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Phylogeny V
BIO2093 –Metagenomics and HGT
Darren Soanes
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 γProteobacteria
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 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.
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 protein
src - 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.1
64400.113.abstract
Gene capture by Helitron TEs
Helitrons are a class of
transposable elements
that replicate by a socalled "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 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
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. triticirepentis.
• 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. triticirepentis, 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
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.
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
HGT of secondary metabolic cluster
HGT of secondary metabolic cluster
HGT can account for the
observed distribution of the
ETP clusters
BMC Evol Biol. 2007 Sep 26;7:174.
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 reading
Mehrabi 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