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Originally published In Press as doi:10.1074/jbc.M204613200 on June 18, 2002

J. Biol. Chem., Vol. 277, Issue 36, 32606-32615, September 6, 2002
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5'-Adenosinephosphosulfate Lies at a Metabolic Branch Point in Mycobacteria*

Spencer J. WilliamsDagger §, Ryan H. Senaratne, Joseph D. MougousDagger ||, Lee W. Riley, and Carolyn R. BertozziDagger **

From the Dagger  Howard Hughes Medical Institute and Departments of Chemistry and Molecular and Cell Biology, University of California, Berkeley, California 94720 and the  School of Public Health, University of California, Berkeley, California 94720

Received for publication, May 10, 2002, and in revised form, June 17, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bacterial sulfate assimilation pathways provide for activation of inorganic sulfur for the biosynthesis of cysteine and methionine, through either adenosine 5'-phosphosulfate (APS) or 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as intermediates. PAPS is also the substrate for sulfotransferases that produce sulfolipids, putative virulence factors, in Mycobacterium tuberculosis such as SL-1. In this report, genetic complementation using Escherichia coli mutant strains deficient in APS kinase and PAPS reductase was used to define the M. tuberculosis and Mycobacterium smegmatis CysH enzymes as APS reductases. Consequently, the sulfate assimilation pathway of M. tuberculosis proceeds from sulfate through APS, which is acted on by APS reductase in the first committed step toward cysteine and methionine. Thus, M. tuberculosis most likely produces PAPS for the sole use of this organism's sulfotransferases. Deletion of CysH from M. smegmatis afforded a cysteine and methionine auxotroph consistent with a metabolic branch point centered on APS. In addition, we have redefined the substrate specificity of the B. subtilis CysH, formerly designated a PAPS reductase, as an APS reductase, based on its ability to complement a mutant E. coli strain deficient in APS kinase. Together, these studies show that two conserved sequence motifs, CCXXRKXXPL and SXGCXXCT, found in the C termini of all APS reductases, but not in PAPS reductases, may be used to predict the substrate specificity of these enzymes. A functional domain of the M. tuberculosis CysC protein was cloned and expressed in E. coli, confirming the ability of this organism to make PAPS. The expression of recombinant M. tuberculosis APS kinase provides a means for the discovery of inhibitors of this enzyme and thus of the biosynthesis of SL-1.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mycobacterium tuberculosis is a major pathogen of global importance. This scourge has afflicted humanity for at least 5000 years, and we are still far from reliable and cost-effective treatments (1, 2). The limitation of current therapy originates from the widespread occurrence of antibiotic resistant strains and also the requirement for prolonged and uninterrupted administration of antibiotics (3). Thus, new classes of antibiotics are desperately needed. Fortunately, the recent sequencing of the M. tuberculosis genome offers a wealth of possible avenues to explore for new antibiotic therapies (4). To this end, we have chosen to examine the sulfate assimilation pathway of this organism with the aim of defining features that may represent targets for therapeutic intervention.

Our attention was attracted to reports, dating back over 40 years, of a number of novel sulfated molecules isolated from various mycobacteria (5). The first of these to be characterized was a lipidated trehalose 2-sulfate, sulfolipid 1 (SL-11; Fig. 1) (6, 7). In subsequent studies, this molecule has been ascribed a variety of intriguing attributes including a correlation with increasing strain virulence (8). There have been two reports of sulfated glycopeptidolipids in Mycobacterium avium and Mycobacterium fortuitum. In M. avium, the sulfate group was found attached to a 6-deoxy-L-talose residue (9), whereas in M. fortuitum the sulfate was attached to a dimethyl-L-rhamnose moiety (10). Interestingly, each of these molecules resides in the cell wall and thus may be important mediators of intercellular dialog. Consequently, the enzymes that install these sulfate groups, the sulfotransferases, are of great interest. Recently, the Cummings laboratory has shown that the M. tuberculosis gene Rv1373 encodes a sulfotransferase capable of sulfating several glycolipids, including crude glycolipids from the avirulent M. tuberculosis strain H37Ra (11). Whereas a definitive function has not been assigned to any mycobacterial sulfotransferase, in all well characterized cases to date, sulfotransferases exclusively use 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the activated source of sulfate (12). Due to the intriguing properties reported for the sulfatides of M. tuberculosis and the occurrence of at least one sulfotransferase in its genome, we were interested in defining the PAPS biosynthesis and sulfate assimilation routes used by this organism. Our immediate aim was to find a key enzyme involved in PAPS synthesis that could be interrupted, thereby providing a route to inhibiting the biosynthesis of all sulfated molecules, including SL-1. Also, it was hoped that such an enzyme was exclusively devoted to producing PAPS for the sulfotransferases and consequently would have no effect on the pathway used to produce cysteine and methionine.


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  		Fig. 1.   Structurally characterized sulfated molecules isolated from various mycobacteria. a, SL-1 from M. tuberculosis; b, sulfated glycopeptidolipid from M. avium; c, sulfated glycopeptidolipid from M. fortuitum.

The prototype sulfate assimilation pathway is that of Escherichia coli (Fig. 2) (13, 14). In this organism, sulfate is first activated by reaction with ATP to form adenosine 5'-phosphosulfate (APS). The enzyme that performs this task, ATP sulfurylase (CysD), forms part of a heterodimer. The other component is a GTPase (CysN) that couples the hydrolysis of GTP to the sulfurylation of ATP, thereby providing energy to shift the equilibrium so as to favor the formation of the product (15, 16). APS kinase (CysC) then converts APS to PAPS. Following this reaction, PAPS is acted upon by a thioredoxin-dependent reductase (CysH) that converts PAPS to sulfite and 3'-phosphoadenosine 5'-phosphate. Sulfite, in turn, is reduced by the later enzymes in the Cys regulon, forming first sulfide before incorporation into cysteine and, ultimately, methionine (17).


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  		Fig. 2.   Sulfate assimilation pathways in plants and bacteria. Plants appear to universally use the upper, APS reductase pathway. Bacteria follow either the upper APS reductase pathway or the lower PAPS reductase pathway. Common enzyme designations are given below each arrow.

In recent years, it has been discovered that several organisms subvert this classical sulfate assimilation route. In these organisms, CysH or its equivalent reduces APS rather than PAPS, thereby bypassing the requirement for the formation of PAPS (Fig. 2). APS reductases were first identified in plants (18) (i.e. Arabidopsis thaliana (19-21) and, soon after, Lemna minor (22) and the marine algae Enteromorpha intestinalis (23)). In the case of these plant enzymes, glutaredoxins rather than thioredoxins are utilized as cofactors and are frequently found attached to the APS reductase as a separate domain (24). Interestingly, recent reports have shown that APS reductases are not limited solely to plants but are also found in several bacteria including Sinorhizobium meliloti (25) and Pseudomonas aeruginosa (26). The bacterial APS reductases are thioredoxin-dependent, and both APS and PAPS reductases share a high level of sequence homology to one another. Indeed, the strong sequence similarity between these different enzymes has dissuaded some investigators from making clear statements about the predictive power of sequence comparisons (26). Recently, Kopriva et al. (27) have described a detailed classification of APS and PAPS reductase genes that relies heavily on the presence of two pairs of cysteine residues in APS reductases and their absence in PAPS reductases. According to this analysis, M. tuberculosis probably contains an APS reductase, and there is a single report ascribing some APS reductase activity to a crude extract of an untyped mycobacterial strain (26). However, there still remains some doubt over the use of such a simple classification scheme for the identification of the substrate specificity of APS and PAPS reductases, especially given the unknown role of such a sequence motif in substrate binding or catalysis. This is particularly important, since there are conflicting reports in the literature about the substrate specificity of the reductase of the Bacillus subtilis that has been reported as both a PAPS (28) and an APS reductase (27) and that possesses the same pair of cysteine residues. Our preliminary analysis of the genomic organization of the Cys regulon of M. tuberculosis shows that it differs from that of other well studied organisms (see below). Until now, the function of the M. tuberculosis reductase had been predicted, but it was not known with certainty.

In the present study, we have functionally defined the sulfate assimilation pathway of M. tuberculosis and Mycobacterium smegmatis. We have cloned the genes involved in the manipulation of APS and have characterized their specificity by genetic complementation. This study has revealed that the M. tuberculosis CysH is an APS reductase and, thus, that APS kinase (CysC) produces PAPS for the exclusive use of sulfotransferases. Genetic complementation was used to define a functional APS kinase domain from the CysN/CysC gene of M. tuberculosis. Deletion of CysH from M. smegmatis afforded a cysteine and methionine auxotroph, providing strong evidence for a metabolic branch point centered about APS. In addition, the M. tuberculosis CysC was overexpressed in E. coli and purified. The heterologously expressed enzyme was characterized to extract apparent first order kinetic parameters of the substrates APS and ATP. Finally, we have redefined the substrate specificity of the Bacillus subtilis CysH, previously characterized as a PAPS reductase, as an APS reductase (28). Taken together, these results provide confirmation of sequence-based identification of APS and PAPS reductases. Moreover, the clarification of the sulfate assimilation pathway of M. tuberculosis provides opportunities for the study of the biosynthesis of sulfated metabolites in this and other mycobacteria. Notably, APS reductases are not found in humans and may therefore represent unique targets for antibiotic therapy.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Pfu DNA polymerase was obtained from Stratagene. Restriction enzymes were from New England Biolabs or Amersham Biosciences. Calf intestinal alkaline phosphatase (CIAP) was from Amersham Biosciences. Plasmid miniprep kits and the QIAquick kit for DNA extraction from agarose gel were from Qiagen. T4 DNA ligase was from New England Biolabs. Reagents and enzymes for the assay of CysC were from Sigma. Plasmids used in this work are shown in Table I, and oligonucleotide primers are shown in Table II. Preliminary sequence data for M. smegmatis and M. avium were obtained from the Institute for Genomic Research Web site (www.tigr.org). Sequence data for Mycobacterium bovis were produced by the M. bovis sequencing group at the Sanger Institute and can be obtained from the World Wide Web at www.sanger.ac.uk/Projects/M_bovis. E. coli JM81A and JM96 were obtained from the E. coli genetic stock center at Yale University.

                              
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Table I
Bacterial strains and plasmids used in this study
Ampr, Kanr, and Hygr denote resistance to ampicillin, kanamycin, and hygromycin, respectively. CGSC, E. coli genetic stock center (Yale University).

                              
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Table II
Oligonucleotide primers used in this study
Sequences in boldface type indicate relevant restriction sites.

Preparation of CysC Expression Vector-- The C-terminal portion of the cysNC gene encoding CysC (cysC) was amplified from genomic DNA by PCR. The PCR mixture contained 10 µM oligonucleotide primers (MYCYSCF and MTCYSCR; Table II), 0.25 mM dNTPs in 50 µl of Pfu polymerase buffer, 10% dimethyl sulfoxide, and 100 ng of M. tuberculosis genomic DNA. After heating to 95 °C, the reaction was initiated by adding 5 units of Pfu DNA polymerase. The PCR was performed in a thermal cycler (PerkinElmer Life Sciences, GeneAmp PCR System 2400). The following PCR program was used: 25 cycles (20 s at 94 °C, 30 s at 50 °C, and 55 s at 72 °C) and then incubation for 7 min at 72 °C. Agarose gel electrophoresis of the PCR mixture revealed a single DNA fragment of ~500 bp. This fragment was cut from the gel and purified using the QIAquick kit. The product was ligated into pCR4Blunt-TOPO according to the manufacturer's instructions (Invitrogen). Isolated colonies were grown overnight in liquid media, and plasmid DNA was isolated by miniprep. Plasmids containing insert were identified by restriction digest and confirmed by sequencing. The insert was excised by digestion with NdeI/XhoI, separated by agarose gel electrophoresis, and purified using the QIAquick kit. The product was ligated into the NdeI/XhoI-digested pET24b(+) vector (treated with CIAP) using T4 DNA ligase. After incubation at 16 °C for 2 h, 8 µl of the reaction mixture was used to transform 100 µl of E. coli DH5alpha . After growth on LB amp, colonies were selected and grown overnight. Plasmid DNA minipreps were screened by restriction digest to afford pET28b(+)CysC.

Preparation of E. coli Complementation Vectors-- The gene encoding CysH (cysH) was amplified from genomic DNA as described above using primers CYSHPUCF and CYSHPUCR (Table II) and cloned into pCR4Blunt-TOPO as above. After sequencing, the insert was excised by digestion with NcoI/BamHI and ligated into CIAP-treated NcoI/BamHI-digested pUC18/RBS, to generate pUC18/RBS/MtCysH.

The gene encoding CysC (cysC) was amplified as above using primers CYSCPUCF and MTCYSCR (Table II) and cloned into pCR4Blunt-TOPO as above. After sequencing, the insert was excised from this vector by digestion with NcoI/XhoI, and the two fragments generated were separated by gel electrophoresis and purified as above. The longer NcoI/XhoI fragment was ligated into NcoI/XhoI-digested pUC18/RBS/MtCysH from above and transformed into E. coli DH5alpha . Colonies containing the correct insert were verified by restriction digest. This vector was digested with NcoI, treated with CIAP, and ligated to the second, smaller NcoI/NcoI fragment. After transformation and plasmid isolation, the plasmid minipreps were screened for correctly oriented insert with EagI/XhoI, affording pUC18/RBS/MtCysC.

The gene encoding the M. smegmatis CysH (cysH) was amplified from M. smegmatis mc2155 genomic DNA using primers pUCMsHFor and pUCMsHRev (Table II) and cloned into pCR4Blunt-TOPO as above. After sequencing, the insert was excised by digestion with NcoI/XhoI and ligated into CIAP-treated NcoI/XhoI digested pUC18/RBS to yield pUC18/RBS/MsCysH.

The gene encoding the B. subtilis CysH (cysH) was amplified from pBS170 using primers pUCCYSHBsF and pUCCYSHBsR (Table II) and cloned into pCR4Blunt-TOPO as above. After sequencing, the insert was excised by digestion with NcoI/XhoI and ligated into CIAP-treated NcoI/XhoI-digested pUC18/RBS to yield pUC18/RBS/BsCysH.

Genetic Complementation in E. coli-- E. coli JM81A and JM96 were grown in Oxoid CM1 medium (1 g of Oxoid Lab Lemco powder, 2 g of yeast extract, 5 g of peptone, and 5 g of NaCl per liter). Plasmid DNA was transformed into cells by electroporation (Bio-Rad Gene-Pulser, following the manufacturer's protocol). Transformants were grown on CM1-agarose containing 100 mg/liter ampicillin before transfer to M9 minimal medium supplemented with thiamine (0.0005%), mannitol (0.2%), glucose (0.2%), and 18 amino acids excluding cysteine and methionine (each 25 mg/liter) and containing MgSO4 (0.01%) as the sole sulfur source.

Construction of CysH M. smegmatis Deletion Mutant-- The cysH deletion mutant was constructed using the allelic replacement method of Parish and Stoker (29). Oligonucleotide primers were used to amplify 2-kb regions upstream and downstream of the cysH gene. The upstream region was generated using primers CysHKO#3 and CysHKO#4 (Table II), which generate a NotI/KpnI fragment, and the downstream region was generated using primers CysHKO#1 and CysHKO#2, which generate a KpnI/HindIII fragment. The PCR products were gel-purified, digested with the relevant restriction enzymes, and ligated into a similarly digested p2NIL vector that was pretreated with CIAP. A hygromycin resistance marker was inserted between the two fragments into the KpnI restriction site. The final delivery vector, p2NIL_MsCysH, was generated by adding the PacI cassette (PAg85-lacZ Phsp60-sacB) from pGOAL17 to the vector bearing the mutated allele. This cassette contains the lacZ reporter gene and the sacB-negative selection marker. sacB, encoding levan sucrase, confers toxicity to the cell when grown on a sucrose-containing medium. The delivery vector was pretreated with UV light (100 mJ cm-2) and used to electroporate M. smegmatis mc2155. Transformants were selected on Middlebrook 7H11 medium containing 20 mg/liter kanamycin and 50 mg/liter hygromycin. After 5 days, colonies were tested for the presence of the lacZ gene, and positive colonies were grown in 7H9 medium containing 50 mg/liter hygromycin overnight. Serial dilutions were plated onto 7H11 plates containing 2% sucrose (50 mg/liter), X-gal (50 mg/liter), and 2 mM methionine. Colonies that did not turn blue were tested for kanamycin sensitivity and were then subjected to genotypic analysis.

Genotypic Analysis-- Genomic DNA was prepared from M. smegmatis by standard methods. Southern blotting analysis was carried out using the DIG detection kit (Roche Molecular Biochemicals). Two probes were generated by PCR (5'-probe, CysHprFor and CysHKO#3; 3'-probe, CysHprRev and CysHKO#2) with Expand polymerase (Roche Molecular Biochemicals) using DIG-labeled dNTPs; one probe was specific for the upstream region of the gene (5'-probe), and one probe was specific for the downstream region (3'-probe). Genomic DNA was digested with the restriction enzymes SphI, NotI, and AscI, which generated unique bands for the wild-type and mutant strains. The digested genomic DNA was separated by electrophoresis on two 0.8% agarose gels (20 V, 12 h), and each digest was transferred to HyBond-N+ membrane (Amersham Biosciences) by capillary action. After cross-linking the DNA to the membrane (UV light, 120 mJ), each blot was hybridized (68 °C) with DIG-labeled probes specific for regions upstream and downstream of the interrupted gene. After washing and blocking, the hybridized bands were detected using an anti-DIG antibody-alkaline phosphatase fusion to hydrolyze bromochloroindolyl phosphate.

Construction of Mycobacterial Complementation Vectors-- Complementation of the mutant strain was performed using the vector pMS3GS. This vector was constructed by inserting a 400-bp region containing the M. tuberculosis glutamine synthetase promoter into pMS3 and will be described in detail elsewhere. A cloning site was introduced that allowed the insertion of a PacI-BamHI fragment. The M. tuberculosis cysH gene was amplified from genomic DNA using CYSHPMSF and CYSHPMSR (Table II) and cloned into pCR4Blunt-TOPO as described above. After sequencing, the insert was excised from this vector by digestion with PacI/BamHI and ligated into CIAP-treated PacI/BamHI-digested pMS3GS to afford pMS3GSMtCysH.

Expression of M. tuberculosis CysC-- pET28b(+)CysC was transformed into BL21 STAR and grown on LB-agarose containing 50 mg/ml kanamycin. An isolated colony was picked and grown in 2 ml of LB medium containing 50 mg/ml kanamycin. When this culture had reached an A600 = 0.5, 1 ml was used to inoculate 500 ml of 2YT medium containing 50 mg/ml kanamycin. The culture was grown at 37 °C with shaking until an A600 = 0.5, and then the suspension was cooled to 20 °C, and isopropyl 1-thio-beta -galactoside was added to a final concentration of 0.4 mM. The culture was allowed to grow overnight. Cells were collected by centrifugation (10 min at 4000 rpm) and suspended in lysis buffer (20 mM Tris buffer containing 100 mM NaCl and 10 mM imidazole) before disruption by ultrasonication. The cell lysate was cleared by centrifugation (10 min at 10000 rpm), and the supernatant was applied to a column of Ni2+-nitrilotriacetic acid-agarose resin and eluted with 20 mM Tris buffer (pH 7.8) containing 100 mM NaCl and a gradient of imidazole up to 250 mM. The fractions containing protein were concentrated and stored in the same buffer. Total yield was ~25 mg of protein per liter of culture. Further characterization was performed using a PerkinElmer Life Sciences Sciex API III electrospray mass spectrometer that gave a mass of 23,192 Da. This compared satisfactorily with the calculated mass predicted for the protein with the loss of the N-terminal methionine (calculated, 23,168). Protein concentrations were measured using the Pierce Micro BCA analysis kit.

Assay of M. tuberculosis CysC-- Kinase activity was measured for the formation of PAPS from APS using the pyruvate kinase plus lactate dehydrogenase plus nuclease P1-coupled spectrophotometric assay as described previously (30). In this assay, formation of ADP from ATP is monitored by coupling its production to consumption of NADH. The large excess of nuclease P1 allows the continuous regeneration of APS from PAPS, thus allowing the measurement of initial velocities at low concentrations of APS. Reaction rates were measured at 25 °C using a 50 mM Tris buffer (pH 8.0) containing 1 mM KCl and 0.1% bovine serum albumin. Each sample contained 700 µl of buffer, 25 units of lactate dehydrogenase, and 35 units of pyruvate kinase (from rabbit muscle, 50% suspension in glycerol), 25 units of P1 nuclease, 3 µM phosphoenolpyruvate, 4 µM NADH, 5 mM ATP, 5 mM MgCl2, 100 mM Tris base, and various amounts of APS. Reaction progress was monitored using a Varian CARY100 UV-visible spectrophotometer at 340 nm. Prior to the addition of APS kinase, the background rate was measured and typically was -0.001 A340 units/min. Reactions were started by the addition of M. tuberculosis CysC, and measurements of the decrease of absorption at 340 nm per min in a continuous assay yielded reaction rates. The decrease was linear during all measurements. The concentration of APS was determined by measuring the total change in absorbance at 340 nm in a reaction catalyzed by APS kinase but omitting P1 nuclease (to allow the complete consumption of APS) and using an extinction coefficient for NADH of 6.22 mM-1 cm-1. Michaelis parameters (Vmax and Km) were extracted from these data by best fit to the Michaelis-Menten equation using the program Grafit (31). Km and Vmax/E0 values were obtained by measuring rates in a series of cells at a range of substrate concentrations (6-10 concentrations), which encompassed the Km value ultimately determined, generally from 0.2 × Km to 5 × Km.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of cysH, cysC, and cysN Homologs in M. tuberculosis and M. smegmatis-- cysH and cysC homologs were identified in M. tuberculosis, M. smegmatis, and M. avium by BLAST analysis, and for M. tuberculosis the genes corresponded to those annotated in the published genome (4). Interestingly, amino acid sequence comparison of these CysH proteins shows that they align well with both PAPS and APS reductases from a variety of organisms (Fig. 3). However, the mycobacterial CysH proteins each contain two pairs of cysteine residues in the C-terminal half of the sequence. These two pairs of cysteine residues are common to all of the known APS reductases and are absent in all but one of the proven PAPS reductases (that of B. subtilis; see below).


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  		Fig. 3.   Multiple sequence alignment of cloned and characterized (P)APS reductases. Consensus of reductases is shown below with uppercase letters indicating entirely conserved residues and lowercase letters indicating similar residues. Dark shading indicates highly conserved residues, and light shading indicates conserved similar residues. The sequences were aligned with ClustalW, and shading was performed with BoxShade Version 3.21. The abbreviations used, and data bank accession numbers are as follows: Myctub_CysH, APS reductase from M. tuberculosis (GenBankTM identifier P71752); Sinmel_CysH, APS reductase from S. meliloti (GenBankTM identifier AAD55759); Burcep_CysH, APS reductase from Burkholderia cepacia (GenBankTM identifier AAD50979); Aratha_APR2, APS reductase from A. thaliana (GenBankTM identifier S71242); Lemmin_Lapr, APS reductase from L. minor (GenBankTM identifier CAB65911); Entint_EAPR1, APS reductase from E. intestinalis (GenBankTM identifier AAC26855); Pseaer_CysH, APS reductase from P. aeruginosa (GenBankTM identifier O05927); Bacsub_CysH, APS reductase from B. subtilis described here (GenBankTM identifier H69611); Saccer_Met16, PAPS reductase from S. cerevisiae (GenBankTM identifier S59826) Esccol_CysH, PAPS reductase from E. coli (GenBankTM identifier P17854); Thicap_CysH, PAPS reductase from T. roseopersicina (GenBankTM identifier CAA80690). The amino and carboxyl termini of A. thaliana APR2, L. minor Lapr, and E. intestinalis EAPR1 have not been shown for the sake of brevity. Asterisks indicate the two pairs of cysteine residues, and open circles indicate the other residues fully conserved among APS reductases. A closed circle indicates the completely conserved cysteine residue implicated in the catalytic mechanism (41).

The M. tuberculosis CysC gene is fused to the C terminus of CysN, a GTPase that forms a heterodimer with CysD. This is not uncommon. For example, similar fusions are found in the functionally equivalent NodQ genes of S. meliloti and in CysN/CysC of P. aeruginosa (Fig. 4). Unlike these organisms, M. tuberculosis contains only single copies of each domain of cysH, cysC, and cysN, thereby representing a much simpler sulfate assimilation system than that of many other bacteria. The CysN/CysC gene overlaps the CysD gene by four nucleotides and appears to be part of the same operon. A putative ribosomal binding site (RBS) upstream of the start of the CysN/CysC gene was identified that lies within the C terminus of the preceding gene, cysD.


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  		Fig. 4.   Genomic organization of selected sulfate assimilation genes in several bacteria. *, APS reductase; #, PAPS reductase; **, in S. meliloti cysHDN are chromosomally located, NodPQ1 are located on megaplasmid 1, and NodPQ2 are located on megaplasmid 2.

Genetic Complementation of E. coli CysC and CysH Knockout Strains-- Given the large degree of sequence similarity between the PAPS and APS reductases, we chose to confirm the function of the M. tuberculosis and M. smegmatis cysH genes using genetic complementation in E. coli. The cysH genes were amplified by PCR from genomic DNA using primers complementary to the N and C termini. The PCR products were ligated into a pUC18-based vector containing an RBS upstream of the insertion point. Due to the high copy number of the pUC18 plasmid in E. coli (>100 copies/cell) and the low copy number of the lac repressor protein (~10/cell) (32), this plasmid allows the constitutive expression of proteins in the absence of a chemical inducer. The plasmids bearing the M. tuberculosis and M. smegmatis cysH genes were separately transformed into E. coli JM81A (a mutant strain lacking APS kinase) and JM96 (a mutant strain lacking PAPS reductase) and grown on ampicillin-containing CM1 medium (a rich medium able to support the growth of these knockout E. coli strains). Isolated colonies were plated onto M9 minimal medium supplemented with 18 amino acids (not cysteine or methionine), containing sulfate as the sole metabolizable sulfur source. Complementation of JM96, an E. coli strain capable of the synthesis of PAPS but not its reduction, confirmed that the gene product has either PAPS or APS reductase activity. Complementation of JM81A, an E. coli strain capable of the synthesis of APS but not PAPS showed that the gene encodes an APS reductase. pUC18/RBS/MtCysH and pUC18/RBS/MsCysH were able to complement both E. coli JM81A and JM96 strains to cysteine prototrophy (Fig. 5). This result is consistent with both the M. tuberculosis and M. smegmatis cysH encoding APS reductases.


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  		Fig. 5.   Genetic complementation of M. tuberculosis CysH in E. coli strains deficient in sulfate assimilation. Left, complementation performed in E. coli JM81A (CysC minus). Right, complementation performed in E. coli JM96 (CysH minus). -, E. coli strain without plasmid; +, E. coli strain bearing pUC18/RBS/CysH; BioB, E. coli strain bearing pUC18/RBS/BioB as a negative control. Cells were grown for 2 days on CM1 medium containing sulfate as the sole metabolizable sulfur source.

The assignment of APS reductase activity to both the M. tuberculosis and M. smegmatis CysH enzymes is in agreement with the observation that all proven APS reductases contain two pairs of conserved cysteine residues. However, as noted above, there is one CysH, that from B. subtilis, that has been assigned PAPS reductase activity but contains these same two pairs of cysteines (28). We were concerned with the assignment of this gene product as a PAPS reductase, which was made on the basis of its ability to complement the E. coli mutant JM96, a strain lacking PAPS reductase activity (28). Whereas the ability to restore this strain to cysteine prototrophy is consistent with the gene product being a phosphosulfate reductase, it does not show whether the enzyme is an APS or PAPS reductase. Consequently, we obtained the plasmid used in the original study by Mendoza and colleagues (28), pBS170, and confirmed its ability to complement JM96 but also tested its ability to complement JM81A. Interestingly, we were able to repeat the original result of Mendoza and co-workers with JM96 but found that the plasmid did not restore prototrophy to JM81A. However, of particular concern here was the low growth rate seen with the complemented JM96. While colonies could be seen with JM96 transformed with pUC18/RBS/MtCysH after 24 h of growth, similar sized colonies with JM96 transformed with pBS170 did not appear until 96 h of growth. It was thought that the expression of the B. subtilis gene could be limiting from the pBluescript SKII(+) vector, particularly since this construct used the native B. subtilis RBS (-14 to -8, AGGAGAA) (28). Consequently, we subcloned the B. subtilis cysH into the pUC18/RBS vector (which uses a typical E. coli RBS, -13 to -8, AGGAGG) and tested its ability to complement JM96 and JM81A. Both mutant cell lines were transformed to cysteine prototrophy, thereby confirming an APS reductase activity of the B. subtilis enzyme.

Identification of an Active CysC Domain-- As is illustrated by the presence of numerous sulfated molecules in mycobacteria and the recent work of the Cummings laboratory, M. tuberculosis and other mycobacteria possess numerous sulfotransferases. In this work, we have shown that in M. tuberculosis, APS is used directly for the production of sulfite; it appears that PAPS is produced for the sole use of these putative sulfotransferases. Given this newly defined sulfate assimilation pathway for M. tuberculosis, we identified APS kinase, CysC, as a possible target for generating a functional knockout of PAPS biosynthesis and therefore of all sulfotransferase activity in this organism. As discussed above, CysC is fused to CysN, thereby complicating the generation of a knockout. In order to construct a defined knockout of CysC, the APS kinase domain of CysN/CysC needed to be identified. We confirmed the identity of the CysC domain by using genetic complementation. The C-terminal domain of CysN/CysC was identified by alignment to the CysN and CysC proteins of E. coli. According to our analysis, the CysN and CysC domains of M. tuberculosis are separated by a short linker with the sequence TPSP. The C-terminal domain of CysN/CysC was amplified from genomic DNA, and the product was subcloned into pUC18/RBS and tested for its ability to complement the E. coli strain JM81A. Transformation of this strain with the plasmid restored it to cysteine prototrophy, confirming that we had identified an active CysC domain.

Construction of a CysH Deletion Mutant of M. smegmatis-- In order to confirm the route proposed herein for sulfate assimilation of M. smegmatis, we constructed a deletion mutant of cysH. This gene was interrupted using the allelic replacement method of Parish and Stoker (29). Briefly, a delivery vector containing the interrupted allele was constructed in the plasmid p2NIL by replacing the middle portion of the gene with a hygromycin resistance marker. After irradiation with UV light, a pretreatment that promotes homologous recombination, the delivery vector was transformed into M. smegmatis mc2155, and kanamycin/hygromycin-resistant transformants were obtained. Since the vector p2NIL is unable to replicate in M. smegmatis, resistant colonies must arise either from incorporation of the vector into the genome or as a result of spontaneous resistance. Homologous recombination was confirmed by the detection of LacZ (which is also present in p2NIL) by treatment with X-gal. These putative single crossover colonies were grown in liquid media overnight, to allow for a second crossover, and then on solid media containing hygromycin, sucrose, and X-gal. Sucrose acted as a negative selection marker and allowed only those cells that had lost the sacB gene to grow. The loss of this gene was confirmed by the absence of the lacZ gene. Colorless colonies represented potential knockouts and were confirmed by Southern analysis of isolated genomic DNA (Fig. 6). The M. smegmatis mc2155::cysH mutant was tested for auxotrophy in liquid media containing 1 mM cysteine or 1 mM methionine (Fig. 7). This strain was found to be a cysteine and methionine auxotroph. Complementation of the cysH knockout strain with M. tuberculosis cysH in the complementation plasmid pMS3GSMtCysH restored this strain to prototrophy, confirming the role of the M. tuberculosis cysH gene in the reduction of APS.


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  		Fig. 6.   Southern analysis of the M. smegmatis mc2155::cysH mutant. Top, genomic organization of wild-type, mc2155, and mutant, mc2155::cysH, strains of M. smegmatis. The arrow depicts the cysH gene, and the block labeled Hyg indicates the deletion of the center of the cysH gene and replacement with a hygromycin resistance cassette that introduces a new NotI site. Bars labeled 5'probe and 3'probe indicate the probes used for Southern blot analysis. Solid bars, regions used to generate the knockout construct p2NIL_MsCysH. Note that the restriction sites used to perform Southern analysis lie outside regions used to generate the knockout construct and therefore confirm the genomic context of the deleted region. Bottom, Southern blot analysis of genomic DNA from parental and mutant strains of M. smegmatis mc2155. Genomic DNA was digested with SphI, NotI, and AscI. The probes used are shown in the top panel. Left, 5'-probe; right, 3'-probe. W, wild type; M, mutant; MII, marker II from Roche Molecular Biochemicals; MVII, marker VII from Roche Molecular Biochemicals.


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  		Fig. 7.   Deletion of cysH from M. smegmatis mc2155 confers cysteine and methionine auxotrophy. Growth of M. smegmatis strains in Middlebrook 7H9 medium containing sulfate as the sole metabolizable sulfur source. , wild-type mc2155 bearing control plasmid pMS3GSGFP; , mc2155::CysH bearing control plasmid pMSGS1373; triangle , mc2155::CysH bearing complementation plasmid pMS3GSMtCysH; black-square, mc2155::CysH bearing control plasmid pMS3GS1373 in 1 mM methionine; open circle , mc2155::CysH bearing control plasmid pMS3GS1373 in 1 mM cysteine.

In Vitro Assay of M. tuberculosis CysC-- CysC was cloned into pET28b(+) and overexpressed in E. coli as an N-terminal His6 fusion. Purification was achieved through affinity chromatography on Ni2+-nitrilotriacetic acid resin. The purified protein had an apparent molecular mass of 23 kDa by SDS-PAGE and a mass as determined by electrospray ionization mass spectrometry of 23,192 Da. This compares favorably with the predicted molecular mass of 23,168 Da for the protein with the loss of the N-terminal methionine, presumably effected by the endogenous methionyl aminopeptidase of the expression host. The enzyme was tested for its ability to phosphorylate APS using the coupled assay of Burnell and Whatley (33) with the modifications of Renosto et al. (34). This assay allows the direct monitoring of rates by coupling the production of ADP (from ATP) to pyruvate kinase and the lactate dehydrogenase-catalyzed reduction of pyruvate by NADH. The decrease in the concentration of NADH may be continuously monitored at 340 nm. P1 nuclease is also included to regenerate APS from the product, PAPS, thereby enabling the measurement of very low Km values. Using this assay, we measured an apparent Km value of 0.64 ± 0.10 µM and an apparent Vmax/E0 value of 0.85 ± 0.04 mM-1 s-1 (for an apparent (Vmax/E0)/Km = 1334 mM-1 s-1) for APS in the presence of saturating ATP (Fig. 8A). In the presence of 75 mM sulfate, the apparent Km value increased nearly 4-fold to 1.7 ± 0.1 µM with little change in the Vmax/E0 value (1.05 s-1), leading to a 2-fold decrease in the value of Vmax/E0 (611 mM-1 s-1) (Fig. 8B). The higher Km value for the enzyme in 75 mM sulfate simplifies the kinetics somewhat and, as has been suggested previously, provides closer mimicry of the intracellular ionic strength (34). To confirm that the assay was being run at a saturating concentration of ATP, the kinetic parameters were measured in the presence of saturating APS (10 µM) in buffer containing 75 mM sulfate. The kinetic parameters confirmed that the concentration of ATP was saturating (apparent Km = 0.91 ± 0.10 mM, apparent Vmax/E0 = 0.95 ± 0.03s-1, apparent (Vmax/E0)/Km = 1.04 mM-1 s-1) (Fig. 8C). This Km value is similar to that seen for the APS kinase of E. coli (Km = 0.25 µM). However, the M. tuberculosis enzyme differs from that of E. coli in that the former does not appear to suffer from the potent substrate inhibition seen for the latter (35).


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  		Fig. 8.   Kinetic characterization of M. tuberculosis CysC. A, rates versus APS concentration at saturating MgATP (5 mM), [sulfate] = 0 mM (inset, Lineweaver-Burk replot); B, rates versus APS concentration at saturating MgATP (5 mM), [sulfate] = 75 mM (inset: Lineweaver-Burk replot); C, rates versus ATP concentration at saturating APS (5 µM), [sulfate] = 75 mM (inset, Lineweaver-Burk replot).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Sulfate Assimilation Pathway of M. tuberculosis, M. smegmatis, and B. subtilis-- Fig. 3 shows a sequence alignment of all of the APS and PAPS reductases for which the substrate specificity has been experimentally determined. In this work, we have redefined the substrate specificity of the B. subtilis CysH from a PAPS to an APS reductase and defined the M. tuberculosis and M. smegmatis CysH genes as APS reductases. Thus, in every proven case to date, APS reductases contain the sequence CCXXRKXXPL followed by another pair of cysteine residues, SXGCXXCT, whereas PAPS reductases lack both of these signature sequences. We propose that the presence or absence of this set of conserved motifs be used to assign the substrate specificity of APS and PAPS reductases, respectively. The role of these residues in the catalytic activity of the enzyme remains unclear, although it has been shown that they form a (4Fe-4S)2+ iron-sulfur cluster in several plant and bacterial APS reductases (27, 36).

Mycobacteria Possess an APS Reductase That Is Required for Sulfate Assimilation-- The E. coli complementation experiments described herein with the M. tuberculosis and M. smegmatis cysH genes serve to define these enzymes as APS reductases. To provide further evidence for the essentiality of the cysH gene in the sulfate assimilation pathway, we constructed an M. smegmatis mc2155 mutant strain containing a deletion of this gene. As expected, this mutant strain was a cysteine and methionine auxotroph but could be restored to prototrophy by a complementation plasmid bearing the M. tuberculosis cysH gene. Together, these data confirm that cysH encodes APS reductase and that this enzyme and APS are essential parts of the pathway for the assimilation of sulfate. Additionally, these data also suggest that M. smegmatis does not harbor an unidentified PAPS reductase elsewhere in the genome. Thus, on the basis of these results, APS kinase does not reside in the pathway for the biosynthesis of cysteine but instead occurs on a separate branch, solely concerned with the production of PAPS for alternative purposes.

This interpretation is illuminated in Fig. 9, which shows that these two mycobacteria produce APS for the use of two enzymes, APS reductase and APS kinase. APS reductase reduces APS to sulfite (and AMP) for the eventual incorporation into cysteine and methionine. On the other hand, APS kinase converts APS to PAPS, presumably for the sole use of the sulfotransferases of these mycobacteria. CysH homologs were identified in the unpublished M. avium, M. paratuberculosis, and M. tuberculosis CDC1551 genome sequences that possess both the CCXXRKXXPL and SXGCXXCT motifs, suggesting that these enzymes are also APS reductases (Fig. 10). Additionally, M. smegmatis and M. avium each possess open reading frames with high levels of similarity to the CysN/CysC genes of M. tuberculosis, and thus the pathway for the production of PAPS appears to be intact. Consequently, the sulfate assimilation pathway we have defined for M. tuberculosis seems to be common to these other two mycobacteria. Interestingly, the genome sequence for M. leprae does not possess homologs of CysH or several other genes in the sulfate assimilation pathway, suggesting that this organism is a cysteine and methionine auxotroph, consistent with the widespread genome decay that has given rise to over 1000 pseudogenes and its obligate in vivo habitat (37, 38).


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  		Fig. 9.   Sulfate assimilation pathway used by M. tuberculosis, M. smegmatis, M. avium, and M. paratuberculosis.


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  		Fig. 10.   Sequence alignments of CysH proteins from M. tuberculosis, M. avium, and M. smegmatis. Myctub_CysH, APS reductase from Mycobacterium tuberculosis (GenBankTM identifier P71752). Preliminary sequence data for Mycavi_CysH (putative APS reductase from M. avium) and Mycsme_CysH (putative APS reductase from M. smegmatis) was obtained from the Institute for Genomic Research Web site (www.tigr.org). The sequence from M. tuberculosis CDC1551 is identical to that of CysH from M. tuberculosis H37Rv; the corresponding sequence from M. paratuberculosis is identical to that of M. avium. M. leprae does not contain a CysH homolog. The key for the symbols (*, open circle , and ) is given in the legend to Fig. 3.

Implication of the Sulfate Assimilation Pathway for the Biosynthesis of SL-1 and Other Sulfated Metabolites-- The potential for multiple sulfotransferases in M. tuberculosis presents a problem in defining the substrate specificity of these enzymes and the identity of the enzyme that produces the sulfated glycolipid, SL-1, particularly if these enzymes have overlapping substrate specificities. By defining the sulfate assimilation pathway used by this organism, we have identified a possible route to generating a complete knockout of all sulfotransferase activity in this organism and therefore of SL-1. An M. tuberculosis knockout of CysC should in effect generate a global knockout of all sulfotransferase activity by interrupting the biosynthetic pathway for PAPS. Such a knockout could be of great use in studying the importance of sulfation in this organism; for example, the phenotype of a global sulfotransferase knockout could be compared with that of an individual sulfotransferase knockout.

Recently, Sirakova et al. (39) reported on the disruption of pks2 in M. tuberculosis, suggesting that this gene is required for the synthesis of the phthioceranic acid lipid moieties that occur on the sulfatides, including SL-1. Three of the lipids of SL-1 are based on the phthioceranoyl core, and disruption of pks2 appeared to completely abolish SL-1 synthesis. Further studies on the biological and physiological consequences of this knockout have not been reported. The studies reported herein identifying an active CysC domain and showing that CysH is an APS reductase provide an alternative approach to generating a knockout of SL-1. In addition, the results herein enhance our understanding of the biosynthesis of this intriguing molecule as well as other, poorly characterized sulfated metabolites.

A knockout of CysC in M. avium could also be of great value, particularly since the sulfotransferases of this organism have not yet been described. Khoo et al. (9) compared isolates of a clarithromycin- and ethambutol-resistant M. avium isolate from an infected AIDS patient with an ethambutol-susceptible strain. One notable difference between the strains was the expression of a sulfated glycopeptidolipid. The importance of this compound in the drug resistance of M. avium could be conveniently addressed by the deletion of cysC, thereby inactivating all of this organism's sulfotransferases. In addition, the successful cloning and expression of the CysC domain of CysN/CysC in E. coli provides a manifold for the discovery of new inhibitors of this enzyme. To date, no inhibitors of APS kinases have been described. With a suitable cell-based assay now in hand, the way is clear for the discovery of inhibitors of this enzyme and, thus, of inhibitors of sulfation in M. tuberculosis. This work also provides a route to generating a cysteine auxotroph of M. tuberculosis. Knockout of cysH should affect the sulfate assimilation pathway, preventing the biosynthesis of cysteine, without affecting the biosynthesis of sulfated metabolites such as SL-1 that require PAPS as a precursor. Notably, humans do not possess PAPS or APS reductases, so such a result would be of great value in defining a potential target for new therapeutics. Additionally, M. tuberculosis auxotrophs are of great interest in the development of new attenuated vaccine strains, especially given the less than complete efficacy of the current vaccine strain BCG (40). Whether disruption of cysteine biosynthesis is sufficient to attenuate the virulence of this organism will be reported in due course.

    ACKNOWLEDGEMENTS

We thank Dr. Diego de Mendoza for generously providing samples of pBS170 and pBS173 and Drs. Dominic Campopiano and Derek MacMillan for the kind gift of pUC18/RBS/BioB. Preliminary sequence data were obtained from the Institute for Genomic Research Web site at www.tigr.org.

    FOOTNOTES

* This work was supported by National Institutes of Health (NIH) Grants GM59907 and AI51622. Sequencing of M. avium and M. smegmatis was accomplished with support from NIAID, NIH.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ A Howard Hughes Medical Institute Fellow of the Life Sciences Research Foundation.

|| Recipient of a Ford Foundation graduate Fellowship.

** To whom correspondence should be addressed. E-mail: bertozzi@cchem.berkeley.edu.

Published, JBC Papers in Press, June 18, 2002, DOI 10.1074/jbc.M204613200

    ABBREVIATIONS

The abbreviations used are: SL-1, sulfolipid 1 from M. tuberculosis; APS, adenosine 5'-phosphosulfate; PAPS, 3'-phosphoadenosine 5'-phosphosulfate; CIAP, calf intestinal alkaline phosphatase; DIG, digoxigenin; RBS, ribosomal binding site; X-gal, 5-bromo-4-chloro- 3-indolyl beta -D-galactopyranoside.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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J. D. Mougous, R. H. Senaratne, C. J. Petzold, M. Jain, D. H. Lee, M. W. Schelle, M. D. Leavell, J. S. Cox, J. A. Leary, L. W. Riley, et al.
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