Article presentation

Anaerobic endosymbiont generates energy

for ciliate host by denitrification

Wikipedia

Link to the article

Quick introduction
 We shall go through the main topics of the article
 Background will be given through the lecture
 Zug lake
 Zug lake
 Switzerland, right next to Zurich
 200 m deep
 Fresh water lake

Photo by - Alan Smithers Google maps

Zug lake
 Freshwater lake – typical layers
 Hypolimnion -> ‘deep water’
 Anoxic :
 Oxygen depletion
Common characteristics
 Nitrate repletion

Epilimnion
 What nitrate is ?

Metalimnion

Hypolimnion

Wikipedia
 The Nitrogen (N) cycle

https://www.youtube.com/watch?v=gF8rZVmuipw
 Nitrogen (N) cycle
 Key ‘players’:

 Basic reactions :
Fixation Nitrification De-Nitrification

https://www.youtube.com/watch?v=gF8rZVmuipw
Nitrogen (N) cycle

Campbell biology, 2014.

…Coming back to lake zug
Deep water’ Chemical profiles‘
 Chemical tests - Decrease of nitrate concentration ()

 Copious unicellular eukaryotes movement (video)

 The eukaryotes resembled to be ciliates

 What is a ciliate ?
(‫)ריסנית‬
Ciliates
Ciliates
 Single cells eukaryotes
 Water leaving organisms
 Bacteria - main ‘diet’

Wikipedia
In order to understand ciliates evolution we need to refresh –

Endosymbiosis

B.C: A New Theory On How Complex Life Evolved (prehistoricict.blogspot.com)

Endosymbionts evolution
 The “oxygen revolution” : 2.7-2.4 billion years ago
 Eukaryotic life evolved at ~ 1–1.9 billion years ago
 ‘right’ after earth atmosphere gradual oxygenation
 Prokaryotic -> endosymbiosis -> mitochondria

Campbell biology, 2014.

Campbell biology, 2014.

Campbell biology, 2014.

Endosymbionts evolution
 What happened to the symbiont (the bacteria) :
 Lost most of its genes
 Only - translation and energy making genes

 Eukaryotes that transfer to an anoxic environment :

 Fermentation -> energy
 Mitochondria -> reductive evolution
 Un-functional mitochondrial remnants

 Mitochondria -> hydrogenosomes

…Back to Ciliates

 Before, The movement to anoxic environment…

 Mitochondria -> hydrogenosomes
Summarize - So far

 Unicellular organism (might be ciliates…)

 Their abundances increases with depth (‘deep water’ core – ciliates only)

 Obligated to an an-aerobic environment (total absence adjacent to oxygen)

 The lake shows - Oxygen depletes with depth, and so do nitrate!

? Are their Unique DNA forms
 ‘DAPI’ test :
 What is DAPI test ?
 Multiple intracellular DAPI signals

 Questions so far :
 Does intracellular DAPI signals is a reminiscent of an endosymbionts ?
 To which organism does this DNA belong to ?
 What nitrate has to do with all of this ?
Identifying the symbiont
Identify the symbiont
 Elimination :
 No cofactor F420 was found - unlikely to be mitochondria who turned to be hydrogenosome
 No hybridization with archaeal oligonucleotide probe – unlikely to involve archaea

 Positive hybridization to bacteria oligonucleotide probe – probably bacteria ?

…sequencing
 Metagenomic sequencing (‘deep water’ layer) :
 290 kb (small) circular genome

 16S rRNA :
 86% identity with:
 Legionella clemsonensis
 Candidatus Berkiella cookevillensis
 Naturally, both attack protists (Amoeba)
 Gammaproteobacteria class

 Proved to be bacteria !

 Named - Candidatus Azoamicus ciliaticola

Identifying the host
…sequencing
 18S rRNA test - Ciliophora phylum -> Plagiopylea Class
 Plagiopyleans :
 Anaerobic ciliates
 Found in anoxic freshwater or marine habitats
 Thought to be rely on hydrogenosomes for energy

 Proved to be ciliate !
Lecturer change

? Is that an endosymbiosis
Symbiont (Candidatus Azoamicus ciliaticola) genome

 Very small – 292,520 bp

 Low G/C content (24%)
 High protein-coding density (94%)
 Few coding protein genes (310)
Universal symbiont features
 Lack of coding genes for :
 Cell wall
 Membrane
 Most amino-acids synthesis or transportation

 Remarks :
 Smallest protist endosymbiont reported
 Those features – Been found only in insects obligate endosymbionts
Genomes comparisons
 Prokaryotic full genomes
 Gene bank data

Non-obligate ciliate
endosymbiosis

Mitochondria

Obligate insect
endosymbiosis
Protein coding genes comparisons
Jakobid
Ca. A. mitochondria
ciliaticola
Protein coding genes comparisons
 On the basis of all of the above – the searchers concluded :
 Candidatus Azoamicus ciliaticola is an obligative endosymbiont !
.’Theorized metabolic potential and activity of ‘Ca. A. ciliaticola
 Metatranscriptome coverage of two ‘Ca. A. ciliaticola’ genomes
 Correlation of ciliate abundance and denitrification rates

Number of ciliates per litre

Hypothesized evolution of ‘Ca. A. ciliaticola’–ciliate symbiosis