Tag Archives: Phylogenetics

Carbohydrate binding gene family expansion in the amphibian pathogen Batrachochytrium dendrobatidis

You’d have to be living under a rock – as some amphibians do – to not be aware of the massive extinction facing our vertebrate friends living within aquatic habitats.  Researchers still don’t fully understand what is causing the amphibian mass-extinction – stress from habitat loss, increased chemical concentrations in the environment, and an auto-immune degrading infection have all been proposed.  What is known is that the chytrid fungus Batrachochytrium dendrobatidis – opportunistic or not – is infecting and killing a large number of amphibians.

What is not fully understood about B. dendrobatidis is its pathogenicity and what mechanisms it employs to cause infection.  A recent paper, “Species-Specific Chitin-Binding Module 18 Expansion in the Amphibian Pathogen Batrachochyrium dendrobatidis”, published in the mBio journal by John Abramyam & Jason Stajich at UC Riverside, begins to address this pathogenicity.  As the authors point out – more than 100,000 species of fungi have been described to date and very few of them are pathogenic.  This means that the ability to be pathogenic is derived from somewhere: genome expansion events, gene family duplication and diversification events – and we’re only starting to understand horizontal gene transfer events in fungi. This paper addresses the expansion of a gene family across two B. dendrobatidis genomes that are associated with pathogenicity.

When comparing the genomes of B. dendrobatidis with the genomes from other chytrid fungi there has been an expansion of genes within the family Carbohydrate-Binding Module Family 18 (CBM18).  The CBM18 family is a large group of proteins that have been implicated in other fungal pathogenic infections on both plants and animals.  The authors here question whether this interesting lineage specific expansion of CBM18 in B. dendrobatidis could be associated with the virulence of its pathogenicity on amphibians.

The authors used the CBM18 protein family domain HMM to search across the B. dendrobatidis genomes and found an increase in the number of domains when comparing it to genome of its closest relative.  When constructing phylogenetic trees of the CBM18 gene family, three monophyletic and strongly supported clades emerged.  When focusing on divergence of the protein domains, the authors determined that individual domain groups were monophyletic and showed a general pattern with regards to their genome locations.

More specifically, clades of the CBM18 family appears to possess different gene functions, some of which appear to be similar to lectins (LL), tyrosinase/catechol oxidases (TL), and chitin deacetylases (DL).  The function of these genes has yet to be experimentally determined, but the authors make some deductions based on DNA sequences.  The lectin-like genes may be involved in the sequestering of chitin, which could then be disrupting the amphibian immune response.  The tyrosinase/catechol oxidase gene family is associated with melanin synthesis, which could be disrupting the electron transport of the infected amphibians.  Lastly, chitin deacetylases may be involved in suppressing defense mechanisms in place to suppress the fungal infection of the host.  The authors plan to continue to elucidate the pathogenicity of B. dendrobatidis in an attempt to understand the ecology and evolution of its infection on amphibians.

Human Pathogenic Types of the Fungus Fusarium Detected in Plumbing Drains

I recently wrote about a paper that surveyed the diversity of bacteria in public restrooms using metagenomic techniques.  While that paper focused on bacteria on bathroom surfaces, another recent paper – “Widespread Occurrence of Diverse Human Pathogenic Types of the Fungus Fusarium Detected in Plumbing Drains”, authored by Dylan Short and colleagues – focused specifically on probing the diversity of the large Ascomycete genus Fusarium found in sink drains, with specific focus on isolates that are human pathogens.

The authors sampled 471 drains – more than 95% of which were from public bathroom sinks – from 131 buildings throughout the mid-eastern to southern United States (and California too).  They selectively isolated Fusarium species from sink drains using cotton swabs and then streaked petri plates of Nash-Snyder Agar, which is a semi-selective medium containing the fungicide pentachloronitrobenzene.  The plates were inspected after the fungi had some time to grow, were propagated, and then verified as Fusarium species using microscopic morphology and DNA sequencing.

Six different loci – translation elongation factor (TEF), the internal transcribed spacer region (ITS) into the large ribosomal subunit (LSU), the nuclear rDNA intergenic spacer region (IGS), the RNA polymerase II large subunit (RPB2), portion of the alpha-tubulin (TUB) gene, and calmodulin (CAM) – were identified using Sanger sequencing to assess the diversity of Fusarium in the sink drains.  The sequence data was compared to an extensive database of the genus Fusarium maintained by the Geiser Lab and others.

Fusarium species were extremely common in sink drains; 66% of the sink samples – and 82% of all the buildings sampled – yielded at least one isolate.  These isolates could largely be placed within three Fusarium species complexes: the Fusarium solani species complex (62% of samples), the F. oxysporum species complex (28%), and the F. dimerum species complex (8.5%).  Sink drains from 91% of private residences and 80% of public buildings yielded Fusarium isolates.  Of all the buildings that yielded Fusarium within sink drains, approximately 80% contained one of the six major isolates recognized from human infections.

It is interesting to note that human infections from Fusarium species are rare, but the six most common Fusarium isolates found in sink drains are also the six most common involved in human infection.  The authors note that it’s apparent that people are in constant contact with these fungi within indoor environments.  It’s also notable that novel species complexes were identified using these techniques and that there was a wide phylogenetic breadth to the Fusarium isolates that were sampled from sink drains.

This paper is a substantial contribution to the growing literature documenting the indoor environment for fungi.  The next step would be to use metagenomic techniques – and marker loci for fungi to encompass a meta-taxonomic assessment – to identify all the fungi found in sink drains.

Horizontal Gene Transfer In Ascomycete Fungi

Horizontal Gene Transfer (HGT) goes against what we typically consider the normal transfer of genetic material from parent to offspring.  HGT involves the transfer of genetic material from one organism to another.  Within the bacteria, whose mode of survival typically depends on phagocytosis, there is a fairly amount of HGT.  Events of HGT have been rarely observed in Eukaryotes because numerous barriers exist to prevent foreign nucleotides from entering a cell’s nucleus.  Some of these barriers in the Fungi include a substantial cell wall made of chitin, multiple cell and nuclear membranes to cross, and the secretion of metabolic enzymes to the outside of the cells and subsequent uptake of the nutrients.  Despite these barriers, there is now evidence of multiple occurrences of HGT in the fungi.

In a recent article published in the journal Current Biology, Jason Slot and Antonis Rokas, both of Vanderbilt University, provided evidence of HGT in two Ascomycete clades.  In this study, the authors identified a 23-gene cluster from the genus Aspergillus which relocated to the genus Podospora.  Genes that are in this cluster synthesize the toxic compound, Sterigmatocystin, which is a precursor to aflatoxins, noted for their production in Aspergillus.  Both genera are located in the subphylum Pezizomycotina, so each clade is not distantly related, but HGT was observed using different methods.

While it’s easy to observe genetic material passed from generation to generation, recognizing HGT is a little more difficult.  The main way the researchers have identified HGT is using phylogenetic methods to identify gene clusters whose homology cannot be explained by lineage alone.

Thomas Richards points out in his commentary on the Slot & Rokas paper (also in Current Biology), that because fungi do not phagotrophically consume their food they are less likely to incur HGT event.  There are two notable hypotheses to why we do see HGT in the fungi.  First, many secondary pathway genes in Eukaryotes are encoded in gene clusters, and the fungi have a fair amount of these clusters.  Gene clusters, which are more functional in a natural selection sense, are therefore more likely to persist upon transmission, as opposed to individual genes.  Data from HGT studies in fungi support this hypothesis.  Second, fungi are naturally, from the basis of their biology and natural history, intimately tied to other organisms, and fulfill roles as saprobes, pathogens, or symbionts.  This close intimacy increases the opportunity for genes to transfer from one organism to another.  Data suggests that this hypothesis is true also, as many of the recorded instances of HGT in fungi have been observed in organisms with overlapping environments.

Raiders Of The Lost Domain

Metagenomics, the process of acquiring the genomes – or pieces of genomes – of all the microorganisms in a single environmental sample and then analyzing their composition, has developed in recent years with the advent of next-generation sequencing techniques.  Metagenomic studies are increasing our knowledge about microbial life by providing vast amounts of data on the overall diversity of organisms found in soil, aquatic habitats, the human body, and even what is splattered across car windshields (see here).  Unknown organisms found in metagenomic studies correspond to the three domains of life: Bacteria, Archaea, and Eukaryotes, but scientists have wondered if other domains of life exist, but have gone unnoticed.

A paper authored by Wu et al., entitled “Stalking the Fourth Domain in Metagenomic Data: Searching for, Discovering, and Interpreting Novel, Deep Branches in Marker Gene Phylogenetic Trees“, recently published in the PLoS One journal and from the laboratory of Jonathan Eisen at UC Davis (see here and here), ponders the presence of novel lineages of life by searching for genes with presumed deep origins in the tree of life.  By using metagenomic sequences from Craig Venter’s Global Ocean Sampling (GOS) initiative, the authors searched for novel life by probing for genes – those associate with ribosomal RNA – assumed to have early origins in the evolution of life.  Image link from a commentary from the Economist.

The researchers began looking for novelty across the small subunit rRNA gene, a common gene for phylogenetics at the level of bacteria and archaea, but were unable to resolve these phylogenies at deep levels due to a lack of robust sequence alignments for novel sequences.  The researchers ended up focusing on two rRNA associated genes also with assumed deep origins: RecA, a gene involved in DNA recombination, and RpoB, a gene involved in translating DNA into RNA.  Jonathan Eisen has written a very detailed and elucidating blog post of the background of the methodology, in supplement to the methodology found in this paper. The following figure comes from Norm Pace’s excellent 2009 review article on the tree of life and shows how the basal nodes of many lineages remain unresolved.

When constructing phylogenetic trees of the RecA and RpoB sequences, the authors found specific novel branches that could not be easily identified.  The authors describe four explanations concerning the characterization of these sequences.  One explanation is that these novel clades come from undescribed viruses not previously observed.  A second possibility is that the sequences represent recombinations of previously identified genes, which the authors rule out due to phylogenetic uniqueness.  A third explanation is the presence of ancient paralogous genes from organisms lacking gene data or information.  Lastly, a fourth possibility is that the novel sequences come from yet unknown lineages of organisms and their phylogenetic novelty actually represents novel organisms.  The authors stress that this study needs more data and more rigorous research in order to investigate these possibly novel clades, but this study is the first of, hopefully, many to address this interesting research question.  If you would like to read more about this research there are numerous commentaries available for your reading pleasure (see here, here, and here).

Newly Identified Branch of Marine Eukaryotes on the Tree of Life

We’re only just now starting to get a grasp on the sheer amount of global biological diversity, most of which has been very difficult to observe with conventional observational means.  Changes in technology and sampling strategies have resulting in the acquisition of information regarding many previously undocumented forms of biological life.  Along with microorganisms associated with plant roots – the strict focus on my research interests – phytoplankton represent a large group of organisms that we still know little about.  For selfish reasons I was interested in this study because I wanted to see how these authors addressed ways of learning more about a previously unknown lineage of ocean phytoplankton.  As evidenced by next generation sequencing efforts, there are many unknown and undescribed fungi in soils and there is a huge amount of commonality of the diversity of microbial life in soils and oceans.

Published in the Proceedings of the National Academy of Sciences, a study entitled “Newly identified and diverse plastid-bearing branch on the eukaryotic tree of life”, by Kim et al, describes a recently identified and previously uncultured marine and freshwater microalgal lineage of Eukaryotic organisms.  The researchers title this group of phytoplankton the rappemonads, from the initial paper (authored by Rappé et al 1998) that reported unknown DNA sequences from this lineage.  The researchers designed nucleotide primers and fluorescent probes from initial DNA sequences (from the Rappé et al study) and used these molecular diagnostics to observe marine and freshwater samples for their presence or absence of these unknown organisms.

Phylogenetic analysis of environmental nucleotide sequences revealed that rappemonads are related to both haptophyte and cryptophyte algae but constitute a diverse and independent lineage.  To resolve the phylogenetic position of the rappemonads the authors designed specific nucleotide primers spanning the 18S-ITS1-5.8S-ITS2-28S rRNA genes and sequenced this gene cluster.  The authors used maximum likelihood algorithms to construct a phylogeny, which resolved the rappemonads between the haptophyte and cryptophyte algae.  It should be made clear that there is low branch support (at around 50) for some of these clades, so more data is needed for strict resolution of the red plastid algae.

Probes for fluorescent in situ hybridization were developed to observe rappemonads.  Rappemonads were described to be relatively large in size – approximately 6 µm in diameter versus the smaller picophytoplankton (2 to 3 µm) – significantly larger than open-ocean phytoplankton.  Rappemonads appear to contain two to four plastids and are putatively photosynthetic.

Using quantitative PCR methods, the authors identified high concentrations of rappemonads in late-winter blooms along the surface waters at a site in the Sargasso Sea.  Rappemonads were rare or absent in stratified summertime conditions, when concentrations of chlorophyll containing microorganisms are at their highest in deep waters.  Rappemonads were frequently found in North Pacific anticyclonic eddy samples, which are characterized by colder more nutrient-rich waters that have been brought to the sea surface.  When considering water characteristics (such as depth, salinity, phosphate, nitrate, and nitrite), there were no statistical significance between samples containing rappemonads and those where they were absent.  In addition, rappemonads were found in both marine and freshwater conditions, bringing into question when and where one may find these organisms and which would warrant further study.