Endosymbiosis and the evolution of eukaryotes

Question 1

What is the evidence that endosymbiosis happened?

Answer 1

1. Size and behaviour is bacteria-like
2. Number and arrangement of membranes and presence of nucleomorph
3. Organelles contain ribosomes which are more bacterial than eukaryotic
4. Organelles contain DNA which codes mainly for the transcription/ translation apparatus – all components show strong homology to eubacteria

Question 2

How are mitochondrial and chloroplast ribosomes more bacterial-like than euk-like?

Answer 2

o Sedimentation closer to 70S (bacterial) form
o Inhibited by bacterial antibiotics eg. Streptomycin
o rRNA sequence homologous to bacteria rRNA

Question 3

Endosymbiotic theory

Answer 3

• Mitochondria and chloroplasts did not evolve in situ but were acquired as free-living bacteria
• Two independent types of bacteria engulfed by host cell
- Cyanobacterium formed chloroplast
- A-proteobacterium formed mitochondrion
• Bacterial phylogeny from rRNA sequence only
- Shows that chloroplast and mitochondria are not in any way closely related

Question 4

Things in euks and not in proks

Answer 4

nucleus, endoplasmic reticulum, golgi, organelles with double membranes

Question 5

Why is it still relevant to know when and how it happened?

Answer 5

Understanding endosymbiosis in the past may make the NITROPLAST a more feasible solution to world hunger

Food security RELIES on NITROGEN FERTILISERS
o There’s been an exponential rise in human pop.
o Food production has managed to keep up with demand pretty well, but by
growing more food in the same area ie. increased intensification
o Increased yield has been matched with a much increased demand for N fertilisers

NITROGEN FERTILISERS come from OIL are UNSUSTAINABLE & energetically expensive to manufacture

o N demand so far met w/ the Haber process, but the symbiosis between leguminous plants and bacterial N2 fixers has been explored.

Legumes don’t need fertiliser as the bacteria fix nitrogen in a usable form for them.

WE CAN’T J USE LEGUMES
Legumes don’t have high carbohydrate content so are not very viable as alternatives to staple crops eg. Maize, wheat, rice, potato

Engineering stable crops to produce usable N?
o There has been a long history of trying to GM plants to express all the genes required for a staple crop to fix its own nitrogen, but it has had little success

The nitroplast (nitrogenosome)
o Idea to encourage an endosymbiotic event between rice plant and a free-living N2-fixing bacteria to develop a N2-fixing organelle

Question 6

How are mitochondrial and chloroplast size and behaviour bacteria-like?

• however…

Answer 6

o Organelles in eukaryotes resemble bacteria morphologically
o They replicate by binary fission like bacteria! New organelles only arise from existing ones – they are never formed de novo

However…
 Mitochondria and most chloroplasts lack any cell walls and peptidoglycan
 Mitochondria are surprisingly dynamic, showing morphological plasticity and with extensive fusion and fission (they change shape and fuse together!)

Question 7

How are mitochondrial and chloroplast number and arrangement of membranes and presence of nucleomorph bacteria-like?

Answer 7

o Most organelles (nucleus and lysosome) are surrounded by a single membrane, but mit. have two membranes
• ‘chloroplast’ with 4 membranes is created by the existing double membrane of the red algal chloroplast + the double membrane of the red alga itself

Question 8

What’s a nucleomorph? How formed?

Answer 8

▪ Theory that red alga underwent primary endosymbiosis, engulfing a bacteria and not eating it. This led to the production of a chloroplast with 2 membranes.
▪ Then red alga is itself engulfed by a protoeukaryotic host (cryptomonads) in secondary endosymbiosis.
• The nucleus of the red alga is nearly lost, becoming a nuleomorph
• The mitochondrion of the red alga is lost
• ‘chloroplast’ with 4 membranes is created by the existing double membrane of the red algal chloroplast + the double membrane of the red alga itself

Question 9

name photosynthetic eukaryotes with…

single membrane
double membrane
triple membrane
quadruple membrane

Answer 9

single = cyanobacteria
double = green and red algae after primary endosymbiosis
quadruple = cryptomonod, brown algae, apicomplexa !! after secondary endosymbiosis

triple = two lineages have triple (dinoflagellates, euglenoids)

due to secondary loss of one membrane

Question 10

if you test a cryptomonad with FISH, where would you see the eukaryotic and prokaryotic probes?

Answer 10

fluorescent probes are for in situ hybridisation to rRNA so will target pro/euk ribosomes

prokaryotic rRNA probe
o Gold atoms as stain, electron microscopy for detection
→ Gold label only found within the innermost compartment of the chloroplast, supporting cyanobacterial ancestry for chloroplast

eukaryotic rRNA probe
o There is labelling seen in the cytoplasm of the 1st and 2nd eukaryotic host ie. in the nucleomorph bit of the chloro and in the cytoplasm (as expected)
→ Labelling in both compartments supports eukaryotic ancestry of both hosts

Question 11

How does organelle DNA show evidence of endosymbiosis?

Answer 11

Organelles contain DNA which codes mainly for the transcription/ translation apparatus – all components show strong homology to eubacteria

o The symbiosis has progressed - mitochondria and chloroplasts are now no longer capable of independent existence. The genes from them have migrated exclusively to the nucleus.
o Bacteria – 7000 genes
vs Mitochondrion – 35 genes
The proteins are made in the cytoplasm and are imported into organelles

Question 12

What selective forces encouraged endosymbiotic events?

Name the 4 theories

Answer 12

1. The Great Oxygenation event
2. OxTox model (not correct)
3. The hydrogen hypothesis
4. Sulphur synotrophy

Basically… there are still lots of options on the table and it’s v much still an active research question

Question 13

How did atmospheric oxygen levels over time encourage endosymbiotic events?

Answer 13

Rise in oxygen levels due to cyanobacterial photosynthesis was HIGHLY TOXIC to the surrounding biota, and that this selective pressure drove the evolutionary transformation of an archaeal lineage into the first eukaryotes.

Question 14

The Great Oxygenation event

- what

Answer 14

Earth’s atmosphere and the shallow ocean experienced a rise in oxygen around 2.4 billion years ago

o For most of earth’s history (4.5-2billion years ago) O2 not present in atmosphere
o Life predates oxygen – before there was anaerobic fermentation and non-oxygenic photosynthesis
o There were no fossils for the first 3.5 billion years, until cyanobacterial stromatolites (the first aerobic prokaryotes)

Question 15

The Great Oxygenation event

- caused by

Answer 15

Cyanobacteria produced and released oxygen as a by-product of photosynthesis, converting the early oxygen-poor, reducing atmosphere into an oxidising one

Question 16

The Great Oxygenation event

Why did it take so long for oxygen levels to get high?

Answer 16

There was a huge amount of iron that first was oxidised from Fe2+ to Fe3+

Question 17

The OxTox model

Answer 17

OxTox model - Metabolic integration during the evolutionary origin of mitochondria

o Eukaryotes evolved due to increasing oxygen tension
o Oxygen is actually very toxic – we have evolved a system to detoxify it but they didn’t

Mitochondria
+ provide eukaryotic cells with metabolic advantages (compartmentalisation, increased efficiency of ATP production)
- produce reactive oxygen species that damage the nucleocytoplasm and the mitochondria, causing mutations, diseases, and ageing

o Ancestor of the nucleocytoplasm was an O2-intolerant cell. It became associated with aerobic bacteria that served to consume any O2 present, protecting the cell from O2 toxicity. In return, the host provided fermentation product such as pyruvate to the bacteria
o Over time, bacteria became intracellular, ATP export added etc

Question 18

Why is oxygen toxic?

Answer 18

Mitochondria produce reactive oxygen species that

• damage the nucleocytoplasm and the mitochondria
• causr mutations, diseases, and ageing

Question 19

Criticism of the OxTox model

Answer 19

How could the symbiont shield the host from O2 once it had been internalised, as surely the O2 would have to pass through the cytoplasm?

There would be no advantage to internalising the bacteria as the protection would be lost!!
→ This theory ‘oxtox’ is NOT COMPATIBLE with symbiosis!!!!

Question 20

The hydrogen hypothesis

- what

Answer 20

The hydrogen hypothesis – Methanogenic syntrophy. Symbiosis between archaeon and bacterium to allow archaea to use H produced by proteobacterium

o Metabolic symbiosis between methane-producing archaea and -proteobacteria (capable of aerobic and anaerobic resp)
o In the absence of oxygen, protobacterial cell metabolised organic compounds to H2 and CO2, and archaeal methanogen anaerobically converted those products into CH4
o Over time, the methanogenic species becomes increasingly dependent on fermenting bacteria, engulfing it.
o Methanogen evolved into the nucleocytoplasm and the H2-excreting bacteria became mitochondria
o Gene transfer between symbiont (bacteria) and host (archaea) to allow host to take up glucose needed by bacteria

Question 21

Criticism of the hydrogen hypothesis

Answer 21

Methanogenic metabolism is incompatible with O2 – methanogens are strict anaerobes. There is no evolutionary pathway by which a methanogen can become aerobic

Question 22

The Sulphur syntrophy hypothesis

- what

Answer 22

Sulphur syntrophy between archaeon and bacterium

o Neither cell in isolation can extract all the energy from glucose – requires anaerobic/aerobic interface
o Theory that an alpha-proteobacterium oxidised H2S to sulphur and heterotrophic archaeon reduced it back to H2S
o Archaea uses glucose and anaerobically respires sulphate to sulphide. Sulphide used as electron donor for energy
respiratory complexes.

Question 23

The Sulphur syntrophy hypothesis

Evidence and criticism

Answer 23

Evidence:
- Mitochondria are closely related to purple sulphur bacteria (which oxidise H2S to sulphur either photosynthetically or by using O2)

Criticism
- H2S is highly toxic and inhibits mitochondria

Question 24

When did endosymbiosis happen for mitochondrion?

The Old Theory – Mitochondrion-late school

Answer 24

• Theory that eukaryotes evolved and radiated a bit before the mitochondrion was acquired
• Later the mitochondrion was acquired by endosymbiosis by a protoeukaryote and there was further radiation into many different eukaryotic groups

Now seen as WRONG

Question 25

When did endosymbiosis happen for mitochondrion?

Why is the OLD theory now seen as WRONG?

Answer 25

There’s a whole set of proteins (HSP70) that are present in ALL eukaryotes with mitochondria, but INITIALLY thought to be absent in all archaezoa

1) Now, mitochondrial HSP70 proteins are found in all key archezoal groups, suggesting they lost their mitochondria
2) Determined that archezoa actually had organelles surrounded by TWO membranes which were functionally derived from mitochondria – the hydrogenosome and the mitosome.

Now thought that mito were there in archaezoa but got diverted to other processes, evolving into H2 producing hydrogenosomes and mitochondrion-derived mitosomes

Question 26

When did endosymbiosis happen for mitochondrion?

The New Theory – Mitochondrion early model

Answer 26

• Mitochondrial acquisition occurred before eukaryotic radiation
• All basal groups had mitochondria but either lost them or diverted them (hydrogenosomes/mitosomes)

ISSUE
• Only issue is that prokaryotes cannot phagocytose so the bacteria (proto) cannot be engulfed

Question 27

Who participated in endosymbiosis?

chimeric theory

Answer 27

Currently thought
mitochondrion = rhodobacter-like
chloroplast = cyanobacteria-like

(unless their endosymbiosise were not unique events??)

Comparison of sequence homology suggest that eukaryotes are a chimera of bacteria AND archaea

o There are two sets of genes that imply different lineages (ie. Which are more closely related to eukaryotes?? We dunno)
o Recent evidence suggests that informational genes (RNA/DNA) are from archaea and operational (metabolic) genes are from bacteria

Question 28

Problems with chimeric theory

Answer 28

• No prokaryote was known to allow phagocytosis. Therefore there cannot have been early mitochondrial engulfment using this host
• NOT TIME for eukaryotic signature proteins (present in all eukaryotes but not in any bacteria or archaea) to evolve in a common ancestor if mitochondria evolved early and mitochondria are monophyletic.

Question 29

Possible solution to no bacterial/archaeal phagocytosis problem of chimeric theory

Answer 29

RUSSIAN DOLLS
• Current bacteria CAN endosymbiose, although mechanism is unknown
• Bacterial endosymbionts of mealybugs themselves contain bacterial endosymbionts!
• Red fluorescence shows FISH probe to gamma-proteobacteria
• Blue fluorescence shows FISH probe to beta-proteobacteria
• Composite shows and combined, with localised within

Question 30

Solution to eukaryotic signature proteins problem of chimeric theory

Answer 30

• Recent analysis of a group of eukaryotic genes suggests that Eukaryota lie WITHIN the Archaea
ie. Eukaryotes are not a monophyletic group

• Eukaryotes now thought to nestle deeply within the Asgard archaea (found near to hydrothermal vents)
▪ The asgard group contain many genes previously only ever in eukaryotes!! Including genes coding for signature proteins
• This allows time for the development of signature proteins within an archaeal-related lineage

Question 31

Order of events during endosymbiosis

Answer 31

1. chimera ‘protoeukaryote’ formed from archaea engulfing a gram -ve α-proteobacteria

This THEN diversified into red and green algae (they resulted from the same endosymbiotic event)

2. Chloroplasts

Primary endosymbiosis
- red/green algae engulfs ancestral prokaryotic cyanobacterium, forming chloro with 2 membranes

Secondary endosymbioses

• eukaryote engulfs red/green algae, forming chloro with 4 membranes
• within the first set of membranes there’s a nucleomorph and 80S ribosomes, consistent with the remnants of the red/green algae

engulfing red formed cryptomonads, brown algae, dinoflagellates

engulfing green formed apicomplexa, chlorarachniophytes, and euglenoids

Question 32

Did the red and green algae arise from separate endosymbiotic events?

Answer 32

Molecular evidence points to a SINGLE endosymbiotic event

The chloroplast

• has a double membrane
• can self-replicate
• has its own circular DNA lacking introns and histone proteins
• has 70S ribosomes and synthesises some of its own proteins

RED algae have chlorophyll a & c and phycobiliproteins and a spiral thylakoid membrane
GREEN algae have chlorophyll a & b and stacked thylakoids

Question 33

Chromist algae

Answer 33

4 membranes but no nucleomorph

• formed from engulfing of red algae in 2nd endosymbiosis event

Question 34

Apicomplexan parasites

Answer 34

green algal plastid with 4 membranes

eg. T. gondii

Question 35

algae that have chloroplasts with 3 membranes

Answer 35

dinoflagellate (red)
- complex skeleton made from cellulosic plates

euglenoid (green)
- no cell wall but a flexible, protinaceous pellicle

loss of 4th membrane!

Question 36

support for serial endosymbiotic theory

Answer 36

EM ultrastructure

• look at no. membranes down microscope
• presence of nucleomorph

pigment analysis
- use stains

biochemical data

• Different rRNA species can be detected by FISH
• gold label for pro. only present in innermost part of chloroplast

sequence data

• Sequence comparisons group the nucleomorph with the red algae and the main nucleus with acanthamoeba
• substantial number of genes have been transferred to the host nucleus, even in multiple symbiotic associations

Question 37

What are the algae?

Answer 37

photosynthetic autotrophs

• chlorophyll a is the major pigment
• they lack morphological differentiation into roots, stems and leaves
• they lack a sterile covering around their reproductive cells
• they are not monophyletic, but include prokaryote and eukaryote members

Question 38

Endosymbiosis of mitochondria and chloroplasts not unique events??

Answer 38

o Even more evidence in insects of endosymbiotic BACTERIA!!
o Now thought that bacteria aren’t that uncommon as endosymbionts!
o Maybe the nitroplast is worth some research effort??