Abstract
Thus, neisserial competence for transformation is dependent on the expression of several pilus-related components, including a number of pilus biogenesis components (Tønjum & Koomey, 1997) and the minor pilin ComP (Wolfgang et al., 1999). The secretin PilQ is associated with translocation of the pilus fibre across the outer membrane by mediating type IV pilus extrusion and retraction (Tønjum et al., 1998). Neisserial PilQ mutants are non-piliated and non-competent for transformation, and PilQ has previously been shown to bind DNA (Assalkhou et al., 2007). Other components suggested to be involved in neisserial transformation include the competence factors ComE (Chen & Gotschlich, 2001), ComA (Facius & Meyer, 1993) and ComL (Fussenegger et al., 1996, 1997). However, all of the DNA binding components involved in the machineries which drive meningococcus transformation and other forms of horizontal gene transfer have not yet been identified.
In order to identify and characterize meningococcus proteins that directly bind DNA, we have previously employed cellular fractionation and a solid-phase overlay assay in the form of South-Western analysis in combination with MS analysis (Lång et al., 2009). In general, the procedure employed proved to be a useful strategy for identifying DNA binding components. Here, the resolution of the solid-phase overlay assay was improved by using 2D gel electrophoresis. In the meningococcus membrane and soluble fraction, respectively, the competence lipoprotein ComL and the neisserial orthologue of the single-stranded DNA binding protein SSB were predominant. The observed DNA binding activity of ComL and SSB was verified by electromobility shift analysis. We propose 3D models for the structures of meningococcus ComL and SSB and define their protein-interacting counterparts. Thereby, meningococcus DNA dynamics relevant for horizontal gene transfer and recombination are further elucidated.
In this study, we searched cellular fractions from a representative panel of neisserial strains for DNA binding components, using 1D and 2D electrophoresis combined with a solid-phase overlay approach. The DNA binding proteins were identified by MS analysis. Enrichment of the membrane and the soluble fractions were important steps contributing to the generation of target solutions with reduced complexity, as compared with meningococcus whole-cell extracts, for the identification of DNA binding proteins. The 2D solid-phase overlay strategy further increased the spatial resolution of the proteins in comparison with the 1D approach. Employing peptide mass fingerprinting by MALDI-TOF-MS enabled protein identification from small amounts present in the 1D/2D gel spots which exhibited DNA binding.The predominant membrane components exhibiting DNA binding activity were identified as ComL and PilG, as previously reported (Lång et al., 2009). In this context, we wanted to characterize ComL further. In EMSA, ComL was shown to bind DNA in a Mg2+-dependent manner, indicating that Mg2+ facilitates the direct binding of DNA (Fig. 5). A hypothetical 3D structure for ComL was generated and the charge distribution on the ComL molecular surface was predicted, suggesting that there are several regions of positive charge, which may function as specific or non-specific DNA binding regions. ComL was also shown to have structural homology with the P. aeruginosa pilus biogenesis protein PilF (Kim et al., 2006), which is homologous to the neisserial outer membrane lipoprotein PilW engaged in pilus biogenesis (Carbonnelle et al., 2005; Trindade et al., 2008).
The neisserial ComL protein has been suggested to contribute to DNA uptake by cleavage of the peptidoglycan layer during transformation (Fussenegger et al., 1996, 1997). The gonococcal comL gene exists in a single copy, which is transcribed in the opposite direction to the neighbouring comA gene and encodes a periplasmic lipoprotein with a relatively high theoretical pI (9.03). The comL and comA gene pair has a common DUS-containing transcriptional terminator in an appropriate position for joint use (Jose et al., 2003). The ComL orthologue in E. coli, YfiO, is anchored to the outer membrane and is a member of the β-barrel assembly machinery (the BAM complex) (Wu et al., 2005). Recently, ComL was referred to as an orthologue of BamD, which suggests that neisserial ComL is associated with the outer membrane and is involved in outer membrane protein biogenesis (Knowles et al., 2009). This notion was supported by our membrane separation (Fig. 3a). Orthologues of ComL are conserved among both neisserial and other Gram-negative bacterial species (Fussenegger et al., 1996; Malinverni et al., 2006). Bioinformatic inferences suggest that the comL gene encodes TPRs, and that the resulting structure presents several large regions of positive charge, which may act as either ssDNA or dsDNA binding sites. Still, it is not possible to determine from these structural predictions whether the apparent DNA binding capacity is related to the TPRs. TPRs have been predicted for neisserial PilW and Pseudomonas PilF, and have been suggested to serve a functional role in PilQ stabilization (Koo et al., 2008; Trindade et al., 2008). Thus, as previously suggested, the TPRs predicted could be of functional importance in mediating protein–protein interactions between ComL and other BAM proteins (DAndrea & Regan, 2003; Knowles et al., 2009). No potential DNA–protein interactions have been reported for TPR-containing proteins (DAndrea & Regan, 2003), though the predicted positively charged regions of ComL may interact with DNA in a non-specific manner. Site-directed point mutations will enable the elucidation of the potential role of TPRs in ComL-mediated DNA binding or protein–protein interactions.
A significant decrease in the transformation rate of a gonococcal comL mutant has been documented, suggesting a role of the lipoprotein ComL in the neisserial transformation process in interaction with the peptidoglycan layer (Fussenegger et al., 1996). Moreover, neisserial ComL has been suggested to be involved in the folding of outer membrane proteins (Knowles et al., 2009). The true function of ComL is difficult to assess since most mutants of this essential component are lethal. It is of note that Fussenegger and co-workers managed to generate a viable gonococcal comL mutant, expressing a truncated version of the protein, therefore indicating the importance of an intact N terminus in ComL protein expression and function (Fussenegger et al., 1996). Meningococcal comL null mutants were also non-viable, indicating that ComL is essential in neisserial species (Table 1). The pilQ and pilG null mutants, defective in pilus biogenesis, were non-competent for transformation. For these two components, the biological significance of their DNA binding capabilities is complicated by the fact that they participate in type IV pilus biogenesis, which in turn is required for competence. Thus, it is a conundrum as to whether the lack of competence in these mutants is due to a defect in their direct binding of DNA or whether this lack is indirect through pilus biogenesis.
SSB, the predominant DNA binding protein detected in the soluble neisserial cell fractions, is involved in processing ssDNA intermediates during DNA replication, repair and recombination in E. coli (Shereda et al., 2008). Furthermore, SSB proteins are conserved, serve critical functions in genome maintenance and are indispensable for cell survival among both prokaryotes and eukaryotes (Fanning et al., 2006; Shereda et al., 2008; Zou et al., 2006). In our hands, neisserial SSB was found to bind both ssDNA and dsDNA, which is consistent with findings on SSB in other prokaryotes, in which SSB either binds to ssDNA or intercalates itself into dsDNA, thereby disrupting it (Makhov & Griffith, 2006; Makhov et al., 2009; Mapelli et al., 2005). Previously, the genome of Bacillus subtilis was shown to comprise two paralogous SSB genes, ssb and ywpH. Interestingly, the proteins encoded by ssb and ywpH have distinctive expression patterns, with SSB being essential for cell survival, while YwpH is required for natural transformation (Lindner et al., 2004). In the ywpH null mutant, the transformation rate was reduced fivefold, whereas the ssb null mutant was not viable (Lindner et al., 2004; Ogura et al., 2002). In Haemophilus influenzae RD KW20, gene expression analyses have revealed that the SSB orthologue HI0250 is induced 3.4-fold during the competent state (Redfield et al., 2005). Based on the broad conservation of SSB functions and the documented role of SSB in transformation, we suggest that neisserial SSB might also have a functional role in transformation. As in E. coli (Shereda et al., 2008), meningococcus ssb null mutants were non-viable, and meningococcus SSB phenotypes directly associated with transformation could therefore not be assessed (Table 1).
The purpose of this study was to identify and characterize neisserial DNA binding proteins and assess their potential relevance for transformation, putting a special emphasis on the characterization of ComL and SSB and their DNA binding properties. The neisserial DUS has been shown to mediate selective uptake of DNA through transformation; thus the search for DUS-specific DNA binding components was a priority. However, this endeavour turned out to be a difficult task since none of the DNA binding proteins identified bound DNA in a DUS-specific manner, nor contained the DUS sequence within their ORFs. The difficulties and challenges in identifying a potential DUS-specific binding protein are multiple, since functional and technical obstacles obscure the hunt for an unknown or putative component. If it does exist, the cellular location of DUS selectivity is not yet known. This search for DNA binding candidates targeted the inner membrane and cytoplasm, and a DUS-specific protein in these fractions and the neisserial outer membrane has not yet been identified. This negative search result could well be due to technical limitations, in that the in vitro conditions in the solid-phase overlay approach employed do not reflect the DNA binding that goes on in vivo. The solid-phase overlay assay also has limitations in the form of the level of protein expression, protein folding and renaturation abilities after SDS-PAGE. Thereby, not all DNA binding candidates will be detected using this method, and DUS-specific DNA binding might be of such a subtle and transient nature that it is not detected in this assay. Yet, the reproducible identification of PilG, ComL and PilQ DNA binding by this approach (Lång et al., 2009), in addition to other independent assays (Assalkhou et al., 2007), is strong evidence that the method is indeed valid for detecting a number of DNA binding proteins in general.
This study strengthens previous findings on potential direct roles for ComL, PilG and PilQ in transformation. Additional studies are warranted to provide new insights into the functional relationships between these and other proteins involved in the transformation process. Characterization of the physical interactions between ComL and SSB with DNA, in addition to other proteins, will contribute to a better understanding of how transforming DNA is processed in meningococcus cells and will further elucidate the neisserial DNA uptake and genome integration process.
The Medical Research Curriculum and the Institute of Medical Basic Sciences at the University of Oslo are greatly acknowledged for their support to A. V. B. We are grateful to Håvard Homberset for excellent technical assistance and invaluable discussions and to Marit Jørgensen for protein identification at the Proteomics Core Facility at Oslo University Hospital (Rikshospitalet)/University of Oslo. This work was supported by a Centre of Excellence grant from the Research Council of Norway to the Centre for Molecular Biology and Neuroscience (CMBN) and the FUGE platform Consortium for Microbial Science Technology (CAMST).Footnotes
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Received 16 October 2010; revised 18 January 2011; accepted 16 February 2011.
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