More than just a NAP
Signaling by the second messenger c-di-GMP in bacteria has been studied for over 30 years but its many functions keep surprising us. Especially c-di-GMP effectors are very diverse, allowing this second messenger to affect a large number of cellular processes.
The starting point for my work was the search for c-di-GMP binding proteins in our model organism Myxococcus xanthus. We knew that M. xanthus uses c-di-GMP to regulate its social behaviors including motility and development1,2 but we did not know the c-di-GMP binding proteins that would be involved in mediating this regulation. Because known c-di-GMP receptors are highly variable, it is difficult to predict them bioinformatically. Therefore, we decided to use a so-called Capture Compound approach to identify c-di-GMP binding proteins3. In this approach, we fish out c-di-GMP binding proteins directly from cell lysates and identify them using mass spectrometry. Although this experiment provided us with several interesting candidates, most of them belonged to previously known groups of c-di-GMP binding proteins and were no big surprises. However, two small, hypothetical proteins of unknown function were exceptions that particularly caught my attention. They are paralogs, encoded next to each other in the genome and did not show homology to any of the known c-di-GMP proteins. All this made them very interesting and quite mysterious candidates for a completely novel type of effector. Indeed, when we purified the two proteins both turned out to be able to bind c-di-GMP and we decided to call them CdbA and CdbB for c-di-GMP binding protein A and B.
At this point, we did not have any information about the proteins’ structure and function. Luckily, we had great collaboration partners. We teamed up with the research group of Dr. Andrew Lovering at Birmingham University, who solved the crystal structure of CdbA and showed that it belongs to the ribbon-helix-helix family of proteins, which bind DNA and are usually involved in transcriptional regulation. Surprisingly, further experiments demonstrated that CdbA is very different. Unlike other RHH proteins, CdbA binds to many sites on the chromosome with a low specificity and rather than regulating transcription, it contributes to nucleoid organization. Because we could not crystallize CdbA complexed with c-di-GMP we teamed up with the research group of Dr. Gert Bange. Using HDX-MS, they identified regions in CdbA, which responded to the addition of c-di-GMP suggesting their role in c-di-GMP binding. To our surprise, those interfaces perfectly overlapped with the regions involved in DNA binding and indeed, we could show in vitro that c-di-GMP and DNA binding are mutually exclusive.
Figure a. CdbA dimer interacting with DNA with the two interfaces identified by HDX to be involved in c-di-GMP binding colored in red and green. b. Images of representative cells showing a DAPI staining of the nucleoid and ParB-YFP localization as a marker of origin of replication of CdbA depletion strain in comparison with wild-type strain and wild-type strain blocked in cell division with cephalexin. Scale bar, 10 µm.
This motivated us even more to investigate the function of our two new receptors in M. xanthus cells and I continued with in vivo experiments. While inactivation of CdbB did not affect any of the cellular processes that we tested, we found out (after many many trials to inactivate cdbA) that CdbA is essential for survival. Depleting CdbA from the cells caused severe defects in chromosome organization and segregation causing a defect in cell division that ultimately resulted in cell filamentation and eventually lysis. CdbA has many features common with so-called NAPs (nucleoid-associated proteins) but while NAPs are usually regulated by abundance, we propose that CdbA is an unusual, ligand-regulated NAP meaning that its DNA binding activity is regulated by c-di-GMP.
Strikingly, how CdbA is regulated by c-di-GMP in vivo remains a mystery, as a M. xanthus strain with artificially increased c-di-GMP level does not show any obvious defects in chromosome organization and segregation. On top of that, from our previous studies, we know that c-di-GMP level increases significantly during development with fruiting body formation2, suggesting that maybe CdbA is involved in regulating multicellular development. We will keep you posted as soon as we know more.
- Skotnicka, D. et al. c-di-GMP regulates type IV pili-dependent-motility in Myxococcus xanthus. J Bacteriol, doi:10.1128/JB.00281-15 (2015).
- Skotnicka, D. et al. A minimal threshold of c-di-GMP is essential for fruiting body formation and sporulation in Myxococcus xanthus. PLoS Genet 12, doi:10.1371/journal.pgen.1006080 (2016).
- Nesper, J., Reinders, A., Glatter, T., Schmidt, A. & Jenal, U. A novel capture compound for the identification and analysis of cyclic di-GMP binding proteins. J Proteomics 75, 4874-4878, doi:10.1016/j.jprot.2012.05.033 (2012).