Cdvbased cell division and cell cycle organization in the

Cdv-based cell division and cell cycle organization in thethaumarchaeon Nitrosopumilus maritimusmmi_7834 1..12

Erik A. Pelve,1† Ann-Christin Lindås,1†

Willm Martens-Habbena,2 José R. de la Torre,3

David A. Stahl2 and Rolf Bernander1*1Department of Molecular Evolution, EvolutionaryBiology Center, Uppsala University, Norbyvägen 18C,SE-752 36, Sweden.2Department of Civil and Environmental Engineering,University of Washington, 201 More Hall, Seattle, WA98195-2700, USA.3Department of Biology, San Francisco State University,1600 Holloway Avenue, San Francisco, CA 94132-1722,USA.

Summary

Cell division is mediated by different mechanisms indifferent evolutionary lineages. While bacteria andeuryarchaea utilize an FtsZ-based mechanism, mostcrenarchaea divide using the Cdv system, relatedto the eukaryotic ESCRT-III machinery. Intriguingly,thaumarchaeal genomes encode both FtsZ and Cdvprotein homologues, raising the question of their divi-sion mode. Here, we provide evidence indicating thatCdv is the primary division system in the thaumar-chaeon Nitrosopumilus maritimus. We also show thatthe cell cycle is differently organized as compared tohyperthermophilic crenarchaea, with a longer pre-replication phase and a shorter post-replicationstage. In particular, the time required for chromosomereplication is remarkably extensive, 15–18 h, indicat-ing a low replication rate. Further, replication did notcontinue to termination in a significant fraction ofN. maritimus cell populations following substratedepletion. Both the low replication speed and the pro-pensity for replication arrest are likely to representadaptations to extremely oligotrophic environments.The results demonstrate that thaumarchaea, crenar-chaea and euryarchaea display differences not onlyregarding phylogenetic affiliations and gene content,but also in fundamental cellular and physiologicalcharacteristics. The findings also have implications

for evolutionary issues concerning the last archaealcommon ancestor and the relationship betweenarchaea and eukaryotes.

Introduction

Two main evolutionary lineages, the Crenarchaeota andEuryarchaeota phyla (kingdoms), were originally recog-nized within the Archaea domain of life (Woese et al.,1990). The phylum Thaumarchaeota was proposed in2008 (Brochier-Armanet et al., 2008) based on phyloge-netic evidence and gene content analyses, a proposalthat subsequently has gained further support (Spanget al., 2010; Gupta and Shami, 2011; Kelly et al., 2011).Thaumarchaea are ubiquitous in both marine and terres-trial biotopes and are particularly abundant in the vastaphotic zone of ocean environments (Karner et al., 2001;Leininger et al., 2006; Agogué et al., 2008), and thus rankamong the most numerous organisms on Earth. TheThaumarchaeota phylum comprises large and diverseclades of ammonia oxidizers (Francis et al., 2007; de laTorre et al., 2008; Hatzenpichler et al., 2008; Zhang et al.,2010; Urakawa et al., 2011), including the first thaumar-chaeal species to be isolated and grown in pure culture,Nitrosopumilus maritimus (Könneke et al., 2005). Bio-geochemical and physiological studies implicate thaumar-chaea as a key factor in global nitrogen cycling (Wuchteret al., 2006; Beman et al., 2008), and N. maritimus dis-plays a 200-fold higher ammonium affinity than any char-acterized bacterial ammonia oxidizer (Martens-Habbenaet al., 2009).

In all crenarchaeal species so far analysed, the cellcycle is organized into a distinct pattern in which thepost-replicative stage (G2, mitosis and cell division) is thelongest phase during exponential growth (Bernander andPoplawski, 1997; Poplawski and Bernander, 1997; Hjortand Bernander, 2001; Bernander, 2007; Lundgren et al.,2008), possibly as an adaption to life in extreme envi-ronments (Andersson et al., 2010). In contrast, the eur-yarchaeal species that have been investigated do notdisplay a coherent picture in terms of cell cycle organiza-tion (Malandrin et al., 1999; Maisnier-Patin et al., 2002;Majernik et al., 2005; Soppa, 2011). No information has,to date, been reported about the cell cycle characteristicsof thaumarchaea.

Accepted 31 August, 2011. *For correspondence. E-mail [email protected]; Tel. (+46) 18 4714698; Fax (+46) 184716404. †E.A.P. and A.-C.L. contributed equally to this work.

Molecular Microbiology (2011) � doi:10.1111/j.1365-2958.2011.07834.x

© 2011 Blackwell Publishing Ltd

Cell division is an integral part of the cell cycle in allorganisms. A majority of the euryarchaea, as well as mostbacteria, utilize a system based on the tubulin-like FtsZprotein (Bernander and Ettema, 2010). Recently a novelcell division mechanism, the Cdv machinery, was discov-ered in crenarchaea (Lindås et al., 2008; Samson et al.,2008). Two out of the three Cdv proteins display homologyto the eukaryal ESCRT-III vesicle formation system, whichalso has been implicated in cytokinesis (Morita et al.,2010; Guizetti et al., 2011). Intriguingly, genome sequen-cing of N. maritimus (Walker et al., 2010) and of othermembers of the recently recognized thaumarchaeallineage, e.g. Caldiarchaeum subterraneum (Nunouraet al., 2011), Cenarchaeum symbiosum (Hallam et al.,2006), Nitrosoarchaeum limnia (Blainey et al., 2011) andNitrososphaera gargensis (Spang et al., 2010), hasrevealed the presence of both FtsZ- and Cdv-encodinggenes, bringing up the issue of which system is utilized forcytokinesis.

Here, we provide evidence indicating that N. maritimusemploys the Cdv system for cell division, raising the ques-tion of the function of FtsZ in this organism, and presentthe first cell cycle characterization of a thaumarchaeon.

Results

To investigate cell cycle characteristics and division fea-tures, N. maritimus cells were cultivated in batch culturesat 30°C (Martens-Habbena et al., 2009). Growth wasmonitored by measuring nitrite accumulation (Martens-Habbena et al., 2009), and cells were harvested at differ-ent time points to obtain samples representing activelygrowing cultures at different cell concentrations (Fig. 1).Cell cycle and stationary phase characteristics weredetermined by flow cytometry, while division features wereinvestigated by immunofluorescence microscopy, usingpolyclonal antibodies raised against N. maritimus Cdvand FtsZ proteins.

Relative lengths of cell cycle periods

The relative number of cells in different cell cycle phasesin actively growing cultures was determined by flowcytometry analysis. The DNA content distributions (seeexample in Fig. 2A) revealed a major peak correspondingto a single copy of the chromosome (G1 stage), a propor-tion of about half the cell population containing betweenone and two chromosome equivalents (S phase), and aminor peak corresponding to two fully replicated chromo-somes (cells in the G2, mitosis and division stages).

To calculate the length of the cell cycle phases in theindividual cell, all samples collected from actively growingcultures (Fig. 2B; also from cultures not shown) were sub-jected to theoretical simulations (Michelsen et al., 2003),

in which the exponential age distribution was taken intoaccount, i.e. the fact that there are twice as manynewborn as dividing cells. About two-fifths of the cellpopulations in actively growing cultures contained a singlechromosome, resulting in a pre-replicative period lengthof 19–29% of the cell cycle, depending on sample, or6–10 h at a doubling time of about 33 h (calculated fromFig. 1). The proportion of replicating cells was 45–53%,corresponding to an S phase length of 15–18 h, and thepost-replicative period was estimated to 24–30% of thecell cycle (8–10 h). A schematic illustration of the deducedcell cycle is shown in Fig. 3.

Growth cessation and rate of replication fork movement

The overall organization of the cell cycle (above) con-trasted markedly with that observed in hyperthermophiliccrenarchaea (Bernander and Poplawski, 1997; Poplawskiand Bernander, 1997; Hjort and Bernander, 2001;Bernander, 2007; Lundgren et al., 2008), for which therelative peak heights in DNA content distributions areopposite relative to N. maritimus, i.e. most cells reside inthe post-replicative cell cycle stages (cells with two copiesof the chromosome) during exponential growth, while theproportion of pre-replicative cells (single chromosome) issmall. An additional characteristic of hyperthermophiliccrenarchaea is a distinct tendency to rest in the post-replicative cell cycle phase when growth has ceased (Ber-nander and Poplawski, 1997; Hjort and Bernander, 1999;Lundgren et al., 2008). Again, N. maritimus differed fromcrenarchaea, in that the proportion of cells containing two

Time [days]

Nitrite

[µM

]

Fig. 1. Growth of N. maritimus batch cultures. Growth of replicatebatch cultures measured by monitoring nitrite accumulation. Arrowsindicate sampling for flow cytometry.

2 E. A. Pelve et al. �

© 2011 Blackwell Publishing Ltd, Molecular Microbiology

chromosomes decreased when nitrite accumulation (i.e.cell multiplication) declined (Fig. 4), showing that cell divi-sion continued in the cultures during entry into stationaryphase. Interestingly, despite continued incubation forseveral days after cessation of growth, the populationsnever reached a state in which the cells exclusively con-tained fully replicated chromosomes. Many cells that werein S phase at the time when ammonium was depleted andcellular growth ceased did, thus, not complete replicationand arrested with a genome content between one and twochromosomes. This could be related to the extremely slowrate of replication in N. maritimus (below), and might be a

characteristic feature in the extremely oligotrophic envi-ronments in which marine thaumarchaea reside (seeDiscussion).

With a genome size of 1.645 Mb (Walker et al., 2010)and assuming bidirectional replication from a single chro-mosome replication origin, an S phase length of 15–18 hcorresponds to a replication rate of 13–15 bp s-1 for an

A

B1 2

DNA content

(number of chromosomes)

1 2

Rela

tive

nu

mb

er o

f cel

lsRe

lati

ve n

um

ber

of c

ells

Fig. 2. Flow cytometry analysis and cell cycle simulations ofN. maritimus.A. Flow cytometry DNA content distribution corresponding to 9 daytime point in Fig. 1.B. Winflow (Michelsen et al., 2003) simulation of DNA contentdistribution in A.Explanation of designations: diagonal stripes, cell populationscontaining a single chromosome (cells in pre-replicative stage; G1

phase; see Fig. 3) or two chromosomes (post-replicative stage; G2,M and D; see Fig. 3); grey shading, cells containing between oneand two genome equivalents (S phase); solid line, combineddistribution.

Fig. 3. Cell cycle of N. maritimus. Explanation of designations: G1,gap 1 phase; S, DNA synthesis (replication) phase; G2, gap 2phase (may not exist in N. maritimus); M, mitosis (genomesegregation); D, division (cytokinesis). The micrographs wereselected to illustrate different cell cycle stages although the precisephase of a cell with a single fluorescence focus is unknown: an Sstage cell is generally larger and displays a stronger fluorescencesignal (increased DNA content resulting from chromosomereplication) than a G1 cell. Cell perimeters were manually enhancedfor clarity. Blue (DNA) and green (CdvA) fluorescence signals weregenerated as explained in the legend to Fig. 5.

A B

DC

9 days 12 days

14 days 19 days

DNA content

(number of chromosomes)

Rela

tive

nu

mb

er o

f cel

ls

1 21 2

1 2 1 2

Fig. 4. DNA content distributions during substrate deprivation.Flow cytometry DNA content distributions from samples of anN. maritimus batch culture at different time points (see Fig. 1).

Cdv-based cell division in N. maritimus 3


individual replication fork. If the N. maritimus chromosomewould contain multiple replication origins, similar to arange of crenarchaeal species (Lundgren et al., 2004;Robinson et al., 2004; Robinson and Bell, 2007), thespeed of individual fork movement would be even lower.Even assuming a single origin, this is a low replication ratecompared to other, hyperthermophilic, archaeal speciesanalysed (80–330 bp s-1) (Myllykallio et al., 2000;Lundgren et al., 2004), and more similar to rates observedin eukaryotic organisms (Jackson and Pombo, 1998;Conti et al., 2007; de Moura et al., 2010). The low rate islikely to be an effect of the lower temperature at whichDNA polymerization occurs, relative to hyperthermo-philes, combined with adaptation to the low nutrient avail-ability in the natural environment of Nitrosopumilusspecies, with consequent limitations in terms of energyand precursors for DNA synthesis.

Cell division

Intriguingly, thaumarchaeal genomes contain genes asso-ciated with both main cell division systems used by bac-teria and archaea. Antibodies against CdvA (Nmar_0700),three CdvB paralogues (Nmar_0029, Nmar_0061 andNmar_0816), CdvC (Nmar_1088) and FtsZ (Nmar_1262)were used to elucidate which of the two systems is utilizedby N. maritimus. Immunofluorescence microscopy of cellsstained with antibodies against CdvA revealed sharp, cen-trally positioned, band-like structures in a subpopulationconsisting of 20–25% of the cells (Fig. 5A). The structurescorrelated strongly with the presence of segregatednucleoids (Fig. 5B), similar to patterns observed in cre-narchaea from the genus Sulfolobus (Lindås et al., 2008;Samson et al., 2008). Conversely, in cells in which nosegregated nucleoids could be discerned, CdvA bandstructures were rarely observed. Staining with antibodiesagainst CdvC resulted in a similar pattern (Fig. 6), indicat-ing colocalization of the two proteins in a joint central cellstructure during the division stage of the cell cycle(Samson et al., 2011).

Two of the three CdvB paralogues, Nmar_0029 andNmar_0816, also correlated with the presence of segre-gated nucleoids and, further, also with the presence ofCdvA and CdvC in double stainings (Fig. 7A and B), i.e.the CdvB proteins were rarely observed in cells lackingeither of the other two Cdv proteins. In contrast to CdvAand CdvC, however, distinct band structures were not

detected: most cells instead displayed evenly distributedfluorescence across the interior.

Presence of the FtsZ protein was neither temporally norspatially correlated with nucleoid segregation and noband structures were observed (Fig. 8). Instead, FtsZ, aswell as the third CdvB paralogue, Nmar_0061 (Fig. 8C),were evenly distributed in a majority of the cell populationregardless of cell cycle stage, i.e. independent of cell sizeor nucleoid distribution.

In addition to direct observation of division structures,the microscopy analyses provided information aboutoverall cell cycle parameters. Since the fraction of cellscontaining two chromosome equivalents in the flowcytometry analysis was in the order of 25–30%, the factthat 20–25% of the cells displayed segregated nucleoidsin fluorescence microscopy indicated that genome segre-gation occurred within a short time interval after termina-tion of chromosome replication, i.e. that the G2 stage isshort, or absent, in N. maritimus. Finally, cells undergoingnucleoid segregation and cell division displayed a dis-tinctly elongated morphology (e.g. Fig. 5B), showing thatdynamic changes in cell organization occurred during cellcycle progression.

Discussion

We report evidence indicating that N. maritimus utilizesthe Cdv machinery for cell division, and also show that thecell cycle displays strikingly differential characteristicsas compared to species belonging to the crenarchaeallineage, with which thaumarchaea were previouslygrouped.

The CdvA and C proteins formed distinct centrallypositioned structures, presumably ring-shaped, betweensegregated nucleoids, similar to the Cdv machinery ofSulfolobus (Lindås et al., 2008; Samson et al., 2008). Twoout of three CdvB paralogues correlated with the pres-ence of CdvA and CdvC, and also with segregated nucle-oids, indicating participation in the same process. Theabsence of band-like structures for these could reflectmechanistic differences, less stable association with thedivision structure, or a large protein excess that obscuredvisualization. Regardless, we conclude that Cdv proteins,rather than the FtsZ machinery as previously suggested(Makarova et al., 2010; Samson and Bell, 2011), are usedfor cell division in thaumarchaea, and that division is likelyto occur in a similar mode as in crenarchaea. In terms

Fig. 5. Distribution of the CdvA protein in actively growing N. maritimus cells. The cells were incubated with antibodies against the CdvAprotein, followed by staining with secondary antibodies conjugated to Alexa Fluor 568 (red fluorescence). Nucleoids were visualized with theDNA-specific DAPI stain (blue).A. Overview of frequency of cells displaying CdvA-dependent fluorescence.B. Enlarged images of individual cells from A (rows 1–3) or from additional micrographs (rows 4–5).For clarity, cell perimeters have been manually enhanced in the stainings: actual cell perimeters may be observed in the phase contrastimages (first column). Scale bar, 1 mm.



ACell shape Nucleoids CdvA Merged

12

1

2

3

3

BCell shape Nucleoids CdvA Merged

1 1 12 2 2

3 3 3

I

II

III

IV

V



of regulatory features, the cdv genes are cell-cycle-specifically transcribed in S. acidocaldarius, such thatstrong induction occurs shortly before the division stage(Lundgren and Bernander, 2007). The specific correlationbetween Cdv fluorescence foci, elongated cells and seg-regated nucleoids indicate similar regulation of cdvexpression in N. maritimus.

The FtsZ protein, as well as one CdvB paralogue,showed no correlation to nucleoid distribution or cell cyclestage, no band-like structures, and a largely uniform intra-cellular distribution. In Sulfolobus, involvement in viralrelease (Ortmann et al., 2008; Snyder and Young, 2011)and vesicle budding (Ellen et al., 2009) for Cdv proteinshas been discussed, and one or more of the Nitrosopumi-lus CdvB homologues could also be involved in processesother than cytokinesis, in line with their disperse localiza-tion in the genome. Since we find no support in our datafor a cytokinesis function for FtsZ, even if a subtle rolecannot be ruled out, we propose that the protein has

evolved a different function in thaumarchaea. In agree-ment, thaumarchaeal FtsZ sequences form a well-supported distinct branch in phylogenetic analyses(Makarova and Koonin, 2010), separate from bacterialand euryarchaeal clades containing FtsZ proteins shownto be involved in cell division. In further support of analternate role, atypical FtsZ homologues suggested tofunction in other membrane-remodelling processes thancytokinesis have been reported in Sulfolobus (Makarovaet al., 2010), in which division is Cdv-mediated. In thiscontext, it is also of interest that FtsZ homologues (TubZ)in Bacillus species form axial filaments that contribute toplasmid segregation (Ni et al., 2010), suggesting that aninvolvement in chromosome segregation for the N. mar-itimus homologue is conceivable.

The N. maritimus DNA content distributions represent amirror image of those observed in crenarchaea, thus con-stituting a differential feature between organisms from theThaumarchaeota and Crenarchaeota phyla. The fact that

Fig. 6. Distribution of the CdvC protein inactively growing N. maritimus cells. The cellswere incubated with antibodies against theCdvC protein, followed by staining withsecondary antibodies conjugated to AlexaFluor 488 (green fluorescence). Nucleoidstaining and manual enhancement of cellperimeters is described in the legend toFig. 5. Scale bar, 1 mm.

Cell shape Nucleoids CdvC Merged

Fig. 7. Distribution of CdvB proteins in actively growing N. maritimus cells.A. Cells incubated with antibodies against the CdvA and CdvB (Nmar_0029) proteins.B. Cells incubated with antibodies against the CdvA and CdvB (Nmar_0816) proteins.Secondary antibody staining, nucleoid staining and manual enhancement of cell perimeters is described in the legends to Figs 5 and 6. Scalebar, 1 mm.



ACell shape Nucleoids CdvA

CdvB(Nmar_0029)

CdvA+

NucleoidsCdvB+

Nucleoids

BCell shape Nucleoids CdvA

CdvB(Nmar_0816)

CdvA+

Nucleoids

CdvB+

Nucleoids



CdvA FtsZNucleoidsCell shapeA

Cell shape Nucleoids CdvB(Nmar_0061) FtsZ

BCell shape Nucleoids CdvA FtsZ

C

CdvA+Nucleoids

FtsZ+Nucleoids

CdvA+Nucleoids

FtsZ+Nucleoids



the proportion of cells with segregated nucleoids in themicroscopy analyses was similar to the fraction containingtwo genome equivalents in flow cytometry, indicates thatgenome segregation occurs rapidly after termination ofreplication in N. maritimus, and that the G2 phase, conse-quently, is short or absent, also in contrast to the situationin crenarchaea. It should, however, be noted that theseare relative numbers: at a doubling time around 33 h,even a G2 period of only about 3% would last a full hour.

The general tendency towards a genome content of asingle chromosome could reflect the different environ-ment the organism is adapted to, relative to Sulfolobusspecies. While multiple copies of the chromosome mayconstitute an advantage under the highly mutagenicgrowth conditions of hyperthermophilic crenarchaea(Andersson et al., 2010), a single copy of the genomemay suffice in the environmentally stable deep-sea con-ditions characteristic of marine thaumarchaea. We specu-late that a low DNA content even might provide anadvantage, and thus be positively selected for, by reduc-ing cellular costs for chromosome maintenance and DNArepair (cf. Giovannoni et al., 2005), in line with the fact thatthe N. maritimus genome is one of the smallest amongfree-living organisms (Walker et al., 2010).

The most remarkable aspect of the cell cycle organiza-tion is the extensive time, in absolute terms, required forreplication: an N. maritimus cell, thus, spends 15–18 hreplicating a genome of only 1.645 Mb (Walker et al.,2010). This requires continual maintenance of all repli-some components, and supply of DNA precursors, overextended time periods, and also results in long-term genedosage effects, with genes located in the origin-proximalpart of the chromosome being present in two copies formany hours, relative to a single copy for genes closer tothe terminus.

The fact that chromosome replication did not continue tocompletion in many cells after growth arrest could berelated to the slow replication rate. If the time required tocomplete replication after substrate depletion exceededthat during which the cells could supply sufficient metabolicenergy and precursors for DNA synthesis, a fraction of thecell population would arrest during replication. Notably,genome sequence analysis, as well as electron micros-copy, has suggested that N. maritimus may not producesignificant amounts of energy reserves such as glycogen,polyhydroxybutyrate or polyphosphate (Walker et al.,2010; Urakawa et al., 2011). Thus, in contrast to crenar-

chaea and many bacteria, N. maritimus cells may notcontain sufficient energy reserves to complete replicationafter extended periods of substrate depletion. In themarine environment, N. maritimus-like thaumarchaeamay, however, rarely become completely deprived of sub-strate due to their extremely high affinity for ammonium(Martens-Habbena et al., 2009) and, thus, seldom, if ever,experience comparable arrest of growth and replication.

The suggestion that Thaumarchaeota constitutes aseparate phylum is supported not only through phyloge-netic inferences but also by comparative genomics,showing that thaumarchaeal chromosomes contain amixture of genes previously assumed to be specific foreither Crenarchaeota or Euryarchaeota (Brochier-Armanet et al., 2008; 2011; Spang et al., 2010; Walkeret al., 2010; Kelly et al., 2011). We demonstrate that alsoat the cellular and physiological levels, the properties ofthe thaumarchaeon N. maritimus are mixed. The cell divi-sion process is, thus, similar to that of several crenar-chaeal clades while the cell cycle organization differsconsiderably and, although the FtsZ protein is shared witheuryarchaea, it does not play the same pivotal role, if any,in cytokinesis. Yet another differential feature concernsthe mode of interaction between CdvA and CdvB, sinceprotein domains important for binding in crenarchaeaare missing in N. maritimus (Samson et al., 2011). Theabsence of CdvA in Caldiarchaeum subterraneum(Nunoura et al., 2011), in which both CdvB and CdvC, aswell as FtsZ, are present, indicates further cell divisionvariation among deeply branching archaea.

Phylogenomic evidence suggests that the last commonancestor of archaea and eukaryotes contained a combi-nation of cdv and ftsZ genes (Makarova et al., 2010), andthat Thaumarchaeota may be among the archaeal lin-eages most closely related to Eukarya (Foster et al.,2009; Gribaldo et al., 2010; Kelly et al., 2011), furtherstrengthened by our observation of similarities in DNAsynthesis rates. Characterization of the physiological, cel-lular and genomic properties of additional thaumarchaealspecies, in particular soil isolates (Schleper et al., 2005;Schleper and Nicol, 2010), and of other recently discov-ered deep archaeal lineages (de la Torre et al., 2008;Spang et al., 2010; Blainey et al., 2011; Nunoura et al.,2011) will, thus, be necessary to increase our understand-ing of issues related to the origin of the eukaryotic lineage,and of the fundamental evolutionary relationshipsbetween the three domains of life.

Fig. 8. Distribution of FtsZ and Cdv proteins in actively growing N. maritimus cells.A. Overview of frequency of cells displaying CdvA- and FtsZ-dependent fluorescence.B. Enlarged images of individual cells displaying CdvA- and FtsZ-dependent fluorescence.C. Enlarged images of individual cells displaying CdvB (Nmar_0061)- and FtsZ-dependent fluorescence.Secondary antibody staining, nucleoid staining and manual enhancement of cell perimeters is described in the legends to Figs 5 and 6. Scalebar, 1 mm.



Experimental procedures

Growth, harvest and DNA extraction

Nitrosopumilus maritimus strain SCM1 was grown in liquidbatch culture and growth was monitored by accumulation ofnitrite as described previously (Martens-Habbena et al.,2009). For flow cytometry, 50–100 ml samples from four dif-ferent time points in two parallel cultures were deposited byfiltration onto 47 mm polycarbonate membranes (0.1 mm poresize, Whatman) at 4°C by vacuum filtration. Filters wereimmediately transferred to 50 ml tubes (BD Falcon) contain-ing 70% ethanol, and kept on ice. Cells were resuspended bygently rinsing off the filter. The same protocol was used forsampling for microscopy, except that 450 ml duplicatesamples were used. DNA was extracted as described earlier(Urakawa et al., 2010).

Cloning and protein purification

Genes encoding Cdv and FtsZ proteins were amplified using aBIO-X-ACT short PCR kit (BIOLINE), and cloned into thepET-45b(+) vector (Novagen) using T4 DNA ligase (BIOLINE),generating recombinant proteins with an N-terminal hexa-histidine tag. All constructs were verified by DNA sequencing(Big Dye Terminator v3.1 Cycle Sequencing kit, Applied Bio-systems) before electroporation into Escherichia coli RosettaDE3 cells. The histidine-tagged proteins were affinity-purifiedon 5 ml HiTrap NiChelating HP columns (GE Healthcare) andeluted with a 0–500 mM imidazole gradient in 20 mM sodium-phosphate buffer, pH 7.4, containing 0.5 M NaCl. The imida-zole was removed using size exclusion chromatography(PD-10 columns; GE Healthcare).

Antibody generation

Polyclonal antibodies against purified proteins were exter-nally produced (Innovagen, Lund, Sweden). For Nmar_0029,Nmar_0816 and Nmar_1262 antibodies were raised in rabbit,and for Nmar_0061, Nmar_0700 and Nmar_1088 antibodieswere raised in chicken.

Immunostaining and fluorescence microscopy

Ethanol-fixed N. maritimus cells were washed with PBSTbuffer (phosphate-buffered saline containing 0.05% Tween20) and stained with primary and secondary antibodies aspreviously described (Lindås et al., 2008). Immunostainedcells were added to a thin layer of 1% agarose containing5 mg ml-1 of the DNA-specific DAPI (4′,6-diamidino-2-phenylindole) stain on a glass slide, and visualized using aDMRXE epifluorescence microscope (Leica) at 1000-foldmagnification, also as described previously (Lindås et al.,2008).

Flow cytometry and determination of relative lengthsof cell cycle periods

Nitrosopumilus maritimus cells fixed in ethanol were washedwith TE buffer (pH 7.9), stained with 2 mM SYTO 13 green

fluorescent nucleic acid stain (Invitrogen) in TE buffer, andanalysed in a customized Apogee A40 Analyzer flow cytom-eter using a 488 nm wavelength solid-state laser. Alterna-tively, the cells were stained with a combination of ethidiumbromide and mithramycin A and analysed with a 405 nmsolid-state laser in the same instrument, as described previ-ously (Lundgren et al., 2008).

The relative number of cells in the pre-replicative, replica-tive and post-replicative stages in the DNA content histo-grams were simulated with Winflow software (Michelsenet al., 2003), until good fits between the theoretical simula-tions and the experimental data were obtained.

Acknowledgements

We thank Anna Knöppel for artwork in Fig. 3. This workwas supported by grant 621-2010-5551 from the SwedishResearch Council, by a Strong Research Environment grant(Uppsala Microbiomics Center) from the Swedish ResearchCouncil for Environment, Agricultural Sciences and SpatialPlanning to R.B., by NSF award MCB-0604448 to D.A.S. andJ.R.T., by NSF award OCE-0623174 to A. E. Ingalls, D.A.S.,and by A. H. Devol, and NSF Award MCB-0920741 to D.A.S.and M. Hackett.

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