A functional gene approach to studying nitrogen cycling in the sea

Matthew Church (MSB 612 / 6-8779

mjchurch@hawaii.edu)Nov. 20, 2007

Overview

• Brief review of the central dogma• Use of functional genes to study

processes in the marine environment• N2 fixation• Nitrification

• Wealth of information emerging from genome sequencing and metagenomic studies

566 completed: 476 Bacteria, 49 Eukaryotes, 41 Archaea

Ongoing: 1143 Bacteria, 720 Eukaryotes, 56 Archaea

54 from marine organisms with another 100-150 in progress

Year95 96 97 98 99 00 01 02 03 04 05 06 07

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TotalMarine

Link genetic approaches identifying microorganism metabolic capabilities to

biogeochemical determinations of nutrient cycles and energy flow

. . . . | . . . . | . . . . | . . . . | . . .T C C A C A C G T C T T A T T C C G C A C T CT C C A C A C G T C T T A T T C T G C A C T CT C T A C T C G T T T A A T C T T A A A C G C TT C T A C A C G T T T G A T G C T G C A C T C TT C T A C A C G T T T G A T G C T G C A C C C TT C T A C T C g T T T A A T G T T G C A C T CT C T A C T C G T T T A A T G T T G C A C T C

Transcription of DNA by RNA polymerase

mRNA transcriptTranslation of mRNA at the ribosome

5’-UAUUAGGCCAAUCCGC-3’

5’-TATTAGGCCAATCCGC-3’

3’-ATAATCCGGTTAGGCG-5’

Protein

operon

gene

The Central Dogma: Genes to proteinsTATTAGGCC AATCCGC TCCGC AATCC

• RNA polymerases transcribe DNA into single stranded RNA producing:

–Transfer RNA (tRNA)-transfers amino acids to the polypeptide chain at the site of the ribosome.–Messenger RNA (mRNA)-template for coding of proteins.–Ribosomal RNA (rRNA)-a structural component of the ribosome.

•RNA polymerase elongates from the 3’ end of the DNA template, synthesizing 5’ ⇒ 3’ RNA

Small ribosome subunit (30S)

Large ribosomal subunit (50S)

Protein

rRNA(16S)

Protein

rRNA(23S)

rRNA(5S) At the ribosome,

mRNA is translated (with the help of tRNA) to protein.

A functional gene approach to understand biologically controlled ocean processes

• Molecular tools can be applied to understand the role of microorganisms in specific biogeochemical processes.

• Functional genes encode proteins.

• Functional genes can be used to examine the diversity of organisms that possess the genetic capacity to catalyze some process (e.g. photosynthesis, N2 fixation, rhodopsin-driven light harvesting).

• Functional genes can also be used to examine which organisms are actively involved in a process (gene expression).

The nitrogen cycle is maintained by the activities of Bacteria and Archaea

NH4+

NH4+N02-

N02-

The ocean nitrogen cycle

Denitrification: NO3- NO2- NO N2O N2

Nitrification: NH4+ NO2- NO3-

Nitrogen fixation: N2 NH4+

Anammox: NO2 + NH4+ N2 + 2H2O

Nitrogen serves as both an essential

nutrient, and because of its redox potential

also serves as an energy source and electron acceptor.

NO3-NO2-NH4+N2 (*)

NO2-NH4+DON

NO3-NH4+N2

NO3-NO2-NH4+DONN2

Eukaryotes EukaryotesProchlorococcus Diazotrophs Heterotrophs

Nitrogen sources supporting plankton growth

10 µm 100 µm 10 µm10 µm

Not all nutrients are created equal…

Year

89 90 91 92 93 94 95 96 97 98 99 00 01 02 03 04 05 06 07

δ15 N

par

ticul

ate

N e

xpor

t(‰

)

-2

0

2

4

6

8Dore et al. (2004)

NO3-

N2

N2 fixation contributes a major fraction (~30-84%) of

new production in the open

ocean

N2 fixation

4 3.5 3.0 2.5 2.0 1.5 1.0 0.5

EARTH

Continental crust forms

Banded Iron FormationsContinental Weathering

ATMOSPHEREReducing, N2, CH4, NH3

O2 in atmosphere

Present O2

levels

OCEANSAnoxic, Fe2+, S

Phosphorus precipitates

Oxygenated surface, anoxic at depthIncreased SO4Carbon burial

Fe3+ precipitation

LIFEAnaerobes

Cyanobacteria Eukaryotes

METABOLISMAnaerobic metabolism

Autotrophy (carbon fixation)Anoxygenic photosynthesis

Oxygenic photosynthesis

Aerobic Respiration

Billions of years ago

Green-sulfur (photolithotrophs)Heterotrophic sulfate reducerDelta Proteobacteria, iron reducerAlpha proteobacteria, methane oxidizerSpirocheteAerobic heterotrophGram positive firmicutes

Anaerobes

Aerobes

Archaea, methane producers

Facultative anaerobic gamma proteobacteriaFilamentous, non-heterocystous cyanobacteriaUnicellular cyanobacteriaHeterocyst-forming cyanobacteria

nifH gene distance tree- the capacity to fix nitrogen appears in numerous and highly diverse groups of Bacteria and Archaea, including

cyanobacteria, proteobacteria, spirochetes, green and purple sulfur bacteria, methanogens, methanotrophs

nifH gene sequences have been retrieved from some of the following marine habitats: euphotic zone (both free living and symbionts),

mesopelagic, bathypelagic, hydrothermal vents, inside the guts of animals, sediment, biofilms

• N2 + 8H + 8e- + 16ATP + 16H2O → 2NH3 + H2 + 16ADP + 16Pi

• MoFe protein is 220,000Da, formed by 4 subunits that contain 28 ions of Mo

• The Fe protein is 70,000Da, and is formed by 2 subunits which contain 8 atoms of Fe as co-factors

• Nitrogenase activity is suppressed by oxygen

• MoFe encoded by nifd and nifKgenes; Fe protein encoded by nifH gene

Nitrogenase

MoFe

Fe

Fe

Isolating Nitrogenase from Oxygen

• Spatial separation-formation of heterocysts; only photosystem I is active in these specialized cells (light is harvested and NADPH is created, but no H2O split)

• Temporal separation-diel separation of N2fixation and photosynthesis

Heterocyst

NO

N2O

N2

NO2

NH4+

NH2OH

narBNO3

hao

amoAnifH

nosZ

norB

nirKnirS

nirA

aax

DON

Am

monia oxidation

Nitrate assimilationDe

nitri

ficat

ion

N2 fixation

Ammonium assimilationglnA

Genes and processes in the nitrogen cycle

Identifying genes encoding enzymes that catalyze specific biochemical reactions

Methodological approaches

Extract DNA and mRNA.

Amplify genes by PCR and reverse

transcriptase (RT) PCR, clone and

sequence amplified products.

Use sequence information to

develop quantitative approaches

(probes, primers, arrays) to track

dynamics of specific organisms. . . . | . . . . | . . . . | . . . . | . . .

T C C A C A C G T C T T A T T C C G C A C T CT C C A C A C G T C T T A T T C T G C A C T CT C T A C T C G T T T A A T C T T A A A C G C TT C T A C A C G T T T G A T G C T G C A C T C TT C T A C A C G T T T G A T G C T G C A C C C TT C T A C T C g T T T A A T G T T G C A C T CT C T A C T C G T T T A A T G T T G C A C T C

0.10Cluster I

Cluster IICluster IIICluster IV

Phylogenetic relationships among nifH genes

IV: Spirochaetes, Archaea

I: Proteobacteria, Cyanobacteria

II: Proteobacteria, FirmicutesIII: Firmicutes, Spirochaetes

• Are all these microorganisms fixing nitrogen?

• Reverse transcriptase is an enzyme that functions as a RNA-dependent DNA polymerase. Reverse transcriptases copy single stranded RNA into DNA.

• These enzymes are encoded by retroviruses, where they copy the viral RNA genome into DNA prior to its integration into host cells.

mRNA transcript5’-UAUUAGGCCAAUCCGC-3’

Primer5’

Reverse transcription synthesis complementary strand of DNA (cDNA) from the single stranded mRNA template

GCG

ATAATCCGGTTAGGCG5’3’ Single strand of cDNA

5’-TATTAGGCCAATCCGC-3’

3’-ATAATCCGGTTAGGCG-5’Double stranded DNA

PCR amplify cDNA

Diazotrophic bacteria at

Station ALOHA

2-10 µm 10-2000 µm

100 µm100 µm

??

Heterocystous cyanobacteria

Unicellular cyanobacteria

Filamentous cyanobacteria

Proteobacteria

Anaerobes

>0.2 µm >0.2 µm

Group A and B

Trichodesmium

Richelia

Diversity and expression of nifH genes

Chesapeake Bay North Pacific Subtropical Gyre

•Chesapeake Bay-high diversity of organisms with the capability of fixing N2, few organisms actively fixing N2. •Oligotrophic open ocean-low diversity of N2 fixing organisms, but nearly all appear active in N2 fixation

The ocean nitrogen cycle

Denitrification: NO3- NO2- NO N2O N2

Nitrification: NH4+ NO2- NO3-

Nitrogen fixation: N2 NH4+

Anammox: NO2 + NH4+ N2 + 2H2O

Nitrogen serves as both an essential

nutrient, and because of its redox potential

also serves as an energy source and electron acceptor.

Nitrification and Denitrification

Many different groupsNitrosomonas,NitrobacterTaxonomic group

ChemoorganotrophChemolithotrophEnergy source

HeterotrophsAutotrophsC-source

NO3-, NO2-, othersO2Respiratory e-

acceptor

Anaerobic RespirationAerobic RespirationRespiratory

Classification

FacultativeObligate AerobesAerobic status

DenitrificationNitrification

Ocean nitrification• Nitrification is a key step in the global N

cycle.

• 2 step process: 1) oxidation of NH3 to NO2-(ammonia oxidation) and 2) oxidation of NO2-to NO3- (nitrite oxidation).

• Until recently (2005) members of the β- and γ-proteobacteria were considered the predominate nitrifying microorganisms in the sea.

N. Pace’s Tree of Life (1997)

In the early 1990’s, several studies identified 16S rRNA gene sequences from marine environments that derived from Archaea-including both the Crenarchaea and Euryarchaea. Well studied Crenarchaeainclude hyperthermophiles and halophiles, while Euryarchaea include methanogens. The role of these organisms in ocean ecology and biogeochemistry remained unknown until the mid-2000’s.

Green = Eubacteria ProbeRed = Crenarchaea Probe

Photo courtesy of Ed DeLong (MIT)

Using the 16S gene to identify the abundance of ArchaeaFISH —rRNA probes

Distribution of Archaea in the North Pacific Ocean

Karner et al. 2001, Nature

Abundance of Archaea and

Bacteria in the North Atlantic

Bacteria dominate abundances throughout water, accounting for ~50%

of total picoplankton. Crenarchaeaaccount for 18-24% picoplankton

DAPI microautoradiography

FISH

FISH microautoradiography

Microautoradiography combined with 16S-based FISH suggests the Archaea may grow mixotrophically,

assimilating DIC and organic matter.

Herndl et al.

Unlike bacteria, cell walls of Archaea do not contain peptidoglycan; archaeal cell walls contain a molecule similar to peptidoglycan called pseudomurein. Lipids in archaeal membranes contain ether linkages between the glycerol backbone and the fatty acids, instead of the ester linkages found in both bacteria and eukaryotes.

Ester linked lipids found in eukaryotes and bacteria

Ether linked lipids found in Archaea

Ingalls, Anitra E. et al. (2006) Proc. Natl. Acad. Sci. USA 103, 6442-6447

Fig. 1. HPLC chemical ionization-MS chromatograms of ether linked membrane lipids separated from the surface filter (21 m) and deep

sea (670 m). (B) Molecular structures of the ether-linked lipids

Water column properties and ∆14C values for DIC, DOC, sterols, and GDGTs

Conclusion: The predominate (83%) source of carbon fueling archaeal growth in the deep sea is DIC.

21 m

670 m

Potential sources of energyin the deep sea:

Reduced carbon (DOM, methane)Reduced nitrogen (ammonia,

nitrite, urea, amino acids)Reduced sulfur (HS, H2S, S)

Reduced metals (Fe2+)

Energy

What are the energy sources supporting

autotrophic growth in the deep sea?

NO

N2O

N2

NO2

NH4+

NH2OH

narBNO3

hao

amoAnifH

nosZ

norB

nirKnirS

nirA

aax

DON

Nitrification

Deni

trific

atio

n

N2 fixation

glnA

Metagenomics to the rescue!

High diversity of Crenarchaeal ammonia monooxygenase genes

(amoA) found in sediments, water column, and soils

Marine Crenarchaealnitrification

Add NH4+, NO3-, and NO2- and watch which members of the picoplanktonic

assemblage respond.

Time series of nutrients, Crenarchaea, and amoA

gene abundances

Archaea also appear to be the dominate microorganisms involved in ammonium oxidation in soils

Leininger et al. (2006) Nature

Summary on the use of functional genes to study ocean processes

• Helpful for assessing diversity of organisms with specific biogeochemical or metabolic capabilities.

• Useful for understanding environmental controls on the activity of specific processes.

• Can be used to characterize physiology of specific organisms.