The phylum Verrucomicrobia forms a distinct phylogenetically divergent phylum within the domain Bacteria, characterized by members widely distributed in soil and aquatic habitats. Cells of some species such as Verrucomicrobium spinosum and Prosthecobacter dejongeii possess cellular extensions termed prosthecae and cells of other strains occur in an ultramicrobacteria size range 12. Verrucomicrobia are significant for our understanding of both bacterial evolution and microbial ecology. At present, six monophyletic subdivisions (subphyla, classes) are recognized within the phylum Verrucomicrobia on the basis of 16S rRNA gene library studies 34. There are more than 500 different verrucomicrobia 16S rRNA gene sequences in publicly-accessible databases, but only a handful of these represent cultivated strains. The verrucomicrobia pose interesting evolutionary questions – members of at least one genus, Prosthecobacter, possess genes for a homolog of eukaryotic tubulin, unknown in other prokaryotes, along with the bacterial tubulin-like protein FtsZ. Verrucomicrobium spinosum possesses a FtsZ divergent from those in other phyla of the domain Bacteria 5678. In addition, some members of the verrucomicrobia have been recently found to oxidize methane and use methane as a sole source of carbon and energy, making them the only known aerobic methanotrophs outside the proteobacteria, and the only extreme acidophilic methanotrophs known 91011. They are thus significant for our understanding of the evolution of methanotrophy and C1 transfer biochemistry.

It has recently been proposed that the phyla Planctomycetes, Verrucomicrobia and Chlamydiae of the domain Bacteria form a superphylum called the PVC superphylum, which may also include the phyla Poribacteria and Lentisphaerae. The Planctomycetes, Chlamydiae Verrucomicrobia/Lentisphaerae grouping is supported by 16S and 23S rRNA sequence analysis 1213. Another study based on both phylogenetics of concatenated protein datasets and shared conserved inserts in proteins has supported the link between the phyla Verrucomicrobia and Chlamydiae 14. Other studies based on either 16S and 23S rRNA gene sequences 15, or individual or concatenated protein sequences 1617, have shown no specific relationships between the three phyla, Verrucomicrobia, Planctomycetes and Chlamydiae. However, for one of these studies 15 sequences from some superphylum lineages were not yet available and thus sequence selection may have influenced tree topology. In another of these studies 17, the inability to detect the PVC superphylum may have resulted from a loss of resolution due to editing concatenated sequence data to allow inclusion of a wide range of taxa including those of Eukaryotes. It is known that all members of the phylum Planctomycetes so far examined possess a characteristic cell plan involving compartmentalization of the cell cytoplasm by an intracytoplasmic membrane (ICM) separating the cytoplasm into two regions, the inner ribosome-containing pirellulosome and the less central ribosome-free paryphoplasm 1819. The term "pirellulosome" was first introduced to describe a major nucleoid-containing cell compartment of planctomycetes bounded by an internal membrane, the intracytoplasmic membrane (ICM). A ribosome-free "paryphoplasm" region surrounds the pirellulosome and is separated from it by the ICM 18. Based on the proposed relationships between the three lineages, we hypothesized that members of Planctomycetes and Verrucomicrobia might share a similar ultrastructure plan. This is investigated in this study using transmission electron microscopy incorporating techniques such as high pressure freezing, cryosubstitution and freeze fracture, to examine four verrucomicrobia representing three of the six subdivisions.

By applying high-pressure freezing, cryosubstitution and freeze-fracture techniques, internal compartmentalization of the cell has been observed in four representatives of the phylum Verrucomicrobia. The four species examined, Verrucomicrobium spinosum, Prosthecobacter dejongeii, Chthoniobacter flavus, and verrucomicrobia strain Ellin514, represent four genera and three distinct subdivisions (1, 2 and 3) of the phylum. Cells of all four species were examined after high-pressure freezing and cryosubstitution or after preparation of replicas of freeze-fractured cells. Cells of all four displayed features that are consistent with compartmentalization of the cell cytoplasm by internal membranes.

We have demonstrated that all four members of the phylum Verrucomicrobia examined, Verrucomicrobium spinosum, Prosthecobacter dejongeii, Chthoniobacter flavus, and verrucomicrobia strain Ellin514, share a basic cell plan analogous to that found in members of the phylum Planctomycetes. This cell plan is characterized by compartmentalization of the cell cytoplasm by a major cell organelle bounded by a single membrane containing all the cell DNA in a fibrillar condensed nucleoid, as well as ribosome-like particles. This major membrane-bounded organelle is equivalent to the pirellulosome of planctomycetes, and its bounding membrane is equivalent to the intracytoplasmic membrane (ICM) defined in planctomycetes as surrounding the pirellulosome 18. Consistent with the structural analogies between verrucomicrobia and planctomycetes, the ribosome-free region between the ICM of the pirellulosome and the cytoplasmic membrane in verrucomicrobia can be considered equivalent to the paryphoplasm of planctomycetes. The verrucomicrobial cell plan is most similar to the simplest planctomycete cell plan seen in Pirellula staleyi, Blastopirellula marina 18 and Rhodopirellula baltica. There is no indication of a single membrane-bounded organelle not containing a nucleoid such as the anammoxosome of anaerobic ammonium-oxidizing bacteria, a group thought to represent some of the most deep-branching Planctomycetes or even a separate phylum-level lineage within the PVC superphylum 2122 and which share a cell plan including the pirellulosome with planctomycetes 232425. However, the small membrane-bounded regions of ribosome-containing pirellulosome cytoplasm within paryphoplasm in V. spinosum resemble features of a pirellula-like planctomycete cultured from a Mediterranean sponge 26. The cell plan determined in verrucomicrobia was revealed using a cryosubstitution method for preparation of cells before thin-sectioning for electron microscopy, a method comparable to those used previously for establishing the planctomycete cell plan 1827.

Cells of all the species of verrucomicrobia examined here using high-pressure freezing followed by cryosubstitution also possess condensed nucleoids, which is another feature of similarity to the ultrastructure of planctomycetes. All planctomycetes appear to possess condensed nucleoids when cryofixed cryosubstituted cells are examined 18. Cryosubstitution, unlike conventional chemical fixation, is not expected to yield such condensation as an artifact of fixation 282930. This contrasts with the appearance of nucleoids in cryofixed cells of other bacterial species such as Escherichia coli and Bacillus subtilis, where a 'coralline' nucleoid extending through the cell cytoplasm is found 2829. Chromatin-like nucleoids have been reported in "Candidatus Xiphinematobacter", symbionts of nematodes belonging subdivision 2 of Verrucomicrobia 4, and also in epixenosome symbionts belonging to subdivision 4 31, although in both cases these were examined only using chemical fixation. The condensed nucleoids of all the species examined here often contained granules of varying electron density. Such granules within nucleoids have been noted to occur within cryo-fixed cells of Deinococcus radiodurans vitreous sections examined by cryoelectron microscopy 32.

V. spinosum and P. dejongeii are members of subdivision 1 (class Verrucomicrobiae) of the phylum Verrucomicrobia 1. There is another member of the phylum Verrucomicrobia, Rubritalea squalenifaciens, isolated from the marine sponge Halichondria okadai and belonging to subdivision 1 Verrucomicrobia, which seems to possess the planctomycete-like cell plan in an accompanying published figure, but this interpretation was not made by the authors 33. The planctomycete cell plan has also been observed in symbiont bacteria studied directly in sponge tissue 34. Some of those from the sponge Haliclona caerulea include cells with multiple prosthecae and in which both ICM and riboplasm were recognized 35. These bacteria may be verrucomicrobia or prosthecate alphaproteobacteria, with the ultrastructure suggesting the former. At least 3 species of verrucomicrobial subdivision 1 thus appear to possess the planctomycete cell plan. C. flavus is a member of subdivision 2 (class Spartobacteria) 36, and Ellin514 is a member of subdivision 3 37 so that we have determined the planctomycete cell plan to be present in at least 3 distinct subdivisions of the phylum Verrucomicrobia. This cell plan may occur widely among distinct subdivisions of the phylum Verrucomicrobia, which could suggest that the common ancestor of the verrucomicrobial phylum was also compartmentalized and possessed such a plan. The planctomycete cell plan thus occurs in at least two distinct phyla of the Bacteria. These phyla have been suggested to be related phylogenetically in the so-called PVC superphylum 1238. Members of the phylum Poribacteria, also postulated to belong to the PVC superphylum, have been proposed to be compartmentalized 38, and our electron microscopy examination of thin sections of cells of Lentisphaera araneosa, prepared via high-pressure freezing (unpublished data), indicates that at least one member of the phylum Lentisphaerae within the PVC superphylum 39 also possesses compartmentalized cells with the planctomycete plan. This plan seems to be shared by members of the PVC superphylum, and it is possible that a common compartmentalized ancestor of the superphylum may have shared the planctomycete cell plan. Other proposed members of the superphylum, such as members of the phylum Chlamydiae, should also be examined for such a cell plan. Interestingly, Parachlamydia acanthamoeba, a chlamydial organism which occurs as an endosymbiont of free-living amoebae, possesses one stage of its life cycle, the crescent body, which seems to display internal membranes and a cell plan in thin sections consistent with verrucomicrobial and planctomycete plans 40, but this needs to be confirmed using cryo-fixation preparative methods.

Chemically fixed cells of extremely acidophilic methanotrophic members of the phylum Verrucomicrobia forming a new subdivision within the phylum have been reported to possess unusual internal structures, including polyhedral bodies and tubular membranes, when thin sections are viewed by transmission electron microscopy 910. It is not possible from those micrographs to deduce any clear relationship of these structures to a planctomycete cell plan, but it is possible that when these strains are prepared by high-pressure freezing they will also be shown to possess such a plan. The internal membrane structures seen sometimes in cells of the methanotrophic verrucomicrobial strain V4 have been suggested to house particulate methane monooxygenase enzymes, as in other known methanotrophs. However, the occurrence of intracytoplasmic membrane similar to those of planctomycetes and in the chemoheterotrophically-grown verrucomicrobia strains studied here suggest that verrucomicrobial internal membranes need not always be associated with a particular metabolism.

The structure of 'epixenosome' verrucomicrobia symbionts of the ciliate Euplotidium, members of subdivision 4 of verrucomicrobia, is complex and there has been no suggestion of compartmentalization by internal membranes. However, these cells have so far only been examined by chemical fixation 31. The structure of the cells of these organisms should be re-examined via cryo-fixation based techniques to determine their consistency with the model proposed here for the verrucomicrobial cell plan, since it is possible that the complex structures found may be accompanied by internal membranes when methods more suitable for their preservation are used.

A unique cell plan so far found only within the phylum Planctomycetes of the Domain Bacteria, and which challenges our concept of the prokaryote cell plan, has now been found in a second bacterial phylum – phylum Verrucomicrobia. The planctomycete cell plan thus occurs in at least two distinct phyla of the Bacteria, phyla which have been suggested from other evidence to be related phylogenetically as members of the proposed PVC superphylum. This planctomycete cell plan is present in at least 3 of the 6 subdivisions of the Verrucomicrobia, suggesting that the common ancestor of the verrucomicrobial phylum was also compartmentalized and possessed such a plan. The presence of this compartmentalized cell plan in both phylum Planctomycetes and phylum Verrucomicrobia suggests that the last common ancestor of these phyla was also compartmentalized. Cell compartmentalization of this type may thus have significant meaning phylogenetically, and can act as a clue to the meaning of deeper evolutionary relationships between bacterial phyla. Its occurrence in a second phylum of domain Bacteria extends and reinforces the challenge to the concept of prokaryotic organization already posed by planctomycete cell organization. Definitions of the prokaryote depending on absence of membrane-bounded organelles may require further reexamination, a process already underway 414243. Such compartmentalized cell plans may have phylogenetic and evolutionary significance of relevance to such problems as the origin of cell compartmentalization in eukaryotes and the origin of the eukaryotic nucleus. In summary, the cell plan shared by all members of the phylum Planctomycetes so far examined appears also to be shared by several members of the phylum Verrucomicrobia, suggesting that such a plan may be common to these distinct bacterial phyla, and that the common ancestor of these relatively closely related phyla may have also possessed this plan.

K-CL cultured and prepared cells for high-pressure freezing and electron microscopy, and performed electron microscopy. RIW assisted K-CL with expert knowledge of high-pressure freezing cell preparation. TR performed freeze-fracture and production of fracture replicas. PS isolated pure cultures of Ellin strains of verrucomicrobias and shared drafting the manuscript. PHJ supplied pure cultures of Ellin strains and contributed expert knowledge of phylum Verrucomicrobia phylogenetics. JTS supplied pure cultures of Verrucomicrobium spinosum and Prosthecobacter dejongeii and contributed expert knowledge of phylum Verrucomicrobia. K-CL and JAF wrote the manuscript and RIW, PS, PHJ and JTS contributed to drafting the manuscript. JAF conceived of the study, participated in its design and coordination and helped to write the manuscript. All authors read and approved the final manuscript.