4556 Nucleic Acids Research, 2009, Vol. 37, No. 14 The DBD pairs of the two octameric rings found in the asymmetric unit were orientated in such a way that both octamers could bind to the same strand of DNA (Figure 7C). These observations lead us to propose a model whereby CrgA interacts with both LTTR-binding sites in the CrgA/MdaB promoter region and which there- fore reconciles the octameric assembly state of CrgA and its interaction with DNA revealed by footprinting (48). In this model (Figure 7A) we envisage two CrgA octamers stacked against each other, in a comparable manner to that observed in the second crystal form, with DNA bind- ing through a single DBD pair of the two octamers. The LTTR motifs are aligned between the two recognition hel- ices of each pair with S35 contacting the conserved T/A bases. The model satisﬁes the 15-bp spacing between the two LTTR motifs and means that the three bases between CrgA site 1 and 2, which are hypersensitive to DNase, do not contact the protein. Our model does not rule out the possibility that individual CrgA octamers interact with the CrgA/MdaB promoter region in vivo as indicated by MS and AUC in vitro. However, as discussed below, the func- tional consequences in terms of promoter activation/ repression under these circumstances would be diﬀerent to the binding of a double since only single LTTR-binding sites would be engaged at a time. DISCUSSION A large variety of both global and speciﬁc transcription factors are deployed in bacteria to control gene expression in response to diﬀerent environmental signals. One of the largest families of such proteins is the LTTRs, which func- tion to both induce and repress transcription. Despite con- siderable progress in understanding the genetics and biochemistry of LTTRs there remains much to be learnt about the molecular details of how these proteins regulate transcription. In particular, the lack of a strong consensus DNA-recognition sequence has hindered eﬀorts to unam- biguously characterize the relationship between LTTRs and their DNA targets. Combining X-ray crystallography, MS and analytical ultracentrifugation, we have elucidated the structure of the LTTR, CrgA from N. meningitidis and have determined the stoichiometry of binding to DNA. Unlike other LTTRs, which form tetramers, we show that CrgA assembles into an octameric array, which can bind up to four 63-bp DNA oligonucleotides. On the basis of these observations, we have developed a model for the interaction of CrgA with its cognate pro- moter region, which involves the binding of two stacked CrgA octamers to adjacent recognition sites in the target DNA. This contrasts with the current model for tetra- meric LTTRs in which the DBDs of a single LTTR located unilaterally on the tetramer are thought to engage adjacent binding sites (2). However, only in the case of CysB from Salmonella typhimurium has direct experimental evidence been obtained to support this stoi- chiometry (61). In the non-induced state, the transcription of the LTTR is repressed and the divergent regulated gene is not actively transcribed. In response to some environmental cue, a conformational change in the LTTR, propagated through interactions in the C-terminal domain, results in a change in protein–DNA contact. This appears to be asso- ciated with relaxation of the angle of DNA bending within the promoter region and involves, in some cases, translo- cation of the protein from one binding site to another as observed by a change in length and position of the DNA footprints e.g. OxyR, (62) OccR (63) and AtzR (8). It is presumed that this in turn favors RNA polymerase engagement presumably by relieving some steric hindrance and may involve protein–protein interactions between the LTTR and RNA polymerase. In the regulation of cysP by cysB in E. coli, it has been shown that direct interaction between CysB with the C-terminal domain of RNAPa (aCTD) is required for the activation of transcription both in vitro and in vivo (64). Based on the above consid- erations and our model for the CrgA–DNA complex, we propose the following mechanism for how CrgA may switch on mdaB expression, while also repressing its own transcription. During initial adhesion between Neisseria and epithelial cells, expression of crgA is upregulated as part of a regulon, which responds to contact with host cells (17). Increased cellular levels of CrgA leads to the occupancy of both the LTTR-binding sites in the crgA– mdaB promoter region. This, in turn, would repress crgA transcription as RNA polymerase would be sterically blocked from accessing the crgA promoter, resulting in self-limiting autoregulated expression of CrgA. Results from DNA protection assays with dimethylsulfate (DMS) and DNase suggest that the promoter DNA undergoes a conformational change upon CrgA binding (48,65). From our model of a CrgA–DNA complex, occu- pancy of both CrgA-binding sites by the double octamer would cause the DNA to bind inhibiting transcription of mdaB. It is known that expression of mdaB is induced following cell adhesion (66) and also by treatment of cells with MBL, which has been shown to be dependent upon CrgA expression (18). For this to happen CrgA must respond to a further eﬀector signal generated post- adhesion or following exposure to quinones, which induces a conformational change in the protein. This leads to a change in the disposition of the octamers with respect to each other and relaxation of the bound DNA, which in turn leads to the recruitment of RNA polymerase by CrgA and initiation of transcription of the mdaB gene. The proposed sequence of events is necessarily speculative but provokes follow-up experiments including exploring the role of DNA bending in the interaction of CrgA with its target promoters, the possible direct interaction with RNA polymerase and visualization of the assembly of the CrgA-dependent transcription complex. In conclusion, we have shown a new assembly state for the LTTR family of transcription factors, which indicates a role for multimerization in the interac- tion with DNA. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online.