Our reader Bradley K. Sherman commented in the recent 'Tree of Life' post: "Hug has Tenericutes top-center in the tree" (Figure 1). He said: "I don't recall this phylum being discussed here at STC". I replied that we had on several occasions looked at Mycoplasma – which are Tenericutes – but that he was right and we would put them on our to-do list. Here we go...
All known Tenericutes have one morphological feature in common: they lack a peptidoglycan cell wall (they are thus refractory to Gram staining, as Elio had expertly explained here ). Their name tells the story: Tenericutes means 'soft skinned'. Not having peptidoglycan as 'support stocking', many assume the form of blobs or drops, and, with some exceptions, do not come in familiar shapes like rods or cocci. Think of them as sort of prokaryotic amoebae. Also much like amoebae, they have cytoskeletons, with the tubulin-like FtsZ and the actin-like MreB proteins as the best known components. This cytoskeleton enables Spiroplasma, for example, to assume a spiral morphology that supports active swimming, and Haloplasma contractile to dynamically switch between blob-like, largely extended, and spiral morphologies (here's a picture ). However, unlike amoebae that you find in many eukaryotic branches (human macrophages are essentially amoebae; see Talmudic Question #129 ), all Tenericutes belong to one phylum. They branched-off from the Firmicutes an estimated 65 million years ago (no apparent connection to the Chicxulub impact ).
Now some numbers. For the phylum Tenericutes, the NCBI taxonomy lists one class, Mollicutes, together with the genus Candidatus Izimaplasma (with 3 species ), 36 'unclassified Tenericutes', and two 'environmental samples'. Within the class Mollicutes you find the orders Acholeplasmatales, Anaeroplasmatales, Entomoplasmatales, Mycoplasmatales, and the inevitable 'unclassified Mollicutes' and 'environmental samples'. There are presently ~980 listed species in the phylum, with some 85 sequenced genomes. So, this is a modest phylum by numbers, but this might well be an underestimate, or 'undersampling', as most Tenericutes were found only by close inspection of vertebrate and plant cells: they tend to live intracellularly or intercellularly, are difficult to detect due to their small size, and it is often hard to find out whether they are (endo)symbionts, parasites, pathogens, or commensals. But despite their mostly intracellular occurrence in various eukaryotic hosts for which they are notorious among microbiologists (and feared by researchers working with mammalian cell lines ), some free-living Tenericutes are now known, and I introduce here one that was found at an, by all means, unexpected location.
Skennerton et al. isolated anaerobic bacteria from deep-sea methane seep sediments at Hydrate Ridge, at depths of 600 – 776 m, and 100 km offshore Oregon (see here ). Bulk-sequencing of their samples, metagenomic assembly, and binning (few words for >1 year of intense work ) resulted in two distinct, highly complete genome bins, designated Izimaplasma sp. HR1 (1.88 Mb) and Izimaplasma sp. HR2 (2.12 Mb), that belong to the Tenericutes based on phylogenetic analysis (Figure 3). An aside: a third isolate, Izimaplasma sp. ZiA1 (1.88 Mb), was just recently sequenced; it was isolated from a toluene degrading and iron reducing microcosm. The Izimaplasma sp. HR1 and HR2 genomes have a low GC-content (HR1: 34.2%), and show signs of genome streamlining, with high protein-coding percentage and a small number of repetitive elements. The authors reconstructed the metabolic pathways in the two genomes, leading to a model of central carbon metabolism reliant on fermentation of simple sugars, similar to that of other Tenericutes. Both Izimaplasma genomes contain a complete set of genes for the Embden-Meyerhof-Parnas (EMP) pathway of glycolysis and a complete pentose phosphate pathway. The tricarboxylic cycle was incomplete, with lactate as the probable end point. Both genomes encode genes necessary for metabolizing sucrose and maltose. HR1 has, in addition, genes to utilize fructose and trehalose, while HR2 contains genes to metabolize galactose and cellulose. Both genomes also contained the arginine dihydrolase pathway that the Mollicutes use as a source of ATP through the oxidation of arginine to ammonia. Reducing equivalents generated by anaerobic fermentation can be converted to hydrogen as both genomes contain two types of iron hydrogenases and hydrogenase maturation genes. One hydrogenase couples with ferredoxin and could be used to remove excess reducing equivalents during anaerobic fermentation. The other hydrogenase is linked with NADP+ and suggests that it may generate NADPH for the production of biomolecules. Hydrogenase genes are present in Erysipelotrichales, Haloplasma contractile and Turicibacter sanguinis but not in the genomes of the Mollicutes. Both Izimaplasma genomes contain homologs of the sodium translocating Ferredoxin-NAD+ oxidoreductase (RNF complex) and the proton translocating complex I of the respiratory chain. The RNF complex has been described for the genomes of some Acholeplasmatales and many of the Erysipelotrichales; however, complex I has not been found in any other Tenericutes bacterium.
In an attempt to culture Izimaplasma, they used the predicted metabolism and phenotype from both annotated genomes as a guide for designing an enrichment medium, which contained artificial seawater supplemented with vitamins, trace minerals, glucose, yeast extract, the cell wall-synthesis inhibiting antibiotics ampicillin and vancomycin (to supress growth of both Gram-positives and Gram-negatives ), and was reduced by adding Ti(III)‑nitrilotriacetic acid. The enrichment bottles were incubated at 10 °C under N2. After 6 weeks, the community was dominated by two cell types: small coccoid cells (0.5 – 1.0 μm diameter ) that positively hybridized to a Izimaplasma-specific FISH probe, and larger coccoid cells that hybridized to a general bacterial probe (the authors do not mention whether they could identify them ). This community composition remained stable for over five passages, and 16S rDNA Sequencing confirmed the presence of ~60% Izimaplasma cells (98% identity with Izimaplasma sp. HR1 ) in the enriched sample. They could thus show that the Izimaplasma can grow under the chosen conditions, although they did not yet obtain a pure culture.
The electron cryomicrograph of an Izimaplasma cell from the enriched culture with its diameter of ~1 µm shown in Figure 2 is somewhat disappointing in that, due to lacking contrast, no details of the cell's cytoplasm are resolved: no ribosomes, no nucleoid (in contrast to the electron cryomicrographs of the much smaller CPR bacteria ). However, one can clearly see the lack of a cell wall, which is always particularly impressive with this technique. So this is an example for a cell wall-less bacterium of the Tenericutes that thrives extracellularly in its natural habitat.
Despite the Tenericutes being generally accepted as a distinct phylum, their root and branching point from the Firmicutes are not entirely clear. This is a common problem with phylogenies based on molecular data where leaves and smaller twigs are, in general, well separated, yet exact branching points are difficult to nail down the closer one gets to the root(s). It depends on which molecular markers are chosen for the analysis, and how many species/strains are included. In a phylogenetic tree obtained from 38 concatenated protein sequences from single copy genes, Skennerton et al. find their two Izimaplasma isolates in one group together with Acholeplasma and Phytoplasma but separated from the Mollicutes (Mycoplasma, Spiroplasma ) by the Erysipelotrichia (Figure 3; mind the typo in "E(r)ysipelotrichia" ). The Erysipelotrichia (28 named genera, ca. 220 species ) are Gram‑positive, that is, have a cell wall, as has Turicibacter sanguinis at the root of that tree, but not the first branching species, Haloplasma contractile. In contrast, their 16S rRNA-based tree shows the Tenericutes as monophyletic, with the branch point from the Firmicutes within one subgroup of the Erysipelotrichia, but the Gram-positive T. sanguinis is now located right within the Tenericutes. Thus, the quest to find out whether the Tenericutes are truly monophyletic continues. This isn't merely an academic exercise! If they were monophyletic, one would have to assume that doing away with a cell wall was the audacious – but obviously viable – decision of one single ancestral Firmicutes species from the Erysipelotrichia branch. Otherwise, skipping a cell wall would have likely occurred several times during evolution from within a group of the Erysipelotrichia, in which case one would like to learn more about common features that 'primed' some of them to go on living cell wall-less.