CO2 Acquisition In Cyanobacteria: Some Things Do Change – Evolution & diversity

Dean PriceMolecular Plant Physiology Group, Res Sch Biological Sciences, Australian National University

Murray Badger,Fiona Woodger, Ben Long, Loraine Tucker,Megan Shelden,Spencer Whitney,Ben Rae, Susan Howitt (BaMBi)

0.5 μM

carboxysomes

thylakoids

Theory of natural selection

• Wallace & Darwin provided the basic framework for understanding the evolution and diversity in living things; these ideas apply very well to cyanobacteria. • Darwin did not know about DNA, genes & direct transfer of traits, had he done so, he would have certainly suggested rapid periods of evolution by “horizontal gene transfer”. • HGT appears to have played a significant role in cyanobacterial & bacterial evolution, particularly in CO2 acquisition processes.

Evolution & diversity of CO2 acquisition in cyanobacteria: Areas covered

• What are cyanobacteria? • The problems of surviving in aquatic environments.• When did the cyanobacterial lineage begin ?• Evolutionary pressures on Rubisco (primary CO2 fixing enzyme)• Evolution of the cyanobacterial CO2 concentrating mechanism.• Attributes and diversity of cyanobacterial CCMs.• When did the CCM evolve and why did C3 plants “miss out”?

What are cyanobacteria ?• Also known as Blue-Green algae, the cyanobacteria are an ancient linage of photosynthetic bacteria that utilise chlorophyll for light harvesting.•They inhabit an amazing diversity of ecological niches from marine, freshwater, polar, desert crusts, plant or lichen symbioses, soda lakes, hypersaline lakes, hot springs, etc; some are primary nitrogen-fixers.

• Several clades, but essentially filamentous or unicellular (single-celled).

• They evolve Oxygen and use Rubisco to fix CO2 into sugars.

Synechococcus

Trichodesmium

Nostoc

Some cyanobacteria are also opportunists when N & P are in good supply

Algae outbreak hampers Olympic sailing preparations, Qingdao, China (www.abc.net.au)

Chloro

phyll s

olar

cell

RubiscoRubisco

CO2

electrons

Sugars

Biomass / fossil fuels

Light reactions

Dark reactionsO2

H2O

The two sides of photosynthesis

CO2

Scanning EM, The Centre for Microscopy and Microanalysis

Synechococcus

2.8 x 0.8 μM For freshwater Synechococcus species, about 27,000 cells should fit on the head of pin (carefully packed).

- Deep sea Synechococcus ~50,000.- Filamentous strains 5-10,000 cells/pinhead- (3-5 million carboxysomes per pinhead)

How many cyanobacterial cells will fit on the head of pin?

Magnification

Ultrastructure of Synechococcus sp. PCC 7002(formerly Agmenellum quadruplicatum PR-6)

carboxysome

D_Price/7002em.cdr

thylakoid IMOM

Electron-micrographs of ultra-thin sections embedded in epoxy resin & doped with heavy metals.

Ultrastructure of Synechococcus sp. PCC7002 (a halo-estuarine species)

Molecular Biology of the Cell, Fifth Edition, Garland Science 2008

A cyanobacterial ancestor was the progenitor of the modern day chloroplast

The plant chloroplast is the essential primary production factory for sugars & starch, and ultimately biomass.Terrestrial crops & ecosystems would be unsustainable without this ancient endosymbiont.

http://earthobservatory.nasa.gov/Newsroom/NPP/npp.html

Almost 50% of global primary productivity is oceanic** C Field, et al. Science 282, 237 (1998)

2002 Net Primary Production

cyanobacteria, diatoms, macro-algae, seagrass, etc.

cyanobacteria ~ 25% of global productivity. marine foodweb/fisheries.

Limitations on CO2-fixation for cyanobacteria, algae & aquatic plants

• Poor CO2 diffusion in water (104 slower than air)• Temperature• Light• Oxygen• Poor buffering, poor mixing• Nutrient depletion

fluctuations

Inorganic carbon (Ci) species in aquatic environments: a good strategy is to utilise HCO3

- for photosynthesis

H+ + HCO3-

CO2

CO32- + H+H2O + CO2

• CO2 diffusion in water is 10 4 slower than in air (slow supply rate).

• CO2 supply rate impacted by temperature, pH, demand, mixing, etc.

• CO2 HCO3- interconversion is slow at pH 8 (t1/2 ~ 15 sec).

• At high pH, CO2 can be trapped as HCO3- or CO3

=

eg. at pH 8 the CO2air: Ci

aq ratio is ~ 35.

(10 -12 μM)(CO2 + OH - HCO3

-)Carbonic anhydrase

pKa ~ 6.3 pKa ~ 10.3

air

water

When did the cyanobacterial lineage begin ?

Present-day stromatolites, Shark Bay, WA (low tide)

Living fossils

Billions of Years Ago04 3.5 3 2.5 2 1.5 1 0.54.5

Rise in Atmospheric

Oxygen

Fossil stromatolites

Humans

Early land plants

Hard-shelled Animals

First single cells with a

NucleusFirst sedimentary rocks with

isotopic signs of life

Moon forms Tides

Formation of Earth

Earliest fossils

Dinosaurs

Present-day stromatolites

Filamentous Cyanobacteria

Oxygenic photosynthesis

First multi-celled

eukaryotes

Phoenix Lander: water on Mars?

?

A condensed view of cyanobacterial evolution

High CO2/ low O2

low CO2 / high O2

Chemoautotrophs?

Plants cells with

chloroplasts

Limitations imposed by the CO2-fixing enzyme, Rubisco

• Rubisco, Ribulose bisphosphate carboxylase-oxygenase, is a relatively inefficient enzyme.• But it evolved when atmospheric conditions differed greatly form modern atmospheric conditions.

Rubisco L8S8 crystal structure

Side view Top View

CO2

O2

PGA

P-glycolate

Problems for Rubisco• Low affinity for CO2• Oxygen as an alternate substrate• Problems increase with temperature

sugarsproductive

wasteful

productive

wasteful

High CO2& low O2

Low CO2 & high O2

3500 3000 2500 2000 1500 1000 600 500 400 300 200 100 0

ARCHAEAN PROTEROZOIC PHANEROZOIC

Millions of Years Before Present

Rubisco becomes challenged

The problems for Rubisco

CO2

O2

PGA

P-glycolate

Problems for Rubisco• Low affinity for CO2• Oxygen as an alternate substrate• Problems increase with temperature

sugarsproductive

wasteful

productive

wasteful

Evolve a better Rubisco• Higher affinity for CO2• More discerning for CO2• But, lower catalytic efficiency & higher N requirements

Develop a CO2 Concentrating Mechanism (CCM)

• Cyanobacteria• Algae; Aquatic plants• Land plants

Turbo-chargingprotein evolution

Two evolutionary choices to solve the problems

The CO2-concentrating mechanism (CCM) infreshwater/oceanic cyanobacteria

HCO3-

CO2

CO2

Ci pumps

Carboxysome

Rubisco

CA

HCO3-

HCO3-

leak

proteincoat

Thylakoids

HCO3-

NADPH dehydrogenase(cyclic electron flow)

Ci delivered as HCO3-

- up to 1000x accumulation(chemical disequilibria)

Active uptake:multiple systems

specifically localisedcarbonic anhydrase

HCO3- converted to CO2

inside micro-compartmentcontaining Rubisco

CO2 leakage needs to be minimised (carboxysome shell & CO2 pump)

Outcomes: Rubisco operates at catalytic maximum nitrogen-use efficiency & very low photorespiration better ecological fitness.

Plot of Ci response for low and high-Ci cells

External Ci, mM (pH 8)0.001 0.01 0.1 1 10 100 1000 10000

Pho

tosy

nthe

sis,

μm

ol.c

ell-1

.s-1

0

5e-13

1e-12

2e-12

2e-12

3e-12

3e-12

Low-Ci High-Ci No CCM

air equilibrium Ci

How effective is the cyanobacterial CCM?

e.g. Mutants with defective carboxysomes or no CO2 uptake

Porin ?

SbtA

Na+

HCO3-

HCO3-

Na+

Outermembrane

Plasma membrane

HCO3- HCO3

-

CO2

K0.5(HCO3-) K0.5(HCO3

-)

Cytosol

Passive entry

H+

PxcA

D

HCO3-

CmpC

B B

HCO3-

HCO3-

K0.5(HCO3-)

~ 15 μM

ATP

ATP

The suite of Ci-uptake systems in cyanobacteria

Two CO2 uptake systems

Two Na+-dependent HCO3

- transporters

Traffic ATPase(BCT1)

Peptidoglycan layer

HCO3-

Thylakoid membrane

NADPHOr Fd

CO2

H+

ChpY

NdhD3NdhF3

NDH-13complex

NdhB

HCO3-

NADPHOr FdCO2

H+

ChpX

NdhD4NdhF4 NDH-14

complexNdhB

HCO3-

2-5 μM 40-100 μM

Lumen

SbtABicA

Low CO2 induction

Constitutive: lower affinity state

(Ci excess eg. 5% CO2 in air)

Inducible: higher affinity state

(Ci deficiency eg. 20 ppm CO2)

The cyanobacterial CCM has two extreme states:Needed to evolve genetic systems for fine control of expression

Na+ ?

HCO3-

Carboxysome

HCO3-

CO2

Thylakoid

e-

CO2

Na+ ?

ATP

HCO3-

CO2

HCO3-

CA

Rubiscoe-

HCO3-

CO2

HCO3-

e-

P

Ci pool ~ 20 mM max

Net transport affinityK0.5 (Ci) ~ 200 μM

Net transport affinityK0.5 (Ci) ~ 10-15 μM

Ci pool ~ 40 mM max

CO2

HCO3-

CA

Rubisco

Na+

HCO3-

CO2

HCO3-

CA

Rubisco

Ci Transporters probably evolved from existing nutrient transporters or complexes

Respiratory complexNADPH dehydrogenase respiratory complex

Low affinity CO2 uptake; Constitutive;ChpX unique protein

Ndh-I3

Major facilitator superfamily (MFS) transporter

Sodium symporter family(prokaryotes)

High affinity HCO3- uptake;

Na+-dependentSbtA

Respiratory complexNADPH dehydrogenase respiratory complex

High affinity CO2 uptake; Low-CO2 inducible;ChpY (related to ChpX)

Ndh-I4

Proton dependent sulphate uptake

SulP family(present in Eukaryotes & prokaryotes)

Medium affinity HCO3-

uptake; Na+-dependentBicA

High affinity nitrate/nitrite uptake

NrtABCD - NRT1bacterial ABC transporter

High affinity HCO3- uptake;

Traffic ATPaseCmpABCD (BCT1)

Homologue FunctionClosest homologueCCM FunctionUptake System

Two types of NDH-1 complexes in thylakoids

Herranen et al., 2004. Plant Physiology 134: 470-481

Prommeenate et al., 2004. J Biol Chem 279: 28165-28173

D3

F3

NDH-1L NDH-1M NDH-1S

B/C/E/G B/C/E/G

H/J/K

slr1623H/J/K

slr1623

sll1262 sll1262I

A

e- ? e- ?

I

AD1/D2

F1

chpY

CO2 HCO3-

sll1735

e-

H+

Thylakoid membrane

Respiratory CO2 uptake

NDH-1 = NAD(P)H dehydrogenase (Plastoquinone oxidoreductase)

14+ proteins 15+ proteins

Carboxysome function & protein make-up

Anabaena carboxysome PCC6803 carboxysome

50 nm

Icosahedron (20 identical

facets) (Kerfeld et al Science 2005)

• Carboxysomes are micro-compartments (mini-organelles) that are essential for efficient CO2 fixation in cyanobacteria.• Rubisco is packaged into the polyhedral structure

On the basis of Rubisco & carboxysome types the cyanobacteria divide into two basic groups

• α-cyanobacteria (mostly deep oceanic forms); Form 1A.• β-cyanobacteria (freshwater and coastal species); Form 1B.• each have distinctive characteristics.

0.5 μM

carboxysomes

0.1

Syn PCC7942

Syn PCC7002PCC6803

Crocosphaera

Gloeobacter

Thermosynechococcus

Anabaena variabilis

Nostoc PCC7120

Nostoc punctiformeNodularia

Syn JA-2-3B'a

Syn JA-3-3Ab Trichodesmium

Lyngbya

Pro NATL2A

Pro MIT9312Pro CCMP1986

Pro MIT9313Pro CCMP1375

Pro MIT9211

Syn CC9311Syn CC9902

Syn BL107

Syn WH8102

Syn WH5701Syn CC9605Syn RS9917

Syn RS9916

β-cyanobacteriafreshwater/coastal

α-cyanobacteriaoceanic Synechococcus

α-cyanobacteriaProchlorococcus (oceanic)

Form 1A

Form 1A

Form 1B

Two groups of cyanobacteria based on Rubisco & carboxysome types

Form 1B Rubisco = β-cyanobacteria(β-carboxysomes)

ccmKLMNO genes, ccaA

Form 1A Rubisco = α-cyanobacteria(α-carboxysomes)

cso genes

Phylogenetic tree for RbcL protein relatedness

Two types of carboxysomes in cyanobacteria

Synechococcus WH8102

Prochlorococcus marinus MED4

Synechococcus PCC7942ccmK ccmL ccmOccmN rbcL rbcS

β - carboxysomes

Missing

ccmM

Synechococcus PCC7002

Form 1B

Form 1A

ccmK ccmL ccmNccmM rbcL rbcSrbcX

rbcL csoS3csoS2 csoS4ab ndhF4rbcS ndhD4 chpX

ndhF4 ndhD4 chpXcsoS1 rbcL csoS3csoS2 csoS4abrbcS

csoS1

csoS1 orf

orf

cbbX

cbbXHalothiobacillus neapolitanus

rbcL csoS3csoS2 csoS4abcrbcS

csoS1ab

α - carboxysomes

First sequenced bacterial Micro-compartment (BMC) genes

Gene clusters

clues to HGT

Two Carboxysome types have arisen by parallel evolution

α-carboxysomes

Form 1A Rubisco

CA Function

Shell proteins: CsoS1a,b,c

CsoS2 CsoS3CsoS4

CcmKCcmLCcmOCcmM CcmN

Some small proteins are related to each other, and have “bacterial micro-compartment” domain signatures.

CsoS2, CsoS3, CcmM & CcmN have no significant sequence homologies – but there appear to be some structural equivalents.

Specific CA types vary, but the shell protein CsoS3 is a CA, and CcmM has a gamma-CA N-terminus region & CcaA is bound.

CsoS3

CO2HCO3-

Form 1B Rubisco

CA

CO2

CcmM

HCO3-

CcaA

β-carboxysomes

Two carboxysome types in cyanobacteria: some proteins are related

• Some proteins are related at an amino acid sequence and structural level.• prime evidence of HGT

α –cyanobacteria& sulphur bacteria

β -cyanobacteria

CsoS4ab

CsoS1abc

Form-1A Rubisco

CcmL

CcmK,CcmO

Form-1B Rubisco

Salmonella enterosome*

* 1,2-propanediol utilization (pdu)

PduA, PduJ

PduK, N & T

NA

The small shell proteins have similar folded structures

Crystal structure of ccmK1 (hexameric)

Work of S Kerfeld & T Yeates, Berkeley

CsoS1

ccmL homologues (pentameric)

CcmM58 trimer nucleus

Cross-sectional view

50 nm

11 nm

CcmM35

CcaA dimer

SSU binding domain L8-Rubisco

CcmM58 trimer nucleus

11 nm

3 nm

CcmK1 Hexamer

6.7 nm

Icosahedral carboxysome

Proposed that each CcmM58-trimer-nuclei can associate with CccM35 units to form a 7-unit core as the basis for each facet.

A Model for carboxysome shell formation based on CcmM-Rubisco complexes:

Long et al., J. Biol Chem, 2007

CCM Diversity(Resources: 42 fully sequenced genomes)

---Synechococcus CC9902

---Prochlorococcus SS120

---Prochlorococcus MIT9312

---Prochlorococcus MIT9313

---Prochlorococcus MED4

--Synechococcus CC9311

---Synechococcus CC9605

---Synechococcus WH8102

-Crocosphaera watsonii

---Trichodesmium erythraeum

-Synechococcus PCC7002

--Gloeobacter violaceus

-Thermosynechococcus

Nostoc punctiforme

Anabaena variabilis

Nostoc PCC7120

Synechococcus PCC7942

Synechocystis PCC6803

Ndh-13Ndh-14BicASbtABCT1

CO2 uptakeBicarbonate uptakeSpecies

Medium

Low

HighHomology

oceanic

coastal

freshwater

hot springsterrestrial mats

Diversity of Ci-transporters in cyanobacteria

No CO2uptake genes

Verified transport activity

When did cyanobacterial CCMs evolve?

HCO3-

CO2

CO2

Ci pumps

Carboxysome

Rubisco

CA

HCO3-

HCO3-

leak

proteincoat

Thylakoids

HCO3-

NADPH dehydrogenase(cyclic electron flow)

Evolution of Cyanobacterial and Algal CCMsCrunch-time: A marked period during the Carboniferous when a sharp decline in CO2 levels occurred along with a sharp rise in O2 levels.

Maximum selection pressure for CCMs

3500 3000 2500 2000 1500 1000 600 500 400 300 200 100 0

ARCHAEAN PROTEROZOIC PHANEROZOIC

Millions of Years Before Present

Cyanobacteria

O2 evolving photosynthesis

40

20

10

0

RCO2(past/present)

CARB IIImodel

30

O2 %

Start of cyanobacterialand algal CCMs ?

John Raven, U. Dundee

Bob Berner, Yale

CO2

O2

3500 3000 2500 2000 1500 1000 600 500 400 300 200 100 0

ARCHAEAN PROTEROZOIC PHANEROZOIC

Millions of Years Before Present

40

20

10

0

RCO2(past/present)

30

O2 %

CO2Plants

Algae

Oxygen evolving photosynthesisCyanobacteria

O2

High CO2 & low O2 Low CO2 & high O2

Cyanobacteria were the ancestors of plant

chloroplasts

Rubisco

Evolution of photosynthesis & atmospheric side-effects

The potential path of CCM evolution in cyanobacteria

α-Carboxysomes

Low CO2 trigger - 250-400 MYA

Microcompartment geneprecursors and transfers

β-Cyanobacteria α−Cyanobacteria Autotrophicproteobacteria

Form 1A Rubisco and α-Carboxysome gene transfer?

?? ??

Form 1A RubiscoRubisco Form 1A gene transfer

Form 1B Rubisco

β−Carboxysomes

NDH1 CO2uptake systems

Low affinity HCO3-

transporters

High affinity HCO3-

transporters

Proteobacteria

α-Carboxysomes

Multiple HGT events

Transferring parts of the cyanobacterial CCM to C3 chloroplasts??

The goal of improving water and nitrogen-use efficiencies

wheatcanola

Most key crop plants do NOT have a CCM (known as C3 plants)

• C3 crops plants lose around 500 water molecules for every CO2 gained by passive diffusion via stomata (high water cost).

• A large fraction of cellular nitrogen is devoted to the key carboxylase, Rubisco (a large nitrogen cost).

• But, C4 plants such as maize and sugar cane have a complex CCM and use ~ half the water cf. C3 plants

• Introduction of parts of the cyanobacterial CCM into C3 crop plants could be expected to improve water and nitrogen-use efficiency.

Stomatal cavity

Stomata on a leaf surface

Prospects for Genetic Engineering

Rubisco-CA complex

CO2

CO2HCO3

-

HCO3- pump

C3 Chloroplast

Na+

CA

Rubisco

NDH-1 drivenCO2 uptake

NADPH

HCO3-

HCO3-

Goal: improved water & Nitrogen-use efficiencies(akin to the “C4 rice” concept, but wheat and barley are more obvious targets in semi-arid agriculture).

HCO3- uptake

Placing a basal form of the CCM in C3 chloroplasts

Carboxysome or pseudo-pyrenoid