According to our classification, Bce-like systems in B. subtilis belong to three separate groups. All three systems, BceRS-AB, PsdRS-AB, and YxdJK-LM seem to form stand- alone detoxification modules. So far, only a low level of cross-talk has been reported for Bce and Psd systems, most probably with the HK BceS phosphorylating PsdR RR (Rietkötter et al., 2008). However, this cross-talk is weak (induction of the psdA promoter is approximately 30 fold weaker than the bceA promoter) and it is not clear if it plays any physiological role.
BceRS-AB, PsdRS-AB and YxdJK-LM respond to and mediate resistance against various peptide antibiotics, similarly to other Bce-like systems. In our study presented in Chapter 5, we aimed to identify novel inducers for all threedetoxification modules present in B. subtilis. We screened a wide variety of cell envelope-active compounds,including many peptide antibiotics. We also performed an in silico meta-analysis of all previously published stressresponse microarray data sets and searched for additional inducers of these
three systems. In this study, we identified lipid II-binding lantibiotics asthe main group of inducers of PpsdA expression. PpsdA is induced by lantibiotics nisin, subtilin, actagardine and
gallidermin, but also a lipid II-binding lipopeptide, enduracidin. Induction by another peptide antibiotic, bacitracin, is a result of cross-talk, as described by Rietkötter and colleagues (Rietkötter et al., 2008). We also identified two lantibiotics, actagardine and mersacidin, as novel inducers of bceAB expression. This is an interesting finding, as BceAB was previouslythought to be a bacitracin-specific resistance pump (Mascher et al., 2003; Ohki et al., 2003a).We were not able to identify novel inducers ofPyxdL, which is
until now known to respond only to the human peptide LL-37 (Pietiäinen et al., 2005). Results from our study presented in Chapter 5 clearly show that the three detoxification systems are independent modules with only very slightly overlapping inducer spectra, in contrast to other organisms like L. monocytogenes and S. aureus. Interestingly, the three systems show a high level of sequence similarity in all protein components, as well as in their promoter regions. These observations led us to investigate the specificity determinants in Bce-like systems in B. subtilis. In chapter 6 we report the first results on the specificity determinants in the BceR and PsdR dependent promoters. Chapter 7 summarizes the results we obtained from investigating the specificity determinants governing the interactions between the kinases and RR, kinases and ABC transporters, and also specificity determinants in stimulus sensing.
8.3.1. Specificity determinants in BceR and PsdR dependent promoters
Promoters controlled by the BceR-like RR show an unusual architecture (Dintner et al., 2011). Similarly to other 2CS-regulated promoters, these promoters harbour a repeat that is necessary for a RR-dependent induction. Whether this is an inverted or direct repeat cannot be distinguiseh at this point, as both repeats in the psdA and bceA promoters fulfill the criteria of either an imperfect inverted or a direct repeat. Our results clearly show that the core of this repeat is necessary for the induction of the promoter. However, the characteristic feature of the bceA-like promoters is the presence of additional half sites, both upstream and downstream of the main repeat. Results presented in Chapter 6 suggest that these half sites play an important role in regulating the expression of bceA-like promoters in B. subtilis. The upstream half site seems to influence the strength of the promoters, whereas the downstream half site may be responsible for promoter specificity. Based on the first results from the promoter induction assays is conceivable that the the bceA and psdA promoters are subject to a homocooperative activation, where each of the
recognition sites binds the RR with a different affinity. While this hypothesis will have to be validated by independent in vitro and in vivo experiments, this study is a good starting point providing the first insights into the specificity determination in bceA-like promoters.
8.3.1.1. Flexibility (“fuzziness”) of the main binding site. The mutagenesis studies of the
main binding site show that although the main binding site is indispensable for induction, its sequence can be modified to some extent. Our experiments clearly demonstrate that the exact composition of the binding sites does not play an important role in promoter specificity, as long as the overall consensus is not changed. However, it influences the strength of the response.
Motif “fuzziness”, or differences to the optimal consensus sequence can be observed for binding sites of many transcription factors. So called “fuzzy” binding sites differ at various position from the perfect palindrome, and also have a lower affinity towards the transcription factor then the perfect palindrome would have (Francke et al., 2008). The fuzziness of the binding site may be a consequence of a balance between mutation and selection, since the action of the transcription factor may be insensitive to subtle changes in binding affinity, provided they are above a certain threshold (van Hijum et al., 2009). Our results suggest that this may be true for BceR and PsdR. “Fuzziness” of the transcription factor binding sites was also suggested to play a role in cooperative transcription activation and repression ((Hermsen et al., 2006), see below). It is also possible that the binding sites can show a lower level of conservation (and higher level of “fuzziness”) when there are multiple binding sites located in a direct vicinity, as this will cause the local concentration of the transcription factor to be higher than normal (van Hijum et al., 2009).
8.3.1.2. The decisive influence of the downstream half site – homocooperative
activation? The first results obtained from the analysis of chimeric promoters show that
the fragment located between the main binding site and the translation start site is the one that determines the promoter specificity. Its exchange between psdA and bceA promoters leads to either full or partial exchange of specificity. This fragment harbours the downstream half site which is most probably one of the specificity determinants in bceA- like promoters. However, attempts to further narrow down the region that determines the specificity were as yet unsuccessful. In the context of the chimeric promoter, the upstream half site seems to influence the strength of induction. The similarity of the -35 promoter to
the recognition sequences is intriguing, but can be purely accidental and has to be addressed experimentally.
Taken together, our results show that (i) the full repeat is necessary for the induction, but (ii) some substitutions in its sequence that do not change the specificity of the promoter are possible (“fuzziness”). Moreover, (iii) the upstream half site plays a role mainly in modulating the strength of induction, visible only under certain conditions. The most important finding is that (iv) the specificity determinant is located between the main repeat and the translation start site, and possibly involves the downstream half site. Therefore, it seems that these two half sites and the main repeat all play an important, although different roles in the promoter activation.
It is conceivable that these two promoters are subject to a homocooperative activation. In homocooperative activation multiple binding sites can be identified in the promoter region, each binding the transcription factor with different affinity. The sites which bind the transcription factor molecule interacting directly with the RNA polymerase are called primary sites. In case of transcriptional activators, these sites are usually located next to the -35 promoter element (Hermsen et al., 2006). The remaining sites are termed secondary sites. The latter are expected to be more conserved and bind the transcription factor with higher affinity than the primary site in order to maximise the steepness of response to the transcription factor concentration. An interesting scenario is conceivable where the downstream half site (possibly together with the region annotated as the -35 promoter element?) constitutes the primary site, binding the RR with lower affinity, but higher specificity. The main binding site and the upstream half site would play the accessory role and bind the RR with higher affinity. This hypothesis can be supported by the fact that for the bceA promoter a binding of the RR to the region which includes the main repeat as well as the upstream half site was demonstrated with DNase I footprinting assay (Ohki et al., 2003a). The first gel retardation assays with psdA promoter and PsdR RR show different molecular weight complexes, indicative of binding of the RR to more than one recognition site (data not shown). However, until now no cooperative transcription activation was shown for these two promoters and this hypothesis requires further studies.
However intriguing the results discussed above are, it is important to bear in mind that the genetic analysis of regulatory sequences has its limitation, and can rather suggest than prove a model. It has to be also kept in mind that the sequence surrounding the binding site can also have an effect on both the binding affinity and the half-life of RR binding. The higher the affinity of the neighbouring sequence to the RR, the longer the RR will take to
diffuse along the DNA towards its binding site, and the shorter it will stay bound to it (van Hijum et al., 2009). This effect has been demonstrated for the CcpA protein of B. subtilis, which is able to bind with higher affinity to cre boxes located in an AT-rich context that to those located in GC-rich context (Zalieckas et al., 1998). Some regulatory regions – and it cannot be ruled out that this is the case for PbceA and PpsdA – are complex and their action
involves many factors, some with small effects. Therefore, it will be necessary to test the hypotheses using independent biochemical methods.
8.3.2. Specificity determinants in BceRS-AB and PsdRS-AB 2CS
In Chapter 7, we present a complementary study where we investigate the specificity determinants in BceR-like RR. We set out to identify the residues which interact with the important promoter elements and lead to the observed insulation of the three signalling pathways. In the random mutagenesis screen we identified 12 independent mutations in BceR and one in YxdJ. These mutations are located in the receiver and DNA-binding domains, as well as in the flexible linker between them. These results provide a perfect starting point for further studies and functional analyses of the isolated mutants. We also established a random screen that can be easily modified and allows for searching of various other gain-of-function mutations, e.g. in the bceA-like promoters.
In Chapter 7 we also present the first results on stimulus perception in BceRS-like 2CS and their cognate ABC transporters. The question of stimulus perception in BceRS-AB-like systems is an interesting one due to a specific architecture of BceS-like HK. These kinases belong to a family of proteins that has been termed IM-HK, since they lack an obvious input domain and are thought to sense the stimulus in the membrane interface (Mascher, 2006). Homologous HK in in S. aureus and S. epidermidis were suggested to sense the inducing peptides via the short extracytoplasmic loop of the kinase (Li et al., 2007a; Li et al., 2007b). In our study we demonstrate that this is not the case for the BceS-like HK in B. subtilis. We propose that the stimulus (i.e. peptide antibiotic molecule) is sensed by the ABC transporter, which in turn interacts with the HK. This hypothesis is supported by the initial results from bacterial two-hybrid assays in E. coli, whichsuggest that BceS HK and the BceB permease, as well as PsdS HK and PsdB permease are able to interact. Our findings are well in agreement with the results presented in Chapter 4, where we showed the cooccurrence and coevolution of BceB-like transporters and the BceRS-like 2CS.