ThePlanctomycetes represent a remarkable clade within the domainBacteria. They display cellular features once thought to be the sole domain of members ofEukarya. Some species have elaborate membrane-enclosed compartments, at times resembling the eukaryotic nucleus, and the capability to carry out endocytosis using membrane coat-like proteins (3,6,7,9). In addition,Planctomycetes are major players in the global nitrogen and carbon cycles and perform reactions such as the anaerobic oxidation of ammonium with the aid of subcellular organelles known as anammoxosomes (6,11,13). Despite recent progress in revealing the complex subcellular structures of severalPlanctomycetes, in-depth analyses of these unusual features have been hampered by the lack of genetic tools. Since not allPlanctomycetes are available as pure cultures and some species have doubling times of up to 30 days (12), selecting an appropriate model organism in which to develop a genetic toolbox is essential. After analyzing several species (see Table S1 in the supplemental material), we chosePlanctomyces limnophilus to develop methods for gene transfer and mutagenesis. The key criteria for selecting this species were (i) it displays one of the fastest growth rates among the culturedPlanctomycetes (6 to 14 days to detect a colony), (ii) plasmids and bacteriophage have been described (5,16,15), and (iii) the genome sequence is available. However, the unusual cell biology is a key hallmark ofPlanctomycetes, and little was known aboutP. limnophilus in this regard (4,6). Thus, we first set out to perform cell biological analyses of this species using high-pressure cryofixation, freeze substitution, and thin sectioning in conjunction with transmission electron microscopy (TEM) (6). Cryofixation of entire colonies scraped from agar plates was performed with a Leica EM PACT-2 followed by freeze-substitution in acetone with 2% osmium tetroxide at −80°C for 50 h. Afterward, the temperature was increased over a span of 14 h to −20°C and finally brought to +20°C over 22 h. Samples were washed twice for 30 min in fresh acetone and then embedded in TAAB epon. Sections were cut at 50 nm and poststained with uranyl acetate and lead citrate, and subsequent analysis was done with a Tecnai G² Spirit Bio TWIN microscope. The life cycle ofP. limnophilus involves the production of two distinct cell types, somewhat reminiscent of the phylogenetically very distantCaulobacter crescentus. There are sessile cells with a holdfast (hs) and a noncellular stalk that allows surface anchoring (Fig. 1A and B). The second cell type contains a single polar flagellum (Fig. 1C and D). Although these findings had been preliminarily described before (4), negative staining of whole cells points toward two types of crateriform structures (large [LCS] and small [SCS]) on the cell surface ofP. limnophilus. Such structures are common amongPlanctomycetes and had been used for taxonomic purposes in the past (10). Interestingly, our micrographs (Fig. 1C and D) indicate that the putative LCS are distributed across the entire cell, while putative SCS are limited to the pole where the flagellum (FL) is located. The subcellular analysis inFig. 2A shows what appears to be close to a typicalBlastopirellula-type of cell organization (2), with a cytoplasmic membrane (CM) confining the paryphoplasm (Py) and an intracytoplasmic membrane (ICM) dividing it from the pirellulosome (Pi). The ICM and CM both are about 6-nm-wide lipid bilayers (see Fig. S3 in the supplemental material). Interestingly, different cells have different shapes of Py and Pi. Serial sections of individual cells (see Fig. S1 in the supplemental material) revealed that the shapes of Py and Pi change along thez axis, and thusP. limnophilus subcellular structures are not rotationally symmetrical. In summary, the finding thatP. limnophilus displays the characteristic subcellular compartmentalization of thePlanctomycetes provided us with the necessary impetus to develop genetic tools for its manipulation. Such tools would makeP. limnophilus relevant as a model for investigating the molecular basis of planctomycete compartmentalization in general.

We thank John Fuerst, Evgeny Sagulenko, and Rudolf Amann for scientific discussion and Elizabeth Benecci from the HMS EM Facility for technical assistance.

The project was funded by the Deutsche Forschungsgemeinschaft (JO 893/1-1) and the Marie Curie IOF Program (FP7) of the European Union (CoGniSePlanctomyces).