Metabolism IV (Methylotrophy and methanotrophy) rewatch

Question 1

Overview

watch slide 3

Answer 1

• organisms that use C1 compounds.
• very abundant in Nature.
• canonical “pink-pigmented facultative
  methylotrophs” (PPFMs) on leaves of plants and
  bathroom tile grouting. Mostly Methylorubrum spp. and Methylobacterium spp.
• includes other bugs like Hyphomicrobium spp., which have a life-cycle you will get at the end of the module.
• found in guts of termites and mouths of cattle and humans (use the CH4 produced therein).
• key roles in animal and plant pathology.

“PPFMs” on leaf-print on agar plate incubated in methanol vapour (above) and Hyphomicrobium sulfonivorans from garden soil (below) (not coloured).

as plants remodel their lignin, methanol leaks out of stomata, bacteria that eat methanol can have symbiotic relationships/?/?

grouting on bathrooms as bathrooms have carbon???

Question 2

describe

Answer 2

obligate methylotrophs

restricted facultative methylotrophs

facultative methylotrophs

Question 3

definitions

Answer 3

Methanotrophs: These are bacteria that use methane (and sometimes methanol or monomethylamine) as their carbon (C) and energy (E) source for growth. They fall under the category of chemoorganoheterotrophy, meaning they derive energy from organic compounds but need an external source of carbon. Most methanotrophs exclusively use methane, and their names often contain “Methylo-“.

Methylotrophs: These are bacteria that utilize C1 compounds other than methane (like methanol) as their source of carbon and energy for growth. Methylotrophy also falls under chemoorganoheterotrophy. It’s worth noting that methanotrophy is a subset of methylotrophy. The names of methylotrophs also commonly contain “Methylo-“.

Methylotrophic yeasts: These are fungi within the Eukarya domain that use C1 compounds for growth. They have different metabolic pathways compared to bacterial methylotrophs. Methylotrophic yeasts are valuable in industries, especially for expressing human proteins, as methanol is cheaper compared to sugars.

Methanogenic Archaea: These are strictly anaerobic organisms that produce methane. They do so from various sources such as H2/CO2, acetate, or other C1 compounds. Their names often contain “Methano-“.

C1 autotrophs: These are bacteria like Paracoccus spp. and Xanthobacter spp. They perform methylotrophy but assimilate carbon at the level of CO2 via the Calvin-Benson-Bassham (CBB) cycle. They are technically chemoorganoautotrophs.

Methane production by bacteria: This process isn’t true methanogenesis. Some bacteria produce methane by using methyl-phosphorus compounds in the ocean or anaerobically metabolizing methanethiol (CH3SH). This isn’t classified as methanogenesis.

Question 3

C1 compounds: no C-C bonds

Answer 3

• methane (CH4) – decomposition of organic matter, peat bogs etc.
• methanol (CH3OH) – abundant in plants from lignin remodelling.
• formaldehyde (HCHO) – produced mainly in upper atmosphere. (embalming)
• formate (HCOO-) – produced by Formica spp. L. for example.
• methylated amines sensu stricto, e.g. MMA is a gas, in solution it is the monomethylammonium ion (CH3NH3+)
• monomethylamine (MMA, CH3NH2) – abundant in human body and any decomposing organic matter.
• dimethylamine (DMA, (CH3)2NH) – produced in roots of some plants.
• trimethylamine (TMA, (CH3)3N) – rotting fish, vaginal fluid esp. during bacterial vaginosis or just after ovulation, also found in smegma.
• trimethylamine N-oxide (TMAO, (CH3)3NO) – important in gut and osmolyte in fish.
• N,N-dimethylbiguanide (Metformin, C4H11N5) – drug for treatment of PCOS and diabetes.
• methylated sulfur species (continued on next slide!)
• methanethiol (MT, CH3SH, ‘methyl mercaptan’- binds to mercury???) – used to scent natural gas, found in skunk musk and rotting organics.
• dimethylsulfide (DMS, (CH3)2S) – ‘smell of the sea’ – key in the CLAW and Gaia hypotheses of Lovelock.

mono,di, tri are all??

Question 4

more on C1 compounds: no C-C bonds

Answer 4

• methylated sulfur species (cont)
• dimethylsulfoxide (DMSO, (CH3)2SO) – common laboratory and industrial solvent.
• dimethylsulfone (DMSO2, (CH3)2SO2) – found in many Metazoa and in oceans, soils etc. Sold as ‘methylsulfonylmethane’ (MSM) by some predatory shops
• dimethyldisulfide (DMDS, (CH3)2S2) – produced in many Allium spp.
• methanesulfonate (MSA, CH3SO3-)
• MANY other examples! Explosives, plastics, solvents… I will send you some useful chapters to help you with this.

Question 5

some methanotrophs

Answer 5

Methylococcus spp.
Methylomonas spp.
Methylomicrobium spp.
Crenothrix polyspora
“Clonothrix fusca”
Methylocella spp.
Methylocystis spp.
Methylosinus spp.

Question 6

‘workhorse’ methanotrophs

Answer 6

Methylococcus capsulatus Bath
Methylosinus trichosporium OB3bT
Methylocella silvestris BL2T

Question 7

some methylotrophs

Answer 7

Methylorubrum spp.
Methylobacterium spp.
some Paracoccus spp.
Xanthobacter spp.
Hyphomicrobium spp.

many “generalist” organisms can grow methylotrophically too – examples:
some Mycobacterium spp.
some Sphingomonas spp.
some Klebsiella spp.
some Pseudomonas spp.

Question 8

‘workhorse’ methylotroph

Answer 8

Methylorubrum extorquens AM1
Prior to 2018 or so, it was ‘Methylobacterium extorquens’ AM1.
In pre-1980s works, it was ‘Pseudomonas AM1’!!!

Question 9

Methanotrophy: methane oxidation

Answer 9

• methane must be dissimilated to obtain energy and to oxidise it enough that it can be easily assimilated into biomass.
• same pathway as chemical oxidation of an alkane to an alkanoate that some of you will recognise:
  alkane → alcohol → aldehyde → carboxylic acid
  methane → methanol → formaldehyde → formate → CO2
• we will look at this stepwise and the enzymes involved and what happens to each intermediate.
• remember methylotrophic growth on e.g. methanol or formate will be same as what I’m telling you now, just from those steps onwards!

Question 10

methane monooxygenases

Answer 10

• methane can be oxidised to methanol by two separate enzymes.
  particulate methane monooxygenase (attached to membrane), pMMO (EC 1.14.18.3)
  CH4 + QH2 + O2 → CH3OH + Q + H2O
  structure is PmoABC encoded by genes on the pmo operon. pmoA gene used in ecological studies.
  membrane-bound protein.
  contains Cu – requires Cu for function.
  part of the AMO (ammonia monooxygenase, EC 1.14.99.13) protein family.
  very specific to methane.
  fast

soluble methane monooxygenase (attached to periplasm), sMMO (EC 1.14.13.25)
CH4 + NADH + H+ + O2 → CH3OH + NAD+ + H2O
structure is MmoBCXYZ encoded by genes on the mmo operon. mmoX gene used in ecological studies.
soluble protein.
contains Fe – has no requirement for Cu.
part of the soluble di-iron monooxygenase (SDIMO) protein family.
not specific to methane and quite slow versus pMMO.
slow, reacts to lots of things
soluble has soluble donor

Question 11

More on methane monooxygenases

Answer 11

• traditionally, most methanotrophs had pMMO but would switch to sMMO if the copper-to-biomass ratio fell below a threshold – this is called the Daltonian copper switch. Why? Cytochrome-c oxidase from the respiratory chain needs Cu and can’t switch to another enzyme. Switch often
  accompanied by production of a chalcophore: methanobactin (slightly different in each organism).
  [Gr. masc. n. χᾰλκός (khalkós), copper; Gr. v. φέρω (phérō), I carry; N.L. neut. suff. –phore, carrier]
  Methylococcus capsulatus TexasT (from sewage)
  Methylococcus capsulatus Bath (from the Roman Baths at Bath, UK)
  Methylosinus trichosporium OB3bT (from laboratory air)
• we now know about facultative methanotrophs that use only sMMO and can also grow on some fatty acids and sugars. They dont have pMMO
  Methylocella sylvestris BL2T (from forest soil) [cf. work of Dedysh, Thiesen, Crombie…]
  Methylocella palustris KT (from peat bogs)
  Methylocella tundrae T4T (from tundra peatlands)
• historically we used “Type I”,
  “Type II” etc nomenclature until we ended up with more than 10 types so has been slowly abandoned!

Question 12

assaying methane monooxygenases

Answer 12

Two different approaches:
1) quantitative enzyme assays
* give you proper specific activities for the enzymes.
* often requires an in vitro co-factor that can be easily measured.
2) qualitative enzyme assays
* rapid.
* tells us only if pMMO or sMMO is in use, but not how much of either of them.
* can only assay sMMO this way – so we can’t tell if pMMO has stopped being used, only that sMMO has started to be used.

Question 13

quantitative pMMO assay

Answer 13

• in vivo:
  CH4 + QH2 + O2 → CH3OH + Q + H2O[QH2/Q in vivo varies but in Mcc. capsulatus is it menaquinol 8/menaquinone-8 – MKH2-8/MK-8]
• in vitro:
  C3H6 + DQH2 + O2 → C3H6O + DQ + H2O
• 1,2-propylene oxide build-up is easier (faster) to quantify by gas chromatography than methanol AND isn’t oxidised by downstream enzymes!
• can’t measure propylene (or methane) depletion as you have to put in such a huge excess you’re trying to measure a tiny change in a large amount.
• DQH2 used because buying DQ and reducing chemically to DQH2
  is much
  cheaper than purified MK-8/any of the ubiquinols to reduce.
• 1 mL whole cells in a buffer [or a cell-free extract (CFE)] is placed in small (3-mL) serum bottles with septa and DQH2
  added to 1 mM final concentration. 1 mL headspace air removed and replaced with propylene.
• shaken vigorously at the growth temperature of the organism and 50 μL volumes of headspace gas assayed by gas chromatography every 3 min for propylene oxide production, using a calibration curve to determine the
  concentration in the headspace gas, thus the amount produced can be calculated.
• specific activity given in nmol propylene oxide produced/min/(mg protein)

Question 14

quantitative sMMO assay

Answer 14

• in vivo:
  CH4 + NADH + H+ + O2 → CH3OH + NAD+ + H2O
• in vitro:
  CH4 + NADH + H+ + O2 → CH3OH + NAD+ + H2O
• easy to measure NADH depletion photometrically at 340 nm based on change in absorbance (A).
• other enzymes in a CFE can oxidise NADH and will do so until any/all metabolic intermediates that they can
  use are consumed – so the endogenous rate of NADH oxidation has to be measured until it stops and then the
  substrate for the enzyme assay is added.
• to a 3-mL quartz cuvette equipped with a septum, 3 mL air-equilibrated CFE is added and the spectrophotometer blanked using this cuvette at 340 nm.
• NADH is injected into the cuvette to 1 mM final concentration and the decrease in A340 monitored until is
  plateaus. If the value is very low at this stage, a further ‘spike’ of NADH is added to raise it into a range that
  can be measured.
• 1 mL methane is injected into the headspace of the cuvette which is shaken rapidly and returned to the
  instrument. A340 decrease is now due only to methane-dependent NADH oxidation.
• [NADH] in the reaction is calculated from A340 at time-intervals given ε = 6.22 mM-1 cm-1
• specific activity expressed in nmol NADH oxidised/min/(mg protein).

Question 15

qualitative sMMO assay

Answer 15

• in vivo:
  CH4 + NADH + H+ + O2 → CH3OH + NAD+ + H2O
• in vitro:
  C10H8 + NADH + H+ + O2 → C10H7OH + NAD+ + H2O
• purely used to see if sMMO is active in colonies on plates or in liquid cultures.
• does not tell you if pMMO has gone away, just that sMMO is present.
• used to rapidly see if the Daltonian copper switch has happened.
• does not quantify at all – more intense colour does not relate to more sMMO!
• for plates: a few crystals of naphthalene are placed on the inside of the lid and the plate incubated at the growth temperature for 30 min. Plate is flooded with a 10 mM solution of Fast Blue B salt (zinc chloride salt of tetrazotized o-dianisidine). Pink/violet within 5 mins indicates naphthan-1-ol had been produced by the colonies.
• for liquid cultures: an aliquot is removed to a 2-mL Eppendorf tube containing a crystal of naphthalene – this is incubated at the growth temperature for 30 min and then 1 mL 10 mM Fast Blue B salt solution is added. Pink/violet within 5 mins indicates naphthan-1-ol had been produced by the colonies.

Question 16

C-assimilation/types of bug

Answer 16

• methanotrophs from the Alphaproteobacteria assimilate
  carbon at the level of formate using the serine cycle and
  have concentric membranes around the cell margin to give
  a larger surface area for pMMO to attach. e.g.
  Methylosinus, Methylocystis, Methylocapsa…
• methanotrophs from the Gammaproteobacteria
  assimilate carbon at the level of formaldehyde using the
  Quayle pathway (ribulose 5′-monophosphate, RuMP,
  pathway) and have stacked internal membranes instead.
  e.g. Methylomonas, Methylococcus, Methylocaldum. Some
  assimilate a fraction of their C at the level of CO2 using
  the CBB cycle (Methylococcus capsulatus).
• methanotrophs from the Verrucomicrobiota and the
  “Methylomirabilota” assimilate carbon only at the level of
  CO2 using the CBB cycle. e.g. Methylacidiphilum,
  Candidatus Methylomirabilis.

Question 17

methanol dehydrogenases

Answer 17

• first site at which [H] are obtained in the dissimilation pathway methanol dehydrogenase, cytochrome c-linked (EC 1.1.2.7)
  CH3OH + 2 cyt cL (ox) → HCHO + 2H+ + 2 cyt cL (red)
  encoded on mxa operon, and comprises MxaFI.
  (older literature will say ‘mox’ operon and MoxFI).
  Uses Ca2+ and/or Mg2+ and Fe.
  methanol dehydrogenase, lanthanide-dependent (EC 1.1.2.10)
  same reaction so by rights should not have an EC number, should be an isoenzyme!
  encoded on xox operon, and comprises XoxFI.
  Uses La3+, Ce3+, Pr3+ or Nd3+ for activity though controversial (lanthanoid metals) found in glass

Question 18

anaerobic methane oxidation

Answer 18

respiration of methane using oxygen is:
CH4 + 2O2 → CO2 + 2H2O ΔG° = -818.0 kJ/mol methane oxidised recently, we have found Ca. Methylomirabilis, which does:
3CH4 + 8NO2 - + 8H+ → 3CO2 + 4N2 + 10H2O
ΔG° = -1,035.5 kJ/mol methane oxidised
this looks like a form of denitrification, starting from nitrite (NO2-), i.e. respiration of methane using nitrite…but how is the CH4 oxidised…?
2NO2-is (sort of) used to make O2 inside of the cell, which is used to oxidise methane into methanol, in short. We’ve started calling this pseudorespiration of nitrite.
Wu et al. (2011) Biochem. Soc. Trans. 39: 243-248.
Ettwig et al. (2010) Nature 464: 543-548.
[note methylotrophs using nitrate respiration is VERY well understood!]