table 2 oligonucleotide primers and probes used in fish* s1pro634rhliprochlorococcus gcc gat cag ttt...

TABLE 1 Oligonucleotide primers and probes used in dot blots and DGGE analysis

PCR Primers Specific target DNA Sequence 5´-3´

OXY107F* Oxygenic phototroph biased GGA CGG GTG AGT AAC GC G TG R

OXY1313R* Oxygenic phototroph biased CTT CAC GTA GGC GAG TTG CAG C

CYA359F† Oxygenic phototroph specific GGG GAA TYT TCC GCA ATG GG

PRO1017R Prochlorococcus, excluding MIT9303, MIT9313,

MIT9302

TCC CGA AGG CAC CCT CWA AA

27F Eubacterial AGA GTT TGA TCM TGG CTC AG

1224R Eubacterial CAT TGT AGC ACG TGT GTA

Oligonucleotide Probes Specific target DNA Sequence 5´-3´

S1PRO634R HLI Prochlorococcus GCC GAT CAG TTT CCA CTG

S2PRO634R HLII Prochlorococcus GCC TTT CAG TTT CCA CTG

DPRO634R LL Prochlorococcus (except SS120) GCC AAT CAG TTT CCA CTG

SARG634R SS120 GCC CTT CAG TTT CCA CTG

MIT1023R MIT9303 TGC GTT CCC AAA GGC ACT

EUB338 Eubacteria GCT GCC TCC CGT AGG AGT

PRO444R ‡ Prochlorococcus , excluding MIT9303, MIT9313 TAT TCC TCA AGT ACC GTC ATA

MAR572R Marine picophytoplankton clade biased GCC GCC TGC GGA CGC TTT

.

† A GC-clamp consisting of a 40 nucleotide GC-rich sequence (5´-CGC CCG CCG CGC CCC GCG CCG GTC

CCG CCG CCC CCG CCC-3´) was attached to the 5´ end of the primer and used in DGGE analysis.

‡ Based on SAR6R .

TABLE 2 Oligonucleotide primers and probes used in FISH*



DPRO634R LL Prochlorococcus (PAC1/NATL1-MIT cluster) GCC AAT CAG TTT CCA CTG

.


645 HLI* HLI Prochlorococcus ACC ATA CTC AAG CCG ATC

645 HLII* HLII Prochlorococcus ACC ATA CTC AAG CCT TTC

645 LL* LL Prochlorococcus (PAC1/NATL1-MIT cluster) ACC ATA CTC AAG CCA ATC

181 HLI* HLI Prochlorococcus CAT TTC ACC TAT CGG CAT

181 LL* LL Prochlorococcus (not MIT9211 & includes some Syns) CAT TTC ACC TCT CGG CAT

148 LL-MIT* LL Prochlorococcus (MIT9211,MIT9303, MIT9313 only) CCG TTT CCA ACC GTT ATC

405PRO* Gen Prochlorococcus (General, but not MIT9303, MIT9313) AGA GGC CTT CGT CCC TCA

181LL

645HLI 181HLI

645LL1445NATL

645HLII

405PRO

405SYN

0.1

Prochlorococcus sp. MIT9107

Prochlorococcus sp. SB

Prochlorococcus sp. TAK9803-2





Prochlorococcus sp. GP2


Prochlorococcus sp. EQPAC1

Prochlorococcus sp. PCC9511

Prochlorococcus sp. CCMP1378

Prochlorococcus sp. NATL2A

Prochlorococcus sp. NATL2B

Prochlorococcus sp. PAC1

Prochlorococcus sp. NATL1MIT

Prochlorococcus marinus SS120




Synechococcus sp. WH8018




Synechococcus sp. WH8101 Synechococcus sp. PCC6307

Synechococcus sp. PCC6301

181LL

181LL

TABLE 1 Oligonucleotide primers and probes used in dot blots and DGGE analysis

PCR Primers Specific target DNA Sequence 5´-3´

OXY107F* Oxygenic phototroph biased GGA CGG GTG AGT AAC GC GTG R

OXY1313R* Oxygenic phototroph biased CTT CAC GTA GGC GAG TTG CAG C

CYA359F† Oxygenic phototroph specific GGG GAA TYT TCC GCA ATG GG

PRO1017R Prochlorococcus, excluding MIT9303, MIT9313,

MIT9302

TCC CGA AGG CAC CCT CWA AA

27F Eubacterial AGA GTT TGA TCM TGG CTC AG

1224R Eubacterial CAT TGT AGC ACG TGT GTA




DPRO634R LL Prochlorococcus (except SS120) GCC AAT CAG TTT CCA CTG

SARG634R SS120 GCC CTT CAG TTT CCA CTG

MIT1023R MIT9303 TGC GTT CCC AAA GGC ACT

EUB338 Eubacteria GCT GCC TCC CGT AGG AGT

PRO444R ‡ Prochlorococcus , excluding MIT9303, MIT9313 TAT TCC TCA AGT ACC GTC ATA

MAR572R Marine picophytoplankton clade biased GCC GCC TGC GGA CGC TTT

† A GC-clamp consisting of a 40 nucleotide GC-rich sequence (5´-CGC CCG CCG CGC CCC GCG CCG GTC

CCG CCG CCC CCG CCC-3´) was attached to the 5´ end of the primer and used in DGGE analysis.

‡ Based on SAR6R

TABLE 2 Oligonucleotide primers and probes used in FISH*



DPRO634R LL Prochlorococcus (PAC1/NATL1-MIT cluster) GCC AAT CAG TTT CCA CTG

.


645 HLI* HLI Prochlorococcus ACC ATA CTC AAG CCG ATC

645 HLII* HLII Prochlorococcus ACC ATA CTC AAG CCT TTC

645 LL* LL Prochlorococcus (PAC1/NATL1-MIT cluster) ACC ATA CTC AAG CCA ATC

181 HLI* HLI Prochlorococcus CAT TTC ACC TAT CGG CAT

181 LL* LL Prochlorococcus (not MIT9211 & includes some Syns) CAT TTC ACC TCT CGG CAT

148 LL-MIT* LL Prochlorococcus (MIT9211,MIT9303, MIT9313 only) CCG TTT CCA ACC GTT ATC

405PRO* Gen Prochlorococcus (General, but not MIT9303, MIT9313) AGA GGC CTT CGT CCC TCA

181LL

645HLI 181HLI

645LL1445NATL

645HLII

405PRO

405SYN

0.1


Prochlorococcus sp. SB

Prochlorococcus sp. TAK9803-2





Prochlorococcus sp. GP2


Prochlorococcus sp. EQPAC1

Prochlorococcus sp. PCC9511

Prochlorococcus sp. CCMP1378

Prochlorococcus sp. NATL2A

Prochlorococcus sp. NATL2B

Prochlorococcus sp. PAC1

Prochlorococcus sp. NATL1MIT

Prochlorococcus marinus SS120








Synechococcus sp. WH8101 Synechococcus sp. PCC6307

Synechococcus sp. PCC6301

181LL

181LL

• Eastern North Atlantic

– stratified HLI and LL genotypes correlated with mixed layer depth

in agreement with clone libraries, DGGE analysis and FISH data

• Western North Atlantic

– uniform distribution of HLII and LL genotypes in a mixed water column

• Mediterranean Sea

– stratified HLI and LL genotypes as observed in the eastern North Atlantic

• Red Sea

– stratified HLII and LL genotypes

Distribution of Prochlorococcus genotypes

HLII LL

HLILL HLI LL

HLIILL

LL

Location/Depth (m) Relative hybridisation (%)

HLI LL HLII SARG

AM2 15 m (20.4°S, 70.6°W)* 0.2 14.3 0.5 1.8AM1 55 m (20.4°S, 70.6°W)** 0.2 40.8 1.1 0. 6

* above the oxycline** below the oxycline

15m 1.7 5.4 0. 8 7.140m 2.1 40.1 1.4 1.350m 0.4 26.8 0.9 0.860m 0.4 65.2 1.7 0.7

40m - - - -50m 1.4 26.7 1.0 0.960m 3.4 126.9(?) 2.7 5.170m 0.6 50.8 1.3 0.9

Prochlorococcus dot blot data : Coastal Chile (20.4°S, 70.6°W) low oxygen environment

Sept chlorophyll field for Arabian Sea(SeaWiFS)

March chlorophyll field for Arabian Sea(SeaWiFS)

• oligotophy/eutrophy gradient of surface waters along the transect• oxygen-depleted (ca 2-10 µM O2) waters that occur at depths from ca 200 - 2500 metres throughout the basin. • divergent driven, gentle but continual upwelling of the Equator and adjacent downwelling waters

Indian Ocean - Arabian Sea - cruise Sept 2001

Marine Synechococcus strains: Roscoff

Strain Origin Isolator Location PUB:PEB

Minos 1

Minos 2

Minos 11

Minos 12

Max 42

Almo 3

Eum 14

Oli 31

Mediterranean Sea

Mediterranean Sea

Mediterranean Sea

Mediterranean Sea

Sargasso Sea

Alboran Sea

Tropical Atlantic

Equatorial Pacific

F. Partensky

F. Partensky

F. Partensky

F. Partensky

D. Vaulot

D. Vaulot

D. Vaulot

D. Vaulot

39°10’N 6 °10’E

39°10’N 6 °10’E

34°0’N 18 °10’E

34°0’N 18 °10’E

26°18’N 63°26’W

36°11’N 1°51’W

21°02’N 31°0’W

5°N 150° 0°W

PEB>PUB

PEB>PUB

PEB>PUB

PEB>PUB

PEB<PUB

PEB<PUB

PEB<PUB

PEB<PUB

Analysis of Synechococcus strains III : Red Sea

Strain Date of Depth of Colour PUB/PEB Motility N source ntcA RFLP 16S sequenceisolation isolation ratio type

RS9901 29.3.99 0 m Red 0.7 - NO3-/NH4

+ I +RS9902 29.3.99 0 m Orange 1.4 - NO3

-/NH4+ II +

RS9903 11.5.99 10 m Orange 2.0 - NO3-/NH4

+ III +RS9904 14.6.99 10 m Orange 1.5 - NO3

-/NH4+ III +

RS9905 18.7.99 10 m Orange/Red 1.0 - (+) NO3-/NH4

+ IV +RS9906 23.8.99 10 m Green - - NO3

-/NH4+ N/A +

RS9907 23.8.99 10 m Red 0.5 - NO3-/NH4

+ II +RS9908 7.9.99 10 m Red 0.5 - NO3

-/NH4+ II +

RS9909 7.9.99 10 m Green - - NO3-/NH4

+ N/A +RS9910 7.9.99 10 m Red 0.5 - NO3

-/NH4+ II +

RS9911 11.5.99 10 m Red 0.8 - NH4+ V +

RS9912 23.8.99 10 m Red 0.5 - NH4+ N/A +

RS9913 7.9.99 10 m Green - - NH4+ N/A +

RS9914 18.10.99 10 m Green - - NH4+ N/A +

RS9915 18.10.99 10 m Red 0.8 - NH4+ IV +

RS9916 22.11.99 10 m Red 0.7 - NH4+ I +

RS9917 22.11.99 10 m Green - - NH4+ N/A +

RS9918 22.11.99 10 m Green - - NH4+ N/A +

RS9919 22.11.99 50 m Orange NO3- V -

RS9920 22.11.99 150 m Orange NO3- VI -

Synechococcus genes being used for multi-locus sequence typing and community structure analysis

Gene Encoding

rpoC1 DNA-dependent polymerase

petB/D fragments b-type cytochrome & subunit of the photosynthetic

& intergenic region cytochrome b6/f complex, respectively

rnpB ribonucleoprotein enzyme (RNase P)

pstS periplasmic phosphate-binding protein

16S rDNA small subunit rRNA

ntcA global N transcriptional regulator

16S rRNA

petB-D

rpoC1 pstS

Distinguishing alleles

petBD intergenic region - sequence length

rpoC1 pstS 5% divergence

16S rDNA 1% divergence

12C14C

12C

14C

14C labelling of DNA : growth of Prochlorococcus NATL1-MIT on 14C NaHCO3

55,000 x g o/n40,000 x g o/n

Environmental DNA received from partners :

ROSCOFF BLANES HELGOLAND

Name DNA amplifiable

RA000404 x x

RA000609 faint

RA000709 faint x

RA001006


BL000921

BL001221


HE000803

HE001005

HE001206 x x

Full length clone libraries generated : pA (8-28) pHr (1542-1522)

Site Primers Identifier

HE000803 pA/pHr He(H) 25/02/01

Mio (Prosope cruise) pA/pHr Mio(M) 25/02/01

Dyf (Prosope cruise) pA/pHr Dyf (D) 25/02/01

Prosope cruise track

Sample Name DYF MIO Helgoland

Number of clones 70 70 75

% Containing Insert 95.7 98.6 98.6

% Amplified with Primer couple 359F/1313R 72.9 72.9 61.3 (oxygenic-phototroph specific)

% Amplified with Primer couple 359F/1038R 14.3 22.9 9.3(1038r amplifies all marine Syns& Pros, ACE/PS680 cluster, PCC9005 = marine cyano-specific)

Analysis of full length environmental clone libraries

Sequence analysis of Cyanobacterial/plastid sequences from full length clone libraries

Sequence Closest BLAST match identity

DYF13 Prochlorococcus ENATL4, PAC1 99%DYF33 Prochlorococcus ENATL4, PAC1 99%HE24 env.SAR7; SynechococcusMIO12 Prochlorococcus MIT9211 99%MIO31 Prochlorococcus ENATL4, PAC1 99% MIO43 Prochlorococcus ENATL4, PAC1 99%MIO60 Prochlorococcus MIT9211; MIT9313 99%

DYF18 env. Plastid seqs. OCS20; OM283 (cryptomonad) 99%DYF25 env. Plastid seqs. OCS50, OM153; (Haptophyte) 97%DYF38 Skeletonema pseudocostatum, uncult. Bacillariophyte 96%

env. Plastid seq. DiatomDYF45 Chrysochromulina polyepsis, env. Plastid OCS31; 98%

HE3HE3 Chrysochromulina polyepsis, env. Plastid OCS31; 98%

DYF45HE12 env. Seq. OCS50; E. huxleyi plastid 100%HE17 env. Plastid seq. OM81; Ochromonas sp. plastid 93%MIO26 env. Plastid OCS31, OM21; 98%

Analysis of 107F-1038R environmental clone libraries

Site Total no. of clones No. of unique RFLP types No. of unique clones Coverage (%) (HaeIII + EcoRI)

HE000803 67 8 5 92.5

HE001005 100 34 24 76.0

RA000609 80 20 11 86.25

RA001006 72 13 7 90.3

BL000921* 82 13 8 90.2

BL001221 82 16 9 89.0

* of 9 clones partially sequenced 8 are Syns or Pros 1 has closest homology to a magnetitie containing magnetic vibrio but the identity is low (85.2%)

table 2 oligonucleotide primers and probes used in fish* s1pro634rhliprochlorococcus gcc gat cag ttt...

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