Transcript halifax talk

Comparison of Synechococcus and
Prochlorococcus photosynthetic pigments and cell
size
Characteristic
Prochlorococcus
Synechococcus
Primary photosynthetic
pigment
divinyl chl-a, divinyl
chl-b
chl-a
Phycobilisomes
no
yes
Accessory pigments
phycoerythrin (+/-)
chl-c-like pigment
 -carotene
zeaxanthin
Cell diameter (µm)
~ 0.6
phycoerythrin
phycourobilin/
phycoerythrobilin
zeaxanthin
 -carotene
~1
Prochlorococcus : a model system for studying
marine microbial ecology I
• Responsible for ~ 50% of total chlorophyll over a significant
fraction of the world’s oceans
• It inhabits a relatively simple, well mixed environment that covers
70% of the earth
• It is relatively easy to isolate into culture and has minimal growth
requirements
• It is widespread and abundant in the oceans, and is easily identified
and studied in situ using flow cytometry
Prochlorococcus : a model system for studying
marine microbial ecology II
• Its unique form of chlorophyll a allows measurement of its
proportional contribution to photosynthetic biomass
• Its cell division is highly synchronised, simplifying measurements
of in situ growth rates
• There is a rapidly growing molecular database for the genus, which
facilitates the development of probes to study the distribution of
different ecotypes in situ
• It has an extremely small genome size (1.8 -2.0 MBp)
TOTAL DNA
PCR of 16S rRNA
Prochlorococcus
specific primers
DGGE
TOTAL PROTEINS
SINGLE CELLS
Fluorescent In-Situ
SDS PAGE
Single-cell
Hybridisation (FISH) Immunofluorescence
Oxygenic phototroph-biased
primers
Clone libraries
Western-blotting
Dot-blot hybridisation
Interrogation with
PstS/Amt antibodies
Prochlorococcus
genotype-specific
probes
Sequence
diversity
Sequences
RFLP
Quantification of genotypes
Physiological status with
respect to P & N for:
CELLS
GENETIC DIVERSITY
POPULATIONS
P & N STATUS
light
thermocline
euphotic
zone
upwelling
nutrients
light
thermocline
euphotic
zone
upwelling
nutrients
Dot-blot hybridisation with Prochlorococcus
genotype-specific oligonucleotides
10
E. coli
30
Med
40
Natl1
50
Tatl2
60
Mit9303
70
Sarg
90
WH8103
110
Eub338
Sarg
Mit9303
Surface2
Surface1
Control DNA
Deep
Eub338
Sarg
Mit9303
Surface2
Surface1
Depth m
Deep
Depth profile 2
37°N
Geographical and vertical distribution of
Prochlorococcus
Eastern North Atlantic
HLI
HLII
LL
SS120
Sargasso Sea
EUB338
HLI
10 m
5m
30 m
20 m
40 m
40 m
50 m
60 m
60 m
80 m
70 m
90 m
90 m
100 m
110 m
120 m
150 m
300 m
HLII
LL
SS120
EUB338
M+P+
S+T2
Sarg
Tatl2
Natl2A
Med
Denaturing Gradient Gel Electrophoresis
(DGGE)
10 20 30 40 50 60 70 m
36% constant denaturant
Depth profile 1
37°N
Thermocline
0.35
0.3
20
0.25
15
0.2
10
0.15
0.1
5
0.05
0
0
0
10
20
30
40
50
Depth m
60
70
80
90
Chlorophyll mg/m 3
Temperature °C
25
Sarg
Tatl2
Tatl1
Natl1
Med
Natl2A
10 30 40 50 60 70 90 110 m
36% constant denaturant
Depth profile 2
37°N
Thermocline
0.3
0.25
20
0.2
15
0.15
10
0.1
5
0.05
0
0
0
10
20
30
40
50
60
Depth m
70
80
90
100
110
Chlorophyll mg/m3
Temperature °C
25
Flow cytometry data at 37°N, 20°W
250000
Cells ml
-1
200000
150000
100000
50000
0
10
30
40
50
60
70
90
110
Depth (m)
Total picoplankton
Synechococcus
Prochlorococcus
Picoeukaryotes
FISH analysis of natural Prochlorococcus
populations
• North Atlantic
– positive signals with HLI and LL
Depth (m)
Proportion of DAPI stained cells giving
a signal with each probe (%)
645HLI
181LL
CYA664
3
22
<1
23
40
20
<1
23
80
0
13
14
• Red Sea
– positive signals with HLII
Comparison of physiological properties of Prochlorococcus
strains MED4 (HLI) and SS120 (LL)
MED4
chlb2/a2 ratio
optimal growth irradiance
major antenna apoproteins
low (0.05 -0.15)
15-80 mol photons m-2 s-1
~ 32.5 kDa
SS120
high (0.4 -2.4)
8-30 mol photons m-2 s-1**
34-28 kDa
copies of pcb gene
single
multiple (7)
phycoerythrin
absent
present
P inducible protein
present
absent
no
yes(?)
growth on nitrate
* photoinhibited only around 450 mol photons m-2 s-1
** photinhibited at light intensities greater than 37 mol photons m-2 s-1
N.B. MED and SS120 genomes appear to be co-linear; 16S rDNA identity = 98.3%
Conclusions
• Distribution of Prochlorococcus genotypes is dependent on
hydrological conditions and oceanic region
• Molecular techniques e.g. DGGE, dot-blots, or FISH in
combination with TSA, allows the community structure of
natural populations to be rapidly evaluated
• Niche adaptation of specific strains (species?) potentially
involves a response to both gradients of light and nutrients
Future perspectives
• Determination of carbon fixation potential of distinct
Prochlorococcus genotypes in situ
• Correlation of genotype and phenotype with hydrological
properties and nutrients
• - optimisation of single-cell IF assay
• - analysis of FISH and single-cell IF assays
with flow cytometry
• Comparative genome analysis of HL and LL
strains : what are the specific adaptations of these strains
to their niche?
Acknowledgments
Nyree West
FISH
N.Atlantic samples
Red Sea samples
Sargasso samples
Willi Schönhuber
Rudi Amann, Rosi Rippka
Mike Zubkov
Anton Post, Nick Fuller
James Ammerman
Royal Society