Methanotrophs and Methylotrophs Methylotrophs Any organism using one-carbon organic compound for both an electron donor and

d as a source of cell carbon is termed a Methylotroph Methanotrophs These are aerobic organisms that oxidize methane (CH4) using it for both an electron donor and as a source of cell carbon Distribution and features Organisms capable of this type of metabolism are extremely cosmopolitan due to the availability of substrate o Methane Extremely stable compound produced as a product of anaerobic metabolism. Found extensively in nature Sewage treatment Bogs and wetlands Lake basins (especially in recently flooded reservoirs) Ruminants Review implications of methane in global warming issues Many methylotrophs are facultative methylotrophs and may grow on other organic molecules including (Source of Table 12.7: Madigan et al. 2003) o Organic acids o Ethanol o And sugars Examples of organisms classified as methylotrophs but not necessarily methanotrophs o Pseudomonas o Bacillus o Vibrio Cell membranes contain relatively large amounts of sterols

Cells may form resting structures known as exospores or cysts o Exospores are made by Type II genera (see below). They are produced by budding at one pole. A complex wall and a fibrous capsule are laid down. Following budding the mother-cell is incapable of growth or division. (Source of Figure 16.16: Stanier 1986) o Cysts are produced by Type I and Methylocystis. Cyst formation: the cell enlarges, becomes spherical and lays down additional wall layers. PbHA accumulation may occur Some may fix Nitrogen Cells have internal membrane systems o Type I also have incomplete TCA cycles (lack alpha ketoglutarate dehydrogenase) o Type II have complete TCA cycles (Source of Figure 12.15: Madigan et al. 2003)

Type I

Type II

A look at the clusters of methanotrophic bacteria (Source of Table12.8: Madigan et al. 2003)

Why does this occur? Why do we classify Methanotrophs and Methylotrophs? We have to discuss these organisms relative to their metabolism and we must (as we have in the past) separate the metabolism into energetics (electron transporting) and biosynthesis (making cell stuff) BUT in this case it seems a bit more complicated.. Energetics The overall energetics looks like this. Methane CH4 Methanol CH3OH Formaldehyde HCHO Formate HCOOBicarbonate (carbon dioxide) HCO3- (CO2)

The substrate utilization depends on the enzymes you have in this sequence If you oxidize CH4, methane monooxygenase is required (if methane is not oxidized, methane monooxygenase is not present in the cell) CH4 + O2 + NADH+H+ CH3OH + H20 + NAD

To oxidize methanol, methanol dehydrogenase is required. PPQ is pyrroloquinoline quinone. CH3OH + PPQ(ox) HCHO + PPQ(red)

To oxidize formaldehyde, formaldehyde dehydrogenase is required HCHO + NAD + H2O HCOOH + NADH+H+

To oxidize formate, formate dehydrogenase is required HCOOH + NAD HCO3 + NADH+H+

The overall energetics looks something like this.

O2
NADH + H+

CH3OH
Methanol dehydrogenase
PPQ

CH3OH

Methane monooxygenase H2O NAD

CH4
Serine pathway

HCHO
H+

PPQH2

To biosynthesis
NAD O2 H2O NAD

RMP pathway

Cyto c e e Cyto aa3

Formaldehyde dehydrogenase

HCOOH

Formate dehydrogenase NADH + H+

CO2

NADH + H+

H+

H2O

Biosynthesis There are two separate pathways by which cells utilize formaldehyde to make cell stuff, o The serine Pathway Cells using this pathway have functioning TCA cycles and type II internal membranes o Ribulose monophosphate pathway o Cells using this pathway have incomplete TCA cycles and type I internal membranes

The Serine Pathway (Source of Figure 17.59 (below): Madigan et al. 2003; Figure 13.11: White 2000) All carbon atoms for cell material are derived from formaldehyde Formaldehyde is at the same reduction state as cell stuff, so no additional reducing power is used to make it available for further biosynthetic reactions. One unique enzyme: serine hydroxymethylase Some notes on the biochemistry: o In some cells, the serine pathway goes around twice. During the second turn, OAA is condensed with Acetyl-CoA to from citrate, which eventually gets cleaved to succinate. The succinate is actually incorporated in cell stuff

The Ribulose monophosphate pathway (Source of Figure 17.60 (below): Madigan et al. 2003), Figure 13.12: White 2000) Two diagrams for this pathway, the simple and the more complex. The more complex (Figure 13.12) describes three stages to the pathway Two unique enzymes function in stage 1 o Hexulosephosphate synthetase o Hexulose-6P isomerase RMP requires energy for each molecule of G-3P synthesized (stage 2) Some notes on the biochemistry: Following stage one and depending on the organism, different routes for the metabolism of fructose 6 phosphate may occur: o The FBPA pathway (Fructose bisphosphate aldolase reactions) or o the KDPGA pathway which uses the same enzymes present in the EntnerDoudoroff pathway (and is named for 2 keto 3 deoxy 6 phosphogluconate aldolase.) Similar events occur in stage three (stage three regenerates the ribulose monophosphate necessary to begin the cycle again): o fructose 6 phosphate may be metabolized via the TA approach (transaldolase typical of the Pentose Phosphate pathway, 2 carbon transfers) or o by the SBPase (sedoheptulose 1,7 bisphosphate aldolase similar to reactions occurring in the Calvin Cycle) Obligate methanotrophs use the KDPGA-TA combination. Facultative methylotrophs use the FBPA pathway in combination with either the TA or SBPase rearrangement pathways.

References:
Madigan, M.T., J.M. Martinko, and J. Parker. 2003. Brock: Biology of Microorganisms. Prentice Hall. Stanier, R. 1986. The Microbial World, 5th ed. Prentice Hall, Englewood Cliffs.

White, D. 2000. The physiology and biochemistry of prokaryotes. Oxford University Press, New York.