Advancing Science with DNA Sequence Ecology of activated sludge Victor Kunin
Advancing Science with DNA Sequence
Ecology of activated sludge
Victor Kunin
Advancing Science with DNA Sequence
Phil Hugen-
holtz
VictorKunin
FalkWarnecke
Suzan Yilmaz
HectorGarciaMartin
NataliaIvanova
Trina McMahon (UW)Linda Blackall (UQ)
Project powered by DOE
Advancing Science with DNA Sequence
Metagenomes in pipeline
Wastewater sludge (EBPR)
Termite gut
Guerrero Negro Hypersaline mat
Antarctic subglacial lake Vostok
Advancing Science with DNA Sequence
Ecology?
• population structure
• predator-pray interactions
• biogeography
Advancing Science with DNA Sequence
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
Advancing Science with DNA Sequence
• Excessive P affects water quality and ecosystem balance through eutrophication.
Enhanced Biological Phosphorus Removal (EBPR)
Advancing Science with DNA Sequence
• P is the key pollutant.
• P limitations in released water very effective
Enhanced Biological Phosphorus Removal (EBPR)
N,C
N,C,P
Advancing Science with DNA Sequence
Brisbane
Madison
US and Australian (OZ) samples
8 L
2 L
*
*
Advancing Science with DNA Sequence
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?
Advancing Science with DNA Sequence
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?
Advancing Science with DNA Sequence
Any geographic isolation of strains?
Geographic isolationOz
US
Yes No
US
OzUS
OZ
Advancing Science with DNA Sequence
How do they move?
• CAP is globally dispersed
• Never observed outside of sludge
• How do they move?
Advancing Science with DNA Sequence
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
Advancing Science with DNA Sequence
Looking for environmental reservoirs
Contra Costa wastewater treatment plant
**
**
*
*
PCR detection using:16S rRNA genePolyphosphokinase gene
Advancing Science with DNA Sequence
Previous map
CAP environmental reservoirs
*
*Aquatic samples: mostly yesTerrestrial samples: mostly no
Metapopulation
Advancing Science with DNA Sequence
In-strain variationdo
min
ant
stra
in
minor
Dominant strain is near clonal
Advancing Science with DNA Sequence
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
Advancing Science with DNA Sequence
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
Advancing Science with DNA Sequence
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
Advancing Science with DNA Sequence
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
Advancing Science with DNA Sequence
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
Advancing Science with DNA Sequence
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?
Advancing Science with DNA Sequence
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
Advancing Science with DNA Sequence
Acknowledgments
Falk WarneckeHector Garcia Martin
Philip Hugenholtz
Linda Blackall
Shaomei HeS. Brook Peterson
Katherine D. McMahon
Matthew Haynes
Forest Rohwer