Regulation of Cellular Nitrogen Metabolism Carbon and Nitrogen Metabolism in Model Marine Diatoms

Andrew E. Allen (JCVI, Scripps Oceanography, San Diego)

Upwelling Boundary Currents: ~25-50% of Total Marine ProductivityDiatom dominated

J. Schmaltz, NASA (from Falkowski and Oliver (2007), Nature Reviews, Micro.)

Images, A. Allen (Puget Sound Phytoplankton Metatranscriptomics Project) (collaboration with Ginger Armbrust, Micaela Parker, Ahmed Moustafa)

Diatoms are thought to be the source of many modern day oil deposits

glycolate 2-P

glycolate

1,3 bisphosphate

glycerate

glyceraldehy

de

3-Pdihydroacetone

phosphateseduloheptulose

7-P

ribose 5-P

ribulose 5-P

ribulose

1,5 bisphosphate

Xylulose 5-P

Plastid

HCO3 + glutamine

carbamoyl-P

carbamolyl aspartate

dihydrorotate

orotate

UMP

UDP

UTP

CTP

UMP

uridine

uridineuracil

Triose/hexose phosphate/

Phosphate

Glucose 6-P/

Phosphate

acyl carnitinecarnitine

acyl

carnitinecarnitine

L-3-hydroxyacyl CoA

Mitochondrion

glycineserine

glyoxylate

hydroxypyruvate

glycerate

cysteine

alanineS

thiamine

2-ketobutyrate

2-

acetohydroxy

butyrate2,3 hydroxy

3-

methylvalerate2-keto 3-

methyl

valerate

fructose

1, 6 bisphosphatefructose

6-P

erythrose 4-P

hypoxanthine guanine

inosine quanosine

IMP

GMP

adenine

adenosine

AMP

PUFAfatty acid

-acyl CoA

trans enoyl CoA

beta hydroxyacyl CoA

fatty acid acyl CoA

acetyl CoAbeta ketoacyl CoA

OAA

citrate

succinate

glyoxylateacetyl CoA

H2O2

Peroxisome

pyruvateOAA

malate

PEP

2-phosphoglycerate

3-phosphoglycerate

1,3 DPG

glyceraldehyde 3-P

fructose 1,6 diphosphate

DHAPglycerol 3-P

glycerol

fructose 6-P

glucosamine 6-P

glucose 6-P

ribose 5-P + xylulose 5-P

ribulose 5-P

6-phosphogluconate

triose

phosphates

NO3Silicate

lactate

cysteine

cystathione

L-homocysteine

methionine

IMP

XMPInome

xanthosine

AMP

xanthine

uric acid

hypoxathine

urea

urea CO2 + NH4

S-adenosyl-L-

methionine

putrescine

spermidine

S-adenosyl

methioninamine

spermidine

S-adenosyl

methioninamine

glucose

glutamate

acetate

N-acetylglucosamine

1-P

N-acetylglucosamine

6-P

UDP-N-acetylglucosamine

glutamine

NH4

4-α-methylcholestra-

8-en-3-β-ol

anthranilate

erythrose 4-P

DAHP

3-dehydro

shikimate

shikimate

shikimate 3-P

EPSP

chorismate

prephenate

phenyl pyruvate

arogenate

malonyl CoA

acetyl CoA

malonyl ACP

3-

ketobutyryl

ACPbutyryl ACP

3-ketoacyl

ACP

continued cycles

16:0 ACP

18:0 ACP

O-acetyl serine

sulfate

APS

PAPS

sulfite

H2S

Serine

NO2

NO2 NH3

glutamate

glutamate 1-semialdehyde

glutamyl tRNA

5-aminolevulinate

porphobillinogen

preuroporphyrinogen

uroporphyrinogen III

coproporphyrinogen III

protoporphyrinogen IX

protoporphyrin IX

Mg protophorphyrin IX

Mg protoporphyrin

monomethyl ester

monovinyl

protochlorophyllide

chlorophyllide a

pyruvate

2-

acetolactate2,3

dihydroxy

isovalerate2-

ketoisovalerate

3-

isopropylmalate

glutathione ox glutathione red pyruvate

+

glyceraldehyde 3-P

deoxyxylulose 5-P

MEP

4-cytodine-5-diphospho

2-C-methyl-D-erythritol

IPP DMAPP

GGPP

phytoene

lycopene

beta carotene

zeaxanthin

antheraxanthine

violaxanthin

diatoxanthin

diadaxanthin

Acyl CoA

trans enoyl CoA

3-keto acyl CoA

acyl CoA

acetyl COA

acetoacetyl COA

HMG COA

mevalonate

MVPP

IPP

DMAPP

farnesyl

diphosphateprotein

prenylation

presqualene

diphosphate

(S)-squalene-

2,3-epoxide

lanosterol

12-demethyl-

lanosterol

zymosterol

cholesta-7,24-dien-3-β-ol

cycloartenollathosterol

7-dehydro-cholesterol

cholesterol

Cholesterol

-5α6β-epoxide

ergosterol phytosterol

PRPP

PRA

GAR

FGAR

FGAM

AIR

CAIR

SAICAR

AICAR

CAICAR

FAICAR

IMP

SAMP XMP

GMPAMP

Cytosol

3-carboxy-3-

hydroxy-

isocaproate

2-oxo-

isocaproate

pyruvate

OAAcitrate

isocitrate

succinyl CoA

succinate

fumarate

malate

acetyl CoACO2

TCA cycle

apha keto

glutarate

H

H

H

H

H

H

Calvin cycle

Glycolat

e

cyclemalate

H

H

R

Polyamines

Fatty acid

oxidation

Phosphat

e

sugars

Photo

respiratio

n

H

Chitin

NO3

H

H

Triglycerides

H

Creatine-P

H

H

H

Purine

biosynthesi

s

Pyrimidine

biosynthesi

s

Fatty acid

oxidation

H

Isoprenoid

biosynthesi

s

Glycolysis &

Gluconeogene

sis

H

3-phosphoglycerate

Chlorophyll a

Heme

Isoprenoid

biosynthesis

Fatty acid biosynthesis

H

Tyrosine

Phenylalanine Cysteine

Isoleucine

Valine

Leucine

Fucoxanthin

glutamate glutamine

H

H

H

Purine SalvagePyrimidine Salvage

A Functional Urea Cycle !

creatine

guanidino

acetate

citruline

carbamoyl P

CO2 + NH3

NO

ornithine

Urea

cycle

ornithine citruline

ure

a arginine argino

succinate

Armbrust et al. Science (2004)

Metabolic Impact of Mitochondrial CPS Knockdown (RNAi)

Allen et al. (2011) Nature

% 15N Enrichment During Growth on Nitrate or Urea in Wild Type Cells

Dupont et al. in prep

Synthesis pathways in chloroplast

Metabolic Consequences of TALEN-Based Urea Cycle Manipulation in Diatoms

Graham Peers, Colorado State University

Weyman et al. Plant Biotechnology Journal (In Press)

Nitrate Reductase Homologous Recombination Vectors for Gene Inactivation

NR Talen vector recognizes NR target sequence; endonuclease fused to recognition domain make double-stranded cut at target site.

NR homologous recombination vector provides homologous NR sequences for Pt’s DNA repair mechanism. The insertion of the Sh blesequence to the NR gene during recombination inactivates NR gene.

P. tricornutum NR Knock Out Results after Biolistic Transformation

WT cultures were grown simultaneously as a

control. Plates were photographed 10 day

after inoculation: 5 ul NR-KO cells into 2 ml

F/2 media; auto-fluorescence was recorded

daily as a proxy for growth for two weeks.

Experiments were successfully repeated over several months.

NR-KO #9 NR-KO #14NH4 vs NO3 growth assay

NH4 NO3 NH4 NO3

blank #9 WT blank #9 WT blank #14 WT blank #14 WT

1 2 3 4 5 6 7 8

1. Ladder

2. WT-1 NH4 day zero

3. WT-2 NH4 day zero

4. KO-9 NH4 day zero

5. KO-14 NH4 day zero

6. WT-1 NO3 day 3

7. KO-9 NO3 day 3

8. KO-14 NO3 day 3NR

Western blot of WT vs KO-9, KO14

Confocal Images of NR KO-1 Cells Grown on NH4 vs NO3

Amended F/2 Media

[880 uM] ammonium [880 uM] nitrate

Day 0 Day 1

Day 2 Day 7

FTIR Scans of NR-KO #1 vs WT on F/2 NO3

lipid protein carbohydrate lipid protein carbohydrate

Peaks for lipids (~1740 AU), proteins (~1600 range), carbohydrates (~1050 AU) are in the 550 – 2000 Abs units region of the scan

Scaled-Up View of Lipid Peaks –

FTIR scans of NR-KO #1 & #9 vs WT on F/2 (NO3)

Bacterial Conjugation

P. tricornutum

E. coli

Conjugative Plasmid

Bacteria

Yeast Mammalian Cell

?

F pilus

Conjugative Plasmid

Conjugative Plasmid

Conjugative Plasmid

Conjugative Plasmid

Karas et al. in prep

Conjugation vs. Particle Bombardment Transformation Efficiency

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

Conjugation Particle Bombardment

Ave

rage

nu

mb

er

of

tran

sfo

rman

tsp

er

mill

ion

re

cip

ien

t ce

lls

Method of transformation

Rescued cells growing in F/2 media show that

both the N’YFP and NR proteins are active

A conjugation plasmid, containing the above N’YFP-NR cassette, driven by the

NR promoter, was transformed into a conjugating strain of E. coli. Pt WT and KO

lines were incubated with the E.coli and plated on selective media.

NH

4

NO3

Potential KO-complemented conjugates

formed colonies large enough to transfer

to liquid media after 10 days. C1 -6 were

transferred to 24 well plates with NH4 and

NO3 amended F/2 media. Strains growing

in both N-media (C2-5) indicate that the

the nitrate reductase gene on the plasmid

is being expressed.

NR knockout complementation by conjugation of P. tricornutum

with E. coli

251 gene cluster of predominantly chloroplast localized genes

Short Time Sacle Addition or Removal of Nitrogen is Major Transcriptional Trigger

Cluster of 428 genes, in addition to fatty acid functional genes, includes transcription factors, vacuolar, peroxisomal, ubiquitin-conjugating enzymes, kinases, and N-response regulators

p-value 1.0E-07p-value 5.0E-04

N-depleted time points15 min after N-addition

McCarthy et al. in prep

Regulation of Nitrogen Metabolism in the

Marine Diatom Phaeodactylum tricornutum

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

N-freewash

NO3spike Wash*

T15 T45 T-end T15 T45 T-end T15 T45 T-end T15 T45 T-end

RP

KM

s (

no

rma

lize

d m

RN

A r

ea

ds)

nitrate reductase

nitrite transporter

glutamine synthetase

N-

free

NH4+ NO3

- NO2-

Experimental design & key enzymes

McCarthy et al. in prep

Nitrate Reductase R and 34 genes that most closely co-vary in

expression; p=6.0E-04.

Transcripts (annotated) include: nitrate & nitrite transporter, nitrite reductase(s), glutamine synthetase, HIS acid phosphastase, protein kinases, voltage-gated chloride channel, vaculor amino acid transporter, silicon transporter, NO dioxygenase (globin domain protein), mitochondrial carrier protein, among others

Putative Nitrate Sensing Gene ptNIT(NO3 sensitive kinase, gene model manually extended in 3’ direction to find novel

Eukaryotic NIT domain)

NasR and NarX(Boudes et al., 2012; Chueng and Hendrickson, 2008)

Nitrate/Nitrite Sensing Kinase

In K-means cluster 1, NR, Pt54983, clusters with phosphorylated

peptides involved in nitrate assimilation:

CPS I, POT and voltage-gated Cl

channels, kinases, phosphatases,

transcription factors, etc.

Note: NR’s proteomic profile added to phosphorylated-peptide data for tracking purposes

In A. thaliana, Cl- channels (members of the proton-dependent oligopeptide transporter,

POT-family) are vacuolar pumps have been shown transport to NO3– across the

vacuolar membrane. We hypothesize that under certain conditions NR co-localizes with

the POT proteins to access nitrate stored in the vacuole.

Rapid response to

addition of NO3

A cluster of phosphorylated peptides that respond quickly to

addition of nitrate to growth medium.

Pt_47148, a homolog for A. thaliana

chloride channels, is the template used

to produce the cluster

N-responsive, phosphorylated transcription factors clustered by abundance per time point

Cohorts of transcription factor Pt44807 clustered by phosphoryl abundance

under replete NO3 conditions

Cluster includes a 2nd TF:

Pt47256, an ammonium

transporter and the putative

NO3, POT transporter,

Pt47148

NO3 T

end

NO3

T15

NO3

T45

N

free

NO3

spike

N-

free

T15

N-free

T45

N-free

T end

LREGS*VSESSDHVTVGGR

S*ASFAAPIR

SAS*FAAPIR

TTTPATVDPQGHQFS*PTTR

TTTPATVDPQGHQFS*PTTR

SSGVGGAAS*AAPEAIIMGRPK

AGS*VGQLSIR

ARAGS*VGQLSIR

DHVDELMEIRAS*K

RMT*TVDASR

SVS*LANVR

ESEYPS*QSNIDFSR

STS*LSMPGSHQEELR

KNPS*LDGLLR

S*ATVTEPQDR

RPS*EDDVASVLQK

TSPDESEPAFS*YEEQR

47256

44807

44807

292

292

1686

26925

26925

27877

37283

43878

45438

46684

47148

47283

48350

48985

Myb1R

_GAP_||TAZ zinc finger

_GAP_||TAZ zinc finger

Myosin head (motor domain)

Bromodomain

_GAP_||Protein kinase domain

_GAP_||Protein kinase domain

_GAP_||Ammonium Transporter Fam

_GAP_||ATP-grasp domain||_GAP_

_GAP_||Ion transport protein

_GAP_||Histidine acid phosphat

_GAP_||POT family||POT family

_GAP_||Adenylate cyclase

_GAP_||Miro-like protein||_GAP_|

NO3 T

end

NO3

T45

NO3

T15

N

free

NO3

spike

N-

free

T15

N-free

T45

N-free

T end

Changes in phosphorylated protein abundance in response to

N-sources: NH4 and NO2NH4 NO2

S1

7: clu

ste

r 1

S1

7: clu

ste

r 1

4

GREEN = Low Phosphorylation

RED = High Phosphorylation

Phosphorylated

in response to

lack of nitrogen

Phosphorylated in

response to

nitrogen

The appearance of

the ammonium

transporter in these

4 clusters suggests

it plays a complex

role in N-source

transport

Peptide profiles of the ammonium transporter Pt_27877

N free NO3

Diel clustering of functional categories within diel expression clusters: Consistent periodic modulation (P < 0.05, over all replicates)

A. Transcription Factors

4 σ-TF’s (total of 8) induced early morning Discrete peak at 1h light

B. Light Harvesting Complex proteins

Induced early morning Discrete peak at 1h light

Repressed by light (at 1h L), back upregulated in the afternoon

Repressed at dawninduced at dusk or at midnight

Kleeson et al, in prep

Testing compartment and non compartment models

Compartmentalized Metabolic Flux Reconstructions

Sabrina KleesonMax Plank, Potsdam

Alisdair Fernie and Zoran Nikoloski, Max Plank, PotsdamBernhard Palsson, UCSD

Ongoing and Future Experiments & Conclusions

In Vitro Transcription Factor Expression and DNA Porbing Targets for all TFs (DNA Aptamer Protein sequencing (DAP-seq)

Generate Biomass Objective Functions Required for Genome Scale Reconstruction (biochemical and energetic requirements for biomass)

Metabolic and Transcriptional Regulatory Network Reconstruction

Conclusions

Novel Eukaryotic recombination and repartitioning of bacterial metabolism; for example diatom mitochondria biochemistry

The diatom urea cycle represents a critical for anaplerotic carbon (CO2) fixation pathway into key nitrogenous compounds

Cellular compartmentalization is critical for cellular ecology and modeling of integrated carbon and nitrogen metabolism (we lack critical information related to intracellular transporter reactions)

Transcriptome rapidly responds to silicon and nitrogen starvation conditions in a way that “sets the stage” for lipid accumulation. Regulation of intracellular carbon flux appears to be under some degree of transcriptional control

Jonathan Badger (JCVI)John McCrow (JCVI)Flip McCarthy (JCVI)Jeroen Gillard (JCVI)Stephane Lefebvre (JCVI)Chris Dupont (JCVI)Philip Weyman (JCVI)Sabrina Kleeson (MPI, Golm)Bernhard Palsson (UCSD)Jennifer Levering (UCSD)Alessandra Gallina (UCSD)Karsten Zengler (UCSD)Graham Peers (Col. State University)Adam Kustka (Rutgers University)Chris Bowler (ENS)Uma Maheswari (EBI)

Marine Microbiology Initiative

Environmental Genomics, Biological Oceanography, Dimensions of Diversity