Community structure and metabolism

through reconstruction of microbial

genomes from the environment

articlesVenter 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 3A: Scaffold bases vs. GC content

Supplementary Figure 2A: Read average GC content vs. local read depth.

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”.

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.

Gggggggggggggggggg’

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 ShotgunSequencing 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 CollectionSurface Water Samples

RV Weatherbird II(coast of Bermuda)

SV Sorcerer II “Hydrostation

S”

1.66 million reads Avg. length 818 bpTotal ~ 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.