1. BIO2093 –Metagenomics and HGT
Phylogeny V
BIO2093 –Metagenomics and HGT
Darren Soanes

2. 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.

4. 16S rRNA sequencing to analyse diversity in marine picoplankton
Schmidt et al., J Bacteriol Jul; 173(14): 4371–4378.

5. 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 :

6. Shotgun sequencing 6

7. 7

8. 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

9. 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,
DNA sequence often complex and difficult to assemble – dominated by abundant organisms.
Next generation sequencing gives much greater sequence coverage.

10. Environmental Gene Tags
Environmental 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.

11. Metagenome projects

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

13. 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.

14. HGT in prokaryotes

15. Agrobacterium tumefaciens – crown gall: HGT from prokaryote to eukaryote
Also in vitro evidence of gene transfer by conjugation between bacteria and Saccharomyces cerevisiae

16. Retrovirus life cycle

17. 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 protein
src - which encodes a tyrosine kinase

18. 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.

19. 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’.

20. 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.

21. 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.

22. 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.

23. 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.

24. Horizontal gene transfer (purine-cytosine permease)
oomycete
fungi

25. Eukaryotic Tree of Life
Phytophthora sojae
Aspergillus oryzae

26. 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.

27. 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 Mar;17(3):352-61

28. Role of HGT in phytopathogenicity
Phytopathogen – 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

29. 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.

30. 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

31. 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.

32. 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.

33. 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.

34. Host specific toxins (HSTs) in Alternaria alternata
Host range of this fungus determined by HST production.
Genes involved in HST production located on CDCs.
Tomato - AAL-toxin
Strawberry AF-toxin
Hybrid strain containing strawberry-infecting genetic background and CDC from tomato-infecting strain was able to cause disease on both tomato and strawberry.

35. HGT of secondary metabolic cluster
Genes 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.
Core ETP
Sirodesmin gene cluster from Leptosphaeria maculans

36. HGT of secondary metabolic cluster

37. HGT of secondary metabolic cluster
HGT can account for the observed distribution of the ETP clusters
BMC Evol Biol Sep 26;7:174.

38. 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 sojae – stem and root rot of soybeans
Phytophthora ramorum – sudden oak death
Hyaloperonospora arabidopsidis – downy mildew of Arabidopsis thaliana

39. 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

40. HGT fungi to oomycete
Richards et al., 2011 Proc. Natl. Acad. Sci. USA 108:

41. HGT reading
Mehrabi et al., (2011) Horizontal gene and chromosome transfer in plant pathogenic fungi affecting host range. FEMS Microbiol Rev. 35:
Richards et al., (2011) Horizontal gene transfer facilitated the evolution of plant parasitic mechanisms in the oomycetes. Proc. Natl. Acad. Sci. USA 108:
Gardiner et al., (2013) Cross-kingdom gene transfer facilitates the evolution of virulence in fungal pathogens. Plant Science 210:
Soanes and Richards (2014) Horizontal gene transfer in eukaryotic plant pathogens. Annu Rev Phytopathol. 2014: