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Transcript of How to unravel secrets of impossible microbes handouts
19-9-2014
1
How to unravel the secrets of impossible anaerobic microbes by
metagenomic analysis
September 19th
MIKE JETTEN
SOEHNGEN INSTITUTE OF ANAEROBIC MICROBIOLOGY
SIAM
Many very useful microbes
–Wastewater treatment
–Oxygen production
–Nitrogen fixation
–Food and fermentation
–Drugs and Antibiotics
–Degradation of xenobiotics
Very few pathogens
INTRODUCTION@MICROBIOLOGY Microbial Planet
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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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Undiscovered Chemolithoautotrophs
H2 CH4 H2S NH4+ Fe2+
O2
NO3- ??? ???
Fe3+
SO42-
CO2
After 40 years of searching in vain
They were being called
“impossible” microbes
ELECTRON DONORS
EL
EC
TR
ON
AC
CE
PT
OR
S
OXIC
ANOXIC
CS2
5
Undiscovered Chemolithoautotrophs
H2 CH4 H2S NH4+ Fe2+
O2
NO3- ??? ???
Fe3+
SO42-
CO2
After 40 years of searching in vain
They were being called
“impossible” microbes
ELECTRON DONORS
EL
EC
TR
ON
AC
CE
PT
OR
S
OXIC
ANOXIC
CS2
6
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4
HOW TO DISCOVER THESE “IMPOSSIBLE” MICROBES?
• 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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•State of the art microbial & metagenomic methods
•Adequate National and EU funding •International collaboration •Industrial collaboration •Skilled and enthusiastic team members
PRE REQUISITES
How to discover new microbes and their secrets?
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Sampling Bioreactors Bioreactors Bioreactors Bioreactors Bioreactors Bioreactors Metagenomics, bioinformatics, new experiments
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Sanger, illumina,
454, pacbio
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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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
HIGH NO3- due to agricultural run-off /ground water
HIGH CH4 production in the sediment
sampling sites
3CH4 + 8NO2- + 8H+ 4N2 + 3CO2 + 6H2O
Twentekanaal Brunsummer
heide Ooijpolder
Where do we find nitrate/nitrite-AOM?
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Gradient profiles nitrate/methane
-250
-200
-150
-100
-50
0
-100 0 100 200 300 400 500
NO3-
NH4+
CH4d
ep
th [
cm]
concentration[µmol/L]
© Pol et al 2007
Nutrient profile
Consortium of Archaea & Bacteria ?
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Bacteria
Methylomirabilis
NO dismutase?
Archaea
Methanoperedens
Mechanism?
Extract DNA, RNA, Protein
Construct draft assemblies, RNAseq, proteomics
+ Nitrate + Nitrite
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Assembly
2.7 Mb; 58% GC; 40-fold
Metagenome Approach
Community DNA
GS-Flex 90 Mb in 242 bp reads
Complemented with Sanger
Sequencing of
WGS ; Fosmids
2010
MetaGenomics of M. oxyfera bacteria
Genome of Methylomirabilis oxyfera
2010
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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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Possible mechanisms of M. oxyfera
Can oxygen be released? Use of suicide substrates, inhibitors and 15N18O nitrite
MMO enzyme
C3H6
15N18O2-
15N18O
15N18O2- 15N18O 30N2
36O2
Unknown enzyme
C3H6O
acetylene
formate
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36O2
36O2
36O2
36O2 36O2
36O2
Methylomirabilis oxyfera
15N18O experiments show: Oxygen Production
0
10
20
30
40
50
60
70
80
0 60 120 180 240 300 360 420 480
time [min]
O2 r
ele
as
ed
[n
mo
l/e
xe
tain
er]
nitrite & propylene
nitrite, propylene & acetylene
nitrite & methane
nitrite, propylene & oxygen
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Many more (meta)genomes do contain putative
NO dismutase genes
NO
NO N 2
O 2
NO dismutase
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Bacteria
Methylomirabilis?
NO dismutase?
Archaea
Methanoperedens?
Mechanism?
Extract DNA, RNA, Protein
Construct draft assemblies, RNAseq, proteomics
+ Nitrate + Nitrite
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Archaeal Metagenome sequencing
Haroon et al Nature 2013
Full pathway of (reverse) methanogenesis
Archaea with anammox as partner
1 nar cluster
25
ERC AG 2013
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
26
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Conclusions nitrate/nitrite-AOM
- Nitrite dependent AOM: M. oxyfera bacteria
- Putative NO dismutase may produce oxygen
- pmoA,nod and mcrA may be used as diagnostic biomarker
- Nitrate dependent AOM: Archaea plus partner
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SIAM
ERC AG 2013
Undiscovered Chemolitoautotrophs ELECTRON DONORS
EL
EC
TR
ON
AC
CE
PT
OR
S
28
H2 CH4 H2S NH4+ Fe2+
O2
NO3- ??? ???
Fe3+
SO42-
CO2
Do nitrite dependent anaerobic
ammonium oxidizers exist???
OXIC
ANOXIC
CS2
OK
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Calculations in the N cycle
NH4+ + NO2
- N2 + 2H2O ΔG’0 = -358 kJ/mol
Engelbert Broda
1910-1983
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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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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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Anammox Cell Biology
33
ERC AG 2008
Anammox genome lessons
2002-2006 major effort with Sanger sequencing 5 contigs
2012 resequencing ion torrent
2014 resequencing pacbio
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
34
ERC AG 2008
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Genome of more anammox bacteria
Core anammox genes present in all
Major difference: Copper based nirK instead of cd1 nirS or no nir !!
35
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
Metatranscriptome of anammox
“Which genes encode hydrazine synthase”
Gene_ID nt fold 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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ERC AG 2008
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© Pol et al 2007
How does ANAMMOX make the ROCKET FUEL HYDRAZINE ?
Protein purification
15N14N
Protein activity Hydrazine and N2 production
Protein crystalisation
Immunogold labelling hzsABC
Metagenomic information of Anammox, M. oxyfera and other new microbes could save the world
Thank you !
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
38
SIAM
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http://www.ru.nl/english/education/master's-
programmes/
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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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 C1 microbes
- AOM and anammox bacteria
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
Credits
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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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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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Acetate + ATP + HSCoA Ac-CoA + AMP +PPi
CO + H2O CO2 + 2[H]
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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
Need for a very good mentor/supervisor!
If not provided by your supervisor, be assertive and ask
If still not provided, get it someone/where else asap.
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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)
Absolute need for human and leadership skills!
These skills contribute up to 85% of your success!!
Chapter2@MIKE-ROBIOLOGY
Post doc 1991-1994
• 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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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
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
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
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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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Way2independece@MIKE-ROBIOLOGY
Moving back to Europe: Assistant professor 1994-2000
• 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
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??
Responsibilities@MIKE-ROBIOLOGY
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• State of the art microbial methods
• Adequate National and EU funding
• International collaboration
• Industrial collaboration
• Skilled and enthusiastic team members
PRE REQUISITES
How to Discover new microbes?
Sampling Bioreactors Bioreactors Bioreactors Bioreactors Bioreactors Bioreactors Bioreactors Bioreactors Genomic Physiology Microscopy Ecology Biochemistry Application
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• State of the art microbial methods
• Adequate National and EU funding
• International collaboration
• Industrial collaboration
• Skilled and enthusiastic team members
PRE REQUISITES
How to Discover of new microbes?
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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Down side of (too much) success
• 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
Vacancies
Full professor
Tenure track
2 Post docs
10 PhD students
Skilled and enthusiastic team members
and give them the best possible training
in human and leadership skills
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VOLCANIC
MICROBIOLOGY
MICROBIAL
INTERACTIONS
(AN)AEROBIC
OXIDATION OF METHANE
ANAMMOX
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
Symbiotic Microbiology
2014-2019 projects
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Discoveries in CH4 & CS2
Genome seq
Physiology
Proteins
1 PhD thesis
Hot and Acid Volcano Microbiology
2008-2013
CH4 + 2O2 CO2 + 2H2O
At pH < 1 and 55°C
© Pol et al 2007
Methylacidiphilum fumariolicum SolV
Sampling at Solfatara, Naples, Italy Enrichment & isolation
Poor growth without
mudpot water
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Evaporating and ashing still stimulated growth:
which mineral /element ?
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
Ce Sm Eu Gd None or Ca
Gro
wth
ra
te (
h-1
)
Maximum growth rates with Ce and REE with higher atomic
numbers tested individually.
Which cellular
component does
contain Cerium?
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A SDS-PAGE (12%) of the purified methanol dehydrogenase of strain SolV
B the UV-Vis spectrum of the pure enzyme
0.0
0.4
0.8
1.2
1.6
2.0
250 300 350 400 450 500 550
Wavelength (nm)
Exti
nct
ion
(b
lack
lin
e)
0.00
0.04
0.08
0.12
0.16
0.20
Exti
nct
ion
(re
d li
ne
)
28
36
55
72
93
130
kDa
250
1 2
1 = Cell extract (50 μg protein)
2 = Pure MDH (10 μg protein)
A B
a
b c
Thomas Barends
MAX-PLANCK GESELLSCHAFT
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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
VOLCANIC
MICROBIOLOGY
MICROBIAL
INTERACTIONS
(AN)AEROBIC
OXIDATION OF METHANE
ANAMMOX
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Does nitrate/nitrite dependent Anaerobic Oxidation of
Methane (AOM) exist???
ERC AG 2013
Anammox, AAA, SolV, M. oxyfera and
other new C1 microbes could save the world
Thank you !
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!
Never give up !