**1. Introduction**

102 Understanding Tuberculosis – Deciphering the Secret Life of the Bacilli

Song H, Huff J, Janik K, Walter K, Keller C, Ehlers S, Bossman SH, and Niederweis M. 2011

Streenken W, Oatwat WH, and Petroff SA. 1934. Biological studies of the tubercle bacillus.

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Tran SL, Rao M, Simmers C, Gebhard S, Olsson K, and Cook GM. 2005. Mutant of

Uyttebroek M, Vermeir S, Wattiau P, Ryngaert A, and Springael D. 2007.Characterization of

Vandal OH, Nathan CF, and Ehrt S. 2009. Acid resistance in *Mycobacterium tuberculosis*.

Vandal OH, Pierini LM, Schnappinger D, Nathan CF, and Ehrt S. 2008. A membrane protein

Vandal OH, Roberts JA, Odaira T, Schnappinger D, Nathan C, and Ehrt S. 2009. Acid-

Via LE, Fratti RA, McFalone M, pagan-Ramos E, Deretic D, and Deretic V. 1998. Effects of

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suceptibility mutants of *Mycobacterium tuberculosis* share hypersusceptibility to cell wall and oxidative stress and to the host environment. Journal of Bacteriology.

cytokines on mycobacterial phagosome maturation. Journal of Cell Science.

*tuberculosis* PhoPR two-component system regulates genes essential for virulence

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#### **1.1 Cyclic Adenosine Monophosphate (cAMP) metabolism in mycobacteria**

#### **1.1.1 General aspects of Adenylyl Cyclases and their presence in** *M. tuberculosis* **genomes**

Adelynate cyclases (ACs), which catalyze synthesis of cAMP from ATP and yield pyrophosphate as a by-product, can be classified into four different classes according to their common features: Class I cyclases, related to enterobacterial adenylate cyclases; Class II, toxic adenylate cyclases isolated from bacterial pathogens; Class III, a large and probably ancient class that comprises cyclases from both eukaryotes and prokaryotes and is strongly related to guanylate cyclases; and Class IV, with mainly one example that differs entirely from all other classes (McCue *et al.*, 2000).

In class I ACs (the enterobacterial type) no long stretch of hydrophobic amino acid residues is present to explain the membrane-bound localization of the adenylate cyclases. In all cases, the proteins are very rich in cysteine residues, an uncommon feature for proteins located in the cytoplasm or at the cytoplasmic border of the membrane. They are also rich in histidine residues, which could indicate that metal ions take part in the folding and/or activity of the polypeptide chain (Mock *et al.*, 1991).

Class II ACs (the calmodulin-activated toxic class) is represented by *Bordetella pertussis* adenylate cyclase. It is synthesized as a large bifunctional polypeptide chain of 1706 amino acid residues. The N-terminal segment of the protein (400 residues) alone displays calmodulin-activated adenylate cyclase activity, whereas the rest of the molecule is responsible for hemolytic activity and for transporting the toxin. After attempts to isolate other members of this class, several examples of similar proteins have now been discovered in *Bacillus anthracis*, *Pseudomonas aeruginosa*, and in Ye*rsinia* species. Comparison of the catalytic regions of the *B. pertussis* and *B. anthracis* adenylate cyclases identified four conserved regions that are involved in catalysis, calmodulin binding and activation. The first region comprises a sequence, Gly-XXXX-Gly(Ala)-Lys-Ser, similar to the nucleotidebinding motif found in many ATP- or GTP-binding proteins. Analysis of the region conserved between the *B. anthracis* and *B. pertussis* enzymes, indicates that these proteins

Mycobacterium Tuberculosis Signaling via c-AMP 105

hydrophobic, predicted transmembrane domains (Agarwal, 2009). This suggests that a necessary improvement to prediction software must emphasize these particularities of

> Gene name Orthologous (No.) *Rv0386* 7 *Rv0805* 7 *RV0891c* 4 *Rv1264* 13 *Rv1318c* 14 *Rv1319c* 14 *Rv1320c* 14 *Rv1358* 7 *Rv1359* 3 *Rv1625c* 11 *Rv1647* 16 *Rv1900c* 10 *Rv2212* 6 *Rv2564* 3 *Rv2565* 3 *Rv3645* 16

Table 1. Orthologous to H37Rv AC-domain containing genes present in Mycobacteria

that polyphosphates possibly obstructs the catalytic fold of ACs (Guo et al., 2001).

**1.2 cAMP producing (Adenylyl cyclases) and degrading (Phosphodiesterase)** 

As mycobacteria lack G-proteins, it is unknown how bacterial adenylate cyclase AC activities are modulated. However, polyphosphates isolated from *M. bovis* BCG were potent inhibitors of Rv1625c, Rv1264, and Rv3645, and the mechanism of inhibition proposed is

Six *M. tuberculosis* AC-domain containing proteins (Rv0891c, Rv1264, Rv1359, Rv1647, Rv2212 and Rv1625c) contain just a cyclase domain. Rv1647 and Rv1625c are phylogenetically distant and biochemically distinct AC-domain containing proteins (Shenoy & Visweswariah, 2006a). The remaining AC-domain containing proteins have additional domains that presumably allow them to respond to multiple signals, regulate their activity in response to environmental conditions, and/or expand their repertoire with effector function capability. Five of these multidomain AC-domain containing proteins (Rv1318c, Rv1319c, Rv1320c, Rv2435c and Rv3645c) are membrane-associated and contain HAMP (histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins and phosphatases) domains. HAMP domains are often associated with two-component signal transduction pathways and connect the sensing of extracellular environmental signals with

mycobacteria.

**mycobacterial enzymes** 

may form a catalytic center from the cooperation of two halves. The function of calmodulin may be to trigger the appropriate conformational change necessary to form an active catalytic center (Drum *et al.*, 2002).

Class III ACs (the "universal" class**)** form a very diverse collection of enzymes in eubacteria**.**  They comprise two domains: the catalytic domain is carboxy-terminal and the regulatory domain is likely an ion transporter in one case and the phosphorylated moiety of a twocomponent regulatory system in another. Most mammalian ACs are monomeric integral membrane proteins that are catalytically active as pseudoheterodimers (Sunahara *et al.*, 1996),while prokaryotes and lower eukaryotes produce both soluble and membrane-bound nucleotidyl cyclases of variant domain compositions functioning as homodimers (Guo *et al.*, 2001). In general, class III ACs are the most widespread class of cAMP-generating enzymes, and they are further subdivided into four subclasses: IIIa-IIId. Dimerization is required for all class III ACs in order to be active, given the substrate-binding sites are formed at the dimer interface (Abdel Motaal *et al.*, 2006). Class III adenylyl and guanylyl cyclases are proteins with a central four stranded anti-parallel β-sheet structurally similar to the palm domain of DNA polymerases, and α-helices on either side (Shenoy & Visweswariah, 2006b). So far, all Cyclase Homology Domain (CHDs) proteins operate as dimers with mostly two catalytic centers positioned at the dimer interface, where catalysis is based on six highly conserved residues. Two aspartate residues coordinate two metal cofactors (Mg2+ or Mn2+), an asparagine and an arginine stabilize the transition-state and a lysine-aspartate couple specifies ATP as a substrate. Several mycobacterial ACs gene products were early annotated as putative cyclases, but have now been characterized biochemically and structurally.

Class IV ACs was assigned to *Aeromonas hydrophila*, which synthesizes a very small cyclase of 193 residues. This class IV cyclase has an optimal temperature for activity of 65°C and is at least ten times more active than the class I adenylate cyclase in the same organism (Sismeiro *et al.*, 1998). No function has yet been discovered for this protein. Currently, it has been found only in various isolates of *A. hydrophila* and in *Y. pestis.*

Most prokaryotes contain a single adenylate cyclase (AC, e.g. *E. coli*) and some contain none (e.g., *Bacillus* species); however, 16 or 17 genes have been identified as ACs in mycobacteria, and in particular *M. tuberculosis*. AC enzymes require the presence of conserved metal-, substrate-binding (either ATP or GTP) amino acid residues and transition-state stabilizing amino acid residues in their catalytic site (Shenoy & Visweswariah, 2006b).

The genome of *M. tuberculosis* H37Rv encodes 16 class III cyclases while the CDC1551 strain has 17 cyclases. Using the 16 H37Rv class III ACs genes as query, we found they posses different numbers of orthologous genes in the up-to-date sequenced mycobacterial genomes available at MycoDB (http://xbase.bham.ac.uk/mycodb/, Table 1). This suggests that some of these cyclases have more conserved roles than others. Several mycobacterial gene products initially classified as cyclases have now been biochemically and structurally characterized as ACs. In mycobacteria, they retain similar catalytic properties to the mammalian enzymes, in terms of the requirement for divalent cations, and dimerization as a prerequisite to generate the catalytic site.

Bioinformatics analysis has predicted subcellular localization of mycobacterial ACs (Shenoy *et al.*, 2004). Nevertheless, experimental studies have identified proteins like Rv0386 and Rv1358 in mycobacterial cell wall and membranes, despite the absence of

may form a catalytic center from the cooperation of two halves. The function of calmodulin may be to trigger the appropriate conformational change necessary to form an active

Class III ACs (the "universal" class**)** form a very diverse collection of enzymes in eubacteria**.**  They comprise two domains: the catalytic domain is carboxy-terminal and the regulatory domain is likely an ion transporter in one case and the phosphorylated moiety of a twocomponent regulatory system in another. Most mammalian ACs are monomeric integral membrane proteins that are catalytically active as pseudoheterodimers (Sunahara *et al.*, 1996),while prokaryotes and lower eukaryotes produce both soluble and membrane-bound nucleotidyl cyclases of variant domain compositions functioning as homodimers (Guo *et al.*, 2001). In general, class III ACs are the most widespread class of cAMP-generating enzymes, and they are further subdivided into four subclasses: IIIa-IIId. Dimerization is required for all class III ACs in order to be active, given the substrate-binding sites are formed at the dimer interface (Abdel Motaal *et al.*, 2006). Class III adenylyl and guanylyl cyclases are proteins with a central four stranded anti-parallel β-sheet structurally similar to the palm domain of DNA polymerases, and α-helices on either side (Shenoy & Visweswariah, 2006b). So far, all Cyclase Homology Domain (CHDs) proteins operate as dimers with mostly two catalytic centers positioned at the dimer interface, where catalysis is based on six highly conserved residues. Two aspartate residues coordinate two metal cofactors (Mg2+ or Mn2+), an asparagine and an arginine stabilize the transition-state and a lysine-aspartate couple specifies ATP as a substrate. Several mycobacterial ACs gene products were early annotated as putative cyclases, but have

Class IV ACs was assigned to *Aeromonas hydrophila*, which synthesizes a very small cyclase of 193 residues. This class IV cyclase has an optimal temperature for activity of 65°C and is at least ten times more active than the class I adenylate cyclase in the same organism (Sismeiro *et al.*, 1998). No function has yet been discovered for this protein. Currently, it has

Most prokaryotes contain a single adenylate cyclase (AC, e.g. *E. coli*) and some contain none (e.g., *Bacillus* species); however, 16 or 17 genes have been identified as ACs in mycobacteria, and in particular *M. tuberculosis*. AC enzymes require the presence of conserved metal-, substrate-binding (either ATP or GTP) amino acid residues and transition-state stabilizing

The genome of *M. tuberculosis* H37Rv encodes 16 class III cyclases while the CDC1551 strain has 17 cyclases. Using the 16 H37Rv class III ACs genes as query, we found they posses different numbers of orthologous genes in the up-to-date sequenced mycobacterial genomes available at MycoDB (http://xbase.bham.ac.uk/mycodb/, Table 1). This suggests that some of these cyclases have more conserved roles than others. Several mycobacterial gene products initially classified as cyclases have now been biochemically and structurally characterized as ACs. In mycobacteria, they retain similar catalytic properties to the mammalian enzymes, in terms of the requirement for divalent cations, and dimerization as a

Bioinformatics analysis has predicted subcellular localization of mycobacterial ACs (Shenoy *et al.*, 2004). Nevertheless, experimental studies have identified proteins like Rv0386 and Rv1358 in mycobacterial cell wall and membranes, despite the absence of

catalytic center (Drum *et al.*, 2002).

now been characterized biochemically and structurally.

prerequisite to generate the catalytic site.

been found only in various isolates of *A. hydrophila* and in *Y. pestis.*

amino acid residues in their catalytic site (Shenoy & Visweswariah, 2006b).

hydrophobic, predicted transmembrane domains (Agarwal, 2009). This suggests that a necessary improvement to prediction software must emphasize these particularities of mycobacteria.


Table 1. Orthologous to H37Rv AC-domain containing genes present in Mycobacteria

As mycobacteria lack G-proteins, it is unknown how bacterial adenylate cyclase AC activities are modulated. However, polyphosphates isolated from *M. bovis* BCG were potent inhibitors of Rv1625c, Rv1264, and Rv3645, and the mechanism of inhibition proposed is that polyphosphates possibly obstructs the catalytic fold of ACs (Guo et al., 2001).

#### **1.2 cAMP producing (Adenylyl cyclases) and degrading (Phosphodiesterase) mycobacterial enzymes**

Six *M. tuberculosis* AC-domain containing proteins (Rv0891c, Rv1264, Rv1359, Rv1647, Rv2212 and Rv1625c) contain just a cyclase domain. Rv1647 and Rv1625c are phylogenetically distant and biochemically distinct AC-domain containing proteins (Shenoy & Visweswariah, 2006a). The remaining AC-domain containing proteins have additional domains that presumably allow them to respond to multiple signals, regulate their activity in response to environmental conditions, and/or expand their repertoire with effector function capability. Five of these multidomain AC-domain containing proteins (Rv1318c, Rv1319c, Rv1320c, Rv2435c and Rv3645c) are membrane-associated and contain HAMP (histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins and phosphatases) domains. HAMP domains are often associated with two-component signal transduction pathways and connect the sensing of extracellular environmental signals with

Mycobacterium Tuberculosis Signaling via c-AMP 107

catalysis (Shenoy *et al.*, 2007). As this gene is found only in pathogenic mycobacteria, Rv0805 may therefore play a key role in the pathogenicity of mycobacteria, not only by hydrolyzing bacterial cAMP, but also by indicating as a protein that can alter cell wall functioning

Cyclic adenosine 3′,5′-monophosphate (cAMP) is one of the most important second messengers used in bacteria and it has been characterized principally in *Escherichia coli*. However its signaling role in *M. tuberculosis* is beginning to emerge. A large number of AC genes are present in *Mycobacterium tuberculosis* in comparison with other microorganims (Cha *et al.*, 2010, Klengel *et al.*, 2005, Shenoy et al., 2004), and also that *Mycobacterium* just have one class III cNMP phosphodiesterase (Rv0805) that has been identified in *M. tuberculosis* (McCue *et al.*, 2000, Shenoy et al., 2007) with a modest ability to efficiently hydrolyse 3′,5′-cAMP and that responds to H2O2 *in vitro* (Bai et al., 2011, Barba et al., 2010). cAMP may serve as both an extra and intracellular signaling molecule in mycobacteria (Agarwal, 2009). cAMP levels are modulated by stress conditions in *M. smegmatis*, where it has been found in concentrations between 10 µM and 1 mM, suggesting that cAMP participates in signalling events within the bacterial cell, mediating its action by

It appears that Mycobacteria need to maintain a steady level of cytoplasmatic cAMP in many conditions. For example, it has been shown that cytoplasmic cAMP levels in *E. coli* are reduced three- to fourfold when the carbon source is ~0.2% glucose rather than glycerol (Bai et al., 2011). In contrast, a recent study showed no significant change in the cytoplasmic cAMP levels of *M. bovis* BCG incubated with 0.2% glucose (Bai *et al.*, 2009), or carbonstarved bacteria (Dass et al., 2008). cAMP levels decrease in both fast- and slow-growing mycobacteria in response to very high levels of glucose (2%) (Bai et al., 2011). cAMP levels have been suggested to be high in *Mycobacterium* cells, exceeding up to 100-fold levels found in other bacteria (Nambi et al., 2010, Stapleton *et al.*, 2010, Shenoy & Visweswariah, 2006b, Rickman *et al.*, 2005). However, it is difficult to make a comparison between studies, due to cAMP variations presents in the conditions tested, as well as the different normalization and

*In silico* studies predict 10 cNMP binding proteins that encompass a wide range of potential effector functions, suggesting a more complex role for cAMP signalling, probably important during host infection. From these 10 cNMP binding proteins, 7 (Rv0073, Rv0104, Rv2434c, Rv2564, Rv2565, Rv3239c and Rv3728) contain an assortment of putative functional domains, including those associated with transport functions and esterase activities (McCue et al., 2000, Shenoy & Visweswariah, 2006a). Only three cNMP binding proteins have been functionally characterized to date (Bai et al., 2011). Two of these proteins, referred to as CRP (*Rv3676*, for Catabolite Represor Protein) and Cmr (*Rv1675c*, for cAMP and macrophage regulator), contain a HTH DNA binding domains, and belong to the CRP-FNR family of transcription factors (McCue et al., 2000). The third protein from this group, is encoded by

**2. Participation of cyclic adenosine 3′,5′-monophosphate (cAMP) in** 

(Podobnik *et al.*, 2009).

**mycobacterial gene regulation** 

downstream effectors (Dass *et al.*, 2008).

reporting methods used.

**2.1 CRP and cNMP binding proteins** 

responding intracellular signalling domains. Mycobacterial ACs with HAMP domains have six transmembrane regions, followed by the HAMP domain and a C-terminal AC domain. Modulators of *M. tuberculosis* AC activity include pH and fatty acids, which were seen to enhance the pH sensitivity of the holoenzyme, and CO2 levels, and could be signals present during *M. tuberculosis* host infection (Abdel Motaal et al., 2006, Bai *et al.*, 2011, Barba *et al.*, 2010).

An N-terminal autoregulatory domain in Rv1264 is a pH-response element that inhibits cyclase activity above pH 6.0. Additional AC-domain containing proteins (Rv0386, Rv1358 and Rv2488c) posses both ATPase and helix– turn–helix (HTH) domains. Rv1900c contains an -hydrolase domain (Barba et al., 2010, Bai et al., 2011). Rv0386 is the first AC-domain containing protein as a representative of the family of putative DNA-binding domaincontaining cyclases in mycobacteria (Castro *et al.*, 2005). Rv0386 has guanylyl cyclase activity that is 20% of its adenylyl cyclase activity.

Rv1625c adenylyl cyclase is particularly unusual, in that it is catalytically active when expressed in mammalian cells or in *E. coli*. The Rv1264 holoenzyme shows higher AC activity at acidic pH (pH 6.0). Similarly, the Rv1264 AC is activated only at low pH (pH 5.5) whereas Rv1647 is active only at high pH (pH 8.5). The localization of Rv1647 in the cell wall and membrane fractions of *M. tuberculosis* might enable the bacteria to sense and respond to extracellular pH shifts. Because *M. tuberculosis* actively avoids phagosomal acidification, sensing of pH and bicarbonate and/or CO2 could be crucial signaling events in *M. tuberculosis* pathogenesis (Shenoy & Visweswariah, 2006a). In fact, cAMP production in pathogenic mycobacteria increased when the pH of their growth medium was shifted from pH 6.7 to pH 5.5 (Gazdik *et al.*, 2009), thus suggesting activation of the acidresponsive ACs.

In *M. tuberculosis*, Rv0998 was recently shown to regulate protein lysine acetylation in a cAMP-responsive manner (Nambi *et al.*, 2010), where acetylation is enhanced in the presence of either cAMP or cGMP. Recently, it was shown that a cAMP-dependent protein acetyltransferase inactivates ACs through acetylation of a single, specific lysine residue, and Rv1151c is a NAD+-dependent deacetylase that reactivates ACs. This acetylation/deacetylation system in mycobacteria is likely to sense the extracellular environment through cAMP levels and also key intracellular metabolites, including NAD+ and AcCoA, since cAMP, AcCoA, and NAD+ are required for acetylation and deacetylation, respectively (Xu *et al.*, 2011).

Adenylyl cyclase Rv2212 from *M. tuberculosis* has a domain composition identical to the pHsensing isoform Rv1264, an N-terminal regulatory domain and a C-terminal catalytic domain. The maximal velocity of Rv2212 was the highest of all 10 mycobacterial cyclases investigated to date. Unsaturated fatty acids strongly stimulated Rv2212c activity by increasing substrate affinity (Findeisen *et al.*, 2007). In addition, fatty acids greatly enhanced the pH sensitivity of the holoenzyme, thus converting Rv2212c to a pH sensor adenylyl cyclase (Abdel Motaal et al., 2006).

To date, the only identifiable cAMP phosphodiesterase (PDE) in the genome of *M. tuberculosis* H37Rv is the one encoded by the *Rv0805* gene. This enzyme is a dimeric Fe(3+)- Mn(2+) binuclear PDE, where metals coordinated at the catalytic site contribute to dimerization and thus play an additional structural role apart from their involvement in

responding intracellular signalling domains. Mycobacterial ACs with HAMP domains have six transmembrane regions, followed by the HAMP domain and a C-terminal AC domain. Modulators of *M. tuberculosis* AC activity include pH and fatty acids, which were seen to enhance the pH sensitivity of the holoenzyme, and CO2 levels, and could be signals present during *M. tuberculosis* host infection (Abdel Motaal et al., 2006, Bai *et al.*, 2011, Barba *et al.*,

An N-terminal autoregulatory domain in Rv1264 is a pH-response element that inhibits cyclase activity above pH 6.0. Additional AC-domain containing proteins (Rv0386, Rv1358 and Rv2488c) posses both ATPase and helix– turn–helix (HTH) domains. Rv1900c contains an -hydrolase domain (Barba et al., 2010, Bai et al., 2011). Rv0386 is the first AC-domain containing protein as a representative of the family of putative DNA-binding domaincontaining cyclases in mycobacteria (Castro *et al.*, 2005). Rv0386 has guanylyl cyclase activity

Rv1625c adenylyl cyclase is particularly unusual, in that it is catalytically active when expressed in mammalian cells or in *E. coli*. The Rv1264 holoenzyme shows higher AC activity at acidic pH (pH 6.0). Similarly, the Rv1264 AC is activated only at low pH (pH 5.5) whereas Rv1647 is active only at high pH (pH 8.5). The localization of Rv1647 in the cell wall and membrane fractions of *M. tuberculosis* might enable the bacteria to sense and respond to extracellular pH shifts. Because *M. tuberculosis* actively avoids phagosomal acidification, sensing of pH and bicarbonate and/or CO2 could be crucial signaling events in *M. tuberculosis* pathogenesis (Shenoy & Visweswariah, 2006a). In fact, cAMP production in pathogenic mycobacteria increased when the pH of their growth medium was shifted from pH 6.7 to pH 5.5 (Gazdik *et al.*, 2009), thus suggesting activation of the acid-

In *M. tuberculosis*, Rv0998 was recently shown to regulate protein lysine acetylation in a cAMP-responsive manner (Nambi *et al.*, 2010), where acetylation is enhanced in the presence of either cAMP or cGMP. Recently, it was shown that a cAMP-dependent protein acetyltransferase inactivates ACs through acetylation of a single, specific lysine residue, and Rv1151c is a NAD+-dependent deacetylase that reactivates ACs. This acetylation/deacetylation system in mycobacteria is likely to sense the extracellular environment through cAMP levels and also key intracellular metabolites, including NAD+ and AcCoA, since cAMP, AcCoA, and NAD+ are required for acetylation and deacetylation,

Adenylyl cyclase Rv2212 from *M. tuberculosis* has a domain composition identical to the pHsensing isoform Rv1264, an N-terminal regulatory domain and a C-terminal catalytic domain. The maximal velocity of Rv2212 was the highest of all 10 mycobacterial cyclases investigated to date. Unsaturated fatty acids strongly stimulated Rv2212c activity by increasing substrate affinity (Findeisen *et al.*, 2007). In addition, fatty acids greatly enhanced the pH sensitivity of the holoenzyme, thus converting Rv2212c to a pH sensor adenylyl

To date, the only identifiable cAMP phosphodiesterase (PDE) in the genome of *M. tuberculosis* H37Rv is the one encoded by the *Rv0805* gene. This enzyme is a dimeric Fe(3+)- Mn(2+) binuclear PDE, where metals coordinated at the catalytic site contribute to dimerization and thus play an additional structural role apart from their involvement in

2010).

responsive ACs.

respectively (Xu *et al.*, 2011).

cyclase (Abdel Motaal et al., 2006).

that is 20% of its adenylyl cyclase activity.

catalysis (Shenoy *et al.*, 2007). As this gene is found only in pathogenic mycobacteria, Rv0805 may therefore play a key role in the pathogenicity of mycobacteria, not only by hydrolyzing bacterial cAMP, but also by indicating as a protein that can alter cell wall functioning (Podobnik *et al.*, 2009).
