, metagenomics is already revealing unexpected yet fundamental features of the virus world.

New dimensions of the virus world discovered through metagenomics. Trends Microbiol. Nov 24 2009
Metagenomic analysis of viruses suggests novel patterns of evolution, changes the existing ideas of the composition of the virus world and reveals novel groups of viruses and virus-like agents. The gene composition of the marine DNA virome is dramatically different from that of known bacteriophages. The virome is dominated by rare genes, many of which might be contained within virus-like entities such as gene transfer agents. Analysis of marine metagenomes thought to consist mostly of bacterial genes revealed a variety of sequences homologous to conserved genes of eukaryotic nucleocytoplasmic large DNA viruses, resulting in the discovery of diverse members of previously undersampled groups and suggesting the existence of new classes of virus-like agents. Unexpectedly, metagenomics of marine RNA viruses showed that representatives of only one superfamily of eukaryotic viruses, the picorna-like viruses, dominate the RNA virome.

Related:

• A Glimpse Into the World of Metagenomics
• Genomes of unknown marine viruses