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