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Cell division in the archaeon Haloferax volcanii relies on two FtsZ proteins with distinct functions in division ring assembly and constriction

Nature Microbiology volume 6, pages 594–605 (2021)Cite this article

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Abstract

In bacteria, the tubulin homologue FtsZ assembles a cytokinetic ring, termed the Z ring, and plays a key role in the machinery that constricts to divide the cells. Many archaea encode two FtsZ proteins from distinct families, FtsZ1 and FtsZ2, with previously unclear functions. Here, we show that Haloferax volcanii cannot divide properly without either or both FtsZ proteins, but DNA replication continues and cells proliferate in alternative ways, such as blebbing and fragmentation, via remarkable envelope plasticity. FtsZ1 and FtsZ2 colocalize to form the dynamic division ring. However, FtsZ1 can assemble rings independent of FtsZ2, and stabilizes FtsZ2 in the ring, whereas FtsZ2 functions primarily in the constriction mechanism. FtsZ1 also influenced cell shape, suggesting it forms a hub-like platform at midcell for the assembly of shape-related systems too. Both FtsZ1 and FtsZ2 are widespread in archaea with a single S-layer envelope, but archaea with a pseudomurein wall and division septum only have FtsZ1. FtsZ1 is therefore likely to provide a fundamental recruitment role in diverse archaea, and FtsZ2 is required for constriction of a flexible S-layer envelope, where an internal constriction force might dominate the division mechanism, in contrast with the single-FtsZ bacteria and archaea that divide primarily by wall ingrowth.

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Fig. 1: Depletion of FtsZ1 or FtsZ2 results in cell division defects.
Fig. 2: FtsZ1 and FtsZ2 are dispensable for survival but required for normal cell division.
Fig. 3: Overproduction of FtsZ1 or FtsZ2 differentially affects cell division and shape.
Fig. 4: Midcell localization of FtsZ1 and FtsZ2.
Fig. 5: Localization interdependency and the effect of ftsZ mutations on FtsZ1–mCh and FtsZ2–GFP localization.
Fig. 6: Model of FtsZ1 and FtsZ2 function at the archaeal division site.

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Data availability

The NCBI (www.ncbi.nlm.nih.gov/protein/) and UniProt (www.uniprot.org) protein sequence databases were searched to obtain sequences of archaeal tubulin superfamily proteins. Genome sequence data generated in this study have been deposited in the NCBI BioProject database under the identifier PRJNA681931. Source data are provided with this paper. Other data and biological materials are available from the corresponding author upon reasonable request.

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Acknowledgements

This study was supported by the Australian Research Council (FT160100010 to I.G.D.) and UK Biotechnology and Biological Sciences Research Council (BBSRC) and Medical Research Council (U105184326 to J.L.). For technical support, we thank L. Turnbull, M. Johnson and L. Cole (from the UTS Microbial Imaging Facility) and K. Anantanawat and M. Liu (from the UTS DNA Sequencing Facility).

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  1. These authors contributed equally: Yan Liao, Solenne Ithurbide.

Authors and Affiliations

  1. The iThree Institute, University of Technology Sydney, Sydney, New South Wales, Australia

    Yan Liao, Solenne Ithurbide, Christian Evenhuis & Iain G. Duggin

  2. MRC Laboratory of Molecular Biology, Cambridge, UK

    Jan Löwe

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Contributions

I.G.D., S.I., Y.L. and J.L. designed the research. I.G.D., S.I. and Y.L. collected the preliminary data and constructed the strains. The following authors contributed and analysed the data: Y.L. (Figs. 1, 2b–i, 3, 5a and 6, Extended Data Figs. 2, 4 and 5, Supplementary Figs. 1–8 and 10, Supplementary Table 5 and Supplementary Videos 1–10), S.I. (Figs. 2a, 4a,b,d, 5a–d and 6, Extended Data Figs. 6–10, Supplementary Figs. 4 and 9–11, Supplementary Tables 1 and 2 and Supplementary Video 11), I.G.D. (Figs. 4c, 5a,d and 6, Extended Data Figs. 1, 3 and 8, Supplementary Fig. 1, Supplementary Tables 1–5 and Supplementary Videos 9 and 10) and C.E. (Fig. 5d, Extended Data Figs. 7 and 8 and Supplementary Fig. 4). I.G.D., Y.L., S.I., J.L. and C.E. interpreted the results, wrote and reviewed the manuscript and produced the figures. I.G.D. and J.L. managed and supervised the project and acquired funding.

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Correspondence to Iain G. Duggin.

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The authors declare no competing interests.

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Peer review information Nature Microbiology thanks Daniela Barilla, Joe Lutkenhaus, William Margolin and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Molecular phylogeny and comparison of archaeal FtsZ1 and FtsZ2 families.

a, Maximum likelihood phylogenetic tree based on an alignment of the tubulin superfamily proteins identified in 60 diverse archaeal genomes (Supplementary Table 3), and the 13 bacterial and plant sequences used to identify them. Bootstrap support is shown for selected branches (%). b, Domain organization and percent sequence identities for FtsZ1 and FtsZ2. The percentages over the domains (green and purple boxes) indicate the average sequence identity in that region for each H. volcanii FtsZ compared to all of the other members of the same family that were identified in the Archaea domain. The region between FtsZ1 and FtsZ2 represents the percent identity in the region between the H. volcanii FtsZ1 and FtsZ2 (%ID). The approximate location of conserved sequence motifs within the tail regions are indicated by vertical bars, coloured to indicate similarities between the two. c, Aligned sequence regions containing conserved differences between the bacterial/plant FtsZ and the archaeal FtsZ1 and FtsZ2 families, labelled with the secondary structural elements. Boxed residues indicate conserved sites that are displayed in panel (e). d, Crystal structure of FtsZ1 from Methanocaldococcus jannaschii (PDB: 1FSZ), with selected loops (T4-T7) involved in nucleotide binding and hydrolysis shown in pink. GDP is shown in orange, and the main domains are coloured as in panel (b). Boxed regions are expanded in panel (e), which displays some conserved residues that characteristically differ between the FtsZ1 and FtsZ2 families (grey space-filling models, with FtsZ2 consensus residues in parentheses) and cluster around the nucleotide-dependent polymerization surfaces.

Source data

Extended Data Fig. 2 Partial division phenotypes during depletion of FtsZ1 or FtsZ2.

a, H. volcanii ID56 (p.tna-ftsZ1) was cultured in Hv-Cab + 2 mM Trp, and then loaded into a microfluidics platform and cultured with a flow of Hv-Cab (without Trp) over 15 h (0.5 p.s.i) to deplete FtsZ1. Shown is one cell that was identified to divide (unilaterally), even after ~9 h of depletion, and then one cell exhibited a budding-like process (arrows). Scale bars, 2 μm. b, H. volcanii ID57 (p.tna-ftsZ2) was pre-cultured in Hv-Cab + 2 mM Trp, and then loaded into a microfluidics platform and cultured with a flow of Hv-Cab + 2 mM Trp for 3 h, followed by Hv-Cab (no Trp) for 10 h (2 p.s.i) to deplete FtsZ2. The zero timepoint represents the start of medium flow without Trp. During the early stage of depletion of FtsZ2, partial constrictions were sometimes observed, as seen in these two examples (i and ii), but these never completed division and the constriction eventually reversed over several hours (see arrows). Cells, however, retained some apparent ‘memory’ of the initial constriction often manifesting as a somewhat bilobed shape. The data shown is representative of at least two independent experiments. Scale bars, 2 μm.

Extended Data Fig. 3 Cellular DNA content during depletions of FtsZ1 and FtsZ2.

a, SYTOX Green (SG) DNA staining of cells sampled from cultures 18 h after resuspension of mid-log cells in media without Trp. Stained cells were placed on an agarose pad and visualized by differential-interference contrast (DIC) and fluorescence microscopy (lower panels). Scale bars, 5 μm. b, Flow cytometry analyses of cells sampled as per panel (a) (upper three panels), displaying side-scatter (as a proxy for cell size) versus SYTOX Green (SG)-DNA fluorescence. The lower three panels represent cultures treated in the same way, except 0.5 mM Trp was included in the medium. The data shown is representative of at least two independent experiments. The individual datapoints represent the area under the curve of each event detected; events were detected by a threshold of the side-scatter signal. After 18 h of ftsZ1 or ftsZ2 depletion, many very large cells with correspondingly high DNA content were observed, consistent with the images shown in panel (a). This indicates that DNA synthesis continues in proportion to the increase in cell volume during inhibition of cell division caused by depletion of FtsZ1 or FtsZ2.

Extended Data Fig. 4 Complementation of ΔftsZ1 ΔftsZ2.

a, Phase-contrast images (left) and Coulter cytometry (right) of strains based on H. volcanii ID112 (ΔftsZ1 ΔftsZ2), plus pTA962-based plasmids expressing the indicated ftsZ genes, sampled during mid-log growth with the indicated concentrations of Trp. The same dataset for the wild-type control (H26 + pTA962) is shown in all graphs as a reference. Scale bars, 5 μm. b, Corresponding western blot analyses of FtsZ1 and FtsZ2 protein levels in total cell extracts of the indicated strains. The data shown is representative of two independent experiments.

Source data

Extended Data Fig. 5 Comparison of ftsZ mutant cellular phenotypes in log and stationary phases.

The differing functions of FtsZ1 and FtsZ2 were also apparent when cultures of the knock-out, complementation and overexpression strains were compared in mid-log and stationary phases. Phase-contrast images (left) and Coulter cytometry distributions (right) of the wild-type and overexpression strains (a) and the indicated ftsZ knockout and complementation strains b, c, all grown in Hv-Cab with the indicated concentration of Trp and sampled at mid-log and stationary phases. The data shown are representative of at least two independent experiments. Scale bars, 5 μm. Compared to mid-log cells, all the strains except the strains without a copy of ftsZ2, tended towards the wild-type size (smaller) and regular plate morphology in stationary phase. The ΔftsZ2 strains were somewhat smaller in stationary phase, but maintained greatly enlarged giant plate and elongated cells, suggesting a poor recovery as cell growth slows in stationary phase. These findings suggest that FtsZ2 confers a partial ability to divide and recover more normal cell sizes as the cell growth rate slows in stationary phase, whereas cells without FtsZ2 have a much stronger block to division that is maintained even as cells slow or stop growth in stationary phase.

Source data

Extended Data Fig. 6 FtsZ1 and FtsZ2 fluorescent fusions are not fully functional as sole copies but at moderate concentrations have minimal impact on cell division in the wild type.

a-b, FtsZ1 and FtsZ2 fluorescent fusion proteins were functionally tested in their respective ΔftsZ1 or ΔftsZ2 backgrounds by phase-contrast and fluorescence microscopy (left) and by Coulter cytometry (right) for cell size. FtsZ1-GFP and FtsZ1-mCherry (0.2 mM Trp) partially complement ΔftsZ1. b, FtsZ2-GFP was unable to complement the ΔftsZ2 background, whereas the untagged protein achieves full complementation (0.2 mM Trp). The same dataset for the wild-type control is shown in both graphs as a reference. c-e, When expressed in wild-type cells, FtsZ1-mCherry or FtsZ2-GFP, or both, cause minimal effects on cell size and shape at a moderate level of expression (0.2 mM Trp), and show sharp midcell bands. These proteins are therefore useful localization markers for division, although detailed analyses of FtsZ subcellular ultrastructure and dynamics await the development of functional complete labelling. The data shown are representative of at least two independent experiments. Scale bars, 5 µm.

Source data

Extended Data Fig. 7 Cell shape analyses for FtsZ localization interdependency studies.

Cell area and shape (circularity) were determined for individual cells, as per Fig. 5 (0.2 mM Trp), and data were combined from two replicate experiments in for each plot. The plots are labelled with the strain’s relevant genomic background (left) and the ftsZ variant(s) expressed on the plasmid (right). The data shown are representative of at least two independent experiments.

Source data

Extended Data Fig. 8 FtsZ1-mCh localization in ftsZ2-mutant strains.

a, Demonstration of the automated image analysis procedure for determining FtsZ localization parameters. Cell outlines were obtained (red), and the fluorescence (FtsZ1-mCherry in yellow) was quantified by averaging the intensity on the transverse axis to create a longitudinal intensity profile. Gaussian peaks were fitted to the detected localizations and a spline fit to the background. The localization thickness (W) was taken as the width of the fitted Gaussian peaks at half height (μm), and the intensity (I) was taken as the integrated peak area (per μm across the cell). See Methods for further details. b, Histograms for cells with the indicated number of localizations versus cell length for ΔftsZ2 + FtsZ1-mCh. Colored lines indicate the lengths of cells that have the indicated relative number of localizations per unit length. c, Violin plots of the thickness of FtsZ1-mCh localization in the indicated strain backgrounds; the median is indicated by a white dot, the thick bar is the interquartile range, and thin bar is the 9th-91st percentile range. The data shown are representative of at least two independent experiments. Explanation of the experiment using the ΔftsZ2 + FtsZ2.D231A-GFP + FtsZ1-mCh strain is given in the Supplementary results and discussion and Extended Data Fig. 10.

Source data

Extended Data Fig. 9 Localization of FtsZ T7-loop mutants, ftsZ1.D250A-mCh and ftsZ2.D231A-GFP.

a-b, The FP-tagged T7 mutants fail to complement their respective ΔftsZ strain. c-d, Suppression of the severe dominant-inhibitory effects of the T7 mutants by the fluorescent tags, shown by phase-contrast and fluorescence microscopy (left) and Coulter cytometry (right). FtsZ1.D250A-mCh showed aberrant localization and cellular distortions and envelope protrusions associated with the fluorescent filaments; small fluorescent particles detatched from cells are also evident (arrowheads). Yet the cell size distribution was only subtly affected. The data shown are representative of at least two independent experiments. Similar results were obtained with FtsZ1.D250A-GFP (H. volcanii ID153). FtsZ2.D231A-GFP shows similar localization to wild-type (FtsZ2-GFP), with only a moderate increase in cell size increase observed at 1 mM Trp; note that FtsZ2-GFP had a much stronger influence (Extended Data Fig. 6d). Scale bars, 5 μm.

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Extended Data Fig. 10 Co-localization studies of wild-type FtsZ and T7-loop mutants.

Fluorescence microscopy (overlay of GFP and mCherry channels; co-localization appears white) (left) and cell size/shape analysis plots (right) for the indicated strains (grown with 0.2 mM Trp) containing one tagged wild-type protein and the alternate tagged the T7-loop mutant. The data shown are representative of at least two independent experiments. In panels (b) and (d), results with cells grown with 1 mM Trp is shown in the lower insets; the morphology percentages for these were: (b) 95.4% wild-type-like, 3.2% giant plates, 0.9% filaments, and 0.5% debris (n = 439), and (d) 40.7% wild-type-like, 25.5% giant plates, 29.1% filaments, and 5.1% debris (n = 196). Scale bars, 5 μm.

Source data

Supplementary information

Supplementary Information

Supplementary Results, Discussion, Figs. 1–11, Tables 1–5 and references, captions for Supplementary Videos 1–11, and a source data file inventory.

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Peer Review File

Supplementary Video 1

Time-lapse microscopy of FtsZ1 and FtsZ2 depletion and restoration. FtsZ depletion was achieved by first growing H. volcanii ID56 (p.tna–ftsZ1) and ID57 (p.tna–ftsZ2) in media with Trp (2 mM). The cells were then washed in fresh media without Trp, and samples were placed on a soft agarose gel media pad without Trp using the submerged sandwich technique for time-lapse imaging (left two panels). For FtsZ restoration (right two panels), the p.tna–ftsZ strains were initially grown without Trp in batch cultures, then restoration was initiated by adding 0.2 mM Trp (for ftsZ1 induction) or 2 mM Trp (for ftsZ2 induction) to the agarose pad. Depletion caused cells to grow without dividing, and some cells of both strains displayed occasional budding-like events instead. Restoration of division occurred in both strains, with the giant cells dividing at multiple locations, quite asynchronously and occasionally asymmetrically. The cell growth rates decrease in the latter part of the video, possibly due to local depletion of nutrients.

Supplementary Video 2

Time-lapse microscopy of division/fragmentation of FtsZ1-depleted cells. H. volcanii ID56 (p.tna–ftsZ1) that had previously been depleted of FtsZ1 by continuous mid-log culturing in the absence of Trp were time-lapse imaged, and examples of dividing/fragmenting cells were identified. These cells show some division events and unusual ways of generating cell fragments.

Supplementary Video 3

Time-lapse microscopy of fragmentation of FtsZ2-depleted cells. H. volcanii ID57 (p.tna–ftsZ2) that had previously been depleted of FtsZ2 by continuous mid-log culturing in the absence of Trp were time-lapse imaged, and examples of fragmenting cells were identified. These cells show some blebbing-like events and unusual ways of generating cell fragments.

Supplementary Video 4

3D imaging of H. volcanii wild-type and ΔftsZ1 ΔftsZ2 strains. Confocal laser-scanning microscopy of wild-type and ΔftsZ1 ΔftsZ2 double-mutant live cells suspended in soft agarose gel. Wild-type plate (top left) and rod (bottom left) cells showed flattened morphology. Giant plates lost much of their flattened morphology in liquid culture (top right), whereas giant rod-like cells appeared to maintain the flatness, albeit with some apparent flexibility (in this case, a somewhat twisted shape). In this field, a cytoplasmic bridge appears between the pole of a large cell and a smaller adjacent cell. This may be an intermediate state during the late stage of separation.

Supplementary Video 5

Time-lapse microscopy of ΔftsZ1 (expansion of giant plates on agarose). H. volcanii ID76 mid-log cultures were time-lapse imaged. Some cells showed a budding-like process. Note that while the cells showed no clear evidence of division while supported on these soft-gel pads, the confinement at the gel–glass interface can somewhat obscure the detection of individual abutting cells. However, it is useful to compare the results obtained with efficiently dividing cells (Supplementary Video 1; right panels). In some giant cells, the phase-contrast revealed a dynamic cell structure and possible buckling of the cell surface.

Supplementary Video 6

Time-lapse microscopy of ΔftsZ1 (occasional division/fragmentation). H. volcanii ID76 mid-log cultures were time-lapse imaged using the submerged sandwich technique, and cells undergoing occasional division or highly acentral division/fragmentation events were identified.

Supplementary Video 7

Time-lapse microscopy of ΔftsZ2 (expansion of giant cells). H. volcanii ID77 mid-log cultures were time-lapse imaged. Most cells of this strain showed the giant plate morphotype, expanding to form extremely large irregular cells when supported on the gel surface. Fragmentation of one cell can be seen in the central top panel.

Supplementary Video 8

Time-lapse microscopy of ΔftsZ1 ΔftsZ2 (expansion of giant plates and polar tubulation of filaments). H. volcanii ID112 mid-log cultures were time-lapse imaged. Tubulation and fission or budding-like processes were frequently observed at the poles of the filamentous cells.

Supplementary Video 9

Time-lapse microscopy of FtsZ1–GFP during multiple rounds of division. H. volcanii ID16 mid-log cultures (with 0.2 mM Trp) were time-lapse imaged over several generations (30-min frame intervals), revealing almost continuous FtsZ1–GFP localization at the midcell during multiple cycles of division. In this selected field, a rare cell that fails division may also be seen, which exhibits a complex and dynamic network of aster-like central clusters of filaments containing FtsZ1–GFP. This pattern was common in other giant plates observed during this study.

Supplementary Video 10

Time-lapse microscopy of FtsZ1–GFP showing dynamic behaviour in midcell rings. H. volcanii ID16 mid-log cultures (with 0.2 mM Trp) were time-lapse imaged (10 min frame intervals). FtsZ1–GFP showed uneven localization around the ring and fluorescence intensity changes over time, consistent with ongoing polymer assembly and disassembly in the ring. Cells were also clearly seen dividing unilaterally and bilaterally in this field.

Supplementary Video 11

FtsZ1 and FtsZ2 dynamically colocalize at the midcell during division. H. volcanii ID67 was sampled from mid-log cultures (grown with 0.2 mM Trp) and imaged by time-lapse microscopy of FtsZ1–mCh (red) and FtsZ2–GFP (green). The proteins both showed dynamic movement in the ring and generally colocalized. They showed somewhat differing localization intensity within the ring, but both FtsZ rings closed down along with visible constriction of the envelope.

Supplementary Data 1

Numerical data for the graphs and unprocessed western blots in Supplementary Figs. 1–11.

Source data

Source Data Fig. 1

Unprocessed western blots.

Source Data Fig. 2

Numerical data for the graphs in Fig. 2a,c.

Source Data Fig. 3

Numerical data for the graphs in Fig. 3b,c.

Source Data Fig. 3

Unprocessed western blots.

Source Data Fig. 4

Numerical data for the graphs in Fig. 4a,b,d.

Source Data Fig. 5

Numerical data for the graph in Fig. 5d.

Source Data Extended Data Fig. 1

Multiple sequence alignment in FASTA (text) format.

Source Data Extended Data Fig. 1

Phylogenetic tree in Newick (text) format.

Source Data Extended Data Fig. 4

Numerical data for the graphs in Extended Data Fig. 4a.

Source Data Extended Data Fig. 4

Unprocessed western blots.

Source Data Extended Data Fig. 5

Numerical data for the graphs in Extended Data Fig. 5.

Source Data Extended Data Fig. 6

Numerical data for the graphs and scatterplots in Extended Data Fig. 6.

Source Data Extended Data Fig. 7

Numerical data for the scatterplots in Extended Data Fig 7.

Source Data Extended Data Fig. 8

Numerical data for the graphs in Extended Data Fig 8.

Source Data Extended Data Fig. 9

Numerical data for the graphs in Extended Data Fig 9c,d.

Source Data Extended Data Fig. 10

Numerical data for the scatterplots in Extended Data Fig 10.

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Liao, Y., Ithurbide, S., Evenhuis, C. et al. Cell division in the archaeon Haloferax volcanii relies on two FtsZ proteins with distinct functions in division ring assembly and constriction. Nat Microbiol 6, 594–605 (2021). https://doi.org/10.1038/s41564-021-00894-z

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