Phylogenomic analysis of Candidatus 'Izimaplasma' species: free-living representatives from a Tenericutes clade found in methane seeps

Abstract

Tenericutes are a unique class of bacteria that lack a cell wall and are typically parasites or commensals of eukaryotic hosts. Environmental 16S rDNA surveys have identified a number of tenericute clades in diverse environments, introducing the possibility that these Tenericutes may represent non-host-associated, free-living microorganisms. Metagenomic sequencing of deep-sea methane seep sediments resulted in the assembly of two genomes from a Tenericutes-affiliated clade currently known as 'NB1-n' (SILVA taxonomy) or 'RF3' (Greengenes taxonomy). Metabolic reconstruction revealed that, like cultured members of the Mollicutes, these 'NB1-n' representatives lack a tricarboxylic acid cycle and instead use anaerobic fermentation of simple sugars for substrate level phosphorylation. Notably, the genomes also contained a number of unique metabolic features including hydrogenases and a simplified electron transport chain containing an RNF complex, cytochrome bd oxidase and complex I. On the basis of the metabolic potential predicted from the annotated genomes, we devised an anaerobic enrichment media that stimulated the growth of these Tenericutes at 10 °C, resulting in a mixed culture where these organisms represented ~60% of the total cells by targeted fluorescence in situ hybridization (FISH). Visual identification by FISH confirmed these organisms were not directly associated with Eukaryotes and electron cryomicroscopy of cells in the enrichment culture confirmed an ultrastructure consistent with the defining phenotypic property of Tenericutes, with a single membrane and no cell wall. On the basis of their unique gene content, phylogenetic placement and ultrastructure, we propose these organisms represent a novel class within the Tenericutes, and suggest the names Candidatus 'Izimaplasma sp. HR1' and Candidatus 'Izimaplasma sp. HR2' for the two genome representatives.

Figure 1

Maximum likelihood phylogenetic tree of Tenericutes and the firmicute class Eysipelotrichia constructed from the concatenated alignment of 38 single-copy genes; Bacillus subtilis was used as the outgroup. Collapsed wedges represent monophyletic groups of genomes. Nodes with greater than 95% bootstrap support are annotated with a black circle. Scale bar indicates substitutions per site.

Figure 2

Maximum likelihood phylogenetic tree of 16S rRNA gene sequences from cultured Tenericutes, Erysipelotrichia, some other Firmicutes representatives and some Actinobacteria used as the outgroup. Gray wedges represent monophyletic groups of a particular taxonomy. In this tree the class Erysipelotrichia are split into two groups, labeled 1 and 2. Sequences belonging to clade ‘NB1-n' are shown in gray. All sequences are labeled with their NCBI accession numbers. Nodes with greater than 70% bootstrap support are labeled with a black circle. Scale bar represents substitutions per site.

Figure 3

Model predictions of the central metabolism inferred from Izimaplasma genomes. Both genomes illustrate the potential for metabolizing a variety of simple sugar compounds that feed into the Embden–Meyerhof–Parnas glycolysis pathway (symbolized by black circles). Reducing equivalents (NADH) generated through glycolysis can be oxidized either by the conversion of acetate to ethanol, using a membrane-bound, sodium-pumping NADH:Ferredoxin oxidoreductase (RNF complex) or by a proton-pumping NADH:Quinone oxidoreductase (complex I). Both complex I and the cytochrome bd oxidase interact with the quinone pool, however no quinone synthesis genes were identified in the genome. Two types of hydrogenases act in catabolism to regenerate oxidized ferredoxin, or in anabolism to regenerate NADPH used during lipid and nucleotide synthesis. Izimaplasma sp. HR2 contains a protein microcompartment that links the degradation of DNA or RNA to the production of acetyl-CoA and intermediates of glycolysis. 2DR5P, 2-deoxy-d-ribose 5-phosphate; DHAP, dihydroxyacetone phosphate.

Figure 4

Proposed model of DNA/RNA degradation by the protein microcompartment found in Izimaplasma sp. HR2. The operon encoding the structural and catalytic genes of this microcompartment, which we propose to call the nucleotide utilizing operon (ntu), is shown below. The reactions catalyzed by this operon are shown above and labeled with the protein that catalyzes each reaction. Two proteins for cofactor recycling (NtuE, NtuO) are also found; however, the protein requiring a cobalamin cofactor is unknown (represented by ?). AdoCbl, adenosylcobalamin; OH-Cbl, hydroxocobalamin.

Figure 5

Fluorescence in situ hybridization (FISH) micrograph of microorganisms recovered from a methane seep sediment incubation. Cells targeted by the general bacterial probe, EUB338, are shown in green (FITC); cells targeted by the specific probe for Izimaplasma (Izzy659) are shown in yellow (combined FITC and Cy3 fluorescence). The scale bar in both images is 10 μm. (a) FISH image of microorganisms from the initial sediment sample used for the Izimaplasma enrichment. (b) Microbial diversity after 6 weeks of cultivation in glucose-amended enrichment media.

Figure 6

(a) Electron cryomicrograph of an Izimaplasma cell. (b–f) Bacterial cell envelope architectures from slices of cryotomograms. (b) Gram-negative cell wall with S-layer: Caulobacter crescentus; (c) Gram-negative cell wall without S-layer: Vibrio cholera; (d) Gram-positive cell wall: Listeria monocytogenes; (e) cell-wall less: Mycoplasma pneumonia; (f) cell-wall less: Izimaplama. CM, cell membrane; IM, inner membrane; OM, outer membrane; PG, peptidoglycan; SL, S-layer. Scale bars, 100 nm.