BIO2093 –Metagenomics and HGT

Darren Soanes

Phylogeny V

Metagenomics

• Study of genetic material recovered directly from environmental samples.

• Can be used to study organisms that cannot be grown in the lab.

• rDNA sequencing using universal primers can be used to analyse diversity of sample.

• Cultivation based methods find less than 1% of the bacterial and archaeal species in a sample.

16S rRNA sequencing to analyse diversity in marine picoplankton

Schmidt et al., J Bacteriol. 1991 Jul; 173(14): 4371–4378.

Identification of bacterial rhodopsin from marine sample

Rhodopsin gene found on 130kb genomic fragment containing rRNA from uncultivated marine γ-Proteobacteria

Béjà et al., Science. 2000 289: 1902-6.

Shotgun sequencing

Environmental Shotgun Sequencing (ESS).

(A) Sampling from habitat; (B) filtering particles, typically by size; (C) Lysis and DNA extraction; (D) cloning and library construction; (E) sequencing the clones; (F) sequence assembly into contigs and scaffolds

Environmental Shotgun Sequencing (ESS)

• 200 litres of seawater contains over 5000 different viruses – Breitbart et al., (2002) Proc Natl Acad Sci U S A. 99: 14250–14255.

• Sequencing of DNA extracted from an acid mine drainage system produced complete genomes for a handful of bacteria and archaea that had not been able to be cultured - Tyson et al., (2004) Nature 428, 37-43.

• DNA sequence often complex and difficult to assemble – dominated by abundant organisms.

• Next generation sequencing gives much greater sequence coverage.

Environmental Gene TagsEnvironmental gene tags (EGTs) are short sequences from the DNA of microbial communities that contain

fragments of functional genes.

Essential genes will occur frequently regardless of enviroment.

Genes adaptive for a particular environment will be occur frequently in that environment but not in others.

Metagenome projects

Reading

Tringe and Rubin (2005) Metagenomics: DNA sequencing of environmental samples. Nature Reviews Genetics 6, 805-814.Eisen (2007) Environmental Shotgun Sequencing: Its Potential and Challenges for Studying the Hidden World of Microbes. PLoS Biology 5, e82.

Lateral / Horizontal Gene Transfer (HGT)

• Most evolution is via vertical gene transfer, in which an organism receives genes from its ancestor.

• In HGT, genes are passed between individuals of the same generation or members of a different species.

• Common in prokaryotes, rare in eukaryotes.

HGT in prokaryotes

Agrobacterium tumefaciens – crown gall:HGT from prokaryote to eukaryote

Also in vitro evidence of gene transfer by conjugation between bacteria and Saccharomyces cerevisiae

Retrovirus life cycle

Rous Sarcoma Virus

RSV can transform chicken cells (induce cancerous growth).

Genome of RSV contains four genes:gag - which encodes the capsid protein pol - which encodes reverse transcriptase env - which encodes the envelope proteinsrc - which encodes a tyrosine kinase

v-src is a modified version of a cellular gene

RSV has gained v-src by ‘capture’ of c-src due to integration of retrovirus genome next to c-src and ‘read through’ by RNA polymerase II.v-src has subsequently accumulated mutations which have given it oncogenic properties.

HGT of transposable elements in plants

HGT is much rarer in eukaryotes. Mechanisms are unknown, but there is evidence of HGT of transposable elements (TE) between eukaryotic species. It could be speculated that TEs could be involved in HGT of genes involving ‘gene capture’.

http://genome.cshlp.org/content/early/2014/02/10/gr.164400.113.abstract

Gene capture by Helitron TEs

Helitrons are a class of transposable elements that replicate by a so-called "rolling-circle" mechanism.

Genes or parts of genes can be captured and moved during replication.

Helitrons are widespread in eukaryotic genomes and there is evidence they have spread by HGT.

HGT by heterokaryon anastomosis in filamentous fungi

A heterokaryon is a cell that contains multiple, genetically different nuclei.

Anastomosis is the fusion between branches of the same or different hyphae.

Evidence for HGT (1)

• Create phylogenetic tree using protein sequences.– Use BLAST to identify similar sequences to the

sequence of interest.– Take sequences from a wide range of taxonomic

groups.

• Compare to species tree – look for incongruities between the two trees.

Evidence for HGT (2)

• Genes of a species (or sub-group of species) should lie within a clade containing genes from distantly related species.

• There should be strong bootstrap support for the tree topology.

• Alternative hypotheses based on multiple duplication and gene loss should be rejected.

Horizontal gene transfer (purine-cytosine permease)

oomycete

fungi

Eukaryotic Tree of Life

Phytophthora sojae

Aspergillus oryzae

Evidence for HGT (3)

• Other types of evidence for HGT often cited – less robust than phylogenetic evidence:– Abnormal GC content, codon-usage,

oligonucleotide frequency.– Mosaic distribution of genes (presence / absence

of genes in closely related species).– Conservation of intron position, lack of introns in

genes obtained from prokaryotes by HGT.

Gain of genes by HGT may enable organisms to exploit new ecological niches

Glycosyl hydrolases (plant cell wall breakdown)

Rumen bacteria Rumen fungi

Mol Biol Evol. 2000 Mar;17(3):352-61

Role of HGT in phytopathogenicityPhytopathogen – organism that causes a disease of plants (includes fungi, bacteria, oomycetes)

Pyrenophora tritici-repentis – tan spot of wheat

Stagonospora nodorum – Stagonospora nodorum blotch of wheat

Role of HGT in phytopathogenicity

• Pyrenophora tritici-repentis was first described in 1923 as an occasional pathogen of grasses.

• Tan spot of wheat was first described in 1941.• The ability of P. tritici-repentis to cause

disease of wheat is dependent on the production of host-specific toxins (HST) - small secreted proteins that cause necrosis in wheat cells.

Role of HGT in phytopathogenicity• ToxA encodes an HST required for pathogenicity of P. tritici-

repentis.• Gene sequence 98% identical to ToxA from S. nodorum, but

not found in other more closely related fungi.

ToxA

ToxA

Role of HGT in phytopathogenicity

• Evidence suggests P. tritici-repentis gained ToxA by HGT from S. nodorum prior to 1941.

• ToxA sequences identical in all strains of P. tritici-repentis, but in S. nodorum there is a high degree of sequence polymorphism.

• S. nodorum has been recognised as a serious wheat pathogen since 1889.

• Tan spot caused by P. tritici-repentis first recognised in 1941 – prior to that this fungus was not known to cause a disease on wheat.

Conditionally dispensable chromosomes (CDCs)

• e.g Mycosphaerella graminicola – causes Septoria leaf blotch of wheat.

• Essential chromosomes found in all strains, dispensable chromosomes not found in all strains.

CDCs

• Some CDCs have genes linked to pathogenicity – e.g. PEP cluster of genes in pea-pathogen Nectria haematococca.

• PDA1 (part of PEP cluster) encodes pisatin demethylase – a cytochrome P450 that detoxifies an anti-fungal toxin produced by the pea.

• PEP cluster may have been acquired by HGT.• HCT – horizontal chromosome transfer.

Host specific toxins (HSTs) in Alternaria alternata

Strawberry AF-toxinTomato - AAL-toxin

Host range of this fungus determined by HST production.

Genes involved in HST production located on CDCs.

Hybrid strain containing strawberry-infecting genetic background and CDC from tomato-infecting strain was able to cause disease on both tomato and strawberry.

HGT of secondary metabolic clusterGenes responsible for biosynthesis of fungal secondary metabolites are usually tightly clustered in the genome. Epipolythiodioxopiperazines (ETPs) are toxins produced by ascomycete fungi and implicated in several plant and animal diseases.

Sirodesmin gene cluster from Leptosphaeria maculans

Core ETP

HGT of secondary metabolic cluster

HGT of secondary metabolic cluster

BMC Evol Biol. 2007 Sep 26;7:174.

HGT can account for the observed distribution of the ETP clusters

Oomycetes

• Superficially resemble fungi, but are in fact related to brown algae and diatoms.

• Some species are phytopathogenic.

Phytophthora infestans – late blight of potato

Phytophthora ramorum – sudden oak death

Phytophthora sojae – stem and root rot of soybeans

Hyaloperonospora arabidopsidis – downy mildew of Arabidopsis thaliana

HGT fungi to oomycete

• A survey of the genomes of four oomycete phytopathogens showed evidence of 34 HGTs from fungi to oomycetes.

• Many of these confer traits of use to a phytopathogen:– The ability to breakdown and utilise plant cell wall

components.– The ability to acquire nutrients from the host.– The ability to overcome plant defences

HGT fungi to oomycete

Richards et al., 2011 Proc. Natl. Acad. Sci. USA 108: 15258-63

HGT readingMehrabi et al., (2011) Horizontal gene and chromosome transfer in plant pathogenic fungi affecting host range. FEMS Microbiol Rev. 35:542-54.

Richards et al., (2011) Horizontal gene transfer facilitated the evolution of plant parasitic mechanisms in the oomycetes. Proc. Natl. Acad. Sci. USA 108: 15258-63.

Gardiner et al., (2013) Cross-kingdom gene transfer facilitates the evolution of virulence in fungal pathogens. Plant Science 210: 151-158.

Soanes and Richards (2014) Horizontal gene transfer in eukaryotic plant pathogens. Annu Rev Phytopathol. 2014: 583-614