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1 Dipartimento di Biologia, Università Tor Vergata, Roma, Italy
2 Dipartimento di Medicina Interna, Facoltà di Medicina, Università La Sapienza, Roma, Italy
3 Dipartimento di Scienze e Tecnologie Biomediche, Sez. Microbiologia Medica, Cagliari, Italy
4 Istituto di Biologia e Patologia Molecolare del CNR, Roma, Italy
Correspondence
Patrizia Ghelardini
ghelardini{at}bio.uniroma2.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Ayala et al., 1994). This region contains many genes whose products are involved in division and cell wall biosynthesis and is therefore known as the dcw (division and cell wall) cluster (Ayala et al., 1994; Vicente & Errington, 1996). This cluster, which is very similar in organization in many Gram-negative bacteria, shows various differences in Gram-positive bacteria, particularly in cocci (Tamames et al., 2001).
Despite the differences in dcw cluster organization, the major proteins involved in the division process appear to be conserved. In particular, at least eight proteins are present in almost all cell-wall-containing Eubacteria – FtsZ, FtsA, FtsK, FtsQ/DivIB, FtsB/DivIC, FtsL, FtsW and FtsI – although additional proteins may also be present, depending on the bacterial species (Margolin, 2000; Errington et al., 2003; Harry et al., 2006).
It has been shown that in many species, the cell division proteins, organized by the tubulin-like FtsZ, assemble at the division site to form a molecular division machinery, the septosome or divisome. FtsZ, in fact, directs the cytokinesis process, constituting a scaffold for the other division proteins, which, as determined for the best-characterized rod-shaped model organisms E. coli and Bacillus subtilis, are recruited hierarchically at the mid-cell to form the septal machinery (Errington et al., 2003; Margolin, 2003; Weiss, 2004; Goehring & Beckwith, 2005; Harry et al., 2006; Vicente et al., 2006; Erickson, 2007). As proposed recently, the assembly of the divisome components is a multistage process, which proceeds in a concerted way (Goehring et al., 2006; Vicente & Rico, 2006).
In E. coli, the early event is marked by the interactions of FtsZ with FtsA and ZipA, which assemble in the cytoplasmic membrane (for review see Vicente et al., 2006). After the addition of FtsK, which completes the formation of the cytoplasmic ring, a periplasmic connector formed by the complex FtsQ–FtsB–FtsL is added (Buddelmeijer & Beckwith, 2004), followed by the proteins involved in manufacturing the septal peptidoglycan, FtsW and FtsI. Finally, FtsN is recruited to the septosome (Vicente et al., 2006).
Despite the differences observed in some aspects of the division mechanism in different bacteria, one common thread is the role of FtsZ as a cell division organizer. Moreover, the similar conservation of many division proteins suggests that they should all be recruited at the Z-ring by a common mechanism.
Streptococcus pneumoniae is a developing model for studying cell division in Gram-positive pathogens. Since the dcw cluster was identified (Massidda et al., 1998), considerable progress has been made in understanding the basic mechanism of septum formation in this Gram-positive coccus. In particular, a number of cell division proteins have now been identified in S. pneumoniae and shown to localize at the mid-cell to form the septal machinery, consistent with what is known for the rod-shaped model bacteria (Errington et al., 2003; Weiss, 2004; Goehring & Beckwith, 2005; Vicente et al., 2006). These proteins include the cell division initiator proteins, such as FtsZ and FtsA, required at the early stages of the process (Massidda et al., 1998; Morlot et al., 2003; Lara et al., 2005), some of the later players FtsQ/DivIB, FtsB/DivIC, FtsL, FtsW, PBP2x and PBP1A (Massidda et al., 1998; Morlot et al., 2003, 2004; Noirclerc-Savoye et al., 2005; Zapun et al., 2008), which constitute the septal markers in S. pneumoniae cells, and most recently DivIVA, which appears to be required for septum completion and pole maturation (Fadda et al., 2007).
Despite this progress many aspects of cell division in S. pneumoniae still remain to be clarified, in particular the sequence of the events necessary for septum formation, including the order of recruitment of the proteins to the cell division site. In addition, very little is known about the complex pattern of interactions among the different players that constitute the divisome.
In model organisms, particularly E. coli, the progression of knowledge has been achieved through the establishment of the requirement of a protein for the sequential localization of the others that follow. In S. pneumoniae, the unavailability of specific thermosensitive or conditional division mutants has so far limited the possibility of establishing precisely the hierarchical order of recruitment, if any, and the interdependence of the division proteins at the mid-cell, Moreover, although interaction between the streptococcal FtsA and FtsZ has been detected (Lara et al., 2005) and biochemical evidence for a trimeric complex, formed by FtsQ/DivIB, FtsL and FtsB/DivIC, has been reported (Noirclerc-Savoye et al., 2005), no other information is available on protein–protein interaction between the other players.
To clarify the different and common pathways of the division mechanism in S. pneumoniae, we studied the division protein interaction network, using a bacterial two-hybrid assay, applied previously to study protein–protein interaction among the cell division players in E. coli (Di Lallo et al., 2003) and, more recently, to identify the interactive partners of the S. pneumoniae DivIVA protein (Fadda et al., 2007). Moreover, we compared the streptococcal division interaction web with that of E. coli and found that a significant number of the interactions were conserved. Consistently, several cross-interactions between S. pneumoniae proteins and the corresponding E. coli orthologues were observed. Taken together, these results indicate the presence of a minimum division interactome in common between these two, phylogenetically distinct, micro-organisms.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). E. coli K-12 and derivatives, used and generated in this work, are listed in Table 1. Luria–Bertani (LB) broth or agar was used for bacterial culture and plating and SM (salt solution) was used for bacterial dilutions as described by Miller (1972). The following antibiotics (Sigma) were added to the culture media when required: ampicillin (50 µg ml–1), chloramphenicol (34 µg ml–1) and kanamycin (30 µg ml–1).
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). PCR was carried out using the Taq DNA polymerase kit (Promega), according to the recommendations of the manufacturer. Synthetic oligonucleotides for S. pneumoniae and for E. coli, used for gene amplification in the construction of plasmids for the two-hybrid assay, are reported in Fadda et al. (2007) and D'Ulisse et al. (2007), respectively. The upstream and downstream primers, containing the restriction sites for SalI and BamHI (underlined), for amplification of pbp2B were spnSFbp2B (5'-GCGTCGACCATGAGAAAATTTAACAGCCATTC-3') and spnBRbp2B (5'-CGCGGATCCGCAATAGGTGTTGGATAAAGCA-3'); the start and stop codons, respectively, are indicated in bold.
Plasmids construction and bacterial two-hybrid assay.
Recombinant plasmids (listed in Table 1
) were constructed by cloning the entire genes of interest into the SalI and BamHI restriction sites of pcIP22 and pcI434 vectors (Di Lallo et al., 2001). The DNA of the gene of interest was obtained by PCR amplification using specific oligonucleotides carrying compatible restriction sites at the ends. All the different combinations of recombinant plasmids coding for the chimeric repressors, obtained as described above, were cotransformed into the recipient E. coli strain R721, carrying the 434/P22 chimeric operator. β-Galactosidase activity was assayed as described by Miller (1972). Bacterial cultures were grown at 34 °C in LB medium supplemented with 0.1 mM IPTG to OD600 0.5, and the residual β-galactosidase activity was evaluated for each strain. E. coli R721 without plasmids and E. coli R721 with plasmids pcI434434 and pcIP22434 were used as negative and positive controls, respectively. Residual β-galactosidase activity of less than 50 % was judged to indicate that there was repression and hence a protein–protein interaction, whereas when the activity was greater than 50 %, the interaction was uncertain or null. The use of these values has been discussed previously (Di Lallo et al., 2003; D'Ulisse et al., 2007; Fadda et al., 2007).
Construction of GFP and GST derivatives for co-immunoprecipitation studies.
The division proteins fused with GFP (green fluorescent protein of Aequorea victoria) were obtained by in-frame cloning of the PCR-produced DNA fragments of the corresponding gene into PstI and HindIII sites downstream of the gfp gene previously inserted into the EcoRI and PstI sites in the plasmid pTTQ18 to give the plasmid pTTQ18-gfp (D'Ulisse et al., 2007; Fadda et al., 2007; Table 1). Analogously, the GST (glutathione S-transferase of Schistosoma japonicum) derivatives were obtained by cloning the gene of interest into SalI and HindIII sites downstream of the gst gene inserted into the SacI and SalI sites of the plasmid pBAD33 to give the plasmid pBAD33-gst (D'Ulisse et al., 2007; Table 1).
Co-immunoprecipitation experiments.
To prepare the extracts for co-immunoprecipitation experiments, cultures of E. coli DH5
, harbouring the recombinant plasmids containing the genes of interest fused in-frame to GST and GFP, were grown in LB supplemented with the appropriate antibiotics to OD600 0.3–0.4. The expression of the tagged genes was induced by adding 0.1 mM IPTG (for the GFP-fused genes) and 0.2 % arabinose (for the GST-fused gene) and the culture was grown for 4 h. The culture was then centrifuged, washed and resuspended (1/300, v/v) in lysis buffer (1 mM EDTA pH 8, 25 mM HEPES pH 7.6, 0.1 mg lysozyme ml–1), cooled on ice for 30 min, and lysed by freezing and thawing. The protein concentration, determined by the Bradford method after cell lysis, was adjusted to 10 mg ml–1 and 1 ml aliquots of lysed samples were centrifuged at 50 000 g for 45 min. Soluble fractions were obtained by recovering the supernatant, and membrane fractions were prepared as described by Buddelmeijer & Beckwith (2004). Immunoprecipitation experiments were similar to those described by Duong & Wickner (1997). The beads (Protein A-Sepharose) were divided into aliquots of 50–100 µl in microcentrifuge tubes; 10 µl anti-GFP (1 : 100) (Santa Cruz Biotechnology) or anti-GST (1 : 100) (Santa Cruz Biotechnology) mouse monoclonal antibodies were added to each tube. The samples were incubated overnight at 4 °C with gentle mixing on a suitable shaker. After centrifugation, samples were washed with 1 ml washing buffer (20 mM HEPES buffer, pH 7.5, containing 150 mM NaCl, 0.1 % Triton X-100 and 10 %, v/v, glycerol), then 0.1–1 ml cell lysate was added to each tube. Samples were incubated from 90 min to overnight at 4 °C, with gentle mixing on a suitable shaker. Immunoprecipitated complexes were collected by centrifugation at 3000 g for 2 min at 4 °C.
Western blotting.
Electrophoresis and immunoblotting were performed as described by Laemmli (1970) and Towbin et al. (1979), respectively. The nitrocellulose membranes were probed with anti-GST antibodies (Santa Cruz Biotechnology) (1 : 2000) or with mouse anti-GFP monoclonal antibodies (1 : 1000) (Santa Cruz Biotechnology), depending on the specific GFP- or GST-fused protein used in the experiment, and detected with the ECF fluorescence-based detection system (Amersham, Pharmacia). The specific polyclonal antibodies raised against S. pneumoniae division proteins DivIB, FtsL, FtsK, FtsW, PBP2x, PBP1A and PBP2B used in Western blot experiments were generously supplied by A. Zapun and T. Vernet of the Institut de Biologie Structurale, CNRS, Grenoble, France.
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RESULTS |
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), was applied to dissect the analogous pathway in S. pneumoniae. In this assay, the interaction between two proteins, encoded by the corresponding genes cloned in the two recombinant plasmids, is tested by their ability to form a chimeric lambdoid repressor. When the gene products interact they shut down the expression of a chromosomal copy of the reporter gene, lacZ, whose synthesis is governed by a hybrid promoter/operator region, recognized by the chimeric repressor consisting of the N-terminal domain of the lytic phage repressors from either of two lambdoid phages 434 and P22 fused in-frame with the pair of division proteins whose interaction is under investigation (Di Lallo et al., 2001). In particular, we tested the cell division proteins that are known or are thought to participate in divisome formation in S. pneumoniae, like FtsZ, FtsA, ZapA, FtsK, FtsQ/DivIVB, FtsB/DivIC, FtsL, FtsW, PBP2x and PBP1A. In addition, we tested PBP2B, an essential monofunctional transpeptidase, the gene of which in S. pneumoniae is located in the dcw cluster with ftsA and ftsZ (Massidda et al., 1998).
Since S. pneumoniae fusion proteins were expressed in a heterologous system, we first tested the correct expression of those proteins for which specific antibodies were available, namely FtsZ, FtsA, FtsL, FtsQ/DivIB, PBP1A, PBP2B and PBP2x, by Western blot experiments (data not shown). Although the Western blot results cannot be generalized for the other S. pneumoniae proteins for which antibodies were not available, since all the proteins tested interact with at least one division partner, as shown in Table 2, such interaction should account for correct expression of the recombinant protein.
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; D'Ulisse et al., 2007; Fadda et al., 2007).
The results reported in Table 2 highlight that, out of 66 combinations among the S. pneumoniae cell division proteins tested, 37 show homo or hetero-dimerizations, while 29 did not interact, either with themselves or with each other. Of the streptococcal cell division proteins shown in Table 2, 18 (i.e. 14 positive interactions and four non-interactions) were consistent with those established previously for E. coli, by two different THAs (Di Lallo et al., 2003; Karimova et al., 2005) and co-immunoprecipitation experiments (D'Ulisse et al., 2007). Some of these interactions, like those of the S. pneumoniae FtsA with itself and with FtsZ, were expected, confirming and extending our previous results (Lara et al., 2005), by using a yeast two-hybrid system, and thus providing additional reliable positive controls. Moreover, the predicted FtsZ self-interaction, which could not be determined before, due to the activation of the reporter gene of single transformants producing the FtsZ protein when fused to the GAL4-binding domain (Lara et al., 2005), was detected. Besides FtsA and FtsZ, other self-interacting cell division proteins were FtsQ/DivIB, FtsB/DivIC, PBP2x, FtsK, FtsL and ZapA, consistent with the same behaviour shown by the E. coli counterparts (Di Lallo et al., 2003; P. Ghelardini, unpublished results; see below).
As shown in Table 2, a total of 29 hetero-interactions were detected among S. pneumoniae proteins and of these seven, FtsA–FtsK, FtsA–FtsL, FtsZ–FtsW, FtsZ–FtsQ/DivIB, FtsZ–FtsL, FtsK–FtsW, FtsL–FtsI/PBP2x, have not been reported previously in E. coli (Di Lallo et al., 2003; Karimova et al., 2005), although a positive FtsZ–FtsW interaction was found in Mycobacterium tuberculosis (Datta et al., 2002). Interestingly, it has been suggested recently that binding of FtsZ to the C-tail of FtsW may modulate its interactions with FtsI, regulating septal peptidoglycan biogenesis (Datta et al., 2006). It would be interesting to determine if this is also the case for S. pneumoniae, where surprisingly no interaction between FtsW and any of the PBPs tested was detected.
Two other hetero-interactions, FtsZ–FtsB(DivIC) and FtsW–FtsB(DivIC), were identified and seemed to be specific of S. pneumoniae (see below).
Among the PBPs, PBP2B was found to interact strongly with FtsZ and ZapA, further supporting the presence of this PBP at the mid-cell, as reported recently (Zapun et al., 2008; D. Fadda & O. Massidda, unpublished results).
Co-immunoprecipitation of interacting protein pairs identified by THA
To confirm the results of the THA, co-immunoprecipitation was used. Because of the large number of interactions detected between the proteins pairs examined, only some of them were tested with this additional method. In particular, we chose to confirm those interactions that were shown only in S. pneumoniae and not detected previously in E. coli, namely: FtsA–FtsK, FtsA–FtsL, FtsZ–FtsW, FtsZ–FtsQ/DivIB, FtsZ–FtsL, FtsK–FtsW, FtsL–PBP2x, FtsZ–FtsB/DivIC and FtsW–FtsB/DivIC.
Since specific antibodies were not available for all these studied proteins, the assays were performed on the division proteins fused with tags, i.e. the GFP and/or GST for which commercial antibodies are available and the specificity of the assay has already been demonstrated (D'Ulisse et al., 2007; Fadda et al., 2007).
The results, presented in Fig. 1, show that, as expected from the THA, an interaction was observed for each of the eight pairs of proteins tested. On the other hand, control experiments with non-interacting protein pairs, like FtsL–FtsB, FtsK–PBP2x and FtsW–FtsA, did not co-immunoprecipitate (data not shown).
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; Karimova et al., 2005; D'Ulisse et al., 2007), and therefore the only possible reference with which to compare the data from S. pneumoniae described above. As noted, these two species share at least nine division proteins and to compare the two interaction webs, we used the data already published on E. coli (Di Lallo et al., 2003) integrated with the data on new interactions detected for E. coli ZapA and FtsB proteins with themselves and with the other E. coli division partners. The results, reported in Table 3, show that ZapA interacted with itself and with all the other cell division proteins tested except FtsI, while FtsB interacted with itself, ZapA, FtsQ, FtsL and FtsI.
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), the ability of FtsLEC to self-interact was also detected, given the 22 % residual β-galactosidase activity, thus correcting the value reported previously (Di Lallo et al., 2003).
As shown in Fig. 2, in both organisms it is possible to identify a complex interaction network, in which each protein interacts with at least one, but more often with several partners. Although it was clear that in S. pneumoniae the interactions are more numerous than those in E. coli, 71 % vs 64 %, respectively, a closer look revealed a high similarity. Indeed, a large part of the interaction pattern between these two organisms seemed to be conserved. Out of 45 protein pairs tested for each bacterium, 30 show the same behaviour: 23 interacted and seven did not. Notably, all eight homo-interactions were conserved in both organisms. On the other hand, nine interactions appeared specific to S. pneumoniae (Fig. 2a), and six to E. coli (Fig. 2b).
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Unexpectedly, the S. pneumoniae FtsW did not interact with PBP2x, or any other PBP tested. However, it should be noted that ftsW is absent from the dcw cluster of many Gram-positive organisms, in particularly cocci (Massidda et al., 1998). Furthermore, it has been reported recently that in Staphylococcus aureus the recruitment of PBP2 to the division site is dependent on its substrate (Pinho & Errington, 2005). If this is also the case for S. pneumoniae PBPs, it could explain the lack of this interaction, although this hypothesis needs to be confirmed.
Heterologous interactions between E. coli and S. pneumoniae division proteins
Heterologous interactions between cell division proteins were first reported by Wang et al. (1997) using a yeast THA. These authors showed that both FtsZ and FtsA of B. subtilis were able to interact with FtsZ of E. coli, despite the phylogenetic distance between the two species.
The common pattern of interactions, conserved between two organisms as phylogenetically distant as E. coli and S. pneumoniae, suggests that, in this case, the interactions between these proteins (or their domains/structures) could have been maintained over the course of evolution, being fundamental to the division process. It was therefore very interesting to see whether the single members of each pair could cross-interact with the respective orthologous protein, i.e. if a selected S. pneumoniae protein would interact with its interactive E. coli counterpart and vice versa. To test this possibility, cross-interactions among heterologous proteins were tested with the THA for all those proteins that showed a conserved interaction profile in the species-specific assay.
The results, presented in Fig. 3, indicate that most of the shared common interactions detected previously, on the basis of the comparison of the two interaction webs, were also conserved in cross-interaction experiments. The cross-interactions, illustrated in the figures, concern the protein pairs FtsZEC–FtsZSPN, FtsAEC–FtsZSPN, FtsQEC–DivIBSPN, FtsQEC–FtsLSPN, FtsLEC–FtsLSPN, FtsLEC–FtsWSPN, FtsKEC–FtsZSPN, FtsKEC–DivIBSPN, FtsKEC–FtsLSPN and vice versa, with the exception of the pairs FtsK–FtsZ and FtsK–FtsQ. In these cases we observed an interaction only unidirectionally between FtsKEC–FtsZSPN and FtsKEC–DivIBSPN; in the opposite orientation, i.e. FtsKSPN–FtsZEC and FtsKSPN–FtsQEC, no interactions were detected. Finally, five cross-interactions were observed between ZapAEC and the streptococcal proteins FtsKSPN, DivICSPN, DivIBSPN, FtsLSPN and FtsWSPN. In this case, however, analysis of the reverse interactions was not performed.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In this organism, at least 12 proteins are known or believed to be involved in the process and, among them, nine, FtsZ, FtsA, ZapA, FtsK, FtsQ/DivIB, FtsB/DivIC, FtsL, FtsW and FtsI/PBP2x, are common with both other Gram-positive and Gram-negative bacteria. A function has been attributed to only a few division proteins. This identification of function is difficult because often the inactivation of the respective genes is not lethal in conditions of overexpression of other division proteins. A common property of all these proteins is their co-localization at the division site. In the last few years, substantial progress has been made in elucidating several aspects of cell division in various bacterial models, such as identification of genes, and structure and activity of the proteins involved in the process, their cytological localization, Z-ring formation, etc. All this knowledge cannot however give us information on the assembly of the division proteins or the regulation and the structure of the divisome.
Studies on division proteins' interactions in various bacterial species, such as E. coli, B. subtilis, S. pneumoniae and M. tuberculosis (Tormo et al., 1986; Wang et al., 1997; Ma & Margolin, 1999; Haney et al., 2001; Datta et al., 2002, 2006; Haeusser et al., 2004; Lara et al., 2005; Fadda et al., 2007) indicate that they engage in many interactions between each other. These results, however, refer to sporadic tests of a few interactions performed in the species reported above, whereas the only whole interaction web currently available is that of E. coli (Di Lallo et al., 2003; Karimova et al., 2005; D'Ulisse et al., 2007).
It is now generally accepted that knowledge on interactions conserved in other organisms, or ‘interologs’ (Walhout et al., 2000) ought to represent useful information that will allow the formulation and testing of biological hypotheses (Walhout et al., 2000; Wojcik & Schachter, 2001). On this basis, in this work we identified the interaction web amongst 11 division proteins of S. pneumoniae in their 66 possible pairwise combinations and compared the results obtained with those for E. coli.
The results showed the existence of a close net constituted of at least 37 homo- and/or heterodimeric protein associations in which each protein generally interacts with more partners, up to 10 for FtsL and FtsZ, whereas the mean number of partners is two or three. PBP1A is the only case described in our results in which the protein interacts with a single partner, i.e. FtsL.
The comparison of this network with the one already established for E. coli (Di Lallo et al., 2003) showed, for the nine common proteins, that among these 45 possible pairs of proteins, 30 show an analogous pattern in both micro-organisms: 23 interact and seven do not. The similarity of these two interactomes is not surprising since various ‘comparative proteomics’ strategies applied to the search for pairs of potential orthologues of known interacting protein partners in Saccharomyces cerevisiae have been performed to identify potentially conserved interactions, or ‘interologs’ (Walhout et al., 2000), in Caenorhabditis elegans. At least 16 % of the protein interactions in these networks are indeed conserved.
Our data raise some considerations: (i) the maintenance (conservation) of analogous interactomes between two phylogenetically distant species could be indicative of the biological significance of each interaction between the protein and its partners; (ii) the presence of interaction overlaps in the two networks suggests the existence of a common core of divisome proteins; (iii) if this interpretation is correct we should expect to find cross-interactions among S. pneumoniae and E. coli division proteins.
This hypothesis is supported both by the results shown in Fig. 3 and by the observation of Wang et al. (1997) of heterologous interactions between division proteins (FtsZ and FtsA) of B. subtilis and E. coli. Notably, the cross-interactions described here were observed between proteins of two bacterial species with a discrete sequence homology. The percentage of identical amino acid residues determined by Lipman–Pearson protein alignment between S. pneumoniae and E. coli proteins FtsA, FtsZ, DivIB and PBP2x (FtsI) is 33.0, 54.7, 17.1 and 24.1 %, respectively (Massidda et al., 1998).
The cross-interaction ability described in this paper may reflect a functional inter-exchangability from one organism to another. This is the case for S. pneumoniae DivIB protein, which phenotypically suppresses the defects of an E. coli FtsQ null mutant (D'Ulisse et al., 2007). Heterologous complementation among division functions of phylogenetically distant bacteria have also been described by other authors (Ma et al., 1997; Gaikwad et al., 2000; Momynaliev et al., 2002; Dziadek et al., 2003; Osawa & Erickson, 2006). We are presently performing other complementation assays to establish whether other S. pneumoniae division proteins can substitute for their E. coli orthologues.
It is generally admitted that various levels of prudence need to be considered when analysing the results obtained by THA. This last notion comes from the application of the Saccharomyces cerevisiae THA, the first and most widely used tool in protein–protein interaction. This assay often highlights false positive or negative interactions. False positives are due to the overexpression of the reporter protein fusions, which could result in magnifying potentially weak interactions whereas the false negatives could be due, for example, to low expression levels and/or instability of the molecules tested. Presently, we have no data that allow evaluation of the limit of the THA used in this work. However, although the number of interactions tested with the phage THA is very low compared with that obtained with the yeast THA, some data suggest that our system excludes the false (or indirect) interactions: formally, if a protein X interacts with both A and B, false positives could be due to indirect interaction between A and B mediated by X in a complex such as A–X–B. Besides the examples reported in previous papers (Di Lallo et al., 2003; D'Ulisse et al., 2007), the absence of interaction between ZipA and FtsA in E. coli suggests that the bridge eventually formed with FtsZ, which interacts with both proteins, is not revealed. Analogously, the assay does not reveal interactions between the E. coli proteins FtsZ–FtsW and FtsZ–FtsB although all of them also interact with ZapA. In addition, the interactions described in this paper were observed in a heterologous system, S. pneumoniae proteins tested in E. coli. The formation of false positives should need an E. coli ‘bridge’ protein able to interact with both the S. pneumoniae partners. Other kinds of false positives are, however, possible, such as the one due to the overexpression of proteins magnifying potentially weak interactions and leading to false signals. In these cases, levels of residual β-galactosidase synthesis close to 50 % could represent weak or transient interactions amplified by protein overexpression. In any case, the S. pneumoniae species-specific interactions have already been confirmed by co-immunoprecipitation, similarly to previous reports (D'Ulisse et al., 2007; Fadda et al., 2007). Finally, as far as false negatives are concerned, this aspect can be related to the expression level and/or stability of the proteins under investigation. Generally, it is easily solved by observing whether there are two molecules, X and Y, that do not interact with each other, but each of which is able to interact with other partners; thus the lack of interaction between them is real. This condition was verified for 29 protein pairs that did not shown any interaction between them.
In conclusion, the comparative analysis of the division protein interactomes reported in this work allows the identification of protein–protein interaction representatives of a minimal common divisome in Bacteria. For this reason, these results may be helpful for both a deeper comprehension of some aspects related to bacterial divisome formation and the elucidation of the cell division process in the Gram-positive coccus S. pneumoniae, where some genetic tools are as yet underused. Finally, given the importance of the cell division proteins as primary targets for the development of new broad-spectrum antibacterial inhibitors, the knowledge of the conserved protein–protein interactions, between phylogenetically distant species, should reinforce their attractiveness in this respect.
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ACKNOWLEDGEMENTS |
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Edited by: W. Margolin
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Received 20 March 2008;
revised 30 May 2008;
accepted 12 June 2008.
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