| Bacteria have to adapt the changes of environment when subjected to environmental stress and nutrition starvation. A special adaptive response induced by (p)ppGpp is called "stringent response". It is of great significance to master bacterial survival mechanism as well as the pathogenesis of infected animals. In bacteria, (p)ppGpp is mediated by RelA/SpoT homologue (RSH) proteins. Streptococcus suis serotype 2(SS2) encodes two (p)ppGpp synthetases RelA and RelQ, RelA contains one (p)ppGpp synthesis structure domain (RSD), one (p)ppGpp hydrolysis structure domain (HD) and two regulation related structure domain (TGS and ACT), but RelQ only contains one (p)ppGpp synthesis structure domain (RSD). In the early studies in our lab, RelA is the only source of (p)ppGpp synthesis during glucose starvation, while RelA and RelQ were both involved in the role of the (p)ppGpp synthesis during iron limitation and amino acid starvation. Generally speaking, it is presumed that RelA and RelQ regulate activity of hydrolysis and synthesis of (p)ppGpp through interaction with ribosomes. Given the function difference between RelA and RelQ, we speculated that they might interact with different ribosomal subunits. Thus, we carried out the research about protein interactions between the (p)ppGpp synthetase RelA/RelQ and the ribosome subunits in SS2. The main results were as follows:RelA and RelQ were respectively used as bait proteins for the ribosomal subunits by BacterioMatch Ⅱ two-hybrid system. We found that only six ribosomal subunits didn’t interact with RelA and RelQ. There were forty-two ribosomal subunits interacting with RelQ, among which twenty-eight subunits interacted with RelA. Thus, there were fourteen subunits interacting with RelQ but not with RelA, including seven subunits which would be stably phosphorylated in the protein synthesis. And the rest subunits play an important role in serving as peptidyl transferases, molecular chaperones or signal recognition particles. The results showed that RelA and RelQ indeed interact with different ribosomal subunits. It could be the possible mechanisms for RelA and RelQ under different conditions of strigent response.In order to further study the interactions between RelA/RelQ and ribosome We used (p)ppGpp synthesis structure domain (RSD) as bait protein for the ribosome subunits. There are only eight subunits that could interact with RSD. However, both RelA and RelQ could interact with this eight subunits. These results suggest that besides RSD, the other domains are also involved in the interactions between RelA and ribosome subunits. Compared with RSD, there are more different subunits interacting with RelQ. This may be related to the 30% homology between RSD structure domain and RelQ. It further illustrates that structural differences between RelA and RelQ determine their interactions to the ribosomes, which could regulate different strigent response under different condotions.In the prievous study, we already got △relA strains. To further study the regulation mechanism on RelA and RelQ, We hope to construct the ArelQ and ArelAArelQ strains. We have already constructed the suicide recombinant plasmid pSET4s-△relQ. Then the plasmid was electrotransformed into SC-19 strains and ArelA strains respectively. And we have already screened 8000 and 5000 colonies respectively. Now we have already got the single exchange strain. The target mutant strain is still under further screening. |
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