towards ‘environomics’ uptake and molecular studies of nitrate assimilation
DESCRIPTION
Towards ‘Environomics’ Uptake and Molecular Studies of Nitrate Assimilation by Marine Heterotrophic Bacteria. Marc E. Frischer Skidaway Institute of Oceanography. Preparation. 1985 A.B. Washington University in St. Louis. (Microbial Genetics). - PowerPoint PPT PresentationTRANSCRIPT
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