Metabolism II: CO2 capture and assimilation in autotrophic Bacteria and Archaea

Question 1

CO2 assimilation

Answer 1

autotrophic Bacteria and Archaea assimilate CO2 by at least 5 key pathways:
* Calvin-Benson-Bassham cycle:
- transaldolase variant in chemolithoautotrophic
Bacteria and some photolithoautotrophs, namely the Chromatiales.
- sedoheptulose bisphosphatase variant only in photolithoautotrophs - the “Cyanobacteria” (thus in chloroplasts of the Viridiplantae, rhodoplasts of the Rhodophyta etc)
- Arnon-Buchanan cycle – many
photolithoautotrophic Bacteria including the ‘green sulfur bacteria’ (the Chlorobiota) and
chemolithoheterotrophic Epsilonproteobacteria.
* Wood-Ljungdahl pathway– acetogenic
Bacteria and methanogenic Archaea.
* 3-hydroxypropionate bicycle – ‘green non
sulfur bacteria’ (Chloroflexus etc) in the Chloroflexota, some Archaea,
* hydroxypropionate-hydroxybutyrate pathway in the Archaea:
* one version in e.g. Saccharolobus spp., Metalosphaera spp.
* one version in e.g. Nitrosopumilus spp.

Question 2

C1 autotrophs – an aside

Answer 2

• there are also a subset of methylotrophs (L09) that assimilate CO2 using the Calvin-Benson-Bassham cycle.
• we call them ‘C1 autotrophs’– examples:
• Paracoccus denitrificans growing on methanol or formate
• Paracoccus versutus ditto.
  [Rhodobacteraceae < Rhodobacterales < Alphaproteobacteria < Pseudomonadota]
• Xanthobacter tagetidis growing on methanol or formate
  [Xanthobacteraceae < Hyphomicrobiales < Alphaproteobacteria < Pseudomonadota]
• they dissimilate their C/E source to CO2 (see L09) then assimilate CO2 rather than assimilating from more reduced levels per the organisms in L09.
• there are also some legitimate chemolithoautotrophs than can use C1 compounds like dimethylsulfide (DMS, (CH3)2S) and dimethyldisulfide (DMDS, (CH3)2S2)
  but they are really using the S(-II) part of the molecule as electron donor and energy source and by virtue of oxidising it to sulfate (S(+VI)), the C atoms end up as CO2 and are assimilated with exogenous CO2 by CBB cycle. Examples:
• Thiobacillus thioparus Tk-m on DMS or methanethiol (CH3SH)
• Thiobacillus thioparus E6 on DMDS, DMS or methanethiol
  [Thiobacillaceae < Nitrosomonadales < Betaproteobacteria < Pseudomonadota]

Question 3

why so many cycles?

Answer 3

• short answer: things live in different kinds of ecosystem so different cycles have evolved over time to suit them.
• we will only consider the key enzymes of each cycle in our considerations of habitat and CO2 capture mechanisms and their need.

Question 4

CBB cycle: RuBisCO

Answer 4

• CO2assimilation into biomolecules occurs at ribulose 1,5-bisphosphate
  carboxylase/oxygenase (RuBisCO, EC 4.1.1.39 – has at least 9 isoenzymes across all 3 Domains of Life) which catalyses:
  D-ribulose 1,5-bisphosphate + CO2 + H2O → 2 × 3-phospho-D-glycerate + 2H+
• Next 2 steps of cycle convert 3-phospho-D-glycerate (3PGA) further:
  
  • phosphoglycerate kinase (EC 2.7.2.3):
    3-phospho-D-glycerate + ATP → 1,3-bisphosphoglycerate + ADP
  • glyceraldehyde 3-phosphate dehydrogenase (NADP+, phosphorylating, EC 1.2.1.59):
    1,3-bisphosphoglycerate + NADPH + H+ → D-glyceraldehyde 3-phosphate + NADP+ + Pi
• 1/6th of D-glyceraldehyde 3-phosphate (GAP) produced (rest is used up in the cycle) is used to make hexose sugars (e.g. fructose, glucose etc) which are fed into e.g. glycolytic pathways and Krebs’ cycle to make building blocks for amino acid biosynthesis.
• isoenzyme subunit codes: all Form I subtypes are CbbLS; Form II is CbbM
  Note many subtypes of each. Older studies may say RbcLS or RbcM– same thing!
  Bassham et al. (1950) J Biol Chem 185: 781-787.

I would ignore anything you find online re: Prof Fong discovering it first – highly contentious matter done long after the fact.

Question 5

When RuBisCO goes wrong…

Answer 5

• ribulose 1,5-bisphosphate carboxylase/oxygenase also catalyses another reaction – the oxygenation of RuBP:
  D-ribulose 1,5-bisphosphate + O2 → 3-phospho-D-glycerate +
  2-phospho-D-glycolate + 2H+
• This means autotrophs can waste half of RuBP given 2-phospho-D-glycolate can’t continue in the cycle (2C lost) and no CO2 was assimilated (net 3C lost)!
• Several carbon concentrating mechanisms (CCMs) have evolved e.g.
  
  • Many “Cyanobacteria” (and chemolithoautotrophic Bacteria) contain carboxysomes (protein
    ‘houses’ for RuBisCO and carbonic anhydrase) to keep pCO2 high around RuBisCO and to exclude oxygen.
  • The Streptophyta have various solutions including the Hatch-Slack pathway (in C4 plants of the Poales) and crassulacean acid metabolism (in CAM plants– cacti and succulents).
  • Some Bryophyta (viz. Anthocerotophyta) and Chlorophyta etc use pyrenoids to concentrate
    CO2 similarly to carboxysomes in general principles but not in structure or function!

Question 6

Carboxysomes

Answer 6

• proteinaceous cell compartments that share an evolutionary ancestor with the enterosome.
• first seen in the “Cyanobacteria” (Drews and Niklowitz (1956) Arch. Mikrobiol. 24:147-162*) and were referred to as “polyhedral bodies” until “carboxysomes” was proposed
  (Shively et al. (1973) Science 182: 584-586) when studied in Halothiobacillus neapolitanus ParkerXT.
  [Halothiobacillaceae < Chromatiales < Gammaproteobacteria < Pseudomonadota]
• contain a specific isozyme of RuBisCO (more on that in a
  moment).
• two types of pores in the protein sheets of the shells – BMC-H (for bicarbonate) and BMC-T (for organic substrates – open and close for them).
• interior is packed with RuBisCO and carbonic anhydrase (EC
  4.2.1.1), which catalyses cleavage of carbonic acid (H2CO3):
  H2CO3 → CO2 + H2O

Question 7

Carboxysomes

Answer 7

• bicarbonate ions (HCO3-) are dominant form of DIC (dissolved inorganic carbon) at pH 7.2 (cytoplasm pH).
• carbonic anhydrase forces the conversion into carbonic acid (H2CO3) which dissociates spontaneously:
  H2CO3→ 2H++ CO2
  (those protons help keep carboxysome pH slightly low to keep carbonic anhydrase from being bypassed by chemical dissociation to bicarbonate instead of CO2).
• thus pCO2 inside carboxysome is much higher than in cytoplasm (or ever could be) and forces RuBisCO to do the carboxylation reaction and not oxygenation!
• CO2 is the ONLY substrate for RuBisCO– can’t use carbonate, bicarbonate or carbonic acid!

Question 8

Carboxysome types

Answer 8

• α-carboxysomes (on cso operon) found in some of the “Cyanobacteria”, chemolithoautotrophic Bacteria (Thiobacillus thioparus, Halothiobacillus neapolitanus, Thiomicrospira spp., Thiomicrorhabdus spp., Hydrogenovibrio spp.).
• 100-160 nm diameter.
• contain Form IAc RuBisCO (Form IAq is cytoplasmic).
• β-carboxysomes (on csm operon) found in some “Cyanobacteria”.
• 200-400 nm diameter.
• contain Form IBc RuBisCO (Form IBq is cytoplasmic).

Question 9

how does DIC get to carboxysomes?

Answer 9

• several steps – transport into the cell, often including changing which species the DIC is and then protecting it as bicarbonate and flipping it back if it changes species
  within the cell.
  One v good recent study that has a really useful introduction
  and reference list etc to help you with this:
  Scott et al. (2019) Appl. Environ. Microbiol. 85: e02096-18.

Question 10

Arnon-Buchanan cycle

Answer 10

• sometimes called “rTCA”, “reverse TCA”, “reverse Krebs’ cycle”, “reductive
  TCA” etc. All have issues with trueness and so Arnon-Buchanan cycle has superseded them
  [there are many TCA cycles of which one is Krebs’ cycle, which operates clockwise; Arnon-Buchanan cycle is another TCA cycle (not the only reverse or reductive one) that operates anticlockwise]
• same intermediates as Krebs’ cycle but not the same enzymes!
• cycle found in many thermophilic organisms near to the root of the Bacteria may be an ancient cycle? (Chlorobium, Thermotoga etc).
  [Chlorobiaceae < Chlorobiales < “Chlorobiia” < Chlorobiota]
  [Thermotogaceae < Thermotogales < Thermotogae < Thermotogota]
• some steps can happen at high temperature and low pH with metal catalysts and Life is not required (Muchowska et al. (2017) Nature Ecol. Evol. 1: 1716-1721)
  compare and contrast with the Krebs’ cycle origin theory of Wächtershäuser (1988) Syst. Appl. Microbiol. 10: 207-210; Wächtershäuser (2000) Science 289: 1307-1308.

Question 11

Arnon-Buchanan cycle enzymes

Answer 11

• DIC fixation steps:
  2-oxoglutarate synthase (EC 1.2.7.3, OorABCD and ForABGDEisoenzymes) is not found in Krebs’ cycle and uses ferredoxin (Fd) as cofactor to convert C4 to C5 species by adding C from CO2:
  succinyl-CoA + CO2 + Fd(red) →
  2-oxoglutarate + CoA + Fd(ox)
  isocitrate dehydrogenase (NADP+, EC 1.1.1.42, Icd1) is found in Krebs’ cycle and converts C5 to C6 species by adding C from CO2):
  2-oxoglutarate + CO2 + NADPH + H+ →
  isocitrate + NADP+

Question 12

Wood-Ljungdahl pathway enzymes

Answer 12

• DIC fixation steps are the same in both cycles found in:
  
  • homoacetogenic Bacteria (Acetobacterium woodii, for example).
    [Eubacteriaceae < Eubacteriales < Clostridia < Bacillota < Bacteria]
  • autotrophic methanogenic Archaea (Methanosarcina thermophila, for example).
    [Methanosarcinaceae < Methanosarcinales < “Methanomicrobia” < “Methanobacteriota” < Archaea]
    CO2 is first reduced to carbon monoxide (CO) by anaerobic carbon-monoxide dehydrogenase
    (EC 1.2.7.4)
    CO2 + 2Fd(red) + 2H+ → CO + H2O + 2Fd(ox)
    CO is then fixed into acetyl-CoA by carbon-monoxide-methylating acetyl-CoA synthase (EC 2.3.1.169)
    CO + CoA + [(Co(III))] → acetyl CoA + [Me(Co(I))] (where [stuff in brackets] are corrinoid cofactors (bit like heme but Co not Fe) inside of a protein – v common in methyl-accepting enzymes)

Question 13

3-hydroxypropionate bicycle enzymes

Answer 13

• found in Archaea like Acidianus spp. and in Bacteria like Chloroflexus spp. [latter is genes only, no evidence they can or do use it]
  [Sulfolobaceae < Sulfolobales < Thermoprotei < Thermoproteota < Archaea]
  [Chloroflexaceae < Chloroflexales < Chloroflexia < Chloroflexota < Bacteria]
  bicarbonate is fixed into malonyl-CoA by acetyl-CoA carboxylase (EC
  6.4.1.2):
  HCO3- + acetyl-CoA + ATP → malonyl-CoA + ADP + Pi
  …and also into (S)-methylmalonyl-CoA by propionyl-CoA carboxylase
  (EC 6.4.1.3):
  HCO3- + propionyl-CoA + ATP →
  (S)-methylmalonyl-CoA + ADP + Pi