19-9-2014
How to unravel the secrets of
impossible anaerobic microbes by
metagenomic analysis
September 19th
MIKE JETTEN
SOEHNGEN INSTITUTE OF ANAEROBIC MICROBIOLOGY
SIAM
INTRODUCTION@MICROBIOLOGY
Microbial Planet
Many very useful microbes
–Wastewater treatment
–Oxygen production
–Nitrogen fixation
–Food and fermentation
–Drugs and Antibiotics
–Degradation of xenobiotics
Very few pathogens
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19-9-2014
INTRODUCTION@MICROBIOLOGY
unexplored biodiversity
•
30,000
strains in ATCC & DMSZ
2,639,157
16S rRNA genes in RDP
10,000,000,000,000,000,000,000,000,000,000
nonillion
microbial cells on Earth
Terra incognita
INTRODUCTION@MICROBIOLOGY
metabolic diversity
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19-9-2014
Undiscovered Chemolithoautotrophs
ELECTRON ACCEPTORS
ELECTRON DONORS
H2
CH4
H2S
CS
2
NH4+ Fe2+
O2
???
NO3-
???
OXIC
ANOXIC
Fe3+
SO42-
After 40 years of searching in vain
They were being called
CO2
“impossible” microbes
5
Undiscovered Chemolithoautotrophs
ELECTRON ACCEPTORS
ELECTRON DONORS
H2
CH4
H2S
CS
2
NH4+ Fe2+
O2
NO3-
???
???
OXIC
ANOXIC
Fe3+
SO42CO2
After 40 years of searching in vain
They were being called
“impossible” microbes
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19-9-2014
HOW TO DISCOVER THESE
“IMPOSSIBLE” MICROBES?
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Survey of selected ecosystems
Bring Best Samples to Lab
Design optimal bioreactors
Enrichment under optimal conditions
Grow enough cells
Use of the molecular toolbox to
unravel their secrets
Back to the ecosystem
Application of the new microbes
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How to discover new microbes and their secrets?
PRE REQUISITES
•State of the art microbial & metagenomic
methods
•Adequate National and EU funding
•International collaboration
•Industrial collaboration
•Skilled and enthusiastic team members
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19-9-2014
Sampling
Bioreactors Bioreactors Bioreactors
Bioreactors Bioreactors Bioreactors
Metagenomics, bioinformatics, new experiments
Sanger, illumina,
454, pacbio
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Important PRE REQUISITE
Skilled and enthusiastic team members
that receive the best possible training
also in human and leadership skills
You can be next:
www.ru.nl/microbiology/vacancies
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19-9-2014
How to unravel the secrets of impossible anaerobic
microbes by metagenomic analysis
Anaerobic oxidation of methane (AOM)
Discovery
Involved organisms
Metagenome
Transcriptome
New experiments
SIAM
Where do we find nitrate/nitrite-AOM?
sampling sites
3CH4 +
Brunsummer
heide
8NO2-
+
8H+
Ooijpolder
 4N2 + 3CO2 + 6H2O
Twentekanaal
HIGH NO3- due to agricultural run-off /ground water
HIGH CH4 production in the sediment
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19-9-2014
Gradient profiles nitrate/methane
0
-100
0
100
200
300
400
500
© Pol et al 2007
depth [cm]
-50
-100
NO3-
-150
NH4+
CH4
-200
concentration[µmol/L]
-250
Nutrient profile
Consortium of Archaea & Bacteria ?
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19-9-2014
+ Nitrate
Archaea
Methanoperedens
Mechanism?
+ Nitrite
Bacteria
Methylomirabilis
NO dismutase?
Extract DNA,
RNA, Protein
Construct draft assemblies, RNAseq, proteomics
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19-9-2014
MetaGenomics of M. oxyfera bacteria
Metagenome Approach
Community DNA
GS-Flex 90 Mb in 242 bp reads
2010
Complemented with Sanger
Sequencing of
WGS ; Fosmids
Assembly
2.7 Mb; 58% GC; 40-fold
Genome of Methylomirabilis oxyfera
2010
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19-9-2014
Genome of Methylomirabilis oxyfera
2010
pathway of (aerobic) methane oxidation
Incomplete denitrification
Methylomirabilis oxyfera
Localisation key enzymes pmoB and nirS
on basis of genome = synthetic peptides for immunization
pmoB antibodies
nirS antibodies
Wu et al FEMS letters 2012
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19-9-2014
Possible mechanisms of M. oxyfera
Can oxygen be released?
Use of suicide substrates, inhibitors and
15N18O
15N18O 2
15N18O 2
30N
2
36O
2
36O
2
36O
2
36O
2
36O 36O
2
2
C3H6O
MMO
enzyme
formate
nitrite
36O
2
Unknown
enzyme
15N18O
15N18O
C3H6
acetylene
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Methylomirabilis oxyfera
experiments show: Oxygen Production
80
nitrite & propylene
nitrite, propylene & acetylene
70
nitrite & methane
nitrite, propylene & oxygen
O2 released [nmol/exetainer]
15N18O
60
50
40
30
20
10
0
0
60
120
180
240
300
360
420
480
time [min]
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19-9-2014
Many more (meta)genomes do contain putative
NO dismutase genes
NO dismutase
NO
N2
NO
O2
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+ Nitrate
Archaea
Methanoperedens?
Mechanism?
+ Nitrite
Bacteria
Methylomirabilis?
NO dismutase?
Extract DNA,
RNA,
Protein
Construct draft assemblies, RNAseq, proteomics
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19-9-2014
Archaeal Metagenome sequencing
Haroon et al Nature 2013
Full pathway of (reverse) methanogenesis
Archaea with anammox as partner
1 nar cluster
ERC AG 2013
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Are the nitrate CH4 archaea important in the environment?
Nitrate generally is more abundant than nitrite, so large potential
Many surveys do contain related mcrA and 16S rRNA genes
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19-9-2014
Conclusions nitrate/nitrite-AOM
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Nitrite dependent AOM: M. oxyfera bacteria
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Putative NO dismutase may produce oxygen
-
pmoA,nod and mcrA may be used as diagnostic biomarker
-
Nitrate dependent AOM: Archaea plus partner
SIAM
ERC AG 2013
27
Undiscovered Chemolitoautotrophs
ELECTRON ACCEPTORS
ELECTRON DONORS
H2
CH4
H2S
CS
2
NH4+ Fe2+
O2
NO3-
OK
???
???
OXIC
ANOXIC
Fe3+
SO42CO2
Do nitrite dependent anaerobic
ammonium oxidizers exist???
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19-9-2014
Calculations in the N cycle
Engelbert Broda
1910-1983
NH4+ + NO2-  N2 + 2H2O
ΔG’0 = -358 kJ/mol
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Anaerobic pilot plant, TU Delft, the Netherlands
Influent
Effluent
Mulder , van de Graaf et al FEMS Ecology 1995
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19-9-2014
ANAMMOX MILESTONES
1995 ANAEROBIC PILOT PLANT
Mulder et al FEMS Ecol
1998 SBR ENRICHMENT CULTURES
Strous et al AMB
1999 PHYLOGENETIC IDENTITY
Strous et al Nature (MJ =37y)
2002 LADDERANE LIPIDS
Damste et al Nature
2003 BLACK SEA
Kuypers et al Nature
Anammoxoglobus
propionic
Jettenia asiatica
KSU-1
© Jetten et al 2009
ANAMMOX MILESTONES
2006 GENOME ASSEMBLY
Strous et al Nature
2008 CELL BIOLOGY
Van Niftrik et al Mol Mic
2011 METABOLISM
Kartal et al Nature
2013 CHINESE WETLANDS
Zhu et al Nature Geoscience
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19-9-2014
Anammox Cell Biology
ERC AG 2008
33
Anammox genome lessons
2002-2006 major effort with Sanger sequencing 5 contigs
2012 resequencing ion torrent
2014 resequencing pacbio
ERC AG 2008
200 respiratory genes
61 ‘cytochrome C genes’
Central role for nitric oxide (NO) and hydrazine (Hzs)
E-acceptors: nitrate; nitrite; Fe (III); Mn(IV)
E-donors: ammonium; formate; acetate; propionate; Fe (II)
Acetyl CoA pathway for CO2 fixation
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19-9-2014
Genome of more anammox bacteria
ERC AG 2008
anammox
Journal year
Nitrite reduction by
Kuenenia
Nature 2006
nirS or ???
Scalindua
EMI2012
nirS
Brocadia
BST 2012
???
KSU-1
FEBS Lett 2012
nirK
Jettenia
Frontiers 2012
nirK
Kuenenia CH1
Frontiers 2012
nirS or ???
Scalindua arabica
Frontiers 2014
nirS
Brocadia
In press
???
Scalindua pacifica
Submitted
nirS
Anammox in the wild
In pogress
Core anammox genes present in all
Major difference: Copper based nirK instead of cd1 nirS or no nir !!
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Metatranscriptome of anammox
“Which genes encode hydrazine synthase”
Gene_ID
nt
fold
ERC AG 2008
reads description
Kuste2859 1161 74.7 2711 hzsC, hypothetical protein
Kuste2560 1062 52.7 1748 hzsB , diheme protein
Kuste2861 2430 51.4 3902 hzsA , diheme protein
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19-9-2014
How does ANAMMOX make the
ROCKET FUEL HYDRAZINE ?
Protein purification
Protein crystalisation
© Pol et al 2007
15N14N
Immunogold labelling hzsABC
Protein activity
Hydrazine and N2 production
Metagenomic information of Anammox, M. oxyfera and
other new microbes could save the world
Unique bacteria hiding out in a witches’ brew
of anoxic water not only thrive in cold
wetlands and oceans but also chow down its
ammonium and methane
Thank you !
SIAM
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19-9-2014
http://www.ru.nl/english/education/master'sprogrammes/
A scientific life of discovering
new anaerobic C1 microbes
while trying to keep young
(wo)men in science
MIKE JETTEN
SOEHNGEN INSTITUTE OF ANAEROBIC MICROBIOLOGY
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19-9-2014
Table of content
Pivotal career steps/advise
- PhD and supervision
- post doc and family
- tenure and independence
- full professor and responsibilities
intertwined with C1 examples
- Acetoclastic methanogens
- Hot and acid volcano C 1 microbes
- AOM and anammox bacteria
Credits
Curt Rice, Tromso 6 steps to gender equality
Petra Rudolf, RU Groningen Roosevelt Foundation
Katty Kay and Claire Shipman, The Confidence Gap
http://www.genderinscience.org/
http://www.informatics-europe.org/images/documents/
more-women-in-informatics-research-andeducation_
2013.Pdf
My supervisors AJB, AJS, JGK, SG
My co workers, collaborators and funding agencies
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19-9-2014
Academic careers seem to be unappealing to both man and woman,
The impediments are perceived as disproportionate
The sacrifices are great.
Research career? PhD 1y
PhD 3y
Career in
Academia at 3y
M
61%
59%
21%
F
72%
37%
12%
Why is academia not appealing?
Hunt for projects/money; all consuming & competitive
String of post docs w/o prospects =
Lack of security
F Lack of self confidence
F Lack of good F role models
INTRODUCTION@MIKE-ROBIOLOGY
PhD Microbiology 1991
Wageningen University
Post doc 1991-1994
Assistant professor 1994-2000
Professor Microbiology 2000-
ERC AG 2008
ERC AG 2013
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19-9-2014
INTRODUCTION@MIKE-ROBIOLOGY
PhD Microbiology 1987 - 1991
Acetate Metabolism in Methanothrix soehngenii
- Optimally prepared by best anaerobic training in experts lab
(Georg Fuchs, Ulm 86)
- State-of-the-art research facilities
- Coherent team and colleagues
- Combination high level sport & study is
foundation 4 ambition & perseverance
- Get your papers out asap
- Excellent supervisors
Alex Zehnder
Prof Microbiology WUR
Director EAWAG CH
Presiden ETH Zurich
“fat rod” Methanothrix soehngenii
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19-9-2014
Acetate + ATP + HSCoA  Ac-CoA + AMP +PPi
CO + H2O  CO2 + 2[H]
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19-9-2014
Need for a very good mentor/supervisor!
Apart from giving scientific guidance, she/he should
• provide training in the art of presentation science
• teach how to write fast and how to write good papers
• find funding / help you find funding
• teach you how to write successful proposals
• teach you the “rules of the game” and how to change them
• give you challenging assignments and opportunities
• provide constructive feedback on unsuccessful endeavours
• introduce you to important professional contacts
• give you credit, and advocate you in the academic community
If not provided by your supervisor, be assertive and ask
If still not provided, get it someone/where else asap.
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19-9-2014
Absolute need for human and leadership skills!
Apart from giving scientific guidance, she/he should teach importance of human
and leadership skills
- Educational skills to transfer knowledge
- Project and time management to be as effective as possible
- Scientific communication skills to become an articulate speaker/writer
- Interaction with people 4 networking & collaborating
- Personal happiness 4 balance work/private life
- Career management
- Leadership skills (vision, coaching, delegating, team building, supervision)
These skills contribute up to 85% of your success!!
Chapter2@MIKE-ROBIOLOGY
Post doc 1991-1994
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Bring your own money & freedom
Husband/Spouse program
Complementary and synergy in skills
Quality of supervision
Nose for feasibility & serendipity
practice make perfect but 95% is more effective!
Publish asap & amap
Train human and leadership skills
Move on
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19-9-2014
Challenges in combining family life and career
• Family/career : there is never an easy equilibrium
• 2 body problem: not each location offers opportunity for both
• if your heart allows marry/partner outside your profession
• 50% shared responsibilities
• learn to live with little sleep asap
• (European) labour law = raise kids asap in PhD
• reality most academians have a family after 32
Challenges in combining family life and career
Emancipation is still a long way to go
Ladies be assertive and do not take more than
50% share in family responsibilities
Only 1 in 3000 female physicists had a husband who was not
professionally active but took care of the home and the family
25% of the male physicists are in an equivalent situation.
USA: http://www.physics.wm.edu/dualcareer.html
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19-9-2014
THE CONFIDENCE GAP
For many young women in academia it is difficult to reach out to older
male colleagues to ask for advice
Women tend to underestimate their potential for success in Science.
Women apply when the meet 120% of requirements (M at 50%)
Women do gain confidence from role models!
Women in middle management are more inspiring F role models.
Affirmative action stimulates F in competition
Learning to own victory and survive defeat in sports is apparently good
training for owning triumphs and surviving setbacks at work
Curt Rice, Katty Kay and Claire Shipman
What should universities do:
- Use alumni network to help finding jobs for partners
- Consider partners as internal candidates for university vacancies
- Excellent child care facilities
- Reimbursement for babysitting service for unusual situations
- Family friendly working conditions:
- No meetings at hours that create conflict with family obligations….
- Mentor programs with good F role models
- Special lunch meetings/support groups
- gender neutral advertisements / balanced selection committees
- Many more………….
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19-9-2014
Way2independece@MIKE-ROBIOLOGY
Moving back to Europe: Assistant professor 1994-2000
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Bring your own money & personal grant
Develop nose for feasibility of projects
Practice make perfect but 95% is more effective!
Be at right place and time (serendipity)
Publish HI papers (1st or last author)
Raise more money and build a team
Joint Family responsibilities
Complementary and synergy in skills
Quality supervision/mentoring
Train human and leadership skills
Be assertive and/or move on
Responsibilities@MIKE-ROBIOLOGY
Professor Microbiology 2000• Bring your own money/projects
• Refine nose for feasibility and serendipity
• Practice make perfect but 90% is even more effective!
• Raise a lot more money & build a large team
• Publish more HI papers
• Joint Family responsibility
• Complementary and synergy in skills
• Quality of Supervision & mentoring
• Networking / collaborations
• Become stronger and more assertive
New stable state or tipping point
• Transfer of knowledge
• Transfer of human and leadership skills
and become eligible for dean, VP, VC, etc
or move on??
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19-9-2014
How to Discover new microbes?
PRE REQUISITES
• State of the art microbial methods
• Adequate National and EU funding
• International collaboration
• Industrial collaboration
• Skilled and enthusiastic team members
Sampling
Bioreactors Bioreactors Bioreactors Bioreactors
Bioreactors Bioreactors Bioreactors Bioreactors
Genomic
Microscopy
Biochemistry
Physiology
Ecology
Application
30
19-9-2014
How to Discover of new microbes?
PRE REQUISITES
• State of the art microbial methods
• Adequate National and EU funding
• International collaboration
• Industrial collaboration
• Skilled and enthusiastic team members
ERC AG 2008
ERC AG 2013
Added value of Awards
•
•
•
•
•
•
•
•
Possibility to do research w/o restrictions
Recognition @home university
Many Invitations
New collaborations
Exclusive partnership with industry
Job offers
More awards and prizes (Matthew 25:29)
Many student opportunities
ERC AG 2008
ERC AG 2013
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19-9-2014
Down side of (too much) success
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Harsher peer review (from such PI we expect….)
Difficult to obtain additional grants
Revenge taken out on younger staff & students
Jealous colleagues
Messy university politics
To big for collaboration
Where should phd/post docs move to?
Hardly any friendly colleagues left
It can be very lonely at the top
Ne iupiter quidam placet omnibus
Added value / Down side ≥ 1
Most important PRE REQUISITE
Skilled and enthusiastic team members
and give them the best possible training
in human and leadership skills
Vacancies
Full professor
Tenure track
2 Post docs
10 PhD students
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19-9-2014
ANAMMOX
(AN)AEROBIC
OXIDATION OF METHANE
VOLCANIC
MICROBIOLOGY
MICROBIAL
INTERACTIONS
Symbiotic Microbiology
2014-2019 projects
Methane cycle in wetlands
Kosten & Ettwig
Symbiotic seagrass fields
Hooiberg, vd Heiden
van Alen, Op den Camp
N2 fixation peat lands
Kox, vd Elzen
Lamers, Ettwig
Rhizobiome of wetlands
Vaksmaa, Lueke
Delia Gut microbiome
Welte
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19-9-2014
Hot and Acid Volcano Microbiology
2008-2013
Discoveries in CH4 & CS2
Genome seq
Physiology
Proteins
1 PhD thesis
Methylacidiphilum fumariolicum SolV
CH4 + 2O2  CO2 + 2H2O
At pH < 1 and 55°C
© Pol et al 2007
Sampling at Solfatara, Naples, Italy Enrichment & isolation
Poor growth without
mudpot water
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19-9-2014
Evaporating and ashing still stimulated growth:
which mineral /element ?
0.09
0.08
Growth rate (h-1)
0.07
0.06
0.05
0.04
0.03
Which cellular
component does
contain Cerium?
0.02
0.01
0
Ce
Sm
Eu
Gd
None or Ca
Maximum growth rates with Ce and REE with higher atomic
numbers tested individually.
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19-9-2014
2.0
A
55
36
28
1 = Cell extract (50 μg protein)
2 = Pure MDH (10 μg protein)
1.6
0.16
1.2
0.12
0.8
0.08
0.4
0.04
0.0
250
Extinction (red line)
1 2
250
130
93
72
Extinction (black line)
kDa
0.20
B
0.00
300
350
400
450
500
550
Wavelength (nm)
A SDS-PAGE (12%) of the purified methanol dehydrogenase of strain SolV
B the UV-Vis spectrum of the pure enzyme
a
Thomas
Barends
MAX-PLANCK GESELLSCHAFT
b
c
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19-9-2014
Methane cycle in volcanoes
SolV isolated
Growth dependent on REE
Genome sequenced
Rubisco as CO2 fixation pathway
N2 fixation
Transcriptome
Cell Biology in progress
Nitrosative stress in progress
ANAMMOX
VOLCANIC
MICROBIOLOGY
(AN)AEROBIC
OXIDATION OF METHANE
MICROBIAL
INTERACTIONS
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19-9-2014
Does nitrate/nitrite dependent Anaerobic Oxidation of
Methane (AOM) exist???
ERC AG 2013
Never give up !
Anammox, AAA, SolV, M. oxyfera and
other new C1 microbes could save the world
Unique bacteria hiding out in a witches’ brew of anoxic
water not only thrive in cold wetlands and oceans but
also chow down its ammonium and methane
Microbial science = Fun
Leadership skills = 85% success!
The future = bright for Women in Science!
Thank you !
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