ABSTRACT
The recently discovered seventh order of methanogens, the
Methanomassiliicoccales
(previously referred to as “
Methanoplasmatales
”), so far consists exclusively of obligately hydrogen-dependent methylotrophs. We sequenced the complete genome of “
Candidatus
Methanoplasma termitum” from a highly enriched culture obtained from the intestinal tract of termites and compared it with the previously published genomes of three other strains from the human gut, including the first isolate of the order. Like all other strains, “
Ca
. Methanoplasma termitum” lacks the entire pathway for CO
2
reduction to methyl coenzyme M and produces methane by hydrogen-dependent reduction of methanol or methylamines, which is consistent with additional physiological data. However, the shared absence of cytochromes and an energy-converting hydrogenase for the reoxidation of the ferredoxin produced by the soluble heterodisulfide reductase indicates that
Methanomassiliicoccales
employ a new mode of energy metabolism, which differs from that proposed for the obligately methylotrophic
Methanosphaera stadtmanae
. Instead, all strains possess a novel complex that is related to the F
420
:methanophenazine oxidoreductase (Fpo) of
Methanosarcinales
but lacks an F
420
-oxidizing module, resembling the apparently ferredoxin-dependent Fpo-like homolog in
Methanosaeta thermophila
. Since all
Methanomassiliicoccales
also lack the subunit E of the membrane-bound heterodisulfide reductase (HdrDE), we propose that the Fpo-like complex interacts directly with subunit D, forming an energy-converting ferredoxin:heterodisulfide oxidoreductase. The dual function of heterodisulfide in
Methanomassiliicoccales
, which serves both in electron bifurcation and as terminal acceptor in a membrane-associated redox process, may be a unique characteristic of the novel order.
ABSTRACT
Uncultivated bacteria that densely colonize the midgut glands (hepatopancreas) of the terrestrial isopod
Porcellio scaber
(Crustacea: Isopoda) were identified by cloning and sequencing of their 16S rRNA genes. Phylogenetic analysis revealed that these symbionts represent a novel lineage of the
Mollicutes
and are only distantly related (<82% sequence identity) to members of the
Mycoplasmatales
and
Entomoplasmatales
. Fluorescence in situ hybridization with a specific oligonucleotide probe confirmed that the amplified 16S rRNA gene sequences indeed originated from a homogeneous population of symbionts intimately associated with the epithelial surface of the hepatopancreas. The same probe also detected morphotypically identical symbionts in other crinochete isopods. Scanning and transmission electron microscopy revealed uniform spherical bacterial cells without a cell wall, sometimes interacting with the microvilli of the brush border by means of stalk-like cytoplasmic appendages, which also appeared to be involved in cell division through budding. Based on the isolated phylogenetic position and unique cytological properties, the provisional name “
Candidatus
Hepatoplasma crinochetorum” is proposed for this new taxon of
Mollicutes
colonizing the hepatopancreas of
P. scaber
.
The symbioses between cellulose-degrading flagellates and bacteria are one of the most fascinating phenomena in the complex micro-ecosystem found in the hindgut of lower termites. However, little is known about the identity of the symbionts. One example is the epibiotic bacteria colonizing the surface of hypermastigote protists of the genusStaurojoenina. By using scanning electron microscopy, it was shown that the whole surface ofStaurojoeninasp. from the termiteNeotermes cubanusis densely covered with long rod-shaped bacteria of uniform size and morphology. PCR amplification of 16S rRNA genes from isolated protozoa and subsequent cloning yielded a uniform collection of clones with virtually identical sequences. Phylogenetic analysis placed them as a new lineage among theBacteroidales, only distantly related to other uncultivated bacteria in the hindgut of other termites, including an epibiont of the flagellateMixotricha paradoxa. The closest cultivated relative wasTannerella forsythensis(<85 % sequence identity). Fluorescencein situhybridization with a newly designed clone-specific oligonucleotide probe confirmed that these sequences belong to the rod-shaped epibionts ofStaurojoeninasp. Transmission electron microscopy confirmed the presence of a Gram-negative cell wall and revealed special attachment sites for the symbionts on the cell envelope of the flagellate host. Based on the isolated phylogenetic position and the specific association with the surface ofStaurojoeninasp., we propose to classify this new taxon ofBacteroidalesunder the provisional name ‘CandidatusVestibaculum illigatum’.