Metabolism I: autotrophs and heterotrophs

Question 1

Autotrophs

Answer 1

Autotrophs use CO2 (or DIC) as their source of C. They MUST HAVE a source of energy (energy source) AND a source of reducing power (electron donor) to grow.
* Thiobacillus thioparus uses thiosulfate as both energy source and electron donor. O2 is the terminal electron acceptor. Product of respiration is H2O.
[Thiobacillaceae < Nitrosomonadales < Betaproteobacteria < Pseudomonadota]
* Thiocapsa roseopersicina uses sulfide as electron donor and electromagnetic radiation as energy source. No terminal electron acceptor as cyclic photophosphorylation used. Product of respiration is S8.
[Chromatiaceae < Chromatiales < Gammaproteobacteria < Pseudomonadota]
* Acidithiobacillus ferrooxidans uses ferrous iron (Fe2+) as energy source and electron donor. O2 is terminal electron acceptor. Product of respiration is H2O.
[Acidithiobacillaceae < Acidithiobacillales < Acidithiobacillia < Pseudomonadota]
* Hydrogenophilus thermoluteolus uses molecular hydrogen (H2) as energy source and electron donor. O2 is terminal electron acceptor. Product of respiration is H2O.
[Hydrogenophilaceae < Hydrogenophilales < Hydrogenophilalia < Pseudomonadota]
* The ‘anammox’ Bacteria use ammonium (NH4+) as both energy source and electron donor.
Nitrite (NO3-) is the terminal electron acceptor. Product of respiration is N2.
[mostly Ca. Brocadiaceae < Ca. Brocadiales < Ca. Brocadiia < Planktomycetota]

Question 2

Heterotrophs

Answer 2

Heterotrophs use organo-C compounds as their source of C-and-E. There are specialist functional guilds thereof:
* methanotrophs use methane (the most reduced C compound) as their C-and-E source.
examples:
- Methylococcus capsulatus TexasT
[Methylococcaecae < Methylococcales < Gammaproteobacteria < Pseudomonadota]
- Crenothrix polyspora (uncultivated)
[Crenotrichaceae < Sphingobacteriales < Sphingobacteriia < Bacteroidota]
NB: above is probably really in Gammaproteobacteria!
- Methylosinus trichosporium OB3bT
[Methylocystaceae < Hyphomicrobiales < Alphaproteobacteria < Pseudomonadota]
* methylotrophs use C1 compounds other than methane as their C-and-E source. A C1 compound = compound with no C-C bonds. It can be multi-C though, like trimethylamine ((CH3)3N).
examples:
- Methylorubrum extorquens AM1 and Methylorubrum podarium FM4T
[Methylobacteriaceae < Hyphomicrobiales < Alphaproteobacteria < Pseudomonadota]
- Hyphomicrobium sulfonivorans S1T
[Hyphomicrobiaceae < Hyphomicrobiales < Alphaproteobacteria < Pseudomonadota]
* diazotrophs use N2 gas as their N source but a wide-range of C-and-E compounds.
example:
- Azotobacter chroococcum DSM 2286T
[Azotobacteriaceae < Pseudomonadales < Gammaproteobacteria < Pseudomonadota]
* fermenters use fermentation rather than respiration – I’m only including here for completeness.
example:
- Clostridium acetobutylicum WT [Clostridiaceae < Eubacteriales < Clostridia < Bacillota]

Question 3

autotrophs have energetic issues

Answer 3

• all of the NADH and succinate entering the respiratory chain of heterotrophs is from the glycolytic pathways (Embden-Meyerhoff pathway in most Eukarya and many Bacteria; Entner-Doudoroff pathway in the Viridiplantae and many Bacteria; oxidative pentose-phosphate pathway in many Bacteria) and from Krebs’ cycle.
• not all heterotrophs have these – more on that in a moment.
• autotrophs don’t have either. Their C-assimilation does not use these pathways. Their electron donor/energy source dissimilation is usually very direct and uses only short pathways in which NAD+ is not reduced to NADH.
• NADH is required to make NADPH, which is needed for all biomass formation.
• as such, autotrophs have to be able to make NADH – they use reverse electron transport.
• Following slides show forward and then reverse electron transport in Thiobacillus thioparus from the Betaproteobacteria.

Question 4

autotrophs have energetic issues

Answer 4

• SO forward electron transport translocates protons into the periplasmic space and builds Δp (proton-motive force) and thus ATP can be made.
• BUT reverse electron transport does the reverse and lowers Δp thus if you need to make NAD(P)H at any given time, you can make less ATP as a result.
• Life is a constant balancing act for them!
• how do they gain their electrons? We will look at one key example – the Lu-Kelly cycle in Paracoccus versutus A2T. There are other variations of it in other
  organisms – we don’t care about them! You may see it called “the sox cycle” or “the Kelly-Friedrich cycle” but Wei-Ping Lu discovered it with Kelly in the early 1980s and deserves credit. All Friedrich’s group did was discovered the genes decades later…
  [Paracoccus < Paracoccaceae < Rhodobacterales < Alphaproteobacteria < Pseudomonadota]
• encoded by the sox operon, so each polypeptide has a name like SoxA, and if e.g. SoxAand SoxX form a protein, it is SoxAX.
• I will show it to you firstly all written out with proper equations and balancing and then drawn as a cycle to show how it all fits.

Question 5

energy conservation in Paracoccus spp.

Answer 5

• thiosulfate (S2O32-) is the energy source.
• it is oxidised (at the expense of water) to sulfate (SO42-).
• 8 electrons are conserved and transferred to cyt c.

1) thiosulfate binds to SoxZY, the thiosulfate-binding protein.
SoxYin SoxZY has a pendent cysteine residue (-SH group on side-chain). I will represent this as “SoxZY-SH” and underline the cysteine S so you remember
which it is moving forwards. This reaction is catalysed by SoxAX, L-cysteine S-thiosulfotransferase (EC 2.8.5.2):
S2O32- + SoxZY-SH + 2cyt cox → SoxZY-S-S-SO3- + 2cyt cred + H+

2) the terminal –SO3- group (sulfonate group) is oxidised into sulfate and liberated by SoxB, S-sulfosulfanyl-L-cysteine sulfohydrolase (EC 3.1.6.20).
The sulfate formed is extruded from the cell. The remaining part of the thiosulfate molecule is –S-, a sulfane group:
SoxZY-S-S-SO3- + H2O → SoxZY-S-S- + SO42-+ 2H+

Question 6

energy conservation in Paracoccus spp.

Answer 6

• thiosulfate (S2O32-) is the energy source.
• it is oxidised (at the expense of water) to sulfate (SO42-).
• 8 electrons are conserved and transferred to cyt c.

3) the pendent sulfane moiety is oxidised to sulfonate by SoxCD, S-sulfanyl-L-cysteine oxidoreductase (EC 1.8.2.6):
SoxZY-S-S- + 6cyt cox + 3H2O → SoxZY-S-SO3- + 6cyt cred + 6H+

4) the terminal –SO3- group (sulfonate group) is oxidised into sulfate and liberated by SoxB, S-sulfosulfanyl-L-cysteine sulfohydrolase (EC 3.1.6.20) same as step 2! Only difference is that only one H+ is produced because the other H from the water reacts with the –S- left from the original cysteine residue, restoring it. The sulfate formed is extruded from the cell.
SoxZY-S-SO3- + H2O → SoxZY-SH + SO42-+ H+
If you count the cyt c (each one can carry 1 ε- when in reduced state), you can account for the 8 ε- which have entered the respiratory chain – this cyt c is the respiratory cyt c!