Ruthenium red and alcian blue effects on outer structures of methanotrophic bacteria

Methane, a contributor to the “greenhouse effect”, is oxidized in the natural environment by methanotrophic bacteria. As part of a comprehensive research effort, we have been examining the ultrastructure of methanotrophs. These microorganisms have complex outer cell wall structures similar to those frequently found in other chemol itho- trophic bacteria. (1,2)In our work, we have focused on the “type” strains of Methylomonas albus BG8 and Methylosinus trichosporium OB3b. Between Spurr and LR White embedding resins, we found a difference 1n the preservation of an outer cup layer of BG8 external to the peripheral membranes. Cells from the same sample embedded in Spurr consistently lacked this feature (FIG. 1). This effect was overcome by an en bloc ruthenium red (RR) protocol that resulted in successful retention of the cup layer in Spurr resin (FIG. 2). For OB3b cells, the en bloc RR protocol resulted in an exterior bead feature distinguishable in thin section (FIG. 4) that previously was seen only by SEM.

Indirect immunogold labeling of methanol dehydrogenase in a methanotrophic and a methylotrophic bacterium

Methanol dehydrogenase (MDH), is used by methanotrophic and methylotrophic bacteria to oxidize methanol to formaldehyde. (1) The enzyme is thought to exist both in a soluble form and in association with membrane. (2) Antisera prepared against Methylomonas albus BG8 MDH and Methylobacterium sp. strain AM1 MDH was used in an indirect Immunogold procedure to demonstrate localization of MDH. Following a modification of the method of De Mey (3), thin sections were incubated with primary antibody before exposure to gold-labeled secondary antibody.BG8, an obligate methanotroph, grows on methane and converts it to methanol. Methanol is then further oxidized by MDH to formaldehyde. A distinguishing feature of BG8 are extensive Intracytoplasmlc membranes (ICM) that form bundles or vesicular stacks located away from the peripheral membranes and extending into the cytoplasm (Fig. 1). (4) It is theorized that MDH is associated with ICM.

Comparative surface and ultrastructural views of methylotrophic bacteria by TEM, STEM, SE, and freeze-etch electron microscopy.

Methylotrophic bacteria play an Important role in the environment in the oxidation of methane and methanol. Extensive intracytoplasmic membranes (ICM) have been associated with the oxidation processes in methylotrophs and chemolithotrophic bacteria. Classification on the basis of ICM arrangement distinguishes 2 types of methylotrophs. Bundles or vesicular stacks of ICM located away from the cytoplasmic membrane and extending into the cytoplasm are present in Type I methylotrophs. In Type II methylotrophs, the ICM form pairs of peripheral membranes located parallel to the cytoplasmic membrane. Complex cell wall structures of tightly packed cup-shaped subunits have been described in strains of marine and freshwater phototrophic sulfur bacteria and several strains of methane oxidizing bacteria. We examined the ultrastructure of the methylotrophs with particular view of the ICM and surface structural features, between representatives of the Type I Methylomonas albus (BG8), and Type II Methylosinus trichosporium (OB-36).

The soluble methane mono-oxygenase of Methylococcus capsulatus (Bath). Its ability to oxygenate n-alkanes, n-alkenes, ethers, and alicyclic, aromatic and heterocyclic compounds

1. Methane mono-oxygenase of Methylococcus capsulatus (Bath) catalyses the oxidation of various substituted methane derivatives including methanol. 2. It is a very non-specific oxygenase and, in some of its catalytic properties, apparently resembles the analogous enzyme from Methylomonas methanica but differs from those found in Methylosinus trichosporium and Methylomonas albus. 3. CO is oxidized to CO2. 4. C1-C8 n-alkanes are hydroxylated, yielding mixtures of the corresponding 1- and 2-alcohols; no 3- or 4-alcohols are formed. 5. Terminal alkenes yield the corresponding 1,2-epoxides. cis- or trans-but-2-ene are each oxidized to a mixture of 2,3-epoxybutane and but-2-en-1-ol with retention of the cis or trans configuration in both products; 2-butanone is also formed from cis-but-2-ene only. 6. Dimethyl ether is oxidized. Diethyl ether undergoes sub-terminal oxidation, yielding ethanol and ethanal in equimolar amounts. 7. Methane mono-oxygenase also hydroxylates cyclic alkanes and aromatic compounds. However, styrene yields only styrene epoxide and pyridine yields only pyridine N-oxide. 8. Of those compounds tested, only NADPH can replace NADH as electron donor.