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Molecular Microbiology (2009) 73(6), 1009–1019 䊏 doi:10.1111/j.1365-2958.2009.06841.x First published online 28 August 2009 Cell division ring, a new cell division protein and vertical inheritance of a bacterial organelle in anammox planctomycetes mmi_6841 1009..1019 Laura van Niftrik,1* Willie J. C. Geerts,2 Elly G. van Donselaar,2 Bruno M. Humbel,2 Richard I. Webb,3 Harry R. Harhangi,1 Huub J. M. Op den Camp,1 John A. Fuerst,3 Arie J. Verkleij,2 Mike S. M. Jetten1 and Marc Strous1,4 1 Department of Microbiology, Institute for Water and Wetland Research, Faculty of Science, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, the Netherlands. 2 Cellular Architecture and Dynamics, Utrecht University, Padualaan 8, 3584 CH Utrecht, the Netherlands. 3 Department of Microbiology and Parasitology (JAF)/Centre for Microscopy and Microanalysis (RIW), University of Queensland, Brisbane, QLD 4072, Australia. 4 Max Planck Institute for Marine Microbiology, Celciusstrasse 1, 28359 Bremen, Germany. Summary Anammox bacteria are members of the phylum Planctomycetes that oxidize ammonium anaerobically and produce a significant part of the atmosphere’s dinitrogen gas. They contain a unique bacterial organelle, the anammoxosome, which is the locus of anammox catabolism. While studying anammox cell and anammoxosome division with transmission electron microscopy including electron tomography, we observed a cell division ring in the outermost compartment of dividing anammox cells. In most Bacteria, GTP hydrolysis drives the tubulin-analogue FtsZ to assemble into a ring-like structure at the cell division site where it functions as a scaffold for the molecular machinery that performs cell division. However, the genome of the anammox bacterium ‘Candidatus Kuenenia stuttgartiensis’ does not encode ftsZ. Genomic analysis of open reading frames with potential GTPase activity indicated a possible novel cell division ring gene: kustd1438, which was unrelated to Accepted 30 July, 2009. *For correspondence. E-mail l.vanniftrik@ science.ru.nl; Tel. (+31) 24 3652563; Fax (+31) 24 3652830. © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd ftsZ. Immunogold localization specifically localized kustd1438 to the cell division ring. Genomic analyses of other members of the phyla Planctomycetes and Chlamydiae revealed no putative functional homologues of kustd1438, suggesting that it is specific to anammox bacteria. Electron tomography also revealed that the bacterial organelle was elongated along with the rest of the cell and divided equally among daughter cells during the cell division process. Introduction Anaerobic ammonium-oxidizing (anammox) bacteria perform an important process in the global nitrogen cycle and produce a large part of the atmosphere’s dinitrogen gas (Arrigo, 2005). They are deep-branching members of the phylum Planctomycetes, a group known to possess a unique shared cell plan in which intracellular compartments are bounded by membranes (Lindsay et al., 2001; Fuerst, 2005). In anammox bacteria a major compartment, the anammoxosome (Fig. 1A and B), is the locus of anammox catabolism (van Niftrik et al., 2004; 2008a). This bacterial organelle is bounded by a single bilayer membrane mainly consisting of unique cyclobutane-ring ladderane lipids (Sinninghe Damsté et al., 2002). Membrane-bounded organelles and vacuoles are highly unusual among prokaryotes, the absence of such structures being one of their defining features. The biogenesis of organelles by bacteria is still largely unexplored. How anammox bacteria divide is unknown altogether, apart from the fact that it is a very slow process. The doubling time of these bacteria is in the order of weeks (Strous et al., 1998), rather than the minutes of model organisms such as Escherichia coli. Cell division is perhaps one of the most complicated tasks in the life cycle of bacteria. It has been studied most extensively in a number of model organisms, such as E. coli, where it is performed and regulated by the divisome. The divisome is a multi-protein complex with FtsZ (fts = filamentous temperature-sensitive) as the key player (Dai and Lutkenhaus, 1991). Driven by GTP hydrolysis, FtsZ assembles into a ring-like structure at 1010 L. van Niftrik et al. 䊏 © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 73, 1009–1019 Cell division of anammox bacteria 1011 Fig. 1. Diagram explaining anammox ultrastructure and transmission electron micrographs showing anammox cell division phases in K. stuttgartiensis. A. Schematic representation of a non-dividing, single cell. The cell plan is proposed to be divided into three cytoplasmic compartments separated by single bilayer membranes (Lindsay et al., 2001; Fuerst, 2005). The outer compartment, the paryphoplasm, is defined on its outer side by the cytoplasmic membrane and cell wall and on the inner side by the intracytoplasmic membrane. The second compartment, the riboplasm, contains ribosomes and the nucleoid. The third and innermost compartment, the anammoxosome, is free of ribosomes, packed with tubule-like structures and iron-rich particles (van Niftrik et al., 2008b), and bounded by the anammoxosome membrane. B. Non-dividing cell. C. Phase 1: appearance of division ring. D. Phase 2: invagination of cell wall. E. Phase 3: doubling in cell size. F. Phase 4: constriction. Arrows and insets: division ring, scale bars: 500 nm. midcell (Bi and Lutkenhaus, 1991; de Boer et al., 1992; RayChaudhuri and Park, 1992; Mukherjee and Lutkenhaus, 1994; Ma et al., 1996), recruits at least 14 other proteins (Vicente et al., 2006) and constricts to separate the two daughter cells. Of the 15 proteins that form the divisome – FtsZ, FtsA, ZipA, ZapA, FtsE, FtsX, FtsK, FtsQ, FtsB, FtsL, FtsW, FtsI, FtsN, AmiC and EnvC – 10 (underlined proteins) are essential for cell division (Goehring and Beckwith, 2005). The 10 essential divisome proteins are proposed to be recruited in a concerted mode of assembly (RayChaudhuri and Park, 1992) and each protein requires all upstream proteins to localize. In this concerted mode of assembly, the so-called FtsAindependent divisomal complex (FtsK, FtsQ, FtsB, FtsL, FtsW and FtsI) is believed to assemble independently from the Z-ring complex (ZipA, FtsA and FtsZ) and FtsN and to be recruited to midcell once the Z-ring complex is established (Goehring et al., 2006; Vicente and Rico, 2006). In E. coli and most other bacteria, six of the divisome proteins are encoded in an operon, the division cell wall (dcw) gene cluster (Carrión et al., 1999), together with genes involved in peptidoglycan precursor biosynthesis (see Fig. 2A). The bacterial FtsZ ring itself has proved difficult to observe directly within cells (in vivo) at molecular resolution under the electron microscope (Margolin, 2000; Vicente et al., 2006), but recently cytoplasmic structures and filaments that could be interpreted as cytoskeletal elements driving cell division (i.e. FtsZ) have been described (Briegel et al., 2006; Zuber et al., 2006; Li et al., 2007). With hundreds of bacterial genomes sequenced at present it appears that the key division genes are almost always conserved. There are a few exceptions, most notably members of the Planctomycetes and Chlamydiae, among bacteria the only phyla with no obvious homologue for the otherwise ubiquitous cell division gene ftsZ (Margolin, 2005; Wagner and Horn, 2006). In the present study we investigated cell and organelle division of two anammox planctomycetes using transmission electron microscopy including electron tomography. We show that during anammox cell division, the bacterial organelle is divided among the daughter cells and that despite the absence of ftsZ in the genome of the anammox bacterium ‘Candidatus Kuenenia stuttgartiensis’, a division ring is present. The genome was searched and, based on the presence of an ATP/GTP binding site and associated synergy loops, a possible novel cell division ring gene was identified that was unrelated to ftsZ. Immunogold localization using an antibody raised against the encoded protein showed that it was indeed part of the division ring. Genomic analyses of other Planctomycetes and Chlamydiae revealed no putative functional homologues of the newly identified gene, suggesting that it is specific to anammox bacteria. Results We used transmission electron microscopy including electron tomography to study cell division in two anammox planctomycetes: ‘Candidatus Kuenenia stuttgartiensis’ and ‘Candidatus Brocadia fulgida’. Both organisms were observed to divide by constrictive binary fission, as no septum was visible (Fig. 1). During the process of cell division, the intracellular anammoxosome compartment was also divided among the daughter cells (Fig. 1). The first sign of cell division was the appearance of a division ring in the outermost compartment, the paryphoplasm (phase 1; Fig. 1C), followed by a slight invagination of the cell wall (phase 2; Fig. 1D, Movies S1.1 and S1.2). The cell then doubled in size by elongation of the two poles (phase 3; Fig. 1E), during which the anammoxosome also became elongated and slightly invaginated. After elongation, the constriction continued until the cells were almost entirely pinched off (phase 4; Fig. 1F, Movies S2.1–S2.6). In this way the anammoxosome was divided equally among the daughter cells. Membrane links between the anammoxosome and paryphoplasm compartment were not observed, either in hundreds of thin sections or in electron tomograms collected from cells at different stages of the cell cycle. This indicates that the anammoxosome remains a separate entity during the entire cell cycle. In all phases of cell division, a division ring was clearly visible as a bracket-shaped, electron-dense structure in the paryphoplasm. The ring was situated at a constant distance of 5–6 nm from the cytoplasmic membrane © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 73, 1009–1019 1012 L. van Niftrik et al. 䊏 Fig. 2. Comparison between K. stuttgartiensis (Strous et al., 2006) and E. coli K-12 MG1655 (Blattner et al., 1997) cell division genes. A. E. coli dcw operon (NCBI Accession Numbers NP_414623-NP_414638). B. K. stuttgartiensis dcw operon (NCBI Accession Numbers CAJ73119-CAJ73133) with per cent identities to E. coli genes. C. Other E. coli divisome genes (NCBI Accession Numbers NP_415410, NP_416907, NP_417228, NP_417294, NP_417386, NP_417919, NP_417920, NP_418070 and NP_418368 respectively). D. Other K. stuttgartiensis divisome genes (NCBI Accession Numbers CAJ75021, CAJ72236, CAJ73638 and CAJ73639 respectively) with per cent identities to E. coli genes. E. K. stuttgartiensis putative cell division ring protein (ORF kustd1438, NCBI Accession Number CAJ72183) showing signal peptide, ATP/GTP binding site, synergy loops, staphylocoagulase repeats, polymorphic membrane protein repeats, bacterial neuraminidase repeats and antibody target. suggesting its association with this membrane. The width of the ring (longest side in transsection) increased significantly during the different cell division phases: from on average 27 nm (phase 1), to 42 nm (phase 2), to 52 nm (phase 3), to 111 nm (phase 4). The thickness of the ring (shortest side in transsection) also increased significantly from on average 5 nm (phase 1), to 6 nm (phase 2), to 8 nm (phase 3), to 9 nm (phase 4). Threedimensional modelling of the presently observed structure in anammox cells indicated that it was a continuous ring without (major) gaps (Fig. 3). The contrast of the paryphoplasm and the varying way the ring was secFig. 3. Snapshots of electron tomogram (right pane) and model (left pane) showing cytoplasmic membrane (in the model in dark grey) and continuous division ring (in the model in white) at the anammox cell division site (simplified model from Movie S2.6 B. fulgida cell division phase 4). © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 73, 1009–1019 Cell division of anammox bacteria 1013 tioned resulted in variation of clarity of the ring in different tomograms. In some tomograms the ring structure was visible but less distinct (Movie S1.1) and in these one might question the continuity of the ring structure. In other tomograms with greater contrast the ring structure was clearly continuous (Fig. 3, Movie S2.3) and in thin sections of dividing cells the ring structure was also always observed. Therefore, we modelled the structure as a continuous ring here (Movie S1.2), even though the occurrence of small gaps could not always be excluded. In tomograms and thin sections of single, non-dividing cells the ring was completely absent. We estimated the duration of the constriction of the ring by counting the proportion of dividing cells in thin sections (369 cells were analysed) and electron tomograms (nine tomograms of what appeared in 2D to be non-dividing cells were analysed). This analysis indicated that time-wise ring constriction took 55 days, over two-thirds of the length of the total cell cycle (66 days). In the search for possible candidates for genes encoding division ring proteins, the Kuenenia stuttgartiensis genome (Strous et al., 2006) was investigated for all 15 known cell division genes (Fig. 2). Of the 15 E. coli divisome genes, putative homologues to eight were found with sequence identities ranging between 20% and 43%. The genes ftsL, ftsI, ftsW and ftsQ were part of a putative dcw operon (see also Pilhofer et al., 2008) as in E. coli (Blattner et al., 1997) (Fig. 2A and B). Other putative orthologues of divisome genes, ftsK, ftsB, ftsX and ftsE, were not organized in operons (Fig. 2C and D). Of the 10 known essential cell division genes, the so-called FtsA-independent divisomal complex (Goehring et al., 2006; Vicente and Rico, 2006) (ftsK, ftsQ, ftsB, ftsL, ftsW and ftsI ) was complete. Clear homologues of genes encoding the Z-ring complex itself (ftsZ, ftsA and zipA) and ftsN were not found in the K. stuttgartiensis genome. More sensitive searches with the FtsZ and tubulin signature domains present in prosite and PFAM were also negative. In search for a protein that might substitute for the function of FtsZ, we focused on open reading frames (ORFs) containing a GTP binding site. One ORF (kustd1438, NCBI Accession Number CAJ72183) in particular drew attention (Fig. 2E). This ORF codes for a 3690-amino acids (aa)-long protein that contains an ATP/ GTP binding site (P loop; PROSITE PS00017), two synergy loops [also called T7 loop, involved in GTPase activity (Dai et al., 1994)] and a 22-aa-long signal peptide (Fig. 2E). The presence of a signal peptide was consistent with the location of the division ring in the paryphoplasm, the outermost compartment of the anammox cell. Like FtsZ, kustd1438 was characterized by a high number of alanine and glycine residues (> 10%). In addition, and unlike FtsZ, kustd1438 also contained large amounts of threonine (19%) and serine (10.4%). Together these four aa comprised over 50% of the kustd1438 protein sequence. This type of skewed aa composition is characteristic for structural proteins that form higher-order structures, such as the division ring, the cytoskeleton and the cell wall. The structural role of kustd1438 was further supported by sensitive Hidden Markov searches (Coin et al., 2003). These searches indicated the presence of four staphylocoagulase repeats (PF04022), three polymorphic membrane protein repeats (PF02415) and three bacterial neuraminidase repeats or Asp-boxes (PF02012). ORF kustd1438 was structurally different from both FtsZ and tubulin: it did not contain the FtsZ protein signatures 1 and 2 (PROSITE PS01134 and PS01135), the latter of which contains the GTP binding region (Díaz et al., 2001) (also called tubulin signature motif or T3 loop). Neither of the two tubulin/FtsZ domains (PF00091 and PF03953) was present. In conclusion, sequence analysis indicated that kustd1438 is a large structural protein with predicted ATP/GTP hydrolysis activity and thus potentially capable of effecting supra molecular motion. However, on the level of their primary structure, kustd1438 and FtsZ are not homologous. To investigate whether kustd1438 could indeed be involved in anammox cell division, we first investigated the presence of kustd1438 mRNA by real-time reverse transcription PCR using specific primers on RNA extracted from K. stuttgartiensis cells. This showed that kustd1438 was indeed transcribed. The transcription of kustd1438 was also confirmed by Illumina sequencing of reverse-transcribed K. stuttgartiensis mRNA (data not shown). Subsequently, we investigated the location of the kustd1438 protein in the K. stuttgartiensis cell using immunogold localization. For this purpose, we expressed 464 aa (including the ATP/GTP binding site) of ORF kustd1438 (Fig. 2E) in E. coli. The identity of the heterologously expressed protein was verified by MALDI-TOF MS peptide mass fingerprinting of a tryptic digest of the Ni-NTA purified protein (five peptides, total coverage 33%). We then used this protein to immunize a rabbit after which the affinity and specificity of the produced antibody, anti-kustd1438, for its target was confirmed by immunoblotting (Fig. 4). Finally, immunogold localization using antikustd1438 unambiguously located the protein to the division ring (Fig. 5A–F). After incubation with antikustd1438, 54% of the division rings were labelled in 50 dividing cells inspected and very few non-division ring labels were observed (Fig. 5E). Incubation with the preimmune serum resulted in no labelling at all. Discussion We investigated anammox cell and organelle division using transmission electron microscopy including electron © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 73, 1009–1019 1014 L. van Niftrik et al. 䊏 Fig. 4. Immunoblot analysis of the antibody (anti-kustd1438) targeting 464 aa of K. stuttgartiensis ORF kustd1438 that were heterologously expressed in E. coli. The SDS-PAGE gels were loaded with 50 mg/lane K. stuttgartiensis protein (cell free extract). A. 10% SDS-PAGE gel blotted onto a cellulose-nitrate membrane. Lane 1, marker (PageRuler Prestained Protein Ladder Plus, Fermentas, St. Leon-Rot, Germany); lane 2, incubation with anti-kustd1438; lane 3, incubation with the pre-immune serum instead of anti-kustd1438; lane 4, incubation with blocking buffer instead of anti-kustd1438. B. 6% SDS-PAGE gel blotted onto a cellulose-nitrate membrane. Lane 1: marker; lane 2, incubation with anti-kustd1438; lane 3, incubation with the pre-immune serum instead of anti-kustd1438. In both the 10% and 6% gels blotted onto cellulose-nitrate membranes, anti-kustd1438 shows specific binding to a protein of the expected size (~370 kDa). tomography. Anammox bacteria were observed to divide by constrictive binary fission with an equal division of the anammoxosome compartment between the two daughter cells. Further, a division ring was present in all dividing anammox cells. The genome of the anammox bacterium K. stuttgartiensis was investigated for known cell division genes and found to contain the FtsA-independent complex (Goehring et al., 2006; Vicente and Rico, 2006) while the Z-ring complex, which includes the division ring gene ftsZ, was absent. Further genome analysis identified a putative novel division ring gene unrelated to ftsZ: kustd1438. Immunogold localization studies using an antibody directed at kustd1438 located the protein to the anammox cell division ring. The bacterial cell cycle proceeds differently in different genera. In the canonical ‘E. coli version’ (Nanninga, 2001), it starts with the build-up of ATP, followed by the duplication of the chromosome and ends with the actual septation and separation of the daughter cells (cell division or cytokinesis). In the anammox case the first sign of cell division was the appearance of the division ring after which cell growth proceeded while the ring contracted, leading to a high prevalence of so-called ‘diplococci’. An estimation of the duration of the ring constriction indicated that time-wise ring constriction took 55 days, over twothirds of the length of the total cell cycle (66 days). Thus, the constriction of the ring was 10 000 times slower than cytokinesis in the rod-shaped E. coli, which takes only a third of the total 20 min cell cycle. The observation that anammox bacteria divide by constrictive binary fission was unexpected because all other members of the phylum Planctomycetes so far examined reproduce by budding (Fuerst, 1995). Further, neither transmission electron microscopy nor electron tomography showed membrane links between the anammoxosome and paryphoplasm compartment, indicating that the anammoxosome remains a separate entity during the entire cell cycle. However, because membrane topology is never preserved completely, it is impossible to entirely rule out the possibility that the anammoxosome membrane is not somehow derived from the intracytoplasmic membrane. The anammox division ring was visible as a bracket-shaped, electron-dense structure in the paryphoplasm with transmission electron microscopy. This 2D appearance was comparable to that of the putative Enterococcus gallinarum FtsZ protein (Zuber et al., 2006), and to that of the eukaryotic organelles chloroplast (plastid) and mitochondrion, where division is performed by ring complexes consisting of FtsZ, plastid-dividing or mitochondrion-dividing apparatus and dynamin rings (Miyagishima et al., 2003). Although small gaps could not be excluded for some tomograms, electron tomography showed that the presently observed structure in anammox cells was a continuous ring, unlike the discontinuous ringlike bundle and the individual filaments recently observed in Caulobacter crescentus (Briegel et al., 2006; Li et al., 2007). Of the 10 essential cell division genes, the FtsAindependent divisomal complex was present in the K. stuttgartiensis genome. This higher-order complex is believed to assemble independently from the Z-ring complex and to be recruited to midcell once the Z-ring complex is established (Goehring et al., 2006; Vicente and Rico, 2006). Clear homologues of genes encoding the Z-ring complex itself, including ftsZ, were not found in Fig. 5. Transmission electron micrographs of cryofixed, freeze-substituted and cryosectioned K. stuttgartiensis cells showing immunogold localization of the antibody directed against ORF kustd1438. The antibody localizes kustd1438 to the division ring (insets). A–F. Sectioned cells blocked with 2% skim milk powder and treated with anti-kustd1438. This antibody targets 464 aa of ORF kustd1438 that were heterologously expressed in E. coli. Scale bars: 250 nm. © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 73, 1009–1019 Cell division of anammox bacteria 1015 © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 73, 1009–1019 1016 L. van Niftrik et al. 䊏 the K. stuttgartiensis genome. The genome of K. stuttgartiensis (an environmental genome) is estimated to be 98% complete so it is still possible that ftsZ was missing from the assembly. However, all related bacteria with finished genomes certainly lack ftsZ and the K. stuttgartiensis dcw operon certainly does not encode it, strengthening the position that K. stuttgartiensis most probably lacks ftsZ. In the search for a protein that might form a higherorder structure, kustd1438 was identified as a likely candidate. An antibody raised against part of kustd1438 located the protein to the division ring in immunogold localization studies. These results show that ORF kustd1438 is part of the K. stuttgartiensis divisome complex and on basis of sequence analysis might actively contribute to ring constriction or assembly via GTP hydrolysis. One could speculate that kustd1438 substitutes for the role of FtsZ, forms a ring structure at the site of anammox cell division and recruits the FtsAindependent complex (Goehring et al., 2006; Vicente and Rico, 2006) to perform anammox cell division. There are large differences between anammox cell division and cell division involving FtsZ. First, on the level of their primary structure, kustd1438 and FtsZ are not homologous; the domain organization of kustd1438 is completely different. For more detailed analyses of kustd1438, X-ray crystallography of purified kustd1438 should be performed. Second, the observed surface area of the anammox division ring increased during constriction (see Fig. 1). This was also observed for the outer plastid-dividing and mitochondrion-dividing rings (Miyagishima et al., 1999). For FtsZ, changes in surface area would not be expected considering its dynamic nature with continuous assembly and disassembly of protofilaments (Stricker et al., 2002). Recently, electron cryotomography showed that the putative FtsZ ‘ring’ of C. crescentus consists of short individual filaments situated randomly near the division site instead of a complete ring-like structure (Li et al., 2007). It is tempting to speculate about a role in cell division of similar proteins in the evolutionarily related ftsZ-less bacteria. However, no kustd1438 homologues, at least on the basis of primary sequence similarity or structure, were detected among the phyla Chlamydiae and Planctomycetes and the more distantly related Verrucomicrobia. Thus together with the recently detected divergent homologues of FtsZ in some members of the phylum Verrucomicrobia (Pilhofer et al., 2007; Yee et al., 2007), our results suggest that in this lineage there may be no common theme in cell division other than the absence of FtsZ. In view of the amazingly different lifestyles among this lineage, from obligate intracellular parasites to the chemolithoautotrophic anammox bacteria, that is perhaps not surprising. Experimental procedures Anammox enrichment cultures Samples containing an 80% enrichment culture of either K. stuttgartiensis or B. fulgida were taken from a 2 l sequencing batch reactor or a 15 l continuous reactor modified from Strous et al. (1998). Sample preparation for transmission electron microscopy: cryofixation, freeze-substitution and Epon embedding Kuenenia stuttgartiensis cells were cryofixed by highpressure freezing, freeze-substituted in acetone containing 2% osmium tetroxide, 0.2% uranyl acetate and 1% H2O, embedded in Epon resin and sectioned as described previously (van Niftrik et al., 2008b). For B. fulgida cell division phase 4 early (Movies S2.1–S2.6) the sample was processed as described previously (van Niftrik et al., 2008b) with the following modifications. Cryofixed by high-pressure freezing in a HPM010 HPF (BAL-TEC, Balzers, Liechtenstein). Freezesubstituted in anhydrous acetone containing 2% osmium tetroxide and 0.5% uranyl acetate. Samples were kept at -90°C for 24 h, -80°C for 24 h, brought to -45°C at 2°C h-1, kept at -45°C for 2 h, brought to 0°C at 22.5°C h-1 and brought to 20°C at 10°C h-1. Samples were washed four times for 20 min with anhydrous acetone. Epon was polymerized for 24 h at 60°C. Sample preparation for immunogold localization: cryofixation, freeze-substitution and cryosectioning (rehydration method) (van Donselaar et al., 2007) Kuenenia stuttgartiensis cells were cryofixed by highpressure freezing and freeze-substituted in acetone containing 0.5% glutaraldehyde and 1% H2O as described previously (van Niftrik et al., 2008b). After freeze-substitution, fixation was continued for 60 min on ice. Samples were rehydrated in a graded acetone series on ice: 95%, 90%, 80% and 70% acetone in water containing 0.5% glutaraldehyde, then 50% and 30% acetone in PHEM buffer (60 mM Pipes, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl2, pH 6.9) containing 0.5% glutaraldehyde, and finally 0.5% glutaraldehyde in PHEM buffer. Samples were rinsed in PHEM buffer and embedded in 12% gelatin in PHEM buffer. The gelatin-embedded cells were cut into small cubes (1–2 mm3) under the stereo microscope, infiltrated overnight at 4°C with 2.3 M sucrose in PHEM buffer and frozen in liquid nitrogen. Samples were cryosectioned using a cryoultramicrotome UC6/FC6 (Leica Microsystems, Vienna, Austria). Cryosections (55 nm) were picked up with a drop of 1% methyl cellulose and 1.15 M sucrose in PHEM buffer and transferred to formvar-carbon-coated copper hexagonal 100 mesh grids for immunogold localization. Transmission electron microscopy Ultra-thin sections of Epon-embedded cells were investigated at 80–120 kV and cryosections were investigated at 80 kV in © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 73, 1009–1019 Cell division of anammox bacteria 1017 a transmission electron microscope (Tecnai12, FEI Company, Eindhoven, the Netherlands). Images were recorded using a CCD camera (MegaView II, AnalySis). Electron tomography Electron tomography was performed as described previously (van Niftrik et al., 2008b). Real-time reverse transcription PCR analysis RNA was isolated from K. stuttgartiensis cells using the Promega RNA extraction kit (Promega, Madison, USA) according to the manufacturer’s protocol. Reverse transcription was performed with a reverse primer on nucleotide position 8487 (see below) and RevertAid M_MulV (Fermentas, St. Leon-Rot, Germany). Transcription products of kustd1438 were detected using specific primers [reverse primer on nucleotide position 8487 (CTCGAGTGTCCCTGTGGTA AAACTC); forward primer on nucleotide position 8217 (CAAGCCCGGCGTATGGCGAC)]. Real-time PCR was done using the iQ custom SYBR Green supermix kit (Bio-Rad, Hercules, USA), according to the manufacturer’s instructions. The PCR programme on the Bio-Rad MyiQ was 3 min 95°C and 40 cycles of 30 s at 95°C, 30 s at 54°C and 30 s at 72°C. Two negative controls were performed: real-time PCR without a template and real-time PCR using the isolated RNA as the template (without the reverse transcription step). As a positive control, real-time reverse transcription PCR was performed with specific primers targeting the K. stuttgartiensis hydrazine hydrolase gene (unpublished results). Antibody production For the K. stuttgartiensis ORF kustd1438 antibody, we expressed and purified the region encoding the ATP/GTP binding site in E. coli as described previously (Harhangi et al., 2002), with the following changes. Two primers were designed on the sequence; a forward primer on nucleotide position 7114 (GGATCCTTTTCATCCG) and a reverse primer on nucleotide position 8487 (CTCGAGTGTCCCTGTGG TAAAACTC). For directional cloning, an XhoI restriction site was included in the reverse primer; the forward primer already contained a BamHI site. As an expression vector, pET30a (Novagen, Darmstadt, Germany) was used, and as the host, BL21 cells (Novagen, Darmstadt, Germany). The heterologous expressed 464 aa protein was purified using the nickel-nitrilotriacetic acid protein purification system (Qiagen, Venlo, the Netherlands) with an 8 M urea, 50 mM phosphate buffer with different pH (5.5, 5.0 and 4.5). The identity of the expressed protein was verified by MALDI-TOF MS peptide mass fingerprinting of a tryptic digest of the Ni-NTA purified protein (Harhangi et al., 2002). The protein was used in a 3 month immunization protocol to immunize a rabbit (SEQLAB Sequencing Laboratories Göttingen GmbH, Göttingen, Germany). This was used as the primary antibody (anti-kustd1438, polyclonal, crude serum) in immunoblotting and immunogold localization as described below. PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) gel and transferred to a cellulose-nitrate membrane (Schleicher and Schuell GmbH, Dassel, Germany) with the semidry transfer cell blotting system (Bio-Rad, Veenendaal, the Netherlands). Blotting was performed at 50 mA for 3 h with a transfer buffer that consisted of 25 mM Tris and 192 mM glycine including 20% methanol in the anode buffer and 0.05% SDS in the cathode buffer. After blotting, the blot was dried and stored at 4°C until further use. Blots stored at 4°C were washed in MilliQ for 30 min and incubated in blocking buffer: 1% bovine serum albumin in Tris-buffered saline (TBS; 10 mM Tris-HCl, 0.9% NaCl, pH 7.4) for 1 h. The blot was then incubated for 2 h in either blocking buffer or rabbit pre-immune serum diluted 250-fold in blocking buffer as the negative controls or primary antibody diluted 250-fold in blocking buffer. The blot was washed three times for 10 min in TBS containing 0.05% Tween20 and incubated for 1 h in monoclonal mouse anti-rabbit IgG alkaline phosphatase conjugate (Sigma, Zwijndrecht, the Netherlands) diluted 150 000-fold in blocking buffer. The blot was washed two times for 10 min in TBS containing 0.05% Tween20 and two times for 10 min in TBS. The blot was incubated with the BCIP/NBT Liquid Substrate System (Sigma, Zwijndrecht, the Netherlands) for 5 min, rinsed in excess amounts of MilliQ and dried. All blots were scanned with the same settings. Immunogold localization Grids containing ultra-thin cryosections of K. stuttgartiensis cells were washed for 30 min at 37°C with phosphate-buffered saline (PBS; 0.1 M phosphate, 137 mM NaCl, 2.7 mM KCl, pH 7.4), incubated for 10 min at room temperature on drops of PBS containing 20 mM glycine and blocked for 15 min on drops of PBS containing 2% skim milk powder. After blocking, the grids were incubated for 60 min with the primary antibody 80-fold diluted in PBS containing 2% skim milk powder and washed for 12 min on drops of PBS containing 0.2% skim milk powder. Grids were incubated for 20 min with the secondary antibody; protein A coupled to 10 nm gold (PAG-10), 80-fold diluted in PBS containing 2% skim milk powder and washed for 14 min on drops of PBS. The cryosections on grids were fixed for 5 min with PBS containing 1% glutaraldehyde and washed for 10 min on drops of water. Cryosections were poststained for 5 min with 2% uranyl acetate in 0.15 M oxalic acid pH 7.4 and embedded for 5 min in 1.8% methyl cellulose containing 0.4% aqueous uranyl acetate on ice after which they were air-dried. Several control treatments were performed. As a positive control, grids were incubated with rabbit anti-anammox hydroxylamine oxidoreductase (Schalk et al., 2000) as the primary antibody. Negative controls were: incubation with pre-immune serum instead of primary antibody, incubation with affinity-isolated rabbit anti-influenza haemagglutinin (anti-HA, H6908, Sigma, Zwijndrecht, the Netherlands) as a primary antibody and incubation with blocking buffer instead of primary antibody. Alignments and signal peptide prediction Immunoblotting Kuenenia stuttgartiensis proteins (cell-free extract prepared using French press) were separated on a 6% and 10% SDS- In the search for possible FtsZ homologues or substitutes, the PIR pairwise alignment tool (http://pir.georgetown.edu/ pirwww/search/pairwise.shtml) was used. For the prediction © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 73, 1009–1019 1018 L. van Niftrik et al. 䊏 of signal peptides, the SignalP tool was used (http://www.cbs. dtu.dk/services/SignalP/) using hidden Markov models and Gram-negative trained models. Reactor doubling time The reactor doubling time was determined with the anammox biomass yield (Strous et al., 1998) (0.07 C-mol/mol NH4+), ammonium consumption (0.19 mmol ammonium/min) and total protein content (19 g). The ammonium consumption was calculated by assuming that per 1.32 nitrite, 1 ammonium is consumed (Strous et al., 1998) and the total protein content was measured with the Biuret protein determination (Stickland, 1951). Relevant accession numbers NCBI (GenBank) (http://www.ncbi.nlm.nih.gov/) K. stuttgartiensis: CAJ72183, CAJ73119 to CAJ73133, CAJ75021, CAJ72236, CAJ73638, CAJ73639 CT030148, CT573071 to CT573074 E. coli K-12 MG1655: NP_414623 to NP_414638, NP_415410, NP_416907, NP_417228, NP_417294, NP_417386, NP_417919, NP_417920, NP_418070, NP_418368 Prosite (http://www.expasy.ch/prosite/) Ps01134, ps01135, ps00017 Pfam (http://www.sanger.ac.uk/Software/Pfam/) Pf02415, pf00091, pf03953, pf04022, pf02012 Acknowledgements We thank Professor Gijs Kuenen for discussions, Katinka van de Pas-Schoonen and Boran Kartal for the anammox enrichment cultures and anammox crude extract protocol, and Cathelène Carrière and Benjamin Lee for structural sequence analysis of kustd1438. References Arrigo, K.R. (2005) Marine microorganisms and global nutrient cycles. Nature 437: 349–355. Bi, E., and Lutkenhaus, J. (1991) FtsZ ring structure associated with division in Escherichia coli. Nature 354: 161– 164. 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