Advancing Science with DNA Sequence Ecology of activated sludge Victor Kunin.

Advancing Science with DNA Sequence

Ecology of activated sludge

Victor Kunin


Phil Hugen-

holtz

VictorKunin

FalkWarnecke

Suzan Yilmaz

HectorGarciaMartin

NataliaIvanova

Trina McMahon (UW)Linda Blackall (UQ)

Project powered by DOE


Metagenomes in pipeline

Wastewater sludge (EBPR)

Termite gut

Guerrero Negro Hypersaline mat

Antarctic subglacial lake Vostok


Ecology?

• population structure

• predator-pray interactions

• biogeography


Microbial ecology

• Local population structure

• Global metapopulation structure– Biogeography– Dispersal mechanisms– Survival strategies of the species

• Phage-host interactions– Can we link an unculturable bacterium to its

phage by (meta)genomic sequence?

• Ecosystem resilience


• Excessive P affects water quality and ecosystem balance through eutrophication.

Enhanced Biological Phosphorus Removal (EBPR)


• P is the key pollutant.

• P limitations in released water very effective

Enhanced Biological Phosphorus Removal (EBPR)

N,C

N,C,P


Brisbane

Madison

US and Australian (OZ) samples

8 L

2 L

*

*


Community composition

Rhodothermus

Cytophaga

Chryseobacterium

Flavobacteriales

Clostridium

Prosthecobacter

Chlorobium

Rhodospirillum

Caulobacter Rhizobiales Thiothrix Xanthomonadales

Acidovorax

Dechloromonas

Rhodocyclus

0.10

Bacteroidetes

Firmicutes

Verruco-microbia

Chlorobi

Proteobacteria

CandidatusAccumulibacter

phosphatis

US

Oz

16S rDNA

What organisms live there?


Bacterial biogeography

Everything is everywhere and the environment selects

Microbial species are ubiquitousBeijerinck, 1913

Microbial populations have astronomical sizes

Microbes travel well 1018 microbes are estimated to cross continents

per year by airlift

Identical 16S rRNA molecules are found as far apart as polar oceans

Undetected biogeography -poor methods

Travel takes time, methods are insufficient to detect divergence

Geographical isolation exists in hot spring bacteria

Whitaker et al, 2003, Papke et al, 2003

What is the biogeography of Accumulibacter?


Any geographic isolation of strains?

Geographic isolationOz

US

Yes No

US

OzUS

OZ


How do they move?

• CAP is globally dispersed

• Never observed outside of sludge

• How do they move?


Metabolic reconstruction of CAP

->Looks like it can grow in

C,N,P-limited habitats

->oligotrophic ancestry or

current lifestyle?

N2 fixing genes

CO2 fixing genes

High affinity P transporters

Flagellargenes


Looking for environmental reservoirs

Contra Costa wastewater treatment plant

**

**

*

*

PCR detection using:16S rRNA genePolyphosphokinase gene


Previous map

CAP environmental reservoirs

*

*Aquatic samples: mostly yesTerrestrial samples: mostly no

Metapopulation


In-strain variationdo

min

ant

stra

in

minor

Dominant strain is near clonal


Monoculture

• Population structure– One clonal strain dominates– No homologous recombination

• The bulk of cells in the system are identical

Vulnerable ecosystem!

• Explains the crashes of the system– Waste water engineers: bad influent– No bad influent in bioreactors, still crashes

Kill the winner


Genomic imprints of virus-host interactions

>95% nt identity across most of the genome

Major differences include:

* EPS gene cassettesdefence against predation

important for settling in EBPR

* CRISPR elements


CRISPR elements

1 GTTTGCCGCCGTGATGGCGGCTTAGAAATCGATGATCATCGACAGGAGCAGATCGCCCAC61 GTTTGCCGCCGTGATGGCGGCTTAGAAACAGTGTCGCTCAGTCCGCCGACCAGATTCTTC121 GTTTGCCGCCGTGATGGCGGCTTAGAAATCAACCGGAATCGCGTCTGCTTGTTCGAGGTC181 GTTTGCCGCCGTGATGGCGGCTTAGAAATCGACGTGCCGAGCGACGACAGTTGCGATGCG241 GTTTGCCGCCGTGATGGCGGCTTAGAAAACATCGTGGCGCGCCTTGATGAGCGCCTGCTC301 GTTTGCCGCCGTGATGGCGGCTTAGAAATGCGCAGGCACCGCAGCGCCCAGGCCACCGAC361 GTTTGCCGCCGTGATGGCGGCTTAGAAAGTGCAGGGCGAGGCGGCACGTGAATATCCCGA421 GTTTGCCGCCGTGATGGCGGCTTAGAAAACGAATCTGGTCTGGCCCAGGCTGCAAGTCCT481 GTTTGCCGCCGTGATGGCGGCTTAGAAATATCATGACCACCAATCGGTATACATGATCCT

CRISPR - a mechanism to keep the record of and destroy invasive elements

EPS - mechanical defence against direct contact with phages

Viral influence:

Genomic - CRISPR/EPS

Population structure - non-recombining, clonal dominant strains


Phage metagenome

• Virion phages metagenome was sequenced

– Loads of phages– Some phages have deep coverage

- high abundance

– CRISPR spacers hit phages• 8 in US sludge• 2 in OZ sludge

– Some phage contigs are hit by multiple bacterial CRISPR spacers

– Linking unculturable host and its phage

While dispersal of the host is global, adaptation to phages is local


Expression studies

• An array with viral and microbial genes was prepared

• The expression was monitored for ~3 months

• Any active viruses? Probe source 30-Oct-06 5-Jan-07 31-Jan-07 Mu-like prophage protein gp29 60299 39397 56948 Bacteriophage tail assembly protein 21786 13617 23252 Phage-related protein, predicted endonuclease 18063 11717 12990 Phage terminase-like protein, large subunit 11817 11000 10039 Phage-related minor tail protein 9763 9279 7048 Acetyl-CoA acetyltransferase 11366 16536 16367 Ribosomal protein L16/L10E 4255 10686 10968 Polyphosphate kinase 4555 5010 3475

Phages apply a constant pressure on the bacterial community


Low complexity engineered systems

• Most cells in ecosystem are virtually clonal, non-recombining cells

• Yogurt production• EPS cassettes & CRISPR are exchanged in yogurt-producing

strains of Streptococcus thermophilus

• Internal instability due to phages - common to all low-complexity engineered systems?


ConclusionsEcogenomics

• Community & population structure– Domination of a clonal asexual strain

• Global population structure– Global dispersal– Dispersal strategies & lifestyle

• Phage pressure– Linking a unculturable host to its phage– Constant phage expression

• Ecosystem vulnerability– Skewed strain abundance– Potential stabilization / diagnostics / cure of

failing plants– May be common to many low-complexity

engineered systems


Acknowledgments

Falk WarneckeHector Garcia Martin

Philip Hugenholtz

Linda Blackall

Shaomei HeS. Brook Peterson

Katherine D. McMahon

Matthew Haynes

Forest Rohwer

Advancing Science with DNA Sequence Ecology of activated sludge Victor Kunin.

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