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1 Mycobacterial Research Group, Centenary Institute of Cancer Medicine and Cell Biology, Locked Bag no. 6, Newtown, NSW 2042, Australia
2 Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461-1926, USA
3 Department of Medicine, University of Sydney, NSW 2006, Australia
Correspondence
James A. Triccas
J.Triccas{at}centenary.usyd.edu.au
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Micro-organisms capable of utilizing sulfate as a sole source of sulfur begin the assimilation by actively transporting sulfate into the cytosol, whereupon it is chemically activated in a reaction in which the adenylyl moiety (AMP
) of ATP is transferred to sulfate to form activated sulfate, or APS (adenosine 5'-phosphosulfate) (Leyh, 1993). This extremely unfavourable reaction is kinetically and energetically linked to the hydrolysis of GTP by the enzyme ATP sulfurylase, which is composed of two types of subunits: an adenylyl transferase (CysD) and a GTPase (CysN) (Leyh & Suo, 1992). APS is then phosphorylated at the 3'-hydroxyl to form PAPS (3'-phosphoadenosine 5'-phosphosulfate) in a reaction catalysed by APS kinase, which is encoded by cysC (Satishchandran & Markham, 1989; Williams et al., 2002). Transfer of the sulfuryl group (–SO3–) of PAPS to various metabolic recipients is used widely by the cell to regulate metabolism (Zhang et al., 1998). Alternatively, the sulfuryl moiety of PAPS can be reduced, in two successive enzymic reactions, to sulfide (S2–) and incorporated into cysteine, and, from there, into other reduced-sulfur metabolites (Leyh, 1993).
The central role of sulfur in many metabolic processes extends to its requirement for the expression of virulence by numerous pathogenic bacteria. For example, cysteine availability regulates expression of Bordetella pertussis toxin (Bogdan et al., 2001), while genes in the sulfur assimilatory pathway are required for Brucella melitensis virulence (Lestrate et al., 2000). In mycobacteria our knowledge of sulfur metabolism is limited and has generally been restricted to the study of sulfolipids, which are exclusive to the pathogenic mycobacterial strains (Middlebrook et al., 1959; Goren, 1970). Purified sulfolipids inhibit activation of and phagosome–lysosome fusion in macrophages (Goren et al., 1976; Pabst et al., 1988), but do not appear necessary for Mycobacterium tuberculosis growth within mice (Rousseau et al., 2003). Mycobacterial sulfolipids are sulfated using PAPS (Williams et al., 2002). Sulfur is also present in other important metabolites such as mycothiol, which plays an important role in protecting mycobacteria from toxic oxidants such as hydrogen peroxide (Rawat et al., 2002; Buchmeier et al., 2003). Further, mycothiol influences the resistance of M. tuberculosis to important anti-tuberculosis antibiotics such as rifampicin and isoniazid (Buchmeier et al., 2003). Together, these data highlight the important role played by sulfur metabolism in mycobacteria and the necessity to further understand these pathways in pathogenic strains.
M. tuberculosis is a facultative intracellular pathogen that resides primarily within the nutrient-limiting environment of host macrophages (Britton et al., 1994). Our previous work has demonstrated that expression of the cysD promoter of M. tuberculosis is augmented within the intracellular milieu of the macrophage (Triccas et al., 1999). Although this finding suggested that regulation of ATP sulfurylase may be required for adaptation of M. tuberculosis to its intracellular environment (Triccas & Gicquel, 2000), it was unknown whether cysD and cysNC encode functional enzymes or whether their expression is linked. In this report, we demonstrate that the M. tuberculosis cysD and cysNC genes form a single operon whose transcription is regulated by stress-stimuli, including sulfur starvation. Further, we provide conclusive evidence that these gene products encode GTPase-dependent ATP sulfurylase and APS kinase activities.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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and BL21(DE3) were grown on liquid or solid Luria–Bertani (LB) medium. M. tuberculosis strain Mt103 was grown in liquid Middlebrook 7H9 medium (Difco) supplemented with ADC enrichment (Difco). When required, the antibiotics kanamycin (25 µg ml–1) and ampicillin (100 µg ml–1) were added.
RNA extraction and RT-PCR.
RNA was extracted from M. tuberculosis using RNA-bee (Friendswood). RT-PCR to determine if two adjoining genes are transcribed together was performed as described previously (Camacho et al., 2001). Briefly, cDNA was prepared by reverse-transcribing 5 µg RNA with the Superscript III Reverse Transcriptase (Invitrogen) using 3' primers within the ‘downstream’ ORF of interest. Two microlitres of the resultant cDNA template was used for PCR amplification (94 °C 30 s, 50 °C 30 s, 72 °C 1 min, 30 cycles) using the same downstream primer and a 5' primer sequence derived from the upstream ORF (Table 1). In order to simulate sulfate starvation, bacteria were grown to mid-exponential phase in complete Proskauer and Beck (PB) medium (Falcone et al., 1995) and then grown for an additional 48 h in the same medium (PB : MgSO4), PB in which MgSO4 was replaced with 0·24 mM MgCl2 (PB : MgCl2), or PB : MgCl2 supplemented with 5 mM L-cysteine. Extracted RNA was converted to cDNA as described above using specific 3' primers within the cysNC and M. tuberculosis ppa genes (Table 1). M. tuberculosis ppa is constitutively expressed under stress conditions and within macrophages (Triccas & Gicquel, 2001). The cDNA product was used in a PCR (94 °C 30 s, 50 °C 30 s, 72 °C 1 min, 30 cycles) containing gene-specific primer pairs for cysDNC or ppa (cysD-cysNC.for/cysD-cyNC.rev or ppa.for/ppa.rev; Table 1). RT-PCR was also performed on RNA extracted from bacteria grown for 48 h in 7H9 medium in the presence of 5 mM H2O2, 500 µM 2,2'-dipyridyl (Sigma) or 0·2 mM of the nitric oxide donor DETA/NO (Sigma). PCR band intensity was calculated by first capturing the image using the Chemi-genius Bio-imaging System (Syngene) and band intensity determined using the MultiAnalyst software (Bio-Rad). Normalized changes in mRNA levels were determined by dividing the cysDNC band intensity by that of the M. tuberculosis ppa transcript at each condition, and then calculating the ratio of the PCR product for the different stress conditions compared to that obtained in normal medium.
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) into the NdeI and BamHI sites of pET22b (Novagen), to obtain pRP12. Expression of CysD and CysNC in E. coli BL21(DE3) was induced by the addition of 0·7 mM IPTG to mid-exponential-phase cultures growing at 37 °C in LB. The culture temperature was shifted to 18 °C upon addition of IPTG, and the cells were harvested 15 h later. The pellet was suspended in 4·2 ml buffer [HEPES (50 mM), DTT (1·0 mM), EDTA (10 mM), PMSF (100 µM), lysozyme (0·10 mg ml–1), pepstatin A (1·5 µM), pH 8·0, T=4 °C] per g pellet, and disrupted by sonication. The sonicate was centrifuged and polynucleotides were removed from the supernatant by precipitation (streptomycin sulfate, 1·0 g per 100 ml) followed by centrifugation. The CysDNC complex was precipitated at 30 % (NH4)2SO4; the pellet was harvested by centrifugation and suspended in buffer [HEPES (50 mM), KCl (75 mM), MgCl2 (1·0 mM), 
-mercaptoethanol (12 mM), glycerol (10 %, v/v), pH 8·0, T=4 °C]. The complex was further purified using size-exclusion chromatography (Sephacryl S-300). The activity of the complex was tested by monitoring the formation of [35S]APS and [35S]PAPS from 
(1·0 mM) and ATP (2·0 mM) in the presence and absence of GTP (1·0 mM) (Satishchandran & Markham, 1989; Leyh & Suo, 1992). |
RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) identified well-conserved homologues of the E. coli cysD, cysN and cysC genes (Fig. 1A). Unlike the situation in E. coli, however, the cysN and cysC genes of M. tuberculosis are fused to encode a single polypeptide. The C-terminal 187 aa portion of the predicted M. tuberculosis CysNC protein shares 50 % identity with the E. coli CysC protein. The remaining N-terminal portion of M. tuberculosis CysNC shows a similar level of identity (49 %) with E. coli CysN. Fusion of the CysN and CysC domains has also been described in Rhizobium meliloti (Schwedock et al., 1994) and Pseudomonas aeruginosa (Hummerjohann et al., 1998). Further inspection of the M. tuberculosis genome sequence revealed that both the cysD and cysNC genes are closely flanked by other ORFs (Fig. 1A). RNA extracted from M. tuberculosis Mt103 was employed in RT-PCR to determine if cysD and cysNC form an operon and if transcription is coupled to surrounding ORFs. When this procedure was performed for the five ORFs shown in Fig. 1(A), only the combination of primers spanning the cysD and cysNC gene resulted in a PCR product of the expected size, which was 529 bp for cysDNC (Fig. 1B). This suggests that the cysD and cysNC genes are transcribed from a polycistronic message, and that the surrounding genes are not part of the cysDNC operon.
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), suggesting that this operon may respond to defined conditions potentially encountered within host cells. As intracellular pathogens must adapt to changes in nutrient levels in vivo to promote survival and virulence, we examined if the cysDNC genes were regulated by sulfur availability. To simulate sulfate starvation, bacteria were grown to mid-exponential phase in media either containing (PB : MgSO4) or lacking (PB : MgCl2) a sulfur source. RT-PCR analysis revealed a marked increase in cysDNC message in the absence of sulfur (Fig. 2A). Addition of cysteine, a major end-product of sulfur activation, to the medium repressed the starvation-induced increase in cysDNC expression (Fig. 2A). To gain a semi-quantitative measure of the level of regulation of the operon, amounts of cysDNC message were normalized to transcript levels of the M. tuberculosis ppa gene. The ppa gene encodes a putative inorganic pyrophosphatase and is constitutively expressed within host cells and upon in vitro exposure to various stress stimuli (Triccas & Gicquel, 2001). Estimation of PCR band intensity by densitometry and normalization with ppa transcript levels indicated that the increase in cysDNC expression in response to sulfate starvation was approximately 2·5-fold (Fig. 2B). These results may explain in part the observed upregulation of the cysDNC promoter within host cells and also the recent identification of cysD in a screen for M. tuberculosis genes induced by nutrient starvation (Betts et al., 2002).
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). This result defines another parameter that may account for the in vivo induction of cysDNC, as oxidative stress is one mechanism employed by host cells to eliminate pathogenic invaders (Triccas & Gicquel, 2000). It is of interest to note that mycobacterial strains lacking mycothiol are sensitive to toxic oxidants such as hydrogen peroxide (Rawat et al., 2002; Buchmeier et al., 2003). As ATP sulfurylase is necessary for the ultimate formation of mycothiol, it is tempting to speculate that upregulation of the cysDNC operon upon exposure to oxidative stress is necessary to enhance activity of anti-oxidant defence mechanisms. The operon did not appear to be globally regulated by stress stimuli as we observed no significant change in cysDNC transcript levels in response to nitric oxide (0·2 mM DETA/NO) or depletion of iron from the medium (500 µM 2,2'-dipyridyl; data not shown). We cannot rule out the possibility that the operon may be regulated by other stress conditions or may be differentially regulated at time points other than those used in this study.
The cysDNC operon encodes a tri-functional sulfate-activating complex
Based on their homology to genes from previously characterized sulfate activating pathways, the M. tuberculosis cysD and cysNC genes appeared likely to encode a multi-functional enzyme complex exhibiting ATP sulfurylase, GTPase and APS kinase activities. To test this hypothesis, the M. tuberculosis cysDNC operon was expressed in E. coli and the proteins purified. SDS-PAGE demonstrated that the CysD and CysNC proteins co-purify to approximately 95 % homogeneity (Fig. 3), The complex consists of two roughly equimolar subunits whose apparent molecular masses are comparable to those predicted for CysD and CysNC, 35 kDa and 68 kDa, respectively.
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and ATP in the presence and absence of GTP (Leyh & Suo, 1992; Schwedock et al., 1994). When GTP is present, the complex converts roughly 40 % of the 
to PAPS – the APS levels are below detection (Fig. 4). When GTP is absent, neither APS nor PAPS is detected. This is precisely the behaviour predicted by the functional signatures present in the cysD and cysNC coding regions. The synthesis of PAPS requires both ATP sulfurylase and APS kinase activities, and the GTP-dependent turnover of the complex reveals that, like the E. coli ATP sulfurylase, the M. tuberculosis complex links the GTPase and sulfate activating reactions.
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to PAPS is considerably higher than that predicted by the combined energetics of the ATP sulfurylase and APS kinase reactions (i.e. 4 % conversion at equilibrium; Satishchandran & Markham, 1989; Leyh, 1993), and the lack of detectable product in the absence of GTP suggests that the rate of activated sulfate synthesis is also linked to the GTPase reaction. These properties mirror the behaviour of the E. coli enzyme well (Liu et al., 1994). At the substrate concentrations used in this experiment, which are at or near saturating in all cases, the turnover of the complex is 0·13 s–1; this value, which is 11 % of kcat for the E. coli ATP sulfurylase (Wang et al., 1995), establishes a lower limit for the kcat of the GTPase-activated turnover of the CysD subunit in the M. tuberculosis complex. It should be noted that ATP sulfurylase activity in crude extracts of E. coli grown on rich media, which potently suppresses expression of the cys regulon, is below detectable limits (Jones-Mortimer et al., 1968).
Concluding remarks
We have shown that the M. tuberculosis cysDNC genes encode a tri-functional sulfate-activating complex whose expression is regulated by the availability of sulfur and oxidative stress, conditions likely to be encountered by pathogenic mycobacteria within the hostile environment of the macrophage. ATP sulfurylase activity is linked to a number of important biosynthetic processes in M. tuberculosis including mycothiol synthesis, which, if sufficiently altered, will impair the cells' ability to respond to toxic oxidants and antibiotics (Rawat et al., 2002; Buchmeier et al., 2003). Hence, ATP sulfurylase may prove a valuable target for affecting the viability of pathogenic mycobacteria. Further characterization of the M. tuberculosis CysDNC complex is needed to provide a more complete picture of the sulfate-activating system and decipher the role of this pathway in mycobacterial virulence.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Cole, S. T., Brosch, R., Parkhill, J. & 39 other authors (1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537–544.[CrossRef][Medline]
Falcone, V., Bassey, E., Jacobs, W. Jr & Collins, F. (1995). The immunogenicity of recombinant Mycobacterium smegmatis bearing BCG genes. Microbiology 141, 1239–1245.[Abstract]
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Hummerjohann, J., Kuttel, E., Quadroni, M., Ragaller, J., Leisinger, T. & Kertesz, M. A. (1998). Regulation of the sulfate starvation response in Pseudomonas aeruginosa: role of cysteine biosynthetic intermediates. Microbiology 144, 1375–1386.[Abstract]
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Rousseau, C., Turner, O. C., Rush, E. & 7 other authors (2003). Sulfolipid deficiency does not affect the virulence of Mycobacterium tuberculosis H37Rv in mice and guinea pigs. Infect Immun 71, 4684–4690.
Satishchandran, C. & Markham, G. D. (1989). Adenosine-5'-phosphosulfate kinase from Escherichia coli K12. Purification, characterization, and identification of a phosphorylated enzyme intermediate. J Biol Chem 264, 15012–15021.
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Received 6 November 2003;
revised 1 March 2004;
accepted 5 March 2004.
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) and absence (
) of GTP. Each point is the mean of three independent measurements. The reaction conditions were: GTP (1·0 mM or 0 mM), ATP (2·0 mM), 
(1·0 mM; 0·05 µCi µl–1, 1·85 kBq µl–1), MgCl2 (4·0 mM), HEPES (50 mM, pH 8·0/K+)], CysDNC (5·0 µM, estimated from calculated absorption coefficient), T=25 (±2) °C.