All organisms must sense and respond to their environment in order to survive. The processes that allow a living cell to sense changes in its environment, and respond appropriately are collectively referred to as ‘signal transduction’. Cyclic AMP is a ubiquitously used second messenger molecule that plays diverse roles from hormone signalling in mammalian cells to catabolite repression in enteric bacteria. In several bacterial pathogens such as Pseudomonas aeruginosa, cAMP has also been found to mediate pathogenesis, usually by regulating the production of several virulence factors aiding in colonisation of the host. Cyclic AMP signalling has been suggested to regulate the virulence of the obligate intracellular Mycobacterium tuberculosis. Mycobacteria, including M. tuberculosis, code for a large number of adenylyl cyclases, enzymes that synthesise cAMP. Of the 16 putative adenylyl cyclases encoded by M. tuberculosis H37Rv, 10 have received extensive biochemical attention. A knockout of one of these cyclases, Rv0386, resulted in compromised virulence of M. tuberculosis. Ten proteins predicted to bind cAMP and mediate its cellular roles have also been identified in M. tuberculosis. Among these are the cAMP-regulated transcription factor, CRPMt, and cAMP-regulated protein acetyl transferase, KATmt. Comparatively little information is available, however, regarding the roles of cAMP-degrading machinery in mycobacteria. Two phosphodiesterases, with modest activity against cAMP in vitro, have been identified from M. tuberculosis, and are encoded by the Rv0805 and Rv2795c loci. Of these, Rv2795c has orthologs in all sequenced mycobacterial genomes. However, Rv0805-like proteins are coded only by slow growing mycobacteria such as the M. tuberculosis-complex, M. marinum and M. leprae, several of which are human or animal pathogens. Rv0805 belongs to the metallophosphoesterase superfamily of proteins, consisting of metal-dependent phosphoesterases with substrates ranging from large polymers like nucleic acids to small molecules like cAMP and glycerophospholipids. Like other metallophosphoesterases, Rv0805 displays promiscuous substrate utilisation and efficient hydrolysis of 2’3’-cAMP in vitro. Rv0805 also hydrolyses 3’5’-cAMP in vitro. Overexpression of Rv0805 is reported to lead to reduction in intracellular cAMP levels in M. smegmatis and M. tuberculosis, suggesting that it is capable of hydrolysing cAMP in the bacterial cell as well. The structure of Rv0805 revealed a sandwich-like α/β fold, typical of metallophosphoesterases, along with a relatively flexible C-terminal domain of unknown function. Despite extensive biochemical and structural information on Rv0805 however, its roles in mycobacteria remain unknown. In this study, the cellular roles of Rv0805 are explored and using information from biochemical and structural analyses, novel activities and interactions of Rv0805 have been identified. Rv0805, when expressed in M. smegmatis, led to a reduction in intracellular cAMP, as previously reported. However, the extent of reduction was modest (~30 %) and limited to the exponential phase of growth when both Rv0805 and intracellular cAMP are at their highest levels. Overexpression of Rv0805 also resulted in hypersensitivity to cell wall perturbants like crystal violet and sodium dodecyl sulphate (SDS) indicative of a change in the properties of the cell envelope of M. smegmatis. Importantly, these effects were independent of cAMP-hydrolysis by Rv0805, as overexpression of catalytically inactive Rv0805N97A also elicited similar changes. Unexpectedly, Rv0805 was localised to the cell envelope, both in M. tuberculosis as well as in M. smegmatis. The ability of Rv0805 to localise to the cell envelope was dependent on it C-terminus, as truncation of Rv0805 in this region (Rv0805Δ10, Rv0805Δ20 and Rv0805Δ40) resulted in progressively greater enrichment in the cytosol of M. smegmatis. Overexpression of Rv0805Δ40, which was localised almost completely to the cytosol, did not result in hypersensitivity to SDS, suggesting that cell envelope localisation, rather than cAMP-hydrolysis, was crucial for the cell envelope modifying roles of Rv0805. A possible mechanism behind the cell envelope-related effects of Rv0805 overexpression was the ability of the protein to interact with the cell wall of mycobacteria in a C-terminus-dependent manner. Purified Rv0805, but not Rv0805Δ40, could associate with crude mycobacterial cell wall as well as purified cell wall core polymer (mycolyl-arabinogalactan-peptidoglycan complex) in vitro. In addition to the C-terminus, the architecture of the active site was also crucial for this interaction as mutations in the active site that compromised metal-binding also resulted in poor interaction with the cell wall. Most significant among these residues was His207, which when mutated to Ala almost completely abrogated interaction with the cell wall in vitro. Further, Rv0805H207A was unable to localise to the cell envelope when expressed in M. smegmatis, even in the presence of the C-terminus, highlighting the importance of this residue in maintaining the structural integrity of Rv0805, and demonstrating that the structure of the C-terminus, rather than its sequence alone, played a role in cell envelope localisation and interaction. In order to verify that the observed sensitivity of Rv0805-overexpressing M. smegmatis to cell wall perturbants was due to a change in cell envelope properties atomic force microscopy was employed. Two distinct modes of operation were used to analyse surface and bulk properties of the mycobacterial cell envelope. These were tapping mode phase imaging, and contact mode force spectroscopy. Using tapping mode phase imaging, it was found that the cell surface of M. smegmatis was inherently heterogeneous in its mechanical properties. Further, contact mode force-spectroscopy revealed that the cell envelope of M. smegmatis in cross-section had at least three layers of varying stiffness. Typically, a middle layer of high stiffness was observed, sandwiched between two lower stiffness layers. This organisation is reminiscent of the current model of the mycobacterial cell envelope, possessing a central polysaccharide rich layer and outer and inner lipid rich layers. Treatment of wild type M. smegmatis with cell wall-perturbing antibiotics isoniazid and ethambutol resulted in markedly altered phase images, as well as significantly lower stiffness of the bacterial cell envelopes, validating that the methodology employed could indeed be used to assess cell wall perturbation in mycobacteria. Further, M. smegmatis harbouring deletions in cell envelope biosynthesis related genes, MSMEG_4722 and aftC, showed significantly lower cell wall stiffness than wild type M. smegmatis, providing evidence that genetic perturbation of the cell wall of mycobacteria could also be studied using atomic force microscopy. While phase imaging revealed similar surface properties of Rv0805-overexpressing and control M. smegmatis, force spectroscopy revealed significantly lower cell envelope stiffness, particularly of the middle layer, of the former. Cell envelope stiffness was, however, unaffected by expression of Rv0805Δ40 in M. smegmatis, providing direct evidence for C-terminus-dependent cell envelope perturbation upon Rv0805 overexpression. Additionally, overexpression of Rv0805N97A, but not Rv0805H207A led to reduced stiffness of the cell envelope of M. smegmatis, demonstrating that the cell wall remodelling activity of Rv0805 was independent of cAMP-hydrolysis, but dependent on cellular localisation and cell wall interaction. Like in M. smegmatis, overexpression of Rv0805 also led to lower cAMP levels in M. tuberculosis. Using a microarray-based transcriptomics approach, pathways affected by Rv0805 overexpression were identified. Rv0805 overexpression elicited a transcriptional response, leading to the down-regulation of a number of virulence associated genes such as whiB7, eis, prpC and prpD. Importantly, Rv0805-overexpression associated gene expression changes did not include genes regulated by CRPMt, the primary cAMP-regulated transcription factor in M. tuberculosis. Further, Rv0805N97A overexpression in M. tuberculosis led to similar changes in gene expression as overexpression of the wild type protein. These observations reiterated that, at least upon overexpression, the effects of Rv0805 were largely independent of cAMP-hydrolysis. Using overexpression in M. smegmatis and M. tuberculosis, cAMP-hydrolysis independent roles of Rv0805 in mycobacteria were identified. To further validate these observations, a knockout strain of the Rv0805 gene was generated in M. bovis BCG, a well-established model to study M. tuberculosis. Curiously, deletion of Rv0805 did not lead to a change in intracellular cAMP levels, demonstrating that cAMP-hydrolysis by Rv0805 may not contribute to the modulation of mycobacterial cAMP levels under standard laboratory growth conditions. Rv0805 deletion led to altered colony morphology and possible reduction in cell wall thickness, reaffirming the roles of this phosphodiesterase in regulating cell envelope physiology of mycobacteria. Additionally, Rv0805 deletion also resulted in compromised growth of M. bovis BCG in fatty acid-deficient media, implicating Rv0805 as a possible regulator of carbon metabolism. In summary, this thesis explores novel links between Rv0805 and the mycobacterial cell wall and elucidates the critical importance of the C-terminus domain of this metallophosphodiesterase in modulating its cellular localisation to, and interaction with, the mycobacterial cell envelope. En route to understanding the effects of Rv0805 overexpression on the cell wall of M. smegmatis, an atomic force microscopy-based methodology to assess perturbation of the cell envelope of mycobacteria was also developed. Finally, using a combination of biochemical and genetic analyses, cellular roles of Rv0805, independent of cAMP-hydrolysis, were identified in slow-growing mycobacteria. This study therefore provides direct evidence against the sole role of this mycobacterial phosphodiesterase as a regulator of intracellular cAMP levels, and opens up new avenues to understanding the cellular functions of Rv0805 and indeed other members of the metallophosphoesterase superfamily.

Mycobacterium tuberculosis is one of the most successful human pathogens, estimated to have infected close to one-third of the global human population. In order to survive within its host, M. tuberculosis utilises multiple signalling strategies, one of them being synthesis and secretion of universal second messenger cAMP. This process is enabled by the presence of sixteen predicted adenylyl cyclases in the genome of M. tuberculosis H37Rv, ten of which have been characterised in vitro. The synthesized cAMP is recognised by ten putative cAMP-binding proteins in which the cyclic AMP-binding domain is associated with a variety of enzymatic domains. The cAMP signal can be extinguished by degradation by phosphodiesterase’s, secretion into the extracellular milieu or via sequestration of the nucleotide by upregulation of a high-affinity cAMP-binding protein. Of the sixteen adenylyl cyclases (ACs) encoded by M. tuberculosis H37Rv, a subset of multidomain adenylyl cyclases remain poorly characterised, primarily due to challenges associated with studying these in vitro. The adenylyl cyclase domain in these proteins is associated with an NB-ARC domain (nucleotide binding domain common to APAF-1, plant R proteins and CED-4), a TPR domain (tetratricopeptide repeat) and an LuxR-type HTH motif (helix-turn-helix). This architecture places these multidomain mycobacterial ACs within a larger group of STAND (Signal transduction ATPase’s with numerous domains) proteins, and hence they will be referred to as STAND ACs. The STAND proteins are a recently recognised class of multidomain ATPases which integrate a variety of signals prior to activation. Activation is accompanied by formation of large oligomeric signalling hubs which facilitate downstream signalling events. While most STAND proteins have a single effector domain followed by an NB-ARC domain and a scaffolding domain, the STAND ACs distinguish themselves by retaining two effector domains, the AC domain and the HTH domain, at the N- and C- termini respectively. The cyclase, NB-ARC, TPR and HTH domains have widely divergent taxonomic distributions making the presence of these four domains in a single polypeptide rare. In fact, proteins with cyclase-NB-ARC-TPR-HTH (C-A-T-H) domain organisation were found to be encoded almost exclusively by slow growing mycobacterial species, a clade that harbours most mycobacterial pathogens, such as M. tuberculosis and M. leprae. Notably, one of the STAND ACs, Rv0386, is the only mycobacterial AC shown till date to be required for virulence of M. tuberculosis in mice. Using phylogenetic, the evolutionary underpinnings of this domain architecture were examined. The STAND ACs appear to have most likely evolved via a domain gain event from a cyclase-ATPase-TPR progenitor encoded by a strain ancestral to M. marina. Subsequently, the genes duplicated and diverged, sometimes leading to frameshift mutations splitting the cyclase domain from the C-terminal domains. Consequently, M. tuberculosis encodes for three ‘full-length’ STAND ACs, namely, Rv0386, Rv1358 and Rv2488c and one split STAND AC. The split STAND AC is made up of Rv0891c, containing the AC domain, and Rv0890c, containing the NB-ARC, TPR and HTH domains. rv0891c and rv0890c were found to be expressed as an operatic transcript, though they were translationally uncoupled. Pertinently, M. Canetti, an early-branching species of the M. tuberculosis complex, contains an orthologue of Rv0891c and Rv0890c where all four domains are present in a single polypeptide. Sequence analysis of the four STAND ACs in M. tuberculosis allowed predictions of significant divergence in function. These proteins showed high sequence conservation in their HTH domains, with substantial sequence divergence in their TPR, NB-ARC and AC domains. Biochemical analysis on the AC domains revealed that Rv0891c and Rv2488c possessed poor or no AC activity, respectively. On the other hand, the cyclase domain of Rv0386 could catalyse cAMP synthesis. Moreover, for both Rv0891c and Rv0386, presence of the C-terminal domains potentiated adenylyl cyclase activity, suggestive of allosteric regulation within the STAND AC module. Studies on Rv0891c also revealed that the protein could inhibit the adenylyl cyclase activity of Rv0386 in trans. This result thus provided a novel mechanism by which proteins harbouring poorly active/inactive adenylyl cyclase domains could contribute to cAMP levels, by acting as inhibitors of other adenylyl cyclases. The STAND ACs were found to be inactive ATPases. Additionally, incubation with nucleotides did not stimulate oligomerisation of these proteins, unlike what has been shown for several other STAND proteins. However, mutations in the NB-ARC domain perturbed the basal oligomeric state of these proteins, indicating that the NB-ARC domain can influence self- association. A subset of NB-ARC domain mutants also showed increased adenylyl cyclase activity, reiterating the inter-domain cross-talk in the STAND ACs. Since the AC activity of these proteins was meagre, the properties of the HTH domain were examined, as an alternative effector domain. Genomic SELEX was performed using the TPR-HTH domains of Rv0890c, and revealed a set of sequences that bound to this protein, though they lacked common sequence features. Further analysis revealed that Rv0890c bound to DNA in a sequence-independent manner, through the HTH domain. This binding was cooperative with multiple protein units engaging in DNA-binding. Due to the cooperative nature of binding and the lack of sequence preference, Rv0890c appeared coat the DNA molecule. This was further proved by the ability of Rv0890c to protect DNA from DNaseI-mediated degradation, and the requirement for long DNA sequences to form stable DNA-protein complexes. Studies also revealed that Rv0890c interacted with RNA and ssDNA. In fact, the protein as purified from heterologously expressing E. coli cells was bound to RNA. RNA-binding by a LuxR-type HTH has not been reported previously, providing a new function for this class of HTHs. Interestingly, nucleic acid-binding by a fusion Rv0891c-Rv0890c protein, similar to the one encoded in M. canetti, was shown to stimulate adenylyl cyclase activity. This was likely due to a relief of inhibitory interactions between the TPR-HTH and the AC domains, on DNA-binding. Given the high sequence similarity between the HTH domains of the STAND ACs, they were expected to bind to DNA in an identical manner. Indeed, the HTH domains of Rv0386 and Rv1358 engaged with DNA with an identical affinity as Rv0890c. Sequence comparisons in the HTH domain enabled identification of conserved basic residues, of which one, R850 was essential for nucleic acid-binding. Surprisingly however, Rv0386 and Rv1358 did not exhibit RNA-binding, pointing towards functional divergence of Rv0890c from its paralogues. Since the HTH domains of the STAND ACs were highly conserved, it was possible that the ability to bind to RNA was instead dictated by the adjacent TPR modules. To examine this possibility, TPR domains were swapped between Rv0890c and Rv0386. Interestingly, both the chimeric proteins showed a reduced ability to bind to DNA, while showing a complete absence of RNA- binding. These results suggested that the TPR domains were critical in modulating nucleic acid-binding. Moreover, the effect of the TPR domain was context-dependent, since the presence of non-cognate TPR domains hampered nucleic acid-binding. However, the ability to bind to RNA was not solely governed by the TPR domain since the Rv0890cTPR-Rv0386HTH chimeric protein did not show RNA-binding, in spite of containing a permissive TPR domain. To further dissect the molecular requirements for RNA-binding, the conservation of basic residues between the HTH domains of Rv0890c versus Rv1358 and Rv0386 was examined. Interestingly the HTH domain Rv0890c contained two additional positively charged residues over Rv1358 and Rv0386. Mutations of these abolished RNA-binding by Rv0890c. Thus the evolution of two basic residues permit Rv0890c to diverge in its nucleic acid-binding properties, a possible example of defunctionalisation following gene duplication. In summary, this thesis attempts to understand the evolution and functions of the STAND ACs, a group of pathogenically relevant and uniquely mycobacterial multidomain proteins. Phylogenetic analysis revealed an expansion of this gene family in slow growing mycobacteria. Biochemical characterisation showed that following gene duplication, the resulting proteins diverge both in their ability to synthesize cAMP and in their association with nucleic acids. Studies on these proteins also revealed novel mechanisms of regulation of mycobacterial cAMP levels. Additionally, these proteins exhibited indiscriminate binding to DNA/nucleic acids indicating that they may be responsible for global functions in the cell which extend beyond cAMP synthesis.