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Keywords:

  • protein–protein interaction;
  • bacterial cell division;
  • two-hybrid assay;
  • fts genes

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

FtsK is a multifunctional protein involved in both cell division and chromosome segregation. As far as its role in cell division is concerned, FtsK is among the first divisome proteins that localizes at mid-cell, after FtsZ, FtsA and ZipA, and is required for the recruitment of the other divisome components. The ability of FtsK to interact with several cell division proteins, namely FtsZ, FtsQ, FtsL and FtsI, by the two-hybrid assay was already shown by our group. In this work, we describe the identification of the protein domain(s) involved in the interaction with the cell division partner proteins. The biological role of some interactions is also discussed.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

The development of the division machinery (divisome) is necessary for bacterial cell division. In Escherichia coli, it requires assembly at the mid-cell of at least 15 different proteins that participate in invagination, constriction of the three envelop layers and separation of the two daughter cells (for a review, see Vicente & Rico, 2006). Among these proteins, FtsK, a multifunctional protein, couples cell division and chromosome segregation (for a review, see Bigot et al., 2007).

FtsK is a polytopic membrane protein that can be divided into three domains. The N-terminal domain (about 200 residues), involved in cell division (Draper et al., 1998; Wang & Lutkenhaus, 1998; Yu et al., 1998), contains four transmembrane helices (Dorazi & Dewar, 2000) and localizes to the septal ring, where it is necessary for the recruitment of other division proteins (Chen & Beckwith, 2001). The remainder of FtsK is cytoplasmic and consists of a long proline- and glutamine-rich linker region (about 500 amino acids), followed by the C-terminal domain (about 500 residues) involved in DNA segregation (Yu et al., 1998; Steiner et al., 1999; Bigot et al., 2004). Although it is known how FtsK acts to facilitate chromosome segregation, by aligning the termini of replicated chromosomes with each other via DNA translocation (reviewed in Bigot et al., 2007), its role in cell division is still poorly understood. FtsK is among the first divisome proteins that localizes at mid-cell, after FtsZ, FtsA and ZipA, and is required for the recruitment of the other divisome components (Wang & Lutkenhaus, 1998; Yu et al., 1998; Chen & Beckwith, 2001). It has been reported that overexpression of some divisome proteins (FtsQ, FtsN) could suppress the lethality due to FtsK deletion (Geissler & Margolin, 2005), leading to the hypothesis that FtsK could primarily stabilize the divisome before the onset of cytokinesis. In addition, there is also evidence that the N-terminal domain of FtsK plays an additional role during septum closure (Weiss, 2004).

The ability of FtsK to interact with several cell division proteins, namely FtsZ, FtsQ, FtsL and FtsI, by a two-hybrid assay (THA) was already shown (Di Lallo et al., 2003) and, for several of them, also confirmed by coimmune precipitation experiments (D'Ulisse et al., 2007; Maggi et al., 2008). As expected, these interactions were localized in the first half of the protein. The aim of this work was to identify the FtsK region(s) involved in the interaction with the cell division partner proteins and to shed some light on the role of these interactions in the division process.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Media and chemicals

Luria–Bertani (LB) broth for bacterial culture and plating and suspension medium (salt solution) for bacteria dilutions were as described by Miller (1972). The antibiotics used (Sigma) were ampicillin (50 μg mL−1), tetracycline (40 μg mL−1) and kanamycin (30 μg mL−1). Synthetic oligonucleotides for E. coli gene amplification are listed in Table 1.

Table 1.   Synthetic oligonucleotides used in this work
No.ftsK OligoSequence
11F SalIgcgtcgacCTTGAGCCAGGAATACATTG
22172R BamHIcgggatccAGCAAGCAACGCTTTCATTGG
32118F SalIgcgtcgacCGAATCCGTTCTCGCTGGAT
43991R BglIIgaagatcTTAGTCAAACGGCGGTGGG
5603R BamHIcgggatccTCATGGCGAAGGTGAGAATGT
61203R BamHIcgggatccTCAACTGGTTGTTGCAGCGGC
71500R BamHIcgggatccTCACTCTTCAACTTCTTCAAAG
81818R BamHIcgggatccCGACGAGTTGCCACGCGG
91822F SacIcgagctcGCGTCTTACGGTATTAAGCTG

Bacterial strains and plasmids

The bacterial strains, E. coli K-12 derivatives and plasmids used in this work are listed in Table 2.

Table 2.   Bacterial strains and plasmids used in this work
 Relevant genotypeSources
E. coli strains
 R721R718 derivative glpT∷p434−P22lacZDi Lallo et al. (2003)
 NB 1298NB 1114 ftsK∷cat-Δ5/pJC85Buddelmeijer & Beckwith (2004)
 R722NB1298 pcIP22ftsKΔ1800−2172This work
Plasmids
 pcI434ftsIpcI434 derivative harboring the E. coli ftsI geneDi Lallo et al. (2003)
 pcI434ftsQpcI434 derivative harboring the E. coli ftsQ geneDi Lallo et al. (2003)
 pcI434ftsLpcI434 derivative harboring the E. coli ftsL geneDi Lallo et al. (2003)
 pcI434ftsZpcI434 derivative harboring the E. coli ftsZ geneDi Lallo et al. (2003)
 pcIP22ftsIpcIP22 derivative harboring the E. coli ftsI geneDi Lallo et al. (2003)
 pcIP22ftsQpcIP22 derivative harboring the E. coli ftsQ geneDi Lallo et al. (2003)
 pcIP22ftsLpcIP22 derivative harboring the E. coli ftsL geneDi Lallo et al. (2003)
 pcIP22ftsZpcIP22 derivative harboring the E. coli ftsZ geneDi Lallo et al. (2003)
 pcIP22ftsK1−2172pcIP22 derivative harboring the FtsK residues 1–724This work
 pcIP22ftsK1−603pcIP22 derivative harboring the FtsK residues 1–201This work
 pcIP22ftsK1−1203pcIP22 derivative harboring the FtsK residues 1–401This work
 pcIP22ftsK1−1500pcIP22 derivative harboring the FtsK residues 1–500This work
 pcIP22ftsK1−1818pcIP22 derivative harboring the FtsK residues 1–606This work
 pcIP22ftsK1822−2172pcIP22 derivative harboring the FtsK residues 608–724This work
 pcIP22ftsKΔ1800−2172pcIP22 derivative harboring FtsK deleted for residues 600–724This work
 pcI434ftsK1−2172pcI434 derivative harboring the FtsK residues 1–724This work
 pcI434ftsK1−603pcI434 derivative harboring the FtsK residues 1–201This work
 pcI434ftsK1−1203pcI434 derivative harboring the FtsK residues 1–401This work
 pcI434ftsK1−1500pcI434 derivative harboring the FtsK residues 1–500This work
 pcI434ftsK1−1818pcI434 derivative harboring the FtsK residues 1–606This work
 pcI434ftsK1822−2172pcI434 derivative harboring the FtsK residues 608–724This work

β-Galactosidase assay

β-Galactosidase activity was assayed as described by Miller (1972). The THA was performed on bacterial cultures grown to OD600 nm 0.5 at 34 °C in LB medium supplemented with 1 × 104 M isopropyl-β-d-thiogalactoside (IPTG), as previously described (Di Lallo et al., 2003).

THA

The interaction of FtsK protein both with itself and with the other proteins was determined by studying the ability to reconstruct a functional 434-P22 chimeric repressor able to bind the chimeric operator O-P434/P22 of bacterial strain R721, where it controls the expression of the lacZ reporter gene. Each pair of plasmids carries the N-terminal domain of 434 phage repressor fused inframe with the coding sequence of genes ftsZ, ftsI, ftsQ and ftsL that code for the FtsK interaction partners and the N-terminal domain of P22 phage repressor fused inframe with different gene fragments coding for truncated FtsK protein. The loss of binding to the operator sequence is deduced by the activation of β-galactosidase synthesis, evaluated by over a 50% increase in the ratio between the Miller units of β-galactosidase produced by the bacterial strain R721 carrying every pair of plasmids and that of R721 without plasmids. Each value was the mean of at least three independent experiments. As already discussed (Di Lallo et al., 2003; D'Ulisse et al., 2007), β-galactosidase percentages <50% represent an interaction between the two proteins under investigation whereas percentages >50% indicate a lack of interaction.

General microbiological and recombinant DNA techniques

Standard microbiological techniques were as described by Miller (1972). Standard procedures were used for small-scale plasmid preparations, endonuclease digestion, ligation, agarose gel electrophoresis, elution of DNA fragments from agarose and bacterial transformation (Sambrook et al., 1989). PCR was carried out using the Taq DNA polymerase kit (Promega), according to the recommendations of the manufacturer.

Preparation of samples for microscopic observation

A bacterial culture of NB1298/pcIP22ftsKΔ1800−2172 was grown at 37 °C in LB supplemented with arabinose 0.2%. At OD600 nm=0.3, aliquots were withdrawn, centrifuged and resuspended in LB supplemented with 1 × 10−4 M IPTG and allowed to grow for 3 h. At this time, 1 μL 4′,6-diamino-2-phenylindole (DAPI, 5 mg mL−1) was added to an aliquot of 100 μL of bacterial culture for 10 min in the dark. The sample was then centrifuged and resuspended in the same volume of saline buffer. Finally, 5 μL was loaded on poly-l-lysine-coated slides and examined with a Delta vision (AppliedPrecision) Olympus 1X70 microscope.

As a control, a bacterial culture of NB1298 was grown under the same conditions. At OD600 nm=0.3, the culture was split into two parts: one was DAPI labeled and processed as described above for the microscopic observation and arabinose was added to the other to reach a final concentration of 1%. The culture was grown for 3 h before DAPI labeling and processing for microscopic observation.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

To identify the FtsK domains involved in the interactions with the other cell division proteins, we constructed recombinant plasmids harboring partial deletions of the ftsK gene encoding truncated forms of the protein. These constructs were used in the prokaryotic THA described in Materials and methods.

The rationale of the assay is that if two chimeric proteins formed by the N-terminal portion of phage 434 cI repressor fused inframe with the division protein X (pcI434-X), and the N-terminal portion of phage P22 cI repressor fused inframe with the division protein Y (pcIP22-Y), forming the heterodimer pcI434-X/pcIP22-Y by interaction of their C-terminal domains, a fully functional repressor will be formed. This repressor will be able to shut down the expression of the lacZ reporter gene under the control of the 434/P22 hybrid promoter/operator region.

In our case, X is constituted by various fragments of the ftsK gene to form the recombinant plasmids (pcI434-X or pcIP22-X), and Y by the various division genes whose products interact with FtsK, namely FtsZ, FtsI, FtsL and FtsQ. Pairs of recombinant plasmids encoding these chimeric repressors transformed the recipient strain R721 carrying the reporter gene lacZ under the control of the hybrid promoter/operator 434-P22. β-Galactosidase synthesis, which is constitutive in the strain without plasmids, is repressed in the presence of the two plasmids only if the two chimeric repressors interact. Residual β-galactosidase activity was evaluated for each strain and compared with that of the parental strain without plasmids. Each value reported in Fig. 1 is the mean of at least three independent determinations. The cut-off criteria have been discussed previously (Di Lallo et al., 2003; D'Ulisse et al., 2007).

image

Figure 1.  (a) Schematic representation of Escherichia coli FtsK protein showing the DNA domains corresponding to the localization of the interactions. (b) Various ftsK fragments (left) were cloned in both pcIP22 and pcI434 and tested for their ability to interact with the other Fts proteins by the THA (Di Lallo et al., 2003). Residual β-galactosidase activity of <50% (in bold) indicates an interaction between the Fts protein and peptide coded by the particular FtsK fragment (right). The reported values are the mean of at least three independent experiments. Similar results were obtained when the genes (or the gene fragments) were cloned in the reciprocal vectors, i.e. pcIP22 or pcI434.

Download figure to PowerPoint

FtsK structural analysis showed that the protein consists of an N-terminal domain (about 200 residues) with several predicted membrane-spanning regions involved in cell division (Draper et al., 1998; Wang & Lutkenhaus, 1998; Yu et al., 1998), a proline–glutamine-rich domain (linker region of about 500 residues) and a C-terminal domain with a nucleotide-binding consensus sequence of about 500 amino acids involved in DNA segregation (Yu et al., 1998; Steiner et al., 1999; Bigot et al., 2004).

As far as this protein organization is concerned, the results described in Fig. 1 suggest the existence of three domains of interaction with the other division proteins: the first 200 amino acids at the N-terminus of the protein are involved in the interaction with FtsQ and FtsL whereas, surprisingly, the other interactions are localized outside the region implicated in the division process: FtsZ and a site for FtsI between residues 608 and 724 and the second site for FtsI in the C-terminal domain. The localization of these interactions in an FtsK region not essential for cell division suggests that they could only have a role in divisome stabilization.

In order to verify this hypothesis, we cloned in pcIP22 an ftsK mutant deleted between residues 600 and 724 and studied its ability to interact with the FtsK division partners and to complement the strain NB1298. In this strain, the chromosomal ftsK gene was deleted and inserted into a plasmid under the control of pBAD. This strain is able to grow only in the presence of 0.2% arabinose, as demonstrated by the fact that the e.o.p. determined in the presence and absence of arabinose is of the order of 10−4. As shown in Fig. 1, the deleted protein behaves as expected as far as its interaction ability is concerned, i.e. it interacts with FtsQ, FtsL and FtsI and not with FtsZ. It is important to note that the interaction with FtsI can be ascribed to the presence of the second FtsI interaction site localized in the C-terminal part of the protein, as shown in Fig. 1.

The ftsKΔ1800−2172 mutant is also able to restore the growth of strain NB1298. In fact, the efficiency of plating of this strain, which is 3.8 × 10−4 in the absence of arabinose, becomes 1 under the same experimental conditions but in the presence of the plasmid pcIP22ftsKΔ1800−2172. However, after 3 h of induction with 1 × 10−4 M IPTG, the cells carrying this deleted protein are filamentous (Fig. 2). The filamentation cannot be ascribed only to FtsK overproduction, because it is not observed upon overexpression of the wild-type protein, coded by the pJC85 plasmid under the control of pBAD, even upon induction with 1% arabinose (Fig. 2). This result suggests that the lack of FtsZ–FtsK interaction, although not essential for bacterial survival under laboratory conditions, may play a role in the division process because its absence perturbs normal cell division.

image

Figure 2.  Growth of strains (a) NB1298 in the presence of 1% arabinose and (b) NB1298/pcIP22ftsKΔ1800−2172 in the absence of arabinose and after induction with 1 × 10−4 M IPTG. The experiments were performed as described in Materials and methods.

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This result suggests that, besides the 200 amino acids at the N-terminal end (Draper et al., 1998; Wang & Lutkenhaus, 1998; Yu et al., 1998), other protein domains are also involved in the cell division process. The different genetic background of the strain, as the temperature-sensitive phenotype of the mutant described in Draper et al. (1998), could explain the discrepancy between our result and that described previously.

As far as the FtsK fragment 725–1329 is concerned (Fig. 1), by the analysis of the crystal structure of Pseudomonas aeruginosa protein (Massey et al., 2006), it was proposed that this region is involved in FtsK oligomerization. According to the structural data, our results confirm that the in vivo homodimerization of FtsK is due to the interaction of the protein C-terminal domains with each other.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

We thank Simona Pietrucci for her help in performing some experiments. This work was partially supported by contributions of the Lions Club of Rome and Chianciano (Italy).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
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