Homeostasis of cyclic-di-AMP in Mycobacterium smegmatis: Functional and structural contributions of c-di-AMP synthase (MsDisA) and hydrolase (MsPDE)

Abstract

In bacteria, cyclic-di-nucleotide based second messengers regulate various physiological processes including the stress response. For the past decades, cyclic diadenosine monophosphate (c-di-AMP) has emerged as a crucial second messenger in the bacterial world. It is an essential molecule implicated in fatty acid metabolism, antibiotic resistance, biofilm formation, virulence, DNA repair, potassium and osmotic homeostasis, sporulation etc. The level of c-di-AMP is maintained within the cell by the action of two opposing enzymes, namely diadenylate cyclases and phosphodiesterases. In mycobacteria, this molecule is essential for its regulatory role in bacterial physiology and host-pathogen interactions. However, such modulation of c-di-AMP remains to be explored in Mycobacterium smegmatis. Here, we systematically investigated the c-di-AMP synthase (MsDisA) and a hydrolase (MsPDE) from M. smegmatis at different pH and osmolytic conditions in vitro. Our biochemical assays show that the MsDisA activity is enhanced during the alkaline stress and c-di-AMP is readily produced without any intermediates. At pH 9.4, the MsDisA promoter activity in vivo increases significantly, strengthening this observation. However, under physiological conditions, the activity of MsDisA was moderate with the formation of intermediates. We also observed that the size of MsDisA is significantly increased upon incubation with the substrate. To get further insights into the structural characteristics, we solved a 3.02 Å cryo-EM structure of the MsDisA, revealing some of interesting properties. Analysis of individual domains shows that the N-terminal minimal region alone can form a functional octamer. Altogether, our results reveal the biochemical and structural regulation of mycobacterial c-di-AMP in response to various environmental stresses. Chapter 1 reviews the available literature in the field of second messengers and provide rational behind this study. The discovery of c-di-AMP was accidental, which later had known to regulate plethora of functions. This chapter stresses upon the need to investigate the significance of c-di-AMP homeostasis in Mycobacteria and the scope of the current study. Chapter 2 reports the homeostasis of c-di-AMP by DisA and PDE in M. smegmatis and the substrate-induced inhibitory mechanism of MsDisA. Promoter study suggested MsDisA is expressed in all the growth phases and the changes in the extracellular pH during mycobacterial growth give valuable hints about the trans-membrane pH regulation by the c-di-AMP molecule. Chapter 3 further elaborates the enzyme-substrate reaction and domain movement. The substrate-induced change of the MsDisA structure, which is demonstrated using biophysical characterization and Transmission Electron Microscopy (TEM) image analysis. The details on the structural characteristics of the MsDisA were obtained by cryo-EM. We reconstructed a 3.02 Å structure of MsDisA by Electron Cryomicroscopy (Cryo-EM). We observed an open-complex formation of this protein, which gives insight into the enzyme's active site. Chapter 4 describes the importance of individual domain of MsDisA in c-di-AMP synthesis and critical residues at catalytic core. This study provides a few interesting observations about the oligomerization and activity of the mutant proteins. Further, this chapter also talks about the second activity of DisA protein which binds to 4-way junctions DNA, resulting in an allosteric inhibition of c-di-AMP synthesis activity in Bacillus subtilis. Interestingly, the cryo-EM open structure of MsDisA shows a unique feature where two monomers stay apart to break D4 symmetry, preventing the protein from directly interacting with branched DNA which helps mycobacterial cell for the continuous synthesis of c-di-AMP under stress. Chapter 5 summarizes the results of the study and points out the future directions for the work.

Appendix Chapters includes the work which I have carried out in my first three years.

In appendix chapter 1, we worked on bacterial RNA polymerase and its smallest subunit ω. Here the emphasis of the work was to understand the mechanistic details of lethality by silent mutant of ω. Wild type ω shows a predominantly unstructured circular dichroic profile and becomes α-helical in the enzyme complex. This structural transition is perhaps the reason for the lack of function. We generated several silent mutants of ω to investigate the role of codon bias and the effect of rare codons with respect to their position in rpoZ. Not all silent mutations affect the structure. RNA polymerase when reconstituted with structurally altered silent mutants of ω is transcriptionally inactive. The Codon Plus strain, which has surplus tRNA, was used to assess for the rescue of the phenotype in lethal silent mutants.

In appendix chapter 2, we have utilized 7-Aza-tryptophan to investigate whether there is binding coupled folding for omega. The photo physical properties of free 7-Aza-tryptophan give a strong red-shifted fluorescence (403 nm), which would allow us to trap the conformational changes that occur during the ω-β' interaction and sigma attachment to the core RNAP. The incorporation of non-natural analogue 7-Aza-Trp in ω was achieved by way of using an E. coli Trp auxotrophic strain (RF12) where we have overcharged the system with Tryptophanyl-tRNA synthetase (trpS) gene for proper incorporation. The interaction of 7-Aza-Trp incorporated ω with the core1 (α2ββ') RNAP is being studied here.