Ecotypic diversity of marine cyanobacteria: Tales from the

pangenome

Gabrielle Rocap

Small (0.6 x 0.8 m) divinyl chlorophylls a and b

Larger (0.8 x 1.8 m) phycobilisomes

Prochlorococcus Synechococcus

ubiquitous throughout the tropical and sub tropical oceans

extremely abundant (up to 4x105 cells/ml)

contribute significantly to primary productivity

Unicellular marine cyanobacteria

Images from http://ccmp.bigelow.org

Durand et al., Deep Sea Research II 2001

Prochlorococcus and Synechococcus have differing seasonal abundances in the Sargasso Sea

Prochlorococcus isolates differ in their photophysiology

0

0.2

0.4

0.6

0.8

1

1 10 100 1000

Growth irradiance (mol Q m-2 s-1)

MIT9312

MIT9313

Gro

wth

ra

te (

da

y-1)

MIT9313MIT9312

0

1

2

Growth Irradiance (mol Q m-2 s-1)

Ch

l b/c

hl a

2

1 10 100 1000

From Moore, Rocap & Chisholm 1998

MED4

MIT9313

WH8102

ENATL2

MIT9201GP2

MIT9107

ENATL6

NATL2ENATL5

SAR6

ENATL1ENATL3

MIT9302MIT9312

MIT9202MIT9215

NATL2A

ENATL7ENATL4

SS120MIT9211

MIT9303

SAR139WH8112

SAR100WH8101

WH8012WH7805

SAR7

Cyanobium PCC 6307

0.01

89

97

92

72

71

10078

66

84

Marine Synechococcus

I

IPAC1

TATL1b

TATL1a

rRNA phylogeny corresponds to physiological differences —”ecotypes”

Adapted from Rocap et al 1999

II

IIIII

IV

Ahlgren et al 2005 Env. Micro.

Prochlorococcus ecotypes partition the water column

Johnson et al. Nature 2006

40°S 20°S 20°N 40°N0°

Prochlorococcus ecotypes have latitudinal gradients

MED4

MIT9313

WH8102

ENATL2

MIT9201GP2

MIT9107

ENATL6

NATL2ENATL5

SAR6

ENATL1ENATL3

MIT9302MIT9312

MIT9202MIT9215

NATL2A

ENATL7ENATL4

SS120MIT9211

MIT9303

SAR139WH8112

SAR100WH8101

WH8012WH7805

SAR7

Cyanobium PCC 6307

0.01

89

97

92

72

71

10078

66

84

Whole genome sequences reveal large

differences among strains

PAC1

TATL1b

TATL1a1.66 Mbp, 31 %G+C

1.75 Mbp, 36 %G+C

2.41 Mbp, 51 %G+C

2.43 Mbp, 60 %G+C

Protein Family domains in 3 Prochlorococcus genomes

SS120 (low light)

MED4 (high light)

2430

865

MIT9313 (low light)

119

11

3521

21 domains in MED4 and MIT9313 but not SS120 include:

Urease_alpha Urease alpha-subunit, N-terminal domainUrease_beta Urease beta subunitUrease_gamma Urease, gamma subunitUreD UreD urease accessory proteinUreE_C UreE urease accessory protein, C-terminal domainUreF UreF

ureC ureB ureA ureD ureE ureF ureG urtA urtB urtC urtD urtE

30 domains in MED4 but not SS120 or MIT9313 include:

Cyanate lyase Cyanate lyase-C-terminal domain

cynS

Cyanate ABC transporter

119 domains in MIT9313 but not SS120 or MED4 include:

Form_nir_trans Formate/Nitrite transporter

deletion

deletion

Prochlorococcus MED4

Synechococcus WH8102narB nirA

Prochlorococcus MIT9313nirA

Rocap et al 2003 Nature

Summary of N source utilization genes

NH4 NO2 NO3 urea cyanate N2

MED4

SS120

MIT9313

+

-+

+

+

+

+

-

-

-

-

-

-

-

+

-

-

-

What is the interplay between “core” and “unique” genes in determining ecotype fitness?

0.1

MIT9301

AS9601

MIT9312

MIT9515

MED4

NATL1A

NATL2A

SS120

MIT9303

MIT9313

WH7803

WH7805

CC9311

RS9916

RS9917

CC9902

BL107

CC9605

WH8102

RCC307

WH5701

21 genomes now available

Low light

Synechococcus

coastal

coastal ?

coastal ?coastal ?

coastal

oligotrophic

oligotrophicmotile

High light

Prochlorococcus

1082

Orthologs defined by reciprocal best blast hits

Paralogs resolved by a combination of computational and manual curation

Defining the marine cyanobacterial core:

High light Pro

eMED4eMIT9312eMIT9313

eNATL2Aclade 4

Low light ProSynechococcus

0

500

1000

1500

2000

2500

3000

3500

WH

5701

RC

C30

7R

S99

17R

S99

16C

C93

11W

H78

05W

H78

03W

H81

02

CC

9605

BL

107

CC

9902

MIT

9313

MIT

9303

SS

120

NA

TL

2AN

AT

L1A

MIT

9301

AS

9601

MIT

9312

MIT

9515

ME

D4

unilogs

core

2-20 orthologs

Nu

mb

er o

f g

enes

Core genes make up only 40-60% of each genome

Ahlgren & Rocap in prep

unilogs

core

2-20 orthologs

9%

27%

64 %

COG categories

Unknown function

Colored pie portions: assignable COG function

The pan genome is dominated by unilogs

Ahlgren & Rocap in prep

Phylogenomics of core genes

consensus

Find tree for each gene set

Ask: Are there trees with conflicting branching order suggesting horizontal transfer of some genes?

Ask: Are there genes with evolutionary distances that are greater than or smaller than avergage?

total protein tree distance

Slow core genes

Fast core genes

All core genesn=1082

1 Translation, ribosome

15 Chaperones, protein modification16 Energy product and conversion

25 Unknown function

Relative rates of evolution vary among core genes

1

346

915

16

1720

2122

23

25

13

45

8 910

1415

1617

18192021

2223

24

25

1 34

6101819

2022

24

25

20 Coenzyme transport and metabolism

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0 3 6 9 12 15 18 21 24 27 30

fre

qu

en

cy

4 Replication, recombination, repair

COG classification

Ahlgren & Rocap in prep

WH5701

RCC307

MIT9303MIT9313

AS9601

MED4

MIT9301

MIT9312

MIT9515

NATL1A NATL2A

SS120

RS9917 RS9916

CC9311 WH7803

WH7805

BL107 CC9605

CC9902

WH8102 0.1

MIT9301AS9601

MIT9312

MIT9515MED4

NATL1A

NATL2A

SS120MIT9303

MIT9313

WH7803WH7805

CC9311RS9916RS9917

CC9902

BL107CC9605

WH8102

RCC307

WH5701

Genome reductions

Gene losses

Unilog gains

Gene gains (non-unilog)

Cluster 1 Syn

Cluster 2 Syn

High light Pro

Low light Pro

Phylogenetic mapping of gene gain/loss events

Ahlgren & Rocap in prep

WH5701 WH5701

RCC307 RCC307

MIT9303MIT9313

MIT9303MIT9313

AS9601

MED4 MIT9301

MIT9312

MIT9515

AS9601

MED4 MIT9301

MIT9312

MIT9515

NATL1A NATL2A

NATL1A NATL2A

SS120

RS9917 RS9916

CC9311 WH7803 WH7805

RS9917 RS9916

CC9311 WH7803 WH7805

BL107 CC9605

CC9902

WH8102

BL107 CC9605

CC9902

WH8102

All other functions

Replication

Cell wall/membrane biogenesis

Amino acid transport & metab.

Carbohydrate transport & metab.Inorganic ion transport & metab.

2’ metabolite transp. & metab.TransductionDefense mechanisms

Selected COG categories

Function of genes gained

Ahlgren & Rocap in prep

Insertions of cell surface gene clusters

%G+C

67

27

51MIT 9313

MED4

13,648 bp

tRNA Gly secA cysE

gyrBmutSPMM1645 PMM1634

MIT 9313

56,445 bpsecA cysE

gyrBmutS tRNA GlyPMT0079 PMT0121

33 gene insertion

WH 8102

22,507 bp

secA cysEgyrBmutS tRNA

GlyWH0078 WH0095

WH5701 WH5701

RCC307 RCC307

MIT9303MIT9313

MIT9303MIT9313

AS9601

MED4 MIT9301

MIT9312

MIT9515

AS9601

MED4 MIT9301

MIT9312

MIT9515

NATL1A NATL2A

NATL1A NATL2A

SS120

RS9917 RS9916

CC9311 WH7803 WH7805

RS9917 RS9916

CC9311 WH7803 WH7805

BL107 CC9605

CC9902

WH8102

BL107 CC9605

CC9902

WH8102

All other functions

Replication

Cell wall/membrane biogenesis

Amino acid transport & metab.

Carbohydrate transport & metab.Inorganic ion transport & metab.

2’ metabolite transp. & metab.TransductionDefense mechanisms

Selected COG categories

Function of genes lost

Ahlgren & Rocap in prep

Archaea: 0.3%

phage: 0.15%

Bacteria: 9% ORFans

n = 963

n = 7241

23%

68%

Possible duplication events

Crenarchaeota

Euryarchaeota

phage

Acidobacteria

Actinobacteria

Bacteroidetes

Chlamydiae

Chlorobi

Cyanobacteria

Deinococcus-Thermus

Firmicutes

Planctomycetes

Alphaproteobacteria

Betaproteobacteria

Deltaproteobacteria

Epsilonproteobacteria

Gammaproteobacteria

Magnetococcus

Spirochaetes

Where do the unilogs come from?

Genomic islands

Coleman et al. 2006, Science

Found in all genomes (core)

Found only in query (unilog)

All genes have an ortholog in subject

No genes have an ortholog in subject

% GC

tRNAs

Inverted and direct repeats

Island 29: phycobilisome genes deleted in RS9917

Genomic island

All genes have an ortholog in subject

No genes have an ortholog in subject

Found in all genomes (core)

Found only in query (unilog)

Island 3, insertion of capsular polysaccharide synthesis.

Islands 33,34: urea operon deleted in WH7803

Islands 36: nitrate operon deleted in RS9917

Isl. 4Isl. 7

Isl. 37

Isl. 18

Isl. 19

Synechococcus

WH8102

RCC307RS9917RS9916CC9311WH7803

BL107CC9902

(query, WH8102)CC9605

Ahlgren & Rocap in prep

“close with a profound quote”

How’s your ma?

Not too good, she’s on her way out

We all are, act accordingly