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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
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Comparison of Synechococcus and Prochlorococcus photosynthetic pigments and cell
size
Characteristic Prochlorococcus Synechococcus
Primary photosyntheticpigment
divinyl chl-a, divinylchl-b
chl-a
Phycobilisomes no yesAccessory pigments phycoerythrin (+/-)
chl-c-like pigment -carotenezeaxanthin
phycoerythrinphycourobilin/phycoerythrobilin
-carotenezeaxanthin
Cell diameter (µm) ~ 0.6 ~ 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)
Prochlorococcus specific primers
TOTAL DNA
PCR of 16S rRNA
Oxygenic phototroph-biased primers
Clone libraries Dot-blot hybridisationDGGE
Sequences RFLP Quantification of genotypes
SINGLE CELLS
Fluorescent In-Situ Hybridisation (FISH)
Prochlorococcus genotype-specific
probes
GENETIC DIVERSITY P & N STATUS
TOTAL PROTEINS
SDS PAGE
Western-blotting
Interrogation with PstS/Amt antibodies
Physiological status with respect to P & N for:
Sequencediversity
CELLS POPULATIONS
Single-cellImmunofluorescence
nutrients
light
thermocline
upwelling
euphotic zone
nutrients
light
thermocline
upwelling
euphotic zone
Dot-blot hybridisation with Prochlorococcusgenotype-specific oligonucleotides
10
30
40
50
60
70
90
110
Sur
face
1
Sur
face
2
Dee
p
Mit9
303
Sar
g
Eub
338
E. coli
Med
Natl1
Tatl2
Mit9303
Sarg
WH8103
Dee
p
Sur
face
1
Sur
face
2
Mit9
303
Sar
g
Eub
338
Depth m
Control DNA
Depth profile 237°N
Geographical and vertical distribution of Prochlorococcus
5 m
20 m
40 m
60 m
80 m
90 m
100 m
120 m
150 m
300 m
HLI HLII LL SS120 EUB338
10 m
30 m
40 m
50 m
60 m
70 m
90 m
110 m
HLI HLII LL SS120 EUB338
Eastern North Atlantic Sargasso Sea
Depth profile 137°N
Med
Nat
l2A
Sarg
Tatl2
10 20 30 40 50 60 70 mM+P
+S+
T2
Denaturing Gradient Gel Electrophoresis (DGGE)
36% constant denaturant
Thermocline
0
5
10
15
20
25
0 10 20 30 40 50 60 70 80 90
Depth m
Tem
pera
ture
°C
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
Chl
orop
hyll
mg/
m 3
Med
Nat
l2A
Sarg
Tatl2
Tatl1
Nat
l1
10 30 40 50 60 70 90 110 m
Depth profile 237°N
36% constant denaturant
Thermocline
0
5
10
15
20
25
0 10 20 30 40 50 60 70 80 90 100 110
Depth m
Tem
pera
ture
°C
0
0.05
0.1
0.15
0.2
0.25
0.3
Chl
orop
hyll
mg/
m3
Flow cytometry data at 37°N, 20°W
10 30 40 50 60 70 90 1100
50000
100000
150000
200000
250000
Cel
ls m
l-1
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 CYA6643 22 <1 2340 20 <1 2380 0 13 14
• Red Sea– positive signals with HLII
MED4 SS120
chlb2/a2 ratio low (0.05 -0.15) high (0.4 -2.4)
optimal growth irradiance 15-80 mol photons m-2 s-1 8-30 mol photons m-2 s-1**
major antenna apoproteins ~ 32.5 kDa 34-28 kDa
copies of pcb gene single multiple (7)
phycoerythrin absent present
P inducible protein present absent
growth on nitrate no yes(?)
* 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%
Comparison of physiological properties of Prochlorococcusstrains MED4 (HLI) and SS120 (LL)
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 Willi SchönhuberRudi Amann, Rosi Rippka
N.Atlantic samples Mike ZubkovRed Sea samples Anton Post, Nick FullerSargasso samples James Ammerman
Royal Society