Beiko ANL Soil Metagenomics presentation

Soil, lateral gene transfer, and hybrid genomes

Robert Beiko20 October 2015

A. microbe

Lateral gene transfer

http://genome.cbs.dtu.dk/staff/dave/MScourse/Lekt_11Feb2003c.html

In the gut

Butyrate synthesis in Lachnospiraceae and other organisms

Meehan and Beiko (2014) Genome Biol Evol

Lachnospiraceae

LGT across habitats

Smillie et al. (2011) Nature

Within-site transfer rates are highest in host-associated (i.e., human) habitats

AR genes are frequently transferred BETWEEN habitats

Villegas-Torres et al. (2011) International Biodeterioration & Biodegradation

Forsberg et al. (2012) Science

Sorangium cellulosum So157-214.8 Mbp; 11,599 coding sequences>1200 putative LGT acquisitions

Han et al. (2013) Sci Rep

An ecological view of genomes

Genes as individuals, Genomes as communities

Key concept mappings:

• Diversity: counts of genes / distribution across functional categories• Community: set of genes and their interactions• Migration: lateral gene transfer

Metacommunity Leibold et al., Ecol Lett, 2004

A set of local communities that are linked by dispersal

Species B

Species A

Habitat

Habitat

Habitat

Pattern and intensityof migration for species A

Genome Metacommunity Hypothesis

• Since…• Genes are agents whose trajectories are not bound to

their host organisms• Genes can evolve and take on new functional roles in

concert with other genes

• A genome can be viewed as a community of genes• Related sets of genomes comprise a

metacommunity of genes

Genome Metacommunities Boon et al., Fems Microbiol Rev, 2014

A set of genomes that are linked by LGT

Gene B

Gene A

Genome

Genome

Genome

Pattern and intensityof LGT for gene A

Genome Metacommunities Boon et al., Fems Microbiol Rev, 2014

Related to the pan-genome, but not restricted to specific taxonomic groups

Why is a given gene present in a given genome at a given time?

How are functional roles partitioned across a community?

Soil thinking

How important is LGT in soil communities?

Does it make sense to think of gene metacommunities in the soil context?

Lots of LGTYES

Minimal LGTNO

The procedure

• In the absence of a coherent set of known genomes from a given habitat…

1. Identify an interesting sample2. Select genomes with very high marker-gene (i.e.,

16S) similarity to sequences in the sample (gOTUs)

3. Mine genomes for evidence of LGT, examine patterns of connectivity

Conclusions• Positive relationship between

abundance, diversity and pH• Specific relationships between different

bacterial (notably Acidobacteria) and fungal groups vs. pH

• Fungal OTUs appear to tolerate wider pH ranges

(1)

Chosen sample:http://metagenomics.anl.gov/?page=MetagenomeOverview&metagenome=4455674.3#org_ref (pH = 4.1)

Meet the Sample (MG-RAST)

1277 rRNA gene sequences

Meet the Sample (Matching genomes)

99% 16S identity (e-value < 1e-20):

1211 – No matchBradyrhizobiaceae: 61Pseudomonas: 2Nocardioides: 1Acidithiobacillus: 1Cyanobium: 1Total: 18 genomes covering 8 genera

97% 16S identity:

1100 – No matchBradyrhizobiaceae: 77Pseudoxanthomonas / Cycloclasticus: 25Acidobacteria: 20Other Proteobacteria: 48Other: 10Total: 114 genomes covering 74 genera

114 genomes covering 74 genera

1277 rRNA gene sequences

(1)

gOTUs

16S sequence from sample

Rhodopseudomonas palustris TIE 1

Rhodopseudomonas palustris DX 1

Rhodopseudomonas palustris CGA009

Bradyrhizobium japonicum USDA 6

99% 16S identity

97% 16S identity

141824_31298

Rhodopseudomonas palustris HaA2

Rhodopseudomonas palustris BisA53

Bradyrhizobium BTAi1Nitrobacter winogradskyi

Oligotropha carboxidovorans

Agromonas oligotrophica

Bradyrhizobium ORS278

Weird gOTUs

141824_229613

Bordetella pertussis

Bordetella bronchiseptica

Bordetella parapertussis

Gross et al., 2008

Homology search

• Compare proxy genomes against nr database

• Identify interesting patterns:• Unusual best matches (e.g., best nonself match is to a

completely different group)• Patchy distributions, phylogenetic trees• Linked sets of genes: co-transfer?• Implicated biological processes?

Acidithiobacillus ferrooxidans•A refugee from genus Thiobacillus (a group

shattered by 16S rRNA gene sequencing)

• Loves long walks on the beach, pH < 2.0, oxidizes iron, sulphur, thiosulphate

•Also loves to share genes

https://microbewiki.kenyon.edu/index.php/Acidithiobacillus_ferrooxidans

Beiko (2011) Biol Direct

504 gene trees in which A. ferrooxidans has a unique genus as partnerNot shown: 795 genes w/multiple partnersAlso not shown: 333 other trees with less frequent, unique partner genera

Split by 16S; reunited by genome sequencing?

Genome 1 – Acidithiobacillus ferrivorans(renaming of A. ferrooxidans)

3093 predicted proteins / 3035 with homology matchesObserved / Predicted capabilities:• Facultatively anaerobic• Psychrotolerant• Optimal pH = 2.5• Oxidation of iron and inorganic sulfur• Carbon fixation, nitrate reduction• Trehalose synthesis• “Bioleaching”

Liljeqvist et al. (2011) J Bacteriol

Genome 1 – Acidithiobacillus ferrivorans

Best nonself match is to…(273 non-Acidithiobacillus)

Mobile element signatures dominate• 14 x restriction system-associated• 8 x transposase• 8 x transcriptional regulators (incl CopG, TetR)• Other resistance (LacZ, bleomycin, …)• Integrase, reverse transcriptase, toxin/antitoxin,

bacteriocin, …• Nitrate reductase & related• >90 unknown

1877 found in other Acidithiobacillus + other genera

Best non-Acidithiobacillus match is to…

(only 11 Acidobacteria!)

https://www.jasondavies.com/wordcloud/#

Acidobacterial connections

• short-chain dehydrogenase/reductase SDR • HNH endonuclease • Glycoside hydrolase family 8 (x3)• RES domain protein • Transposase x 5

Phylogenetic profiles# of similar genes (evalue < 10-50)

Min 30 connections

Proteobacteria

Actinobacteria

Cyanobacteria

Planctomycetes

Acidobacteria

Bacteroidetes

Acidithiobacillus

Key observations• Connections to many other groups,

mostly Proteobacteria (not surprising)• No between-group connections outside

Proteobacteria at this threshold• Acidithiobacillus as hub rather than part

of gene-exchange community?

65

Mutual information-based network(do groups co-occur > random?)

Acidithiobacillus

Gammaproteobacteria

Alpha/Betaproteobacteria

Key observations• Connections mostly predictable by

phylogeny• Again, no interesting partners outside of

Proteobacteria• However, many connections between

Alpha/Betaproteobacteria

Phosphate ABC transportersgi 343775109

periplasmic(eval < 10-100)

gi 343775110 inner membranesubunit PstC (eval < 10-50)

gi 343775111 inner membranesubunit PstA (eval < 10-50)

Distribution:• Acidithiobacillus• Acidobacterium• Alpha/Beta/Gamma• Actinobacteria• Firmicutes

Recurrent grouping ofAcidithiobacillus (Gamma)AcidobacteriumThiobacillus (Beta)Defluviimonas (Alpha)Salinisphaera (Gamma)

Genome 2 – Terriglobus roseusEichorst et al (2007) IJSEM

• “Group 1” acidobacterium• Preferred pH: ~6

• Aerobic• Catalase, carotenoids for defense against reactive oxygen• Oligotrophic; can grow on a wide range of carbon sources

• 4245 protein-coding genes (2735 with nr matches, 558 species-specific)

Rousk et al. Eichorst et al.

Best nonself matches (183 non-Acidobacteria)Multidrug resistance / cation efflux / prophage

Best matches outside Terriglobus

Phylogenetic profiles

Profiles are wider and more diverse for Terriglobus than for Acidithiobacillus

LPS O-antigen biosynthesisgi 390412425

CDP-glucose 4,6-dehydratase

gi 390412426 glucose-1-phosphate cytidylyltransferase

gi 390412427 “LPS biosynthesisprotein”

Distribution:• Acidithiobacillus• Acidobacteria• Other proteobacteria• Other

Flavobacteria

Spirochaetes

SpirochaetesCyanobacteria

Contrasting Acidithiobacillus vs Terriglobus relationships:Same partners, different dance

Compare profiles vs Streptomycetaceae (five strains found in sample gOTU)

Acidithiobacillus Common Terriglobus

Polyphosphate kinase

glucose-6-phosphate 1-dehydrogenase

Carbon monoxide dehydrogenase

More glycolytic enzymes

Heavy-metal resistance / export

Multidrug resistance

Ammonium transporter

Catalase / peroxidaseExopolysaccaride

Conclusions

• Different layers of LGT:• Very recent: mostly mobile elements (proxies unsuitable)• Less recent (outside species / genus) (proxies potentially

more justifiable)• Taxonomy is a pain

• What’s the story with gene metacommunities?• Lots of LGT!• Recurrent patterns of sharing among groups not evident• Metacommunities at the pan-genome level?• Need many isolate genomes from single samples

Technical impacts of LGT and gene metacommunitiesMetagenomic read assignment• Recently acquired genes will still look like they belong in

the donor• These are some of the most interesting genes!!

Functional prediction (e.g., PICRUSt)• Phylogeny will fail to accurately predict the distribution of

these genes. Be very careful with extreme or poorly characterized samples!

Phylogenetic beta diversity may be misleading

Key questions in LGT and gene metacommunities• Are gene-sharing networks:• Random?• Driven by shared location / habitat?• Constrained by phylogenetic relatedness?

• Are shared genes:• Neutral or adaptive?• Driven by specific types of mobile element?

Fin