Towards ‘Environomics’Towards ‘Environomics’Uptake and Molecular Studies of Nitrate Assimilation

by Marine Heterotrophic Bacteria

Marc E. FrischerSkidaway Institute of Oceanography

Preparation

1985 A.B. Washington University in St. Louis (Microbial Genetics)

1985 - 1988 Protein Chemist - Sigma Chemical Co.

1988 - 1993 Ph .D. University of South Florida (Marine Science/Microbial Ecology)

1994 - 1996 Postdoc Rensselaer Polytechnic Institute (Molecular Microbial Ecology)

1996 - Present Skidaway Inst. Of Oceanography

Research InterestsResearch Interests

Exploration of microbial diversity and theExploration of microbial diversity and the

elucidation of linkages between the diversityelucidation of linkages between the diversity

of microorganisms, the activity of microbialof microorganisms, the activity of microbial

populations, and the role that microbial diversitypopulations, and the role that microbial diversity

plays in maintaining the stability and functioningplays in maintaining the stability and functioning

of marine/aquatic ecosystems.of marine/aquatic ecosystems.

Development and Use of Novel Molecular Techniques toMeasure Microbial Diversity and Link These Parametersto the Functional Role of Microbes in Aquatic Systems.

Aquatic Molecular Microbial Ecology

Application of Molecular Techniques for theStudy of Eukaryotic Pathogens and PlanktonicBivalve Larvae

Applied Aquatic Molecular Planktonic Studies

Linking N & C CyclesLinking N & C CyclesRole of Molecular Approaches in BiogeochemicalRole of Molecular Approaches in Biogeochemical

StudiesStudies

Case Study: Nitrate AssimilationCase Study: Nitrate Assimilation by Heterotrophic Bacteriaby Heterotrophic Bacteria

Because most marine environments arenitrogen limited, the nitrogen and carbon cycles

are intimately linked

In particular, the pathway of nitrate assimilationinto autotrophic or heterotrophic organismscan have a profound influence on carbon cycling

N2

NH4+

Organic N

NO3-

Nitrification

Assimilation

Nitrogen FixationDenitrification

Decomposition

NitrateAssimilation

CO2CO2

Primary Questions

Do Heterotrophic Marine Bacteria Assimilate Nitrate?If so, How Much? What are the Controls? Are they

Competitive with Phytoplankton

Can Molecular Tools (Gene Based) Be Used to DetermineWho are the Nitrate Assimilators and What Controls Them?

Are Gene Presence and Expression of nasA Quantitatively

Linked to Nitrate Uptake Rates? CO2 Flux?

Are bacteria or the bacterial size class (<0.2µ) taking up a significant

amount of NO3- in marine

environments?

Whole Sea Water> 0.8 µm filtered Sea Water< 0.8 µm filtered Sea Water

15N NO3

15N NH4

Filter onto0.2 µm SilverFilters

MassSpectrometry

Incubate1–3 hours

CalculateNO3 & NH4

Uptake RatesBy Size Class

Estimation of Total and Bacterial N Uptake RatesEstimation of Total and Bacterial N Uptake Rates

Debbie Bronk - VIMS

ICE

Open Water

Barents SeaBarents Sea

Station

I II III IV V% U

pta

ke o

f N

H 4 a

nd

NO

3 b

y <

0.8

0

10

20

30

40

50

NO3

NH4

Bacterial Uptake of DIN, Barents Sea June/July 1999

NCC NorthAtlantic

PolarFront

DriftIce

PackIce

%NH4+ Uptake <0.8

0 10 20 30 40 50 60

%N

O3- U

pta

ke

<0

.8

0

10

20

30

40

50

60

1:1 line

Barents Sea Study

Florida

Georgia

South CarolinaSkIO

% U

ptak

e of

NH

4 an

d N

O3

by <

0.8

0

10

20

30

40

50

NO3

NH4

Estuary Inner-Shelf Mid-Shelf

South Atlantic Bight , April, 2000

Bacteria Appear to Account for SignificantBacteria Appear to Account for Significant

NONO33 Uptake and Utilization Uptake and Utilization

Up to 40% of Total NO3 Utilization May Be Due To

Bacteria Under Some Circumstances, but 10-15% is

Probably a More Reasonable Estimate

However, Experimental Methods are Flawed, ManipulativeHowever, Experimental Methods are Flawed, Manipulativeand Laborious … Can Molecular Approaches be Useful?and Laborious … Can Molecular Approaches be Useful?

Molecular Level Studies Cannot ProvideRate and Flux Estimates, but Can Provide

Information Regarding Genetic capability

Identification

Study Regulation: Transcription into mRNA

Study Regulation: Translation into protein, and

post-translational modification

Protein characterization

Signal Transduction

narB

narB

nasA

Growth

+

Growth

-

Growth on NO3-

As Sole N SourcePCR

+

16

16 0

16

Presence of Presence of nasnasA = Ability to Assimilate NOA = Ability to Assimilate NO33

(32 Isolates from the Barents Sea)

0.1 substitutions/site

Methanobacterium sp. (Formate Dehydrogenase

100

96

72

Marinobacter

Marinomonas

Unknown

Alpha

Vibrio

Alteromonas

Barents Sea Clones100

Cyanobacteria100

Beta 100

PsychrobacterUnknown SAB Clones

100

Unknown SAB Clones100

Unknown Barents Sea Clones100

(45 clones, 2 isolates)

(10 clones, 1 isolate)

(12 clones)

(13 clones, 3 strains)

(14 clones, 3 isolates)

(11 clones, 4 isolates)

(43 clones)

(3 clones)(1 clones, 1 isolate)

(6 clones)(2 clones)

(2 strains)

(6 strains)

159 Clones

10 Clone Libraries

Strain Doubling Time(hours)

Yield(log Increase)

BS-25 5.16 3.13

BS-10 5.48 3.31

BS-4 3.78 3.60

BS-23 No Growth 0.59*

BS-26 No Growth 0.47*

All growth determination in NFG media (Tibbles and Rawlings, 1994)supplemented with 10 mM nitrate (KNO3)

Are Genetic Differences Functionally Meaningful?Are Genetic Differences Functionally Meaningful?

Growth Characteristics

nasA expression regulation in Klebsiella oxytoca

4.903

0.119

1.1806 1.2304

1.878

0

1

2

3

4

5

6

Tp T0 T30 T60Time

O.D

./n

gTota

lRN

A u

sed

in

1st

Rn

d

K. oxytoca NO3- to NH4

+

K. oxytoca NH4+ to NO3

-

15NO3- uptake into Klebsiella oxytoca

0

5000

10000

15000

20000

25000

30000

35000

Tp T0 T60Time

[NO

3- ](

µg

atN

/L/h

r)

Are Genetic DifferencesAre Genetic DifferencesFunctionally Meaningful?Functionally Meaningful?

Gene Regulation

K. oxytoca nasA strictlyregulated

by NO3- and NH4

+

15NO3- uptake in Vibrio diazotrophicus

0.00

500.00

1000.00

1500.00

2000.00

Tp T0 T60

[NO

3-

](µ

gatN

/L/h

r)

Time

nasA expression regulation in Vibrio diazotrophicus

51.36

0 0 01.015 1.043 1.134 1.082

0

10

20

30

40

50

60

Tp T0 T30 T60Time

OD

/ng

Tota

l R

NA

in

1st

Rn

d

V. diazotrophicus NO3- to NH4

+

V. diazotrophicus NH4+

to NO3-

V. diazotrophicus:nasA expression inhibitedBy NH4

+, but not stimulatedBy NO3

-

However, NO3- uptake occurs

In presence of NO3-

(long lived transcripts?)

Are Genetic DifferencesAre Genetic DifferencesFunctionally Meaningful?Functionally Meaningful?

Gene Regulation

P. citrea NO3- to NH4+P. citrea NH4+ to NO3-

nasA expression regulation in Pseudoalteromonas citrea

9.7

7.312

8.509 8.697

6.612

1.793

1.312 1.28561.6198

1.9226

0

2

4

6

8

10

12

Tp T0 T15 T30 T60

Time

OD

/ng

Tota

l R

NA

in

1st

Rou

nd

P. citrea NO3- to NH4

+

P. citrea NH4+ to NO3

-

Pseudoaltermonas citrea:Inhibited by NH4, but notStimulated by NO3

15NO3- uptake in Pseudoalteromonas citrea

0

500

1000

1500

2000

2500

3000

Tp T0 T60

Time

[NO

3-]

(µg

atN

/L/h

r)

P. citrea NO3- to NH4

+

P. citrea NH4+ to NO3

-

Are Genetic DifferencesAre Genetic DifferencesFunctionally Meaningful?Functionally Meaningful?

Gene Regulation

• The nasA Gene is Regulated Differently in Different Bacteria

• Growth Rates of Bacteria with Genetically Distinct nasAGene Sequences Differ

Does Genetic Identity Matter?Does Genetic Identity Matter?

Presumably These are Important Contributing Factors toPresumably These are Important Contributing Factors toThe Ecology & Biogeochemistry of Nitrate AssimilationThe Ecology & Biogeochemistry of Nitrate AssimilationBy Heterotrophic Bacteria in NatureBy Heterotrophic Bacteria in Nature

Molecular Field EcologyMolecular Field Ecology

Community Finger Printing – (TRFLP & RT-TRFLP)

Quantification – Q-PCR & QRT-PCR

Is community composition of nasA containing bacteriacorrelated with nitrate parameters(NO3 concentration & NO3 uptake rates) and otherbiological/chemical parameters?

Is nasA expression correlated with nitrate and other parameters?

Open Water (Station IV)Open Water (Station IV)

Ice (Station I)Ice (Station I)

Back to the Barents SeaBack to the Barents Sea

Barents Sea T-RFLP Patterns

Cluster Analysis

Principal Components Analysis

65

80

79

62

57 Ice

OpenWater

Ice

Open Water

PLS Model – Barents Sea July 1999DNA TRFLP Fingerprints

-0.6 -0.4 -0.2 0.0 0.2 0.4 0.6

-1.0

-0.5

0.0

0.5

1.0

NO3-

NO3-

% Active Cells

NH4+

Bac Abundance

Bac Prod

Chl a

LV1, x & y: 56% & 11%

LV

1, x

& y

: 14

% &

10%

Are nasA-encoding communities and

nasA expressing communities the same?

related? What factors are the diversity

of each related to?

Sequences derivedfrom transcripts

cluster together and distinctly from sequences

derived from total communityDNA

Expressed Sequences from Clone Library

Expressed Sequences Detected byRT-TRFLP

DK14 (14 sequences)GSD10 (12 sequences)

DK18TWS29

GSD10 (3 sequences)GSS39TWS2100015GSD16 (7 sequences)

DK100013DK3.5 DK2

GSS26 (2 sequences)TWS210007 (2 sequences)

DK10003TWS210008

TWS225TWS210005

GSS4 (10 sequences)TWS1 (19 sequences)DK4 (4 sequences)TWS31GSD11 (sequences)

GSD19GSD9GSS33

TWS2100010DK10006

TWS210003 (10 sequences)DK31 (5 sequences)

DK100014DK100012 TWS21006GSD7

TWS216TWS230DK10007DK3h22

DK4h19DK29

DK3.11DK3.1 GSD23

GSD26GSS1GSD27GSS32

TWS2100014TWS220

GSS12GSS13GSS24

GSS27DK9 (5 sequences)GSD34

DK3.3GSS34

DK100011DK10009 (2 sequences)

DK3.10DK23

DK10005DK35

Sargasso DNA- and RNA-derived nasA(RT) TRFLP Studies

800d40d(DCM)

450d

85.9d(DCM)

40r(DCM)

800d

82.9d(DCM)

35d(DCM)

100d

5d

82.9d(DCM)

450r

82.9r(DCM)18r

6-100r

800r

-4

-2

0

2

4

6

8

-6 -4 -2 0 2 4

PC1

PC

2

Can we relate nasA expression measured with our PCR-based

methods to 15N uptake or nutrient concentrations?

2 3 4 5 6 712

14

16

18

20

22

24

26

28

30

Cyc

le T

hres

hold

(C

t)

Log10 Copy Number

Y = -3.416 (log10X) + 36.25r2 = 0.989

Quantification of nasA Transcripts(Skidaway River Estuary – 2001)

Standard

Unknown

Real Time Q-PCRReal Time Q-PCR

0

5

10

15

20

0.000

0.001

0.002

0.003

0.004

0.005

15N Bacterial NO 3- Uptake (<0.8 um size-fraction)

SYBR Green Real-Time PCR

August '00

Ocotber '00

Janurary '01

March '01

May '01

April '01

July '01

June '01

Skidaway River Estuary

NO

3 U

pta

ke (

nm

ole

-N l

-1 d

-1)

(< 0

.8 µ

m)

Mar

ino

bac

ter

sp.

nas

A/1

6S

rR

NA

Gen

e C

op

ies

Marinobacter sp. nasA / 16S rRNA Genes

0.000 0.001 0.002 0.003 0.004 0.005

NO

3- U

ptak

e (n

mol

-N l- d

-1)

(<0.

8 u

m s

ize

fra

ctio

n)

0

2

4

6

8

10

12

14

16

18

20

r2 = 0.77

Skidaway River Estuary

nasA Gene Expression Sometimes Correlates withNO3 Concentration

Barents Sea – Ice StationsSouth Atlantic Bight

Probably Dependent on Many Factors, Available Carbon,Community Composition, etc.

Detection of mRNA transcripts may be transient

Other times with NO3 uptake RatesSkidaway River EstuaryBarents Sea – Open Water StationsSargasso Sea (sometimes)

Sometimes Not With Either ???

Primary Questions & Conclusions

Do Heterotrophic Marine Bacteria Assimilate Nitrate?

Can Molecular Tools (Gene Based) Be Used to DetermineWho are the Nitrate Assimilators and What Controls Them?

Are Gene Presence and Expression of nasA QuantitativelyLinked to Nitrate Uptake Rates? CO2 Flux?

YES – Varies in Space and Time But Can Account for a Significant Fraction of DIN Uptake

Yes – Molecular Tools Provide Unique Insightsand indicate that Genetic Identity Matters andContributes to System Complexity

Sometimes, Incorporation into GCM Models WillBe Interesting!

But, Unsurprisingly, More QuestionsThan Answers … Complex Systems

100’s - 1,000’s of genes per organism involved

Multiple Regulation Pathways per Organism

1,000’s of organisms involved

Lots of Signals

So Where Do We Start???

Identification of and Focus on Simple But Relevant Systemsand Primary Processes (e.g. Nitrogen Cycle)

Focus on Key Functional Genes and Pathways(not just single genes)

Simultaneous Analysis of Suites of Genes

Combine Chemical, Nucleic Acid, and Protein Analyses

HIGH THROUGPUT!!!!

Gene Function

rbcl Primary Production

PEPcase Primary Production

GDC Photooxidation

nir, nos, nor Denitrification

amoA Nitrification

nifH Nitrogen Fixation

dsrA Sulfate reduction

nar, nasA Nitrate Assimilation

pmo Methane Oxidation

mcr Methanogenesis

Microarray Development In ProgressMicroarray Development In ProgressJizhong Zhou (Joe) – Oakridge National LaboratoryJizhong Zhou (Joe) – Oakridge National Laboratory

BlackBlackBoxBox

Chemical Stimuli

Chemical Stimuli

Chemical Stimuli

BiogeochemicalRates

Environomics ???Environomics ???

Combined Molecular & Chemical Approaches AreComplementary and Appear to be Leading to aMore Complete Mechanistic Understanding of Bacterial

Behavior … ENVIRONOMICS

Chemical Stimuli

Gene Response

Gene Expression

Chemical Stimuli

Chemical Stimuli

Proteins

BiogeochemicalRates

AcknowledgementsAcknowledgements

Department of EnergyDepartment of Energy

National Science FoundationNational Science Foundation

Office of Naval ResearchOffice of Naval Research

Andy Allen (Princeton Univ)Peter Verity (SkIO)Peter Verity (SkIO)

Debbie Bronk (VIMS)Debbie Bronk (VIMS)

Jon Zehr (UC Santa Cruz)Jon Zehr (UC Santa Cruz)

Melissa Booth (SkIO/Roanoke)Melissa Booth (SkIO/Roanoke)

Hendi Hendrickson

Christina Archer

Marta Sanderson

Corina Knapp

Sandra Walters