Widespread photosynthesis reaction centre barrel proteins are necessary for haloarchaeal cell division

Abstract

Cell division is fundamental to all cellular life. Most archaea depend on either the prokaryotic tubulin homologue FtsZ or the endosomal sorting complex required for transport for division but neither system has been robustly characterized. Here, we show that three of the four photosynthesis reaction centre barrel domain proteins of Haloferax volcanii (renamed cell division proteins B1/2/3 (CdpB1/2/3)) play important roles in cell division. CdpB1 interacts directly with the FtsZ membrane anchor SepF and is essential for cell division, whereas deletion of cdpB2 and cdpB3 causes a major and a minor division defect, respectively. Orthologues of CdpB proteins are also involved in cell division in other haloarchaea, indicating a conserved function of these proteins. Phylogenetic analysis shows that photosynthetic reaction centre barrel proteins are widely distributed among archaea and appear to be central to cell division in most if not all archaea.

As Francois Jacob famously quipped, “the dream of every cell is to become two cells”. Most archaea use the prokaryotic tubulin homologue FtsZ or the endosomal sorting complex required for transport (ESCRT) (also called the Cdv system) to achieve their dreams^1–10. These polymerizing proteins assemble into ring-like structures to organize the assembly and function of the complete division apparatus. Recent studies found that two FtsZ paralogues, FtsZ1 and FtsZ2, co-assemble into the Z ring and are essential for normal cell division in the euryarchaeon Haloferax volcanii¹¹ with SepF serving as a membrane anchor for FtsZ^12,13. FtsZ and SepF date back to the last universal cellular ancestor (LUCA), suggesting that the FtsZ-SepF-based division system is ancestral in archaea^6,13. However, apart from these findings, relatively little is known about this archaeal division system and its regulation.

The photosynthesis reaction centre (PRC) barrel is a distinct β-barrel domain widely present in bacteria, archaea and plants¹⁴. Bacterial prototypes of this protein superfamily have been shown to play important roles in photosynthesis or RNA processing, leading to the assumption that archaeal PRC barrel proteins are probably involved in RNA processing¹⁴. However, here we show that archaeal PRC barrel proteins play critical roles in haloarchaeal cell division. Moreover, the PRC barrel domain is nearly ubiquitous in archaea and present in the CdvA protein of the ESCRT-based division system, suggesting that this domain is critical for archaeal cell division and widely conserved.

Results

HVO_1691 is a cell division protein in H. volcanii

In a search for H. volcanii proteins involved in cell division, we discovered an insert containing a portion of the HVO_1691 gene (Supplementary Figs. 1 and 2; Methods). Although overexpression of intact HVO_1691 only slightly impaired colony growth (Supplementary Fig. 2), we analysed its subcellular localization because it was predicted to be regulated by CdrS¹⁵, the master regulator of the cell cycle in many archaea^15,16. Unexpectedly, fluorescent protein fusions of HVO_1691 localized to midcell and nearly perfectly colocalized with known cell division proteins, FtsZ1, FtsZ2 and SepF (Fig. 1a and Supplementary Fig. 2d). It should be noted that, although the fluorescent protein fusions were expressed from plasmid systems throughout this study, they did not noticeably distort cell division. To confirm the involvement of HVO_1691 in division, we determined if its localization depended on known cell division proteins using depletion strains in which the expression of these proteins is regulated by the tryptophan-inducible promoter P_tna^11,17. The depletion of FtsZ1 or FtsZ2 (by removing tryptophan from the cultures) caused severe division and morphological defects but did not substantially affect the colocalization of HVO_1691 with the other division proteins (Extended Data Figs. 1 and 2, white arrows). In some cells, HVO_1691 also formed foci that were not associated with FtsZ or SepF (Extended Data Figs. 1 and 2, cyan arrows). However, in the absence of both FtsZ1 and FtsZ2, which resulted in giant misshapen cells, HVO_1691 was mostly diffuse but formed some bright foci in the cytoplasm (Fig. 1b). Similar HVO_1691 localization pattern was observed when SepF was depleted, although faint colocalization with FtsZs was occasionally observed (Fig. 1c and Extended Data Fig. 3, white arrows). Bright foci of HVO_1691 were also observed at a lower frequency in the presence of SepF but their physiological role is unknown (Fig. 1b,c and Extended Data Fig. 3, cyan arrows). Altogether, these results indicate that HVO_1691 depends on FtsZs and SepF for localization and it is probably a component of the FtsZ-based division apparatus. Therefore, we renamed HVO_1691 as CdpB1 (cell division protein B1).

Fig. 2 |. Depletion of CdpB1 does not affect colocalization of FtsZ1, FtsZ2 and SepF.

a, Representative images of FtsZ1-GFP and FtsZ2-mCherry localization in the presence or absence of CdpB1. b, Representative images of FtsZ1-mCherry and SepF-GFP in the presence or absence of CdpB1. c, Representative images of FtsZ2-mCherry and SepF-GFP in the presence or absence of CdpB1. Depletion of CdpB1 was achieved by removal of Trp from the cultures. White arrows indicate cluster of spiral filaments or normal ring-like structures in rod-shaped CdpB1-depleted cells; cyan arrows indicate cluster of filaments in giant cells; magenta arrows indicate the sites of FtsZ1-mCherry where SepF-GFP was absent. Owing to the presence of several GFP/mCherry structures or foci in the filamentous or giant cells, the number of GFP/mCherry structures or foci instead of number of cells was used for the calculation of colocalization. Percentages of colocalization (%) are indicated (n > 200). Scale bars, 5 μm.

Fig. 1 |. Identification of HVO_1691 (CdpB1) as a candidate cell division protein in H. volcanii.

a, Representative images showing the colocalization of CdpB1-GFP with known cell division proteins. Percentages of colocalization (%) are indicated; number of cells (n > 250). b, Representative image of CdpB1 localization in ΔftsZ1 ΔftsZ2 cells. Number of cells (n > 100). c, Representative images of localization of CdpB1-GFP and FtsZ1-mCherry in the presence or absence of SepF. Depletion of SepF was achieved by removal of tryptophan (Trp) from the culture. White arrows indicate faint colocalization of CdpB1-GFP and FtsZ1-mCherry; cyan arrows indicate the CdpB1-GFP foci or aggregates. Percentages of protein colocalization (%) are indicated; number of cells (n > 210). d, Depletion of CdpB1 results in severe division and morphological defects. Strain HZS24 (H98, P_tna::his-cdpB1) was kept in exponential phase in the presence or absence of Trp. Samples were taken at the indicated time points after the removal of Trp and spotted on a BSW agarose pad for microscopy. e, Western blot assay to check the level of His-CdpB1 after the removal of Trp. Samples from d were prepared for SDS–PAGE and western blot. Anti-His mouse mAb was used for detection of His-CdpB1. The lower panel shows Coomassie brilliant blue staining of an SDS–PAGE gel as a loading control. Strain H98, which did not express any His-tagged protein, was run as a negative control. Scale bars, 5 μm.

CdpB1 is important for H. volcanii division and cell shape

Attempts to generate a cdpB1 deletion mutant were unsuccessful; therefore, we constructed His-tagged and non-tagged CdpB1 depletion strains by replacing its promoter with the P_tna promoter¹⁸ so that its expression was regulated by tryptophan (Supplementary Fig. 3a). The two depletion strains grew well in the presence of tryptophan; however, cells gradually became enlarged and misshapen after removal of tryptophan from the cultures (Fig. 1d and Supplementary Fig. 3b), indicating that CdpB1 was critical for division and cell morphology. Quantitative PCR with reverse transcription (RT–qPCR) and western blot analysis confirmed that the cdpB1 transcript and His-CdpB1 protein level were drastically decreased following removal of tryptophan (Fig. 1d and Supplementary Fig. 3c). However, prolonged depletion did not further reduce the His-CdpB1 level, presumably due to leaky expression from the P_tna promoter. This probably explains why cdpB1 could not be deleted and why CdpB1-depleted cells still grew without tryptophan, even though cell division was severely compromised (Fig. 1d and Supplementary Fig. 3d, e). Upon addition of 1 mM tryptophan, the CdpB1-depleted cells resumed division and normal morphology (Extended Data Fig. 4), suggesting that its level is critical for its function. Altogether, these results indicate that CdpB1 is essential and important for normal cell division and cell shape in H. volcanii. We used the non-tagged CdpB1 depletion strain for the rest of this study since the His-tag may affect its function.

CdpB1 is not required for the localization of FtsZs and SepF

To explore the role of CdpB1 in haloarchaeal cell division, we checked if its depletion affected the localization of known cell division proteins. In the filamentous CdpB1-depleted cells, FtsZ1, FtsZ2 and SepF formed both clustered spiral ring-like structures as well as normal-appearing Z rings (Fig. 2, white arrows), whereas in the giant misshapen cells, they mostly formed patches of filamentous structures (Fig. 2, cyan arrows). Occasionally, we observed separate localization of FtsZ1-mCherry and SepF-GFP (Fig. 2b, magenta arrows). Given that FtsZs and SepF could still form apparently normal Z rings in the rod-shaped CdpB1-depleted cells, their abnormal localization in the misshapen cells was probably due to the division block and altered cell morphology. However, it is also possible that CdpB1 directly participates in Z ring formation as its absence results in aberrant FtsZ-containing structures.

Depletion of CdpB1 mimics depletion of FtsZ2 or SepF

To further examine the impact of CdpB1 on division, we compared the cell morphology and FtsZ1 localization in cells depleted of CdpB1, FtsZ2 or SepF. Cells of all three depletion strains gradually became filamentous and misshapen after tryptophan removal (Extended Data Fig. 5a–c). FtsZ1 initially became more diffuse or disorganized in the midcell after removal of tryptophan but then formed several regularly spaced spiral rings or abnormal structures in the cell filaments or giant cells (Extended Data Fig. 5a–c, red arrows). By 21 h after depletion, many giant abnormal cells along with cell debris were observed for all three depletion strains (Extended Data Fig. 5a–c). Thus, depletion of CdpB1, similarly to the depletion of FtsZ2 or SepF, results in a gradual loss of normal FtsZ1 localization, severe impairment of cell division and, as a result, abnormal cell morphology. However, upon the addition of tryptophan, the CdpB1-depleted cells gradually regained their shape and size, accompanied with restoration of normal localization of the FtsZs and SepF (Extended Data Fig. 5d–f, red arrows).

CdpB1 interacts with SepF in vivo and in vitro

To test if CdpB1 interacted with known cell division proteins in vivo, we used the Split-FP (fluorescent protein) assay^19,20, which is based on the reconstitution of the superfolder GFP if two proteins of interest interact. In our case, because the protein pairs are involved in cell division, we would not only detect fluorescence but also localization at the division site. Indeed, we detected strong fluorescence and observed fluorescent rings in cells when CdpB1 and FtsZ1/FtsZ2/SepF were fused to the assay tags but not in cells expressing only the tags or when only one of the proteins was tagged (Fig. 3a,b and Extended Data Fig. 6). These results indicated that CdpB1 and FtsZ1/FtsZ2/SepF strongly interact with each other or are parts of a large protein complex in vivo.

Fig. 3 |. CdpB1 directly interacts with SepF in vivo and in vitro.

a, Representative images of Split-FP assay to access the interaction between CdpB1 and SepF in vivo. Scale bar, 5 μm. b, Quantitation of the interaction signal between CdpB1 and SepF by Split-FP assay. RFU, relative fluorescence unit. Data are presented as mean values ± s.d. Significance in each group was tested by two-sided t-test. ***P < 0.001, n = 3. c, Co-IP experiments show that CdpB1 interacts with SepF in vivo. Cultures of H. volcanii expressing the indicated proteins were lysed by sonication; supernatants were incubated with antibodies coated magnetic beads. Immunocomplexes were eluted with boiling SDS–PAGE loading buffer and then analysed by immunoblot. d, Pull-down assays show that CdpB1 interacts with SepF in vitro. A total of 50 μg of His-SUMO-SepF (upper panel) or His-SUMO (lower panel) was incubated with equal amount of CdpB1 in a total volume of 400 μl and then loaded onto pre-equilibrated Ni-NTA columns, washed twice and eluted with elution buffer according to the pull-down assay described in the Methods. UB, unbound fraction; W1, wash 1; W2, wash 2; Elute, eluate. All fractions were collected and analysed by SDS–PAGE.

To further test the interaction between CdpB1 and FtsZ1, FtsZ2 or SepF, we performed co-immunoprecipitation (Co-IP) experiments with cells expressing tagged versions of these proteins. As shown in Extended Data Fig. 7, CdpB1-His was not detected in immunocomplexes isolated with anti-GFP in cells expressing both FtsZ1-GFP (or FtsZ2-GFP) and CdpB1-His and vice versa. By contrast, CdpB1-GFP was detected in the immunocomplexes isolated with anti-Flag in cells expressing both SepF-Flag and CdpB1-GFP and vice versa (Fig. 3c). These results indicated that CdpB1 directly interacts with SepF but not with FtsZ1 or FtsZ2 in vivo. In agreement with this, pull-down assays with purified CdpB1 and His-SUMO-SepF showed that CdpB1 was retained on Ni-NTA beads and found in the eluate (Fig. 3d). In contrast, when CdpB1 was incubated with the His-SUMO tag, most of the protein was found in the unbound fraction and none was detected in the eluate (Fig. 3d), indicating that retention of CdpB1 on the beads was due to specific interaction with His-SUMO-SepF. Altogether, these results demonstrate that CdpB1 directly interacts with SepF to participate in cell division.

CdpB1 is a member of the conserved PRC barrel protein family

CdpB1 is a small protein of 97 amino acids that belongs to the PRC barrel protein family widely present in archaea, bacteria and plants (Pfam ID: PF05239)¹⁴. Although most PRC barrel proteins remain poorly characterized, four distinct functions have been described: (1) H subunit of PRC²¹; (2) bacterial RimM protein involved in 30S ribosome maturation²²; (3) sporulation proteins YlmC/YmxH in Firmicutes²³ and (4) the CdvA component of the ESCRT-based archaeal division system^24,25. Prompted by the role of CdpB1 in haloarchaeal cell division, we re-examined the distribution and conservation of archaeal PRC barrel proteins (Methods). Initially, five arCOGs (2155, 2157, 2158, 5740 and 8023) were assigned to the PRC barrel family but by extensive sequence comparison (Methods) we identified two additional arCOGs (8931 and 10234) and expanded arCOG04054 (CdvA) by identifying CdvA orthologues in several Thermoproteales genomes (Supplementary Data 1). Altogether, at least one PRC barrel protein was identified in 509 of the 524 searched archaeal genomes (Supplementary Data 1). Phylogenetic analysis of the PRC barrel family revealed three principal branches (Fig. 4a,b and Supplementary Data 2), each of which included representatives of several archaeal lineages from two major phyla (Euryarchaeota and Asgardarchaeota) and two superphyla (TACK and DPANN), suggesting that the last archaeal common ancestor encoded three distinct PRC barrel proteins.

Fig. 4 |. Phylogeny, comparative genomics, domain architectures and gene neighbourhood analysis of PRC barrel family in archaea.

a, Schematic representation of phylogenetic tree of the PRC barrel domain family. The phylogenetic tree was built using the FastTree approximate maximum likelihood method as described in the Methods. Branches corresponding to major archaeal phyla are collapsed and coloured according to the colour key below the dendrogram. Three bootstrap values supporting principal branches are shown. Korarchaeal sequences are highlighted in green. Several distinct arCOGs corresponding to collapsed branches and H. volcanii PRC barrel proteins identifiers, belonging to the respective branches, are indicated on the right. The complete tree in Newick format is available in Supplementary Data 2. b, Numbers of PRC barrel domain proteins in 524 archaeal genomes from the arCOG database. The plot shows the number of distinct PRC barrel domains in all 524 genomes from arCOG database grouped according to their taxonomy and coloured using the same colour code as in a, except for the grey colour, indicating unclassified genomes. c, Selected most-frequent domain architectures of PRC barrel proteins. The proteins are shown to scale as indicated. Distinct domains are shown by coloured rectangles. The respective protein identifier and arCOG number are indicated on the right. d, Selected neighbourhoods of PRC barrel genes. Genes are shown as arrows. Protein name is indicated below each arrow. The numbers inside the arrows indicate the counts of the occurrences of the respective gene in 1,675 PRC barrel gene neighbourhoods (Supplementary Data 3). Genome description, nucleotide accession and coordinates of the neighbourhood are indicated on the right. Nob1, endonuclease Nob1; Cdc48, ATPase of the AAA+ class; NrnA, nanoRNase/pAp phosphatase; Thg1, tRNA-His guanylyltransferase; InfB, translation initiation factor 2; EngB, cell division controlling GTPase; Pth2, peptidyl-tRNA hydrolase; SepF, cell division protein; FtsZ, cell division GTPase; CdvB, cell division protein ESCRT III family; CdvC, Vps4 family ATPase, component of ESCRT cell division system; CdvA, component of ESCRT cell division system; TyrS, tyrosyl-tRNA synthetase; SBH1, preprotein translocase subunit Sec61beta.

The number of genes encoding PRC barrel proteins varies greatly among archaea even within the same lineage, such as Halobacteria or Methanomicrobia, with up to 15 genes in several Methanobacteriales species (Fig. 4b). H. volcanii encodes four PRC barrel proteins: CdpB1 (HVO_1691) and HVO_2019 in Branch 1, HVO_1964 in Branch 2 and a more distant paralogue HVO_1607 (arCOG08931) in Branch 3 (Fig. 4a–c). Although the sequence identity of these four proteins is relatively low (25–35%), the AlphaFold predicted structures of their respective PRC barrels are nearly identical (Extended Data Fig. 8). Most PRC barrel proteins contain a single domain but proteins with duplicated PRC barrel domains or a PRC barrel fused to other domains are also widespread, including CdvA^2,24, in which the PRC barrel is fused to a coiled-coil domain (Fig. 4c). Although most PRC barrel proteins are encoded by standalone genes, many are embedded in conserved neighbourhoods or putative operons (Fig. 4d and Supplementary Data 3). Notably, they are often found in the vicinity of genes encoding translation system components or RNA processing enzymes. Specifically, cdpB1 is encoded next to nrnA, a 5′–3′ exonuclease involved in processing of short RNA substrates²⁶ and HVO_1964 is encoded divergently to infB, translation initiation factor 2. These two genes are located in conserved neighbourhoods in haloarchaea (Supplementary Data 3). In Methanomicrobia, the most highly conserved neighbourhood includes thg1, a transfer RNA-His guanylyltransferase²⁷, whereas in Archaeoglobi, a PRC barrel gene is encoded in a putative operon with pth2 encoding a peptidyl-tRNA hydrolase (Fig. 4d and Supplementary Data 3). Several other, relatively widely spread neighbourhoods include genes coding for a Cdc48 family ATPase and an EngB GTPase previously shown to be involved in cell division control in other organisms^28,29 (Fig. 4d and Supplementary Data 3). CdvA is encoded in the cdv cluster along with other genes involved in cell division (Fig. 4d). Surprisingly, we found only one genome, Micrarchaeota archaeon from the DPANN superphylum, where a PRC barrel protein is encoded in a putative operon with sepF and ftsZ (Fig. 4d and Supplementary Data 3). Overall, our analysis shows that the PRC family is actively evolving in archaea and some subfamilies are likely to be subfunctionalized and neofunctionalized to participate in diverse cellular processes in addition to cell division.

CdpB1 paralogues are involved in cell division in H. volcanii

To test if CdpB1 paralogs were also involved in cell division, we fused them to GFP and examined their subcellular localization. Interestingly, while HVO_1607 (Branch 3) showed a diffuse localization pattern, both HVO_1964 and HVO_2019 (belonging to Branch 2 and Branch 1, respectively) formed midcell ring-like structures (Fig. 5a), suggesting that they were involved in cell division. Thus, we renamed them CdpB2 and CdpB3, respectively. Unlike cdpB1, however, cdpB2 and cdpB3 could be knocked out, although their absence caused severe and minor division and shape defects, respectively (Fig. 5b). The defects of ΔcdpB2 cells appeared less pronounced in the complex medium Hv-YPCTE than in the semidefined Hv-Cab medium where casamino acids were used as the carbon and energy source (Fig. 5b). Notably, overexpression of CdpB2 or CdpB3 alone or in combination did not suppress the defects of the CdpB1-depleted cells (Fig. 5c). Also, the defects of ΔcdpB2 cells could not be suppressed by overexpression of CdpB1 or CdpB3 (Fig. 5d), indicating that the three CdpB proteins have distinct functions in cell division, with CdpB1 playing a dominant role.

Fig. 5 |. Most PRC barrel proteins are involved in cell division in H. volcanii.

a, Representative images of localization of CdpB1 paralogues in H. volcanii. b, Representative images of ΔcdpB2 and ΔcdpB3 cells growing in Hv-YPCTE and Hv-Cab media, respectively. c, Representative images of CdpB1-depleted cells complemented with CdpB1, CdpB2, CdpB3 or CdpB2 and CdpB3 in combination, respectively. d, Representative images of ΔcdpB2 cells complemented with CdpB1, CdpB2 or CdpB3, respectively. e, Representative images of CdpB2-GFP and SepF-mCherry localization in the presence or absence of CdpB1. Depletion of CdpB1 was achieved by removal of Trp from the cultures. White arrows indicate the sites of SepF-mCherry where CdpB2-GFP was absent. f, Representative images of CdpB3-GFP and SepF-mCherry localization in the presence or absence of CdpB1. Depletion of CdpB1 was achieved by removal of Trp from the cultures. White arrows indicate the sites of SepF-mCherry where CdpB3-GFP was absent. g, Representative images of CdpB1-mCherry and CdpB3-GFP localization in ΔcdpB2 cells. White arrows indicate the sites of CdpB1-mCherry where CdpB3-GFP was absent. h, Representative images of CdpB1 and CdpB2 localization in ΔcdpB3 cells. Percentages of protein colocalization (%) are indicated; number of GFP/mCherry structures/foci (n > 200). Scale bars, 5 μm.

To further characterize the functions of the CdpB proteins, we determined their localization interdependency and interactions. We found that they were recruited to the Z ring in a sequential manner: CdpB1 was required for the midcell localization of CdpB2 and CdpB3, whereas CdpB2 was necessary for the localization of CdpB3 but not CdpB1 and the absence of CdpB3 did not affect the localization of CdpB1 or CdpB2 (Fig. 5e–h). Although Split-FP assays showed that the three CdpB proteins interacted with each other (Extended Data Fig. 9a–c), Co-IP experiments revealed that CdpB2 interacted with both CdpB1 and CdpB3 but there was no interaction between the latter two proteins (Extended Data Fig. 9d–f), consistent with the localization dependency. Thus, the CdpB proteins form complexes and localize to the Z ring sequentially.

Functions of CdpBs are conserved in haloarchaea

To test if CdpB orthologues were involved in cell division in other haloarchaea, we checked their subcellular localization in Natrinema sp. J7 (order Natrialbales) and Haloarcula hispanica (order Halobacteriales). As shown in Fig. 6a, three of the four PRC barrel proteins from each species formed midcell ring-like structures in the respective species, indicating that most PRC barrel proteins participate in cell division in these two species. Either ^NJ7GCdpB1 or ^HAHCdpB1, the orthologues of CdpB1 from Natrinema sp. J7 (NJ7G_3475) and H. hispanica (HAH_1390), respectively, restored normal cell division and morphology in CdpB1-depleted H. volcanii cells, indicating that CdpB1 function is broadly conserved in haloarchaea. Attempts to delete the cdpB1 orthologues from Natrinema sp. J7 and H. hispanica failed; thus, we again used the P_tna promoter to replace their native promoter regions to obtain depletion strains (Supplementary Fig. 4a,b). RT–qPCR analysis showed that the transcript level of ^NJ7GcdpB1 and ^HAHcdpB1 after the removal of tryptophan was decreased about 20-fold and 5-fold, respectively (Supplementary Fig. 3b). As a result, depletion of ^NJ7GCdpB1 resulted in a severe cell division and morphological defect, whereas the defects were relatively modest for ^HAHCdpB1-depleted cells (Fig. 6c). Both ^NJ7GCdpB1 and ^HAHCdpB1 depletion strains could still grow on plates without tryptophan (Supplementary Fig. 3c), presumably, due to the leaky expression from the P_tna promoter. Nonetheless, given that these genes could not be deleted from the chromosomes of the respective species and could heterologously complement the CdpB1-depleted H. volcanii cells, CdpB1-like proteins probably play a critical role in cell division in diverse haloarchaea. Lastly, we found that the other two PRC barrel proteins in Natrinema sp. J7 depended on ^NJ7GCdpB1 for midcell localization (Extended Data Fig. 10a), indicating that the recruitment mechanism of CdpB proteins is probably also conserved across haloarchaea.

Fig. 6 |. PRC barrel proteins are widely conserved in haloarchaeal cell division and a proposed model for the functions of CdpB proteins.

a, Representative images of the localization of PRC barrel proteins in Natrinema sp. J7 and H. hispanica cells, respectively. Hv-Cab medium was used for the cultivation of Natrinema sp. J7 and AS-168-M medium was used for H. hispanica DF60. Red arrow indicates the midcell localization of NJ7G_3497-GFP. b, Representative images of CdpB1-depleted H. volcanii cells complemented with CdpB1 or its homologues ^NJ7GCdpB1 and ^HAHCdpB1, respectively. c, Depletion of CdpB1 homologues causes severe and modest division defects in Natrinema sp. J7 and H. hispanica cells, respectively. Scale bars, 5 μm. d, A working model for the functions of CdpB proteins in H. volcanii. FtsZ1, FtsZ2 and SepF assemble into a Z ring at midcell as the cell is about to divide. CdpB1 is first recruited by SepF and itself recruits CdpB2, which in turn recruits CdpB3 to the Z ring. The CdpB proteins form a complex and probably recruit more cell division proteins to form the complete divisome complex. Once the divisome is fully assembled, the cell membrane constricts, producing two daughter cells. The absence of CdpB1, CdpB2 or CdpB3 causes a failure to recruit additional essential or accessory division proteins to the Z ring, ultimately leading to different extents of division and cell shape defects.

Discussion

Compared to the extensive knowledge on bacterial and eukaryotic cell division, relatively little is known about this process in archaea. In this work, we find that three PRC barrel proteins (CdpBs) of H. volcanii play important but distinct roles in cell division. Moreover, the functions of these proteins are highly conserved in halophiles. Importantly, CdpB1 acts as a link between SepF and other CdpBs and possibly other division components. Phylogenetic analysis revealed that the PRC barrel proteins are widely distributed in archaea, including the DPANN superphylum which consists of symbiotic archaea with small genomes. Notably, the highly conserved CdvA protein of the ESCRT-based division system also contains a PRC barrel domain (Fig. 4c and Extended Data Fig. 10b), suggesting that the PRC barrel domain is important for the ESCRT-based division systems in addition to the FtsZ-based system. Similar findings are reported in a complementary study from the Albers laboratory³⁰.

Although this work establishes the CdpB proteins as important components of the FtsZ-dependent division machinery in haloarchaea, detailed mechanisms remain to be elucidated. Our results indicate that the three CdpB proteins perform distinct functions in haloarchaeal cell division, probably as recruiters for other unidentified division proteins. CdpB1 interacts with the FtsZ membrane anchor SepF in vivo and in vitro and its depletion results in the formation of filamentous and giant cells. However, in these abnormal cells, FtsZ and SepF still clearly colocalize and in some cases normal Z rings are formed. Thus, CdpB1 is probably not essential for Z ring formation but important for subsequent division steps. In line with this conclusion, CdpB1 is required for the localization of its two paralogues, CdpB2 and CdpB3, and CdpB2 is required for CdpB3 localization. The function of CdpB1 appears widely conserved in haloarchaea as its orthologues from Natrinema sp. J7 and H. hispanica are also involved in division and complement CdpB1 depletion in H. volcanii. Nonetheless, since the absence of CdpB1 also altered normal Z ring formation, it may have a role in stabilizing Z rings and coordinating them with later steps of division.

Unlike CdpB1, CdpB2 and CdpB3 are not essential for cell division in H. volcanii, although their absence results in severe and minor division defects, respectively. Moreover, their overexpression could not suppress the division and morphological defects of CdpB1-depleted cells. Similarly, neither overexpression of CdpB1 nor CdpB3 could suppress the defects of CdpB2 knockout cells, indicating that they perform distinct functions in archaeal cell division. The localization dependency of the CdpB proteins, along with the results from Split-FP analysis and Co-IP experiments, suggests that CdpB2 directly interacts with CdpB1 and CdpB3, probably via their PRC barrel domains which have been shown to mediate protein–protein interactions in bacterial PRC barrel proteins¹⁴. However, how exactly these proteins interact with each other and regulate cell division remain to be determined.

Phylogenetic analysis of the PRC barrel proteins shows that they are widely distributed in archaea and formed three large branches. In many archaeal genomes, the genes encoding PRC barrel proteins, including cdpB1 and cdpB2, are adjacent to genes for components of the translation system, consistent with previous predictions that the PRC barrel domain is involved in ribosome maturation and RNA metabolism¹⁴. However, given that at least one protein subfamily in each major clade is involved in cell division, the role of PRC barrel domain in archaeal division is probably ancestral. Notably, not all PRC barrel proteins are involved in division despite pronounced similarity to CdpB proteins, such as HVO_1607 in H. volcanii, NJ7G_3454 in Natrinema sp. J7 and HAH_2776 in H. hispanica, all of which belong to arCOG08931 in Branch 3 of the phylogenetic tree of PRC barrel family. Additionally, many genes encoding PRC barrel proteins are adjacent to genes encoding Cdc48, a subfamily of AAA+ ATPases involved in cell cycle regulation and protein degradation³¹. However, it remains to be tested whether the PRC barrel proteins in other archaea lineages are involved in cell division, cell cycle regulation, protein translation or other processes.

Of special note is the presence of a PRC barrel domain in the CdvA protein (Fig. 4 and Extended Data Fig. 10b), an essential component of the ESCRT-based division system. Previous studies showed that the PRC barrel domain of CdvA is not required for its interaction with the ESCRT III-like CdvB protein²⁴. Given the role of the PRC barrel proteins in the FtsZ-based division system, we speculate that the PRC barrel domain of CdvA might recruit other division proteins to the ESCRT-based machinery. Also, the identification of CdvA orthologues in Thermoproteales, which lack orthologues of FtsZ and ESCRT proteins and thus are thought to divide via a distinct, currently unknown mechanism^4,6, implies that PRC barrels might be (nearly) universal components of archaeal cell division systems.

Overall, we identified the PRC barrel proteins as conserved components of the FtsZ-based archaeal division system. These proteins probably function as adaptors for the recruitment of other proteins involved in cell division. The broad distribution of the PRC barrel domain among archaea, and in particular its presence in CdvA, suggests that its role in cell division is ancestral in archaea. Search for interaction partners of the PRC barrel proteins can be expected to advance the exploration of archaeal cell division and shed light on its evolution.

Methods

Strains, plasmids, growth conditions, reagents and chemicals

All strains and plasmids used in this study are listed in Supplementary Data 4 and 5, respectively. The primers used in this study are listed in Supplementary Data 6. All reagents and chemicals are listed in Supplementary Data 7.

Escherichia coli strains were grown in LB medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl and 0.05 mg ml⁻¹ of thymine) at indicated temperatures. When needed, ampicillin was added to a final concentration of 100 μg ml⁻¹. H. volcanii and Natrinema sp. CJ7-F strains were grown aerobically at 45 °C and 200 r.p.m. in Hv-YPC medium or Hv-Ca medium or in media containing expanded trace elements and vitamin solution referred to as Hv-YPCTE or Hv-Cab medium^32,33. When auxotrophic markers were used, media were supplemented with uracil (10 μg ml⁻¹ or 50 μg ml⁻¹) for ΔpyrE2 strains or thymidine and hypoxanthine (40 μg ml⁻¹ each) for ΔhdrB strains. Cultures were generally maintained in continuous logarithmic growth (optical density OD₆₀₀ <0.8) for at least 2 days before sampling for analysis, unless otherwise indicated. To control gene expression via the P_tna promoter, l-tryptophan (Trp) was added at the indicated concentration in cultures. H. hispanica strains were cultured at 45 °C in nutrient-rich AS-168 medium (200 g of NaCl, 2 g of KCl, 20 g of MgSO₄·7H₂O, 3 g of trisodium citrate, 1 g of sodium glutamate, 5 g of bacto casamino acids, 5 g of yeast extract, 50 mg of FeSO₄·7H₂O and 0.36 mg of MnCl₂·4H₂O per litre, pH 7.2) with uracil at a final concentration of 50 μg ml⁻¹. Strains carrying the expression plasmids were cultured in the modified AS-168-M medium without yeast extract to provide selection pressure.

Genomic modification

To construct the depletion strain of cdpB1 in H. volcanii, a non-replicating plasmid was first constructed that can recombine at the cdpB1 locus using two-step homologous recombination, thereby replacing the promoter region of cdpB1 with the specific tryptophan-inducible P_tna promoter¹⁸. The upstream and downstream flanking sequences on either side of the start codon of cdpB1 were PCR amplified from H. volcanii DS70 genomic DNA (upstream flank) and plasmid pZS103 (contains the L11e transcription terminator followed by the P_tna::cdpB1-gfp cassette) with primers listed in Supplementary Data 6, respectively. The upstream and downstream fragments were joined by overlap-extension PCR and the products were digested with HindIII and BamHI and then ligated to pTA131 (ref. 17) (at HindIII-BamHI), giving rise to pZS98. The fragment P_fdx::hdrB from pTA1185 (ref. 11) was inserted between the upstream and downstream fragments of cdpB1 in pZS98 at the SphI site. Clones containing the P_fdx::hdrB oriented with the downstream P_tna::cdpB1 cassette were selected and named pZS111 (pTA131, P_fdx::hdrB P_tna::cdpB1). Demethylated pZS111 was transformed into H. volcanii H98 (ref. 17) (DS70, ΔpyrE2 ΔhdrB) and transformants were selected on agar medium without uracil. The resultant colonies were expected to contain the plasmid integrated between the upstream or downstream of the genomic cdpB1 locus by a single-crossover (pop-in). After growth of single colonies in liquid Hv-YPC medium, cells were plated onto Hv-Ca agar containing 100 μg ml⁻¹ of uracil, 50 μg ml⁻¹ of 5-fluoroorotic acid (FOA) and 1 mM tryptophan to select for excision of the plasmid (pop-out). Single colonies were streaked onto the same medium and arising colonies were then screened by allele-specific PCR and sequencing. Colonies containing P_tna::cdpB1 as the only copy of cdpB1 were saved and named as HZS1 (Supplementary Data 4). The His-tagged version of CdpB1 depletion strain HZS24 (H98, P_tna::his-cdpB1) and the SepF depletion strain HZS2 (H98, P_tna::sepF) were constructed similarly by the pop-in/pop-out approach using the non-replicating plasmids pZS483 (pTA131, P_fdx::hdrB P_tna::his-cdpB1) and pZS99 (pTA131, P_fdx::hdrB P_tna::sepF), respectively.

The construction process for the NJ7G_3475 and HAH_1390 depletion strains in Natrinema sp. CJ7-F (ref. 34) and H. hispanica DF60 (ref. 35), respectively, was similar to the above procedure for cdpB1 in H. volcanii. The upstream fragment (upstream of NJ7G_3475) and downstream fragments (the L11e transcription terminator and P_tna::NJ7G_3475 cassette) were amplified, joined by overlap-extension PCR and the products were digested with BamHI and AflII and ligated to pNBK-F³⁶ (at BamHI-AflII), giving rise to pZS253 (pNBK-F, P_tna::NJ7G_3475). Similarly, the upstream fragment (upstream of HAH_1390) and downstream fragment (containing the L11e transcription terminator and P_tna::HAH_1390 cassette) of HAH_1390 were amplified, joined by overlap-extension PCR and inserted into plasmid pHAR³⁵ (at KpnI and HindIII) to obtain vector pZS280 (pHAR, P_tna::HAH_1390). Demethylated pZS253 and pZS280 were transformed into Natrinema sp. CJ7-F and H. hispanica DF60 strain separately to generate NJ7G_3475 depletion strain HZS4 (CJ7-F, P_tna::NJ7G_3475) in Natrinema sp. CJ7-F and HAH_1390 depletion strain HZS4 (DF60, P_tna::HAH_1390) in H. hispanica, respectively.

The cdpB2 and cdpB3 deletion strains were also constructed by the pop-in/pop-out approach¹⁸ as above. The upstream fragment and downstream fragments of cdpB2 or cdpB3 were amplified, joined by overlap-extension PCR and the products were digested with HindIII and BamHI and ligated to pTA131 (ref. 17) (at HindIII-BamHI), giving rise to non-replicating plasmids pZS398 (pTA131, P_fdx::hdrB up-ΔcdpB2-down) and pZS399 (pTA131, P_fdx::hdrB up-ΔcdpB3-down), respectively. Plasmids pZS398 and pZS399 were transformed into strain H26 (ref. 17) (DS70, ΔpyrE2) to generate the desired deletion strains HZS5 (H26, ΔcdpB2) and HZS6 (H26, ΔcdpB3), respectively.

Plasmid construction

Plasmids used in this study are listed in Supplementary Data 5 and the primers used for plasmid construction are listed in Supplementary Data 6. Plasmids pTA962 (ref. 37), pIDJL40 (containing gfp)³² or pIDJL114 (containing mCherry)¹¹ were used as the backbones to construct plasmids for controlled expression of genes in H. volcanii. Plasmids pFJ6-P_tna and pWL502 (ref. 38) were used as backbone vectors to construct expression plasmids for PRC barrel proteins (including their fluorescent fusions) in Natrinema sp. CJ7-F and H. hispanica strains, respectively. For dual expression of the various division genes in H. volcanii, a fragment containing ftsZ-mCherry or sepF-mCherry was ligated into the NotI-cut (klenow blunt end) of the above plasmids (containing -gfp or -mCherry fusion). To be used in the depletion strains, the P_tna promoter of the above plasmids was replaced by the P_phaR promoter, which is constitutively active or the native promoters of the respective genes.

To detect the interaction between CdpB1 and other cell division proteins, we applied the tripartite split-GFP system²⁰ in H. volcanii. All plasmids for Split-FP assays were constructed using the pTA1228 plasmid as backbone. The sfGFP10 or sfGFP11 fragment was fused to the respective reading frames encoding the cell division proteins. The sfGFP fragments and cell division proteins coding sequences are separated by two kinds of flexible linkers. The longer linkers of 30-mer (GFP10 tag) and 25-mer (GFP11 tag) were used for interaction assays of FtsZ1 and FtsZ2, respectively. The shorter linkers of 15-mer (GFP10 tag) and 17-mer (GFP11 tag) were used for interaction assays of CdpB1 and SepF. We also constructed intermediate vectors carrying sfGFP1–9, sfGFP10 and sfGFP11 with different linkers under the control of P_tna. The three cassettes were separated by several restriction enzyme sites including EcoRI, HindIII, XbaI and NheI. The target genes were cloned to the restriction sites to fuse with the sfGFP10 or sfGFP11 fragments in different directions. A series of plasmids and corresponding control plasmids were constructed to examine their interactions.

Plasmids for protein purification were constructed using pE-SUMO-amp as backbone. All plasmids were demethylated by passage through E. coli JM110 and repurified before transfer to haloarchaea by PEG-mediated spheroplast transformation¹⁷. Detailed construction procedures for every plasmid are provided in the ‘Supplementary Note: Construction of plasmids’ in Supplementary Information.

Construction of the genomic library of H. volcanii

To construct a genomic library of H. volcanii, its genomic DNA was digested with Sau3AI and TaqI and fragments of about 1–5 kilobases were purified and then ligated into a derivative of pTA1228 (carrying the tryptonphan-inducible promoter P_tna) digested with BamHI and ClaI. Ligation products were transformed into competent E. coli and transformants selected on LB plates with ampicillin. The plasmids from ten transformants were isolated and cut with restriction enzymes to verify that most of the plasmids contained genomic DNA fragments of H. volcanii. About 40,000 transformants were pooled together and the plasmids were extracted and saved as the library.

Rationale for the screen for cell division proteins in H. volcanii

In bacteria, overproduction of proteins involved in essential cellular processes often impair cell growth by causing a malfunctioning of the corresponding machineries or a disruption of metabolic pathways. For example, overexpression of cell division proteins or proteins regulating cell division often results in a division block thereby preventing colony formation^39–43. As haloarchaea also divide in an FtsZ-dependent manner^1,4,11, we reasoned that overexpression of haloarchaeal cell division proteins might interfere with cell division and inhibit cell growth. In line with this, overexpression of CdrS, the master regulator of haloarcheal cell cycle, has been shown to cause severe cell division and morphological defects in multiple halophiles¹⁶. Thus, we screened for proteins whose overexpression was toxic to the cell in H. volcanii with a hope that some might cause division and morphological changes. To do this, we transformed the genomic library of H. volcanii into H26 and transformants were screened on plates with or without tryptophan by replica plating. Transformants showing a growth defect on plates with tryptophan were confirmed for the tryptophan-dependent growth defect. The inserts in the selected transformants were determined by sequencing and then recloned into pTA1228 to confirm its toxicity to the cell and the effect on cell morphology in the presence of tryptophan. Using this approach, we found that many DNA segments inserted into pTA1228 blocked colony formation in the presence of tryptophan but few would cause morphological changes to the cells, including one that harboured the first 82 amino acids of SepF. We also found that a fragment containing a part of HVO_1691 (amino acids 1–56) inhibited the growth of H. volcanii, leading to its discovery. This indicated that this approach was working, although not very effective.

Complementation tests

To test if overexpressions of CdpB2 and CdpB3 alone or in combination suppress the division and shape defect of CdpB1-depleted cells, cells of strain HZS1 (H98, P_tna::cdpB1) carrying plasmids pZS236 (P_native::cdpB1), pZS339 (P_native::cdpB2), pZS338 (P_native::cdpB3) or pZS390 (P_native::cdpB2-P_native::cdpB3) were washed with fresh Hv-Cab medium three times to remove tryptophan and then resuspended in fresh Hv-Cab medium. The tryptophan-free culture was then diluted 1:100 in Hv-Cab medium without tryptophan and cultured to OD₆₀₀ ~0.2. A total of 2 μl of the cultures was spotted on a BSW agarose pad for microscopy.

To test if CdpB1 homologues from other haloarchaea could restore normal cell division and shape to CdpB1-depleted cells, exponential phase cultures of HZS1 (H98, P_tna::cdpB1) carrying plasmids pZS236 (P_native::cdpB1), pZS234 (P_native::^NJ7GcdpB1) or pZS276 (P_native::^HAHcdpB1) were treated as above to check cell division and morphology.

To test if overexpression of CdpB1 or CdpB3 suppressed the division and shape defect of ΔcdpB2 cells, cultures of HZS5 (ΔcdpB2) carrying plasmids pZS236 (P_native::cdpB1), pZS339 (P_native::cdpB2) or pZS338 (P_native::cdpB3) were diluted 1:100 in fresh Hv-Cab medium and grown at 45 °C to OD₆₀₀ ~0.2 to examine their impact on cell division and morphology.

Fluorescence microscopy

All phase contrast and fluorescence images were acquired using an Olympus BX53 upright microscope with a Retiga R1 camera from QImaging, a CoolLED pE-4000 light source and a U Plan XApochromat phase contrast objective lens (×100, 1.45 numerical aperture, oil immersion). Green and red fluorescence was imaged using the Chroma EGFP filter set EGFP/49002, mCherry/Texas Red filter set mCherry/49008, respectively. For microscopy, a 2 μl sample of cells was immobilized on 1.5% agarose pads equilibrated with 18% BSW (Hv.Ca medium without casamino acids and CaCl₂) at room temperature and a clean glass coverslip placed on top. Images were processed with Adobe Photoshop (2021) or Adobe Illustrator (2023).

Localization of CdpB proteins and HVO_1607 in H. volcanii.

Overnight cultures of H. volcanii H26 carrying plasmids pZS103 (P_tna::cdpB1-gfp), pZS101 (P_tna::cdpB1-mCherry), pZS336 (P_tna::cdpB2-gfp), pZS337 (P_tna::cdpB3-gfp), pZS422 (P_tna::HVO_1607-gfp) or pZS423 (P_tna::gfp-HVO_1607) were diluted 1:100 in fresh Hv-Cab medium with 0.2 mM tryptophan and grown at 45 °C to OD₆₀₀ ~0.2. A total of 2 μl of the cultures was spot on BSW agarose pads for microscopy.

Colocalization of CdpB1-GFP with FtsZ1, FtsZ2 and SepF.

Exponential phase cultures of H. volcanii H26 carrying plasmids pZS105 (P_tna::cdpB1-gfp-ftsZ1-mCherry), pZS106 (P_tna::cdpB1-gfp-ftsZ2-mCherry) or pZS107 (P_tna::cdpB1-gfp-sepF-mCherry) were treated as above to observe colocalization of protein fusions.

Localization dependency of CdpB1.

Overnight cultures of ID56 (ref. 11) (H98, P_tna::ftsZ1) harbouring plasmids pZS284 (P_phaR::cdpB1-gfp-ftsZ2-mCherry) or pZS285 (P_phaR::cdpB1-gfp-sepF-mCherry), ID57 (ref. 11) (H98, P_tna::ftsZ2) harbouring plasmids pZS239 (P_phaR::c dpB1-gfp-ftsZ1-mCherry) or pZS285 (P_phaR::cdpB1-gfp-sepF-mCherry) and HZS2 (H98, P_tna::sepF) harbouring plasmids pZS239 (P_phaR::cdp B1-gfp-ftsZ1-mCherry) or pZS284 (P_phaR::cdpB1-gfp-ftsZ2-mCherry) were diluted 1:100 in fresh Hv-Cab medium with 1 mM tryptophan and grown at 45°C to OD₆₀₀ ~0.4. Cells were then collected by centrifugation and washed three times with fresh Hv-Cab medium to remove the tryptophan, followed by suspension in the same volume of Hv-Cab medium. The tryptophan-free culture was then inoculated 1:100 in Hv-Cab medium with or without 1 mM tryptophan and cultured to OD₆₀₀ ~0.2. A total of 2 μl of the cultures was spot on BSW agarose pads for microscopy. To check the localization of CdpB1-GFP in the ftsZ1 and ftsZ2 double-deletion strain ID112 (ref. 11) (H98, ΔftsZ1 ΔftsZ2), overnight culture of ID112 carrying plasmid pZS103 (P_tna::cdpB1-gfp) was diluted 1:100 in fresh Hv-Cab medium with 1 mM tryptophan and grown at 45 °C to OD₆₀₀ ~0.2. A total of 2 μl of the cultures was spot on BSW agarose pads for microscopy.

Localization interdependence of CdpB1, CdpB2 and CdpB3.

Overnight cultures of HZS1 (H98, P_tna::cdpB1) harbouring plasmids pZS408 (P_phaR::cdpB2-gfp-sepF-mCherry) or pZS407 (P_phaR::cdpB3-gfp-sepF-mCherry) were treated as in ‘Localization dependency of CdpB1’ to examine the localization dependency of CdpB2 and CdpB3 on CdpB1. To check the localization of proteins in the ΔcdpB2 and ΔcdpB3 cells, overnight cultures of HZS5 (H26, ΔcdpB2) harbouring plasmids pZS417 (P_phaR::cdpB3-gfp-cdpB1-mCherry) and HZS6 (H26, ΔcdpB3) harbouring plasmid pZS418 (P_phaR::cdpB2-gfp-cdpB1-mCherry) were diluted 1:100 in fresh Hv-Cab medium and grown at 45 °C to OD₆₀₀ ~0.2. A total of 2 μl of the cultures was spot on BSW agarose pads for microscopy.

Colocalization of FtsZ1, FtsZ2 and SepF in CdpB1-depleted cells.

Overnight cultures of HZS1 (H98, P_tna::cdpB1) carrying plasmids pZS289 (P_phaR::ftsZ1-gfp-ftsZ2-mCherry), pZS322 (P_phaR::sepF-gfp-ftsZ1-mCherry) or pZS324 (P_phaR::sepF-gfp-ftsZ2-mCherry) were treated as in ‘Localization dependency of CdpB1’ to examine the localization of FtsZ1, FtsZ2 and SepF in CdpB1-depleted cells.

Localization of FtsZ1 upon FtsZ2, SepF or CdpB1 depletion and localization of division proteins upon CdpB1 re-induction.

To check the localization of FtsZ1 upon the depletion of FtsZ2, SepF or CdpB1, overnight cultures of ID57 (H98, P_tna::ftsZ2), HZS2 (H98, P_tna::sepF) or HZS1 (H98, P_tna::cdpB1) harbouring plasmid pZS208 (P_native::ftsZ1-gfp) were diluted 1:100 in fresh Hv-Cab medium with 1 mM tryptophan and grown at 45 °C to OD₆₀₀ ~0.4. Cells were then collected by centrifugation and washed three times with fresh Hv-Cab medium to remove tryptophan, followed by suspension in the same volume of Hv-Cab medium. The tryptophan-free culture was then inoculated 1:100 in Hv-Cab medium without tryptophan. A total of 2 μl of the samples was spot on BSW agarose pads for microscopy every 3 h after the removal of tryptophan until depletion for 24 h.

To check the localization of cell division proteins in CdpB1-depleted cells upon the re-induction of CdpB1, cultures of HZS1 (H98, P_tna::cdpB1) harbouring plasmids pZS208 (P_native:: ftsZ1-gfp), pZS210 (P_native:: ftsZ2-gfp) or pZS212 (P_native:: sepF-gfp) grown in the absence of tryptophan for 24 h were diluted 1:100 in fresh Hv-Cab medium with 1 mM tryptophan and grown at 45 °C to OD₆₀₀ ~0.2. A total of 2 μl of the samples was spot on BSW agarose pads for microscopy every 3 h after the addition of tryptophan until 24 h.

Localization of the PRC barrel proteins in Natrinema sp. CJ7-F and H. hispanica.

To examine the localization of PRC barrel proteins in Natrinema sp. CJ7-F, overnight cultures of CJ7-F carrying plasmids pZS217 (P_tna::NJ7G_3475-gfp), pZS384 (P_tna:: NJ7G_2729-gfp) or pZS475 (P_tna::NJ7G_3454-gfp) were diluted 1:100 in fresh Hv-Cab medium with 0.2 mM tryptophan and grown at 45 °C to OD₆₀₀ ~0.2. A total of 2 μl of the cultures was spot on BSW agarose pads for microscopy. To check the localization of NJ7G_3497, overnight culture of CJ7-F carrying plasmid pZS514 (P_native:: NJ7G_3497-gfp) was diluted 1:100 in fresh Hv-Cab medium and grown at 45 °C to OD₆₀₀ ~0.2. A total of 2 μl of the culture was spot on BSW agarose pads for microscopy.

To check the localization of PRC barrel proteins in H. hispanica DF60, overnight cultures of DF60 carrying plasmids pZS306 (P_tna::HAH_1390-gfp), pZS388 (P_tna:: HAH_0460-gfp), pZS389 (P_tna:: HAH_5240-gfp) or pZS477 (P_tna:: HAH_2776-gfp) were diluted 1:100 in fresh AS-168-M medium with 0.2 mM tryptophan and grown at 45 °C to OD₆₀₀0.2. A total of 2 μl of the cultures was spot on BSW agarose pads for microscopy.

Localization dependency of NJ7G_3475 and NJ7G_2729 on ^NJ7GCdpB1.

To check if the localization of NJ7G_3475 and NJ7G_2729 depended on ^NJ7GCdpB1 in Natrinemasp. CJ7-F, exponential cultures of HZS4 (P_tna::^NJ7GCdpB1) carrying plasmids pZS513 (P_native::NJ7G_3475-gfp) or pZS514 (P_native:: NJ7G_2729-gfp) were washed with fresh Hv-Cab medium three times to remove tryptophan and then resuspended in fresh Hv-Cab medium. The tryptophan-free culture was then diluted 1:100 in Hv-Cab medium with or without tryptophan and cultured to OD₆₀₀ ~0.2. A total of 2 μl of the cultures was spotted on a BSW agarose pad for microscopy.

Split-FP assay.

Overnight cultures of H. volcanii H26 carrying the tripartite Split-FP system plasmids were diluted 1:100 in fresh Hv-Cab medium with 0.2 mM tryptophan and cultivated at 45 °C overnight followed by cultivation at 37 °C for 3 h. A total of 2 μl of the culture was spot on BSW agarose pad for microscopy.

Quantification of fluorescence

To quantify the protein–protein interaction signal between CdpB1 and other cell division proteins by Split-FP, the fluorescence of the H. volcanii transformants was quantified. In each case, 5 ml of culture was cultivated at 45 °C to OD₆₀₀1–1.5. The culture was then brought to OD₆₀₀ of 1 and kept shaking at 30 °C overnight with 0.2 mM tryptophan. A total of 1 ml of the culture was harvested by centrifugation (12,000g, 2 min), washed and brought to OD₆₀₀1 with 18% BSW. A total of 200 μl of sample was analysed in a 96-well plate and evaluated using the Varioskan LUX multifunctional microplate detection system. All experiments were performed with two biological samples and three technical replicates. The P values were calculated using Student’s t-test.

Protein expression and purification

The proteins were produced by heterologous expression in the E. coli strain BL21 (DE3) harbouring plasmids pZS311 (H-SUMO-CdpB1) or pZS288 (H-SUMO-SepF). An overnight culture of each strain grown in LB with ampicillin (100 μg ml⁻¹) was diluted 1:100 into 300 ml of fresh LB medium supplemented with ampicillin (100 μg ml⁻¹) and incubated at 37 °C until OD₆₀₀ reached about 0.4. IPTG was then added to the culture to a final concentration of 1 mM and incubated at 37 °C for another 3 h. Cells were collected by centrifugation, washed with 10 mM Tris-HCl (pH 7.9) and frozen at −80 °C until used. On the day of purification, the cells were thawed and resuspended in 20 ml of high-salt lysis buffer (25 mM Tris-HCl (pH 7.5), 2.5 M KCl, 5% glycerol, 0.1 mM dithiothreitol and 20 mM imidazole) and lysed by sonication. The lysates were centrifuged at 18,000g for 15 min at 4 °C to remove cell debris. The supernatants were loaded onto pre-equilibrated Ni-NTA agarose. The column was washed once with high-salt wash buffer. The bound protein was eluted with elution buffer (25 mM Tris-HCl (pH 7.5), 2.5 M KCl, 5% glycerol, 0.1 mM dithiothreitol and 250 mM imidazole). Fractions were analysed by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) gel and the ones with highest concentration of protein were pooled and dialysed against storage buffer (25 mM Tris-HCl (pH 7.5), 2.5 M KCl, 5% glycerol, 0.1 mM dithiothreitol), aliquoted and stored at −80 °C.

The H-SUMO tag of CdpB1 was cleaved with purified 6×His-tagged SUMO protease (Ulp1) for 1 h at 30 °C in the protein storage buffer with 200 mM KCl. The released tag and protease were removed by passing the reaction mixture through the pre-equilibrated Ni-NTA agarose. Untagged CdpB1 was collected in the flow through, dialysed against protein storage buffer, concentrated and stored at −80 °C.

Pull-down assay

To test the interaction between His-SUMO-SepF and CdpB1, 50 μg of His-SUMO-SepF and 50 μg of purified CdpB1 were mixed in a total volume of 400 μl of equilibrium buffer (25 mM Tris-HCl (pH 7.5), 2.5 M KCl, 5% glycerol, 0.1 mM dithiothreitol), incubated at 4 °C for 2 h and then loaded into a gravity flow column with 200 μl of pre-equilibrated Ni-NTA agarose. After incubation on ice for 10 min without agitation, the mixture was allowed to pass through the column by gravity. The column was then washed with 400 μl of wash buffer (25 mM Tris-HCl (pH 7.5), 2.5 M KCl, 5% glycerol, 0.1 mM dithiothreitol and 20 mM imidazole) twice. Proteins bound to the Ni-NTA beads were eluted with 400 μl of elution buffer (25 mM Tris-HCl (pH 7.5), 2.5 M KCl, 5% glycerol, 0.1 mM dithiothreitol and 250 mM imidazole). A control experiment with His-SUMO and CdpB1 was performed following the same procedure. All fractions were collected during the procedure and analysed by SDS–PAGE.

Western blot of CdpB1

To check the level of His-CdpB1 after the removal of tryptophan, strain HZS24 (H98, P_tna::his-cdpB1) was cultured to middle exponential phase (OD₆₀0.6–0.8) in Hv-Cab medium with 1 mM tryptophan. Cells were then collected by centrifugation and washed three times in fresh Hv-Cab medium to remove tryptophan. Cells were resuspended in the same volume of Hv-Cab medium and inoculated 1:100 in Hv-Cab medium without tryptophan. Samples were taken at different time points after the removal of tryptophan (the OD₆₀₀ of cultures was adjusted to ensure the same amounts of cells were taken for analysis) and crude extracts were prepared by mixing with 2× SDS–PAGE sample buffer. After boiling at 95 °C for 10 min, samples were loaded onto two 12% SDS–PAGE. Proteins from one polyacrylamide gel were transferred onto a nitrocellulose membrane (Pall Corporation), while the other polyacrylamide gel was stained with Coomassie brilliant blue staining. The membrane was incubated in 5% skimmed milk blocking agent for 1 h and then incubated with primary mouse antibodies (anti-His; Transgen-HT501-01; 1:10,000 dilution) at 4 °C overnight. Following 3 × 10 min washing with TBST, the membrane was incubated with secondary antibody (goat anti-mouse IgG-HRP; Transgen-HS201-01; 1:10,000 dilution) for 1 h at room temperature. After washing, signal was visualized using Smart-ECL Enhanced (Smart-Lifesciences) and recorded by ChemiDoc Imaging System (Bio-Rad). Strain H98, which did not express any His-tagged protein, was used as a negative control.

To check the level of His-CdpB1 at different concentration of tryptophan, strain HZS24 (H98, P_tna::his-cdpB1) was cultured in Hv-Cab medium without tryptophan for 24 h. The culture was then inoculated 1:100 in Hv-Cab medium with 0, 0.05, 0.5 or 1 mM tryptophan. After being cultured at 45 °C for another 15 h, samples were taken for Western blot analysis as above.

Immunoprecipitation

Overnight cultures of H. volcanii carrying the expression plasmids were diluted 1:100 in 40 ml of fresh Hv-YPC medium and cultivated at 45 °C to OD₆₀₀ ~1.0. Cells were collected by centrifugation at 15,000g for 10 min and resuspended in 2 ml of high-salt lysis buffer (25 mM Tris-HCl (pH 7.5), 2.5 M KCl, 5% glycerol, 0.1 mM dithiothreitol and 20 mM imidazole) containing an antiprotease cocktail (MCE) and lysed by sonication. The lysates were centrifuged at 18,000g for 5 min at 4 °C to remove cell debris. A total of 400 μl of the supernatant was added to preprepared Ab-coated magnetic beads and incubated overnight at 4 °C. Magnetic beads–Ab-protein complexes were separated by centrifugation and then washed with 400 μl of high-salt wash buffer (25 mM Tris-HCl (pH 7.5), 2.5 M KCl, 5% glycerol, 0.1 mM dithiothreitol, 20 mM imidazole and 0.5% Tween-20) five times. The immunocomplexes were finally eluted with boiling SDS–PAGE loading buffer and were separated by SDS–PAGE. Following transfer onto NC membranes, proteins were revealed by immunoblot. The following antibodies, with their respective dilutions in 5% skimmed milk, were used: anti-GFP (AE078, ABclonal) 1/2,000, anti-Flag (AE004, ABclonal) 1/2,000, anti-His (AE086, ABclonal) 1/2,000, anti-GFP (HT801-01, Transgen) 1/10,000, anti-Flag (HT201-01, Transgen) 1/10,000, anti-His (HT501-01, Transgen) 1/10,000, anti-mouse secondary antibody (HS201-01, Transgen) 1/10,000 and anti-rabbit secondary antibody (HS101-01, Transgen) 1/10,000.

RT–qPCR analysis of CdpB1 transcript level

To determine the transcript level of CdpB1 in the CdpB1 depletion strain upon the removal of tryptophan, cells were incubated to middle exponential phase in Hv-Cab or AS-168-M medium with 1 mM tryptophan and then tryptophan was removed by centrifugation and washing. Afterwards, the culture was inoculated into Hv-Cab medium with or without tryptophan and cultured to logarithmic phase. Total RNA was extracted from 3 ml of the culture with TRIzol reagent (Invitrogen). The degradation of RNA was assessed by 1.5% agarose gel. Before running the reverse transcription-PCR (RT-PCR) reaction, the RNA sample was pretreated with gDNA Eraser Reagent to remove gDNA. RT-PCR was performed according to the manufacturer’s protocol (PrimeScript RT reagent Kit with gDNA Eraser; TaKaRa) using reverse transcriptase and random primers to amplify the complementary DNA. The rpl10, rpoB and 7S RNA genes were used as internal control in H. volcanii, Natrinema sp. CJ7-F and H. hispanica, respectively. The specific primers used for qPCR are listed in Supplementary Data 6. For the reactions, 20 μl of mixtures was prepared containing 5 μl of template, 10 μl of iTaq Universal SYBR green Supermix (Bio-Rad), 1 μl of primer pairs (10 μM) and 4 μl of distilled water. Amplification was performed according to the manufacturer’s instructions. The qPCR data were analysed according to the 2^−ΔCT method. Three independent experiments were performed and error bars indicated the standard deviations.

Sequence comparison, phylogenetic analysis and gene neighbourhood analysis for archaeal PRC barrel domain containing proteins

The arCOG database^44,45 that includes annotated clusters of orthologous genes for 524 archaeal genomes covering all major archaeal lineages is available at https://ftp.ncbi.nih.gov/pub/wolf/COGs/arCOG/tmp.ar18/. PSI-BLAST⁴⁶ search (e-value cutoff of 0.01, effective database size of 2 × 10⁷, no composition-based statistics and no low complexity filtering, five iterations) with several selected query sequences from each arCOG consisting of PRC barrel domain proteins (2155, 2157, 2158, 5740 and 8023) was used to run searches against all proteins in the arCOG database to identify remotely similar homologues. Proteins identified using this approach but currently not annotated as containing the PRC barrel domain were additionally searched against PFAM, CDD and PDB profiles databases using HHpred⁴⁷. If HHpred searches revealed similarity with known PRC barrel protein profiles with probability >80%, then the query sequences and the respective arCOGs were assigned to the PRC barrel family (Supplementary Data 1). Muscle5 programme⁴⁸ with default parameters was used to construct a multiple sequence alignment of archaeal PRC barrel domains. For phylogenetic analysis, several poorly aligned sequences or fragments were discarded and the remaining protein sequences were realigned. Columns in the multiple alignment were filtered for homogeneity value⁴⁹ ≥0.05 and gap fraction <0.667. This filtered alignment was used as an input for FastTree programme⁵⁰ to construct an approximate maximum likelihood phylogenetic tree with the WAG evolutionary model and gamma-distributed site rates (Supplementary Data 2). The same programme was used to calculate support values. HHpred and Marcoil⁵¹ were used to search for sequence similarity and prediction of coiled-coil regions, respectively, for protein domains fused to PRC barrel domain. For genome context analysis and search for putative operons, neighbourhoods containing five upstream and five downstream genes were constructed for all identified genes encoding PRC barrel domain proteins (Supplementary Data 3).

Statistics and reproducibility

All data measurements were plotted and analysed using GraphPad Prism 9 (v.9.9.1). In general, for comparisons of two groups, significance was determined by two-tailed, unpaired Student’s t-test. All experiments were carried out with at least three independent biological replicates. P values <0.05 were considered statistically significant.

Extended Data

Extended Data Fig. 1 |. CdpB1 does not depend on FtsZ1 for colocalization with FtsZ2 and SepF.

a. Representative images of CdpB1-GFP and FtsZ2-mCherry localization in the presence or absence of FtsZ1, respectively. b. Representative images of CdpB1-GFP and SepF-mCherry localization in the presence or absence of FtsZ1, respectively. Depletion of FtsZ1 was achieved by removal of tryptophan from the cultures. Strain ID56 (H98, P_tna::ftsZ1) harbouring plasmid pZS284 (P_phaR::cdpB1-gfp-ftsZ2-mCherry) or pZS285 (P_phaR::cdpB1-gfp-sepF-mCherry) were grown in Hv-Cab medium with or without tryptophan. Percentage of protein colocalization (%) were indicated, number of GFP/mCherry structures or foci (n > 200). Scale bars 5 μm.

Extended Data Fig. 2 |. CdpB1 does not depend on FtsZ2 for colocalization with FtsZ1 and SepF.

a. Representative images of CdpB1-GFP and FtsZ1-mCherry localization in the presence or absence of FtsZ2, respectively. b. Representative images of CdpB1-GFP and SepF-mCherry localization in the presence or absence of FtsZ2, respectively. Depletion of FtsZ2 was achieved by removal of tryptophan from the cultures. Strain ID57 (H98, P_tna::ftsZ2) harbouring plasmid pZS239 (P_phaR::cdpB1-gfp-ftsZ1-mCherry) or pZS285 (P_phaR::cdpB1-gfp-sepF-mCherry) were grown in Hv-Cab medium with or without tryptophan. White arrows indicate the colocalization of the GFP and mCherry fluorescence signal, cyan arrows indicate the CdpB1-GFP foci or aggregates. Percentage of protein colocalization (%) were indicated, number of GFP/mCherry structures or foci (n > 200). Scale bars 5 μm.

Extended Data Fig. 3 |. CdpB1 depends on SepF for correct localization.

a. Representative images of CdpB1-GFP and FtsZ2-mCherry localization in the presence or absence of SepF, respectively. Depletion of SepF was achieved by removal of tryptophan from the cultures. Strain HZS2 (H98, P_tna::sepF) harbouring plasmid pZS284 (P_phaR:: cdpB1-gfp-ftsZ2-mCherry) were grown in Hv-Cab medium with or without tryptophan. White arrows indicate the colocalization of the GFP and mCherry fluorescence signal, cyan arrows indicate the CdpB1-GFP foci or aggregates. Percentage of protein colocalization (%) were indicated, number of GFP/mCherry structures or foci (n > 200). Scale bars 5 μm.

Extended Data Fig. 4 |. Determination of the level of CdpB1 necessary for complementation.

a-b. CdpB1-depleted cells resume normal cell shape and size following restoration of CdpB1 expression. HZS1 (H98, P_tna::cdpB1) or HZS24 (H98, P_tna::his-cdpB1) grown in Hv-Cab (+50 μg/mL uracil) without tryptophan for 24 hours was diluted 1:100 in fresh medium with indicated concentrations of tryptophan. 15 hours later, samples were spotted onto a BSW agarose pad for microscopy. Scale bars 5 μm. c. Western blot to check the level of His-CdpB1 at the indicated concentration of tryptophan. Samples from panel b were prepared for SDS–PAGE and Western blot. The level of His-CdpB1 increased as the concentration of tryptophan in the medium increased. Strain H98, which did not express any His-tagged protein, was run as a negative control. Upper panel, Western blot; lower panel, Coomassie brilliant blue staining.

Extended Data Fig. 5 |. Cell morphology and protein localization upon depletion and repletion of division proteins.

a-c. Representative images of FtsZ1-GFP localization and cell morphology after depletion of FtsZ2 (a), SepF (b) or CdpB1 (c). Strains ID57 (H98,P_tna::ftsZ2), HZS2 (H98, P_tna::sepF) or HZS1 (H98, P_tna::cdpB1) harbouring plasmid pZS208 (P_native:: ftsZ1-gfp) were grown in Hv-Cab medium without tryptophan to deplete CdpB1. Samples were taken at the indicated time points after the removal of tryptophan. Red arrows indicate multiple spiral and large abnormal FtsZ1-GFP localization in rod-shaped and large cells. Scale bars 5 μm. d-f. Representative images of FtsZ1-GFP (d), FtsZ2-GFP (e) and SepF-GFP (f) localization and cell morphology in CdpB1 repleted cells. Strain HZS1 (H98, P_tna::cdpB1) harbouring plasmids expressing fluorescent division protein fusions was grown in Hv-Cab medium without tryptophan for 24 hours and then inoculated into fresh Hv-Cab medium with 1 mM tryptophan. Samples were taken at the indicated time points after the addition of tryptophan. Red arrows indicate the nearly normal localization of FtsZ1-GFP, FtsZ2-GFP and SepF-GFP 9 hours after the addition of tryptophan to induce CdpB1. Scale bars 5 μm.

Extended Data Fig. 6 |. CdpB1 displays interaction with FtsZ1 and FtsZ2 in Split-FP assay.

a. Representative images of Split-FP assay showing the interaction signal between CdpB1 and SepF. b. Representative images of Split-FP assay showing the interaction signal between CdpB1 and FtsZ2. c. Representative images of Split-FP assay showing the interaction signal between CdpB1 and FtsZ1. An exponential culture of strain H26 (DS70, ΔpyrE2) harbouring the Split-FP plasmid expressing the indicated protein(s) was treated as in Fig. 3a and the fluorescence signal and localization were observed by microscopy. Scale bars 5 μm.

Extended Data Fig. 7 |. Co-IP experiments show that CdpB1 does not interact with FtsZ1 and FtsZ2.

The experiment was carried out as in Fig. 3c. a. CdpB1-His did not co-immunoprecipitated with FtsZ1-GFP. b. CdpB1-His did not co-immunoprecipitated with FtsZ2-GFP.

Extended Data Fig. 8 |. Alignment of PRC barrel proteins and their predicted structures.

a. Amino acid sequences of PRC barrel domain containing proteins from H. volcanii, Natrinema sp. J7–1 and H. hispanica were downloaded from Uniprot: https://www.uniprot.org/, aligned by Clustal Omega: https://www.ebi.ac.uk/Tools/msa/clustalo/ and then depicted using ESPRIPT 3.0: http://espript.ibcp.fr/. b. Phylogenetic tree of the PRC barrel domain containing proteins from H. volcanii, Natrinema sp. J7–1 and H. hispanica generated by Clustal Omega. c. Superimposition of the predicted structures of CdpB1, CdpB2, CdpB3 and HVO_1607 of H. volcanii. Structure models were generated by AlphaFold and were downloaded from Uniprot and aligned by PyMOL. CdpB1, green; CdpB2, cyan; CdpB3, magenta; HVO_1607, yellow.

Extended Data Fig. 9 |. Interaction between the CdpB proteins in vivo.

a-c. Interaction between the CdpB proteins determined by Split-FP assay. a. Representative images showing the interaction signal between CdpB1 and CdpB2. b. Representative images showing the interaction signal between CdpB2 and CdpB3. c. Representative images showing the interaction signal between CdpB1 and CdpB3. An exponential phase culture of strain H26 (DS70, ΔpyrE2) harbouring the Split-FP plasmid expressing the indicated protein(s) was treated as in Fig. 3a and the fluorescence signal and localization were examined by microscopy. Scale bars 5 μm. d-f. Co-IP experiments to determine the interactions between CdpB proteins. d. CdpB1-His and CdpB2-GFP immunoprecipitated with each other. e. CdpB2-His and CdpB3-GFP immunoprecipitated with each other. f. CdpB1-His and CdpB3-GFP did not immunoprecipitate with each other. The experiment was carried out as in Fig. 3c. Supernatants were incubated with rabbit antibodies coated magnetic beads, while the following Western blotting analysis used mouse antibodies.

Extended Data Fig. 10 |. Localization dependence of PRC barrel proteins in Natrinema sp. J7 and comparison of the PRC barrel domains of CdpB1 and CdvA from S. acidocaldricus.

a. Midcell localization of NJ7G_2779 and NJ7G_3497 depends on ^NJ7GCdpB1 in Natrinema sp. J7. Strain HZS4 (CJ7-F, P_tna::^NJ7GcdpB1) carrying plasmid pZS513 (P_native::NJ7G_2729-gfp) or pZS514 (P_native::NJ7G_3497-gfp) was grown in Hv-Cab medium with or without tryptophan to check cell morphology and protein localization. White arrows indicate the midcell localization of NJ7G_3479-GFP. Scale bars 5 μm. b. AlphaFold structural models of H. volcanii CdpB1 and CdvA from S. acidocaldricus. Structural models of CdpB1 (D4H036) and CdvA (Q4J923) were downloaded from Uniprot and aligned with PyMOL.

Supplementary Material

Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41564-024-01615-y.

Data availability

Data generated and analysed during this study are presented in the text or in the Supplementary Information and Supplementary Data. Plasmids and strains that support the findings of this study are available from the corresponding authors. Source data are provided with this paper.