The cyanobacterial cell division factor Ftn6 contains an N-terminal DnaD-like domain
© Marbouty et al; licensee BioMed Central Ltd. 2009
Received: 26 May 2009
Accepted: 21 August 2009
Published: 21 August 2009
DNA replication and cell cycle as well as their relationship have been extensively studied in the two model organisms E. coli and B. subtilis. By contrast, little is known about these processes in cyanobacteria, even though they are crucial to the biosphere, in utilizing solar energy to renew the oxygenic atmosphere and in producing the biomass for the food chain. Recent studies have allowed the identification of several cell division factors that are specifics to cyanobacteria. Among them, Ftn6 has been proposed to function in the recruitment of the crucial FtsZ proteins to the septum or the subsequent Z-ring assembly and possibly in chromosome segregation.
In this study, we identified an as yet undescribed domain located in the conserved N-terminal region of Ftn6. This 77 amino-acids-long domain, designated here as FND (Ftn6 N-T erminal D omain), exhibits striking sequence and structural similarities with the DNA-interacting module, listed in the PFAM database as the DnaD-like domain (pfam04271). We took advantage of the sequence similarities between FND and the DnaD-like domains to construct a homology 3D-model of the Ftn6 FND domain from the model cyanobacterium Synechocystis PCC6803. Mapping of the conserved residues exposed onto the FND surface allowed us to identify a highly conserved area that could be engaged in Ftn6-specific interactions.
Overall, similarities between FND and DnaD-like domains as well as previously reported observations on Ftn6 suggest that FND may function as a DNA-interacting module thereby providing an as yet missing link between DNA replication and cell division in cyanobacteria. Consistently, we also showed that Ftn6 is involved in tolerance to DNA damages generated by UV rays.
DNA replication and cell division are probably the most fundamental processes in the cell life cycle. Both proceed through a remarkably conserved general mechanism and are inextricably intertwined to each others and to the cell metabolism .
The DNA replication cycle can be divided into three distinct stages; initiation, elongation, and termination. Replication is initiated by the highly conserved AAA+ superfamily ATPase-member DnaA that binds the oriC, inducing DNA strand melting [2–4]. In E. coli, DnaA also interacts with the ring helicase DnaB and directs the loading of DnaB/DnaC onto the single stranded DNA (ssDNA) region. After binding of DnaB on the ssDNA region of the oriC, DnaC is released in an ATPase dependent manner. Then, DnaB recruits the DnaG primase and DNA polymerase III to form the replication fork . In B. subtilis, two additional essential proteins, called DnaB (be aware of the confusing nomenclature between the E. coli and B. subtilis) and DnaD, are engaged in entry of the ring helicase at oriC. DnaB could function as a membrane anchoring factor for the replication initiation machinery  or, together with DnaI, the functional homolog of E. coli DnaC , in the recruitment of the ring helicase . DnaD interacts with both DnaA and DnaB [8, 9]. It exhibits DNA remodelling activity, enhancing partial melting of the DNA strands, and could, therefore, function in early steps of replication such as initiating the recruitment of the ring helicase [9–15]. During elongation, the replication forks constituted at the oriC travel in opposite directions to achieve replication of the entire chromosome. When the replication forks reach the terminus, terC, the replication complexes are dismantled in a process involving specific termination factors .
The earliest event in bacterial cytokinesis is the definition of the future cell division site. This occurs through the dynamic assembly/disassembly of the Z-ring structure resulting from the self-polymerization of the ubiquitous tubulin-like protein FtsZ [17, 18]. Placement of the Z-ring is mainly dependent on the Min system both in E. coli and in B. subtilis [17, 18]. Once assembled, the Z-ring is believed to serve as a scaffold for recruitment of the cell division machinery to activate septation and physical separation of the daughter cells. In contrast to the model organisms E. coli and in B. subtilis, the molecular basis of cell division has not been as well studied in cyanobacteria. Nevertheless, studies with the two unicellular cyanobacteria Synechocystis PCC6803 and Synechococcus PCC7942 and the filamentous Anabaena PCC7120, have allowed the identification and the characterisation of clear Fts and Min orthologs as well as ZipN/Ftn2 and Ftn6, two cell division factors restricted to cyanobacteria [19–21]. Although ftn6 deletion leads to cell division defects, resulting in cells dramatically elongated in Synechococcus PCC7942 or enlarged in Anabaena PCC7120 [19, 21], the molecular function of Ftn6 remains unclear. Nevertheless, recent data suggest an involvement of Ftn6 in recruitment of FtsZ proteins to the septum or subsequent Z-ring assembly, as cells deleted for ftn6 do not exhibit condensed Z-rings, but rather diffuse localization of FtsZ .
Based on sequence and structure analyses, we here propose that the cyanobacterial-specific cell division factor Ftn6 contains a not hitherto described N-terminal domain related to the DnaD-like domain found in the DnaD chromosomal replication protein family. Identification of the Ftn6 N-terminal Domain, we termed FND (Ftn6 N-terminal Domain), opens up very interesting perspectives about Ftn6 function in cell division and possibly in chromosome segregation as well as on their necessary interplay. Consistently, we also showed that Ftn6-depleted cells are sensitive to DNA damages generated by UV rays.
Results and discussion
Ftn6 orthologs contain a conserved N-terminus domain (FND)
FND is related to the DnaD-like domain
The belonging of FND to the DnaD-like domain family was further supported by fold recognition techniques. Indeed, submission of each FND domains to the PHYRE server constantly returned the two DnaD structures currently deposited in PDB as best hits, i.e. the DnaD-like domain of the replication proteins from Streptococcus mutans (PDB: 2ZC2) and from Enterococcus faecalis (PDB: 2I5U). DnaD-like domains reported from the PHYRE search share low but noticeable similarities with all FND tested (data not shown), suggesting structural similarities between FND and the DnaD-like domain. Then, Streptococcus mutans DnaD-like domain has been included in the alignment shown in Figure 2. As expected for proteins sharing a low level of sequence identity, we noticed that the nature of the hydrophobic residues conserved in DnaD-like domains (noted by red dots below the WebLogo profile  shown in the additional file 2 and shaded in pink background in Figure 2) was not preserved in FND (shaded by light green background in Figure 2). By contrast, their positions were highly conserved. The high degree of conservation (80%; 16 aminoacids residues out of 20; compare positions shaded in pink and green in the bottom of Figure 2) of the hydrophobic pattern between FND and DnaD-like domain strongly argues in favour of a similar fold. This is particularly evident for the helices 3 and 4, in which the hydrophobic pattern is not only conserved in position (83%; 10 out of 12), but is also highly similar, particularly the two alanines of the third helix (A50 and A54 in Syn6803 FND) and the leucine and the tyrosine (L69 and W72 respectively) at the extreme C-terminus of the fourth helix (Figure 2).
Functional prediction for Ftn6
So far, DnaD-like proteins have only been found in some low G+C content gram positive bacteria and their associated phages , where they exhibit pleiotropic functions all related with DNA metabolism. For instance, DnaD was shown to be involved in initiation of chromosome and plasmid replication [25, 26], sporulation , DNA repair  and recombination . Furthermore, the DnaD-related protein from the thermophilic bacteriophage GBSV1 exhibits an unspecific nuclease activity . The exact function of the DnaD-like domain in these processes remains unclear, but the DnaD-like domain from B. subtilis was found to exhibit DNA-binding and DNA-remodelling activities [11–15]. Altogether, these data strongly suggest that the DnaD-like domain does not define a common structural fold occurring in functionally unrelated proteins, but rather that the DnaD-like domain-containing proteins, including Ftn6, share common functions involving DNA.
What is the function of Ftn6? FND could suggest a function in DNA replication for Ftn6. However, this hypothesis is unlikely as Ftn6-depleted mutants do not appear to affect chromosome replication and do not produce anucleate cells . Furthermore, the N-terminal extension in DnaD proteins, which interacts with DnaA , is missing in Ftn6 orthologs (data not shown). Alternatively, Ftn6 could function in the cross talk between chromosome replication and cell division, a fundamental biological process not yet investigated in cyanobacteria. In most bacteria, both processes are intimately co-ordinated, as formation and placement of the future division septum is regulated by nucleoid occlusion and only occurs after replication of a significant portion of the chromosome . By contrast, Z-ring can assemble at nucleoid-occupied sites and nucleoid separation occurs during Z-ring constriction in cyanobacteria . This lack of nucleoid occlusion supposes an efficient mechanism to segregate chromosome trapped at the midcell during Z-ring constriction. It has recently been proposed that Ftn6 could be involved in chromosome segregation in Synechococcus PCC7942 . How Ftn6 is functionally connected to chromosome segregation remains unknown. Nevertheless, identification of the putative DNA-binding domain, FND, strongly supports the involvement of Ftn6 in this pathway and its interplay with cell division.
Although depletion of Ftn6 leads to cell division defects, the molecular function of this cynobacterial-specific divisome component remained unclear. Sequence alignment of Ftn6 orthologs beforehand identified by BLAST allowed us to uncover a new conserved domain localized within the N-terminus of the proteins. Combining several approaches, we then shown that this domain, designated here as FND, exhibits sequence and structure similarities with the DnaD-like domains found in several factors involved in DNA metabolism. The structure similarities between FND and DnaD-like domains together with the sensitivity of the Ftn6-depleted mutant to UV rays, led us to propose that Ftn6 is functionally linked to DNA metabolism, possibly playing a role at the interface between DNA replication and cell division. Whether this function involves or not other cell division factors and what is (are) the DNA target(s) of Ftn6 remain to be determined.
In silico methods
Databases search of Ftn6 and DnaD domain-containing proteins were performed using BLAST (e < 10-4) [33, 34] and PsiBLAST [34, 35] algorithms. Multiple sequence alignment of the DnaD-like-containing proteins or/and Ftn6 orthologs were generated using ClustalW2 [36, 37] (Matrix: BLOSUM, Gap penality: 10 and penality for Gap extension: 0,1), and visualized with Boxshade . Further details are given in the relevant figure legends and in the additional Files 1 and 4. Fold recognition was performed with PHYRE [39, 40]. 3D-structure of Syn6803 FND was modelled using the MODELLER 9v6 program  and visualized with Pymol . Briefly, 10 models of Syn6803 FND were first built based on the alignment shown in Figure 2. All 10 models were then evaluated with DOPE from the MODELLER package and the best chosen as final model. The overall model quality was additionally validated with ProSA-Web [42, 43].
WT Synechocystis PCC6803 and its derivative ftn6Δ::Kmr/FTN6+  were grown as described . Cells were then 4-fold serially diluted in MM medium and then spotted onto MM plates. Finally, the plates were or not exposed to either 250 or 500 J.m-2 UV radiation and incubated 7 days at 30°C under the above described light conditions.
We are particularly indebted to Isabelle Callebaut, Fransisco Malagon and Silvia Jimeno-Gonzalez for their critical reading of the manuscript. This work was supported by grants from the Commissariat à l'Energie Atomique (CEA). CS and MM were supported by CEA post-doctoral and doctoral fellowships respectively.
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