Community structure and
metabolism
through reconstruction of microbial
genomes from the environment
articles
Venter et al.
Outline
• Addressing: uncultivated microbial communities with
unclear roles in natural system
• Source of Study: Acid Mine Drainage (AMD) microbial
biofilm.
• Approach: reconstruction of genomes through shotgun
sequencing of microbial DNA
• Results: near-complete genomes
– Leptospirillum group II
– Ferroplasma type II
– partial recovery of three other genomes.
• Analysis revealed: pathways for Carbon, Nitrogen fixation,
Energy generation and survival strategies.
Biofilms
• Biofilms-collection of
microorganisms
surrounded by the slime
they secrete within a pyrite
(FeS2) ore body12.
• AMD –environmental
problem
• microbial iron oxidation
• acidification due to pyrite
dissolution.
Initial Characterization of a Biofilm
Screening of AMD samples using
fluorescence in situ hybridization (FISH
• Eight samples
• Five-way community sample
– Lecptospirillum- AMD generation
– Ferroplasm- low abundance, well
characterized
– Low eukaryotic abundance
• Probes:
– Leptospirillum group III. bacteria
(green)
– Archaea (blue)
– Leptospirillum genus (red)
– Dominance of Leptospirillum
(yellow).
16rRNA Gene libraries
• 16S rRNA conserved molecule
through evolution
• phylogeny is typically based on
sequence information 16rRNA
16rRNA Gene libraries Cont’d
•
•
•
•
•
•
Polymerase Chain Reaction (PCR)
pCR 2.1 TOPO vector
Primers: bacterial, archaeal, and universal 16S rRNA
384 clones aligned
ARB sequence analysis software (www.arb-home.de)
Phylogenetic tree construction methods
– Distance
– Parsimony
– Bootstrapping
16rRNA Three Phylogeny Results
• 3 bacterial, 3 archaeal lineages
• Groups detected:
–
–
–
–
–
Leptospirillum group III
Sulfobacillus
Ferroplasma
A-plasma
G-plasma
• Abundant clones:
– close relatives of L. Ferriphilum (Leptospirillum grp
II)
– 94% of 17 L. group II clones identical
– 17 minor variants 1.2% 16S rRNA divergence
Community genome sequencing and
assembly
Jazz Shotgun Sequencing
• Shearing DNA
– Blunt-ed repair
– Insert into pUC 18 vector
• Plasmid Amplification
– Rolling circle Amplification
• Plasmid sequencing
– Capillary DNA sequencing
Jazz Shotgun Sequencing Results
• Raw shotgun data:
– bacterial and
archaeal genomes
at sequence
coverage 10x
– Supplementary
information
Supplementary Figure 1: Number of alignments
detected vs. alignment percent identity.
Assignment of Scaffolds to Organism
Types
Supplementary Figure 2A: Read
average GC content vs. local read
depth.
Supplementary Figure 3A: Scaffold bases
vs. GC content
Assignment of Scaffolds to Organism
Types
Supplementary Figure 3B: Scaffold
bases vs. assembly depth
Supplementary Figure 3C: Scaffold bases
vs. assembly depth
Assignment of Scaffolds to Organism
Types
Supplementary Figure 6: Diagram showing the comparison of low GC archaeal
scaffolds vs. the fer1 genome. The peak on the right side (at > 98%) is the fer1
population, that at 77% is the Ferroplasma type II population, and that at 66% is
attributed predominantly to “G-plasma”.
Gggggggggggggggggg’
Supplementary Figure 7: The degree of conservation of gene order within
scaffolds in the Ferroplasma type II consensus genome relative to fer1. In
this plot, the contigs were ordered by reference to the fer1 genome. Plot
constructed using the promer program within the MUMmer3.0 software
package.
Supplementary Figure 8: A 20 kb region of the Ferroplasma type II
consensus genome showing depth (green), polymorphism frequency
(blue) and open reading frames (red).
Mosaic Genome Types
Figure 3 Schematic diagram
illustrating a diversity of mosaic
genome types within that
Ferroplasma type II population
that are inferred to have arisen by
homologous recombination
between three closely related ancestral
genome types (pink, yellow and
green).
Pathways for genetic exchange
• Recombination Achieved?
• Observations:
– Identical reverse transcriptases (LambdaSa1) occur in the Gplasma
and Ferroplasma type II genomes (in very different genomic
contexts)
• suggesting that a single phage type has recently targeted both
lineages.
– retron-type reverse transcriptases with identical adjacent
transposases occur in otherwise different genomic contexts within
the Leptospirillumgroup II and III genomes,
• indicating that a broad host range phage targets both of these
groups
Supplementary Figure 10: Comparison of COG groups found in the
Leptospirillum group II and Ferroplasma type II genomes.
Metabolic analysis
• Five dominant member near-complete gene inventories
• Carbon fixation
– Leptospirillum group II and III have the genes needed to fix carbon by
means of the Calvin–Benson–Bassham cycle
• Nitrogen fixation
– complete nitrogen fixation operon in scaffolds assigned to
Leptospirillum group III and not to Leptospirillum group II
• Energy Source
– Ferrous iron oxidation as an energy source
– Leptosperrilum and Ferroplasma are dominant oxidizer, yet they do
not encode ferrous iron oxidase.
Further analysis
• Leptospirillum group II contains an operon of putative cellulos
synthase genes.
– Cellulose: prevents desiccation and enable biofilms to float
• L. group II and III possess a chemotaxis system and flagellar operon
– Used to respond to ferrous iron and oxygen gradients.
• Resistance to toxic materials
– Ferrosplasma type II has genes for production of isoprenoid-based lipids,
which is highly proton impermeable.
– L. group II has a variaty of genes resulting in a complex cell wall structure
• Light-activated proteins for the repair of UV-damaged DNA
– Present in L. group II, III and G-plasma genomes.
Environmental Genome Shotgun
Sequencing of the Sargasso Sea
Venter, J.C,. et al.
The Sargasso Sea
• interpretation of environmental
genomic data in an
oceanographic context.
• seasonal thermoclines
– Synechococcus and
Prochlorococcus
• Whole-Genome Shotgun (WGS)
Assembly
– DDBJ/EMBL/GenBank
project accession
AACY00000000
– TraceDB trace archive
• Contigs
• scaffolds
• unassembled paired
singletons
• individual singletons
Sample Collection
Surface Water Samples
RV Weatherbird II
(coast of Bermuda)
SV Sorcerer II
“Hydrostation
S”
1.66 million reads
Avg. length 818 bp
Total ~ 1.36 Gbp of
microbial DNA sequence
325,561 sequences
~265 Mbp of DNA sequence.
•
•
Environmental sequencing were
compared to Prochlorococcus marinus
MED4
Outermost concentric circle
– competed genomicsequence of
Prochlorococcus marinus MED4
– Assigned psuedospectrum colors
based on the position of those genes
along the chromosome
– Same color-conserved gene order
– Different color- conserved gene order
w/ MED4 region & chromosomal
rearragements.
– No conserved gene order-black color
• Comparison: Sargassor Sea
Scaffold to Crenarchaeal clone
4B7
– tBLASTx
– BLASTp matches-25%
similarity
Circular Diagrams of 9 Complete Megaplasmids
•The Institute for
Genomic Research
(TIGR)
•structural, functional and
comparative analysis of
genomes and gene
products from a wide
variety of organisms
including viruses,
eubacteria, archaea and
eukaryotes
Figure 5
Conclusion
• shotgun sequencing provides a wealth of phylogenetic markers that
can be used to assess the phylogenetic diversity of a sample
• Important insights
– ultraviolet (UV) light in the surface ocean does not inhibit
ammonia oxidation by chemoautotrophs.
– Sargasso Sea inhabitants may efficiently utilize the available
phosphorus in this extremely phosphorus-limited environment.