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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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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

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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

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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

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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

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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 !!

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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

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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 !