Journal of Bacteriology, September 2004, p . 6050-6058, Vol . 186, No . 18

Targeted Mutagenesis of the Mycobacterium smegmatis mca Gene, Encoding a Mycothiol-Dependent Detoxification Protein

Mamta Rawat,^1,2 Mandeep Uppal,¹ Gerald Newton,³ Micah Steffek,³ Robert C . Fahey,³ and Yossef Av-Gay^1*

Division of Infectious Diseases, University of British Columbia, Vancouver, British Columbia, Canada,¹ Department of Biology, California State University, Fresno, Fresno,² Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California³

Received 9 April 2004/ Accepted 10 June 2004

 
Mycothiol [MSH], a functional analogue of glutathione [GSH]that is found exclusively in actinomycetes, reacts with electrophilesand toxins to form MSH-toxin conjugates . Mycothiol S-conjugate amidase [Mca] then catalyzes the hydrolysis of an amide bondin the S conjugates, producing a mercapturic acid of the toxin,which is excreted from the bacterium, and glucosaminyl inositol,which is recycled back to MSH . In this study, we have generatedand characterized an allelic exchange mutant of the mca geneof Mycobacterium smegmatis . The mca mutant accumulates the S conjugates of the thiol-specific alkylating agent monobromobimane and the antibiotic rifamycin S . Introduction of M . tuberculosis mca epichromosomally or introduction of M . smegmatis mca integrativelyresulted in complementation of Mca activity and reduced levelsof S conjugates . The mutation in mca renders the mutant strainmore susceptible to electrophilic toxins, such as N-ethylmalemide, iodoacetamide, and chlorodinitrobenzene, and to several oxidants, such as menadione and plumbagin . Additionally we have shown that the mca mutant is also more susceptible to the antituberculous antibiotic streptomycin . Mutants disrupted in genes belonging to MSH biosynthesis are also more susceptible to streptomycin, providing further evidence that Mca detoxifies streptomycinin the mycobacterial cell in an MSH-dependent manner.

 
After AIDS, tuberculosis [TB], caused by Mycobacterium tuberculosis, is the second leading cause of death from infectious agents worldwide [26] . The resurgence of TB as a potential publichealththreat due to its synergy with human immunodeficiency virusand the emergence of multidrug-resistant TB strains has resultedin renewed interest in this gram-positive actinomycete . M . tuberculosisis a facultative intracellular pathogen residing in macrophagesand in the granuloma, where it is faced with constant assaultfrom toxic agents . Survival in such hostile environments is dependent on detoxification mechanisms that allow M . tuberculosis to persist in the host despite the host immune response.

Detoxification processes rely on either phase I or phase II detoxification reactions, or both . In phase I reactions, cytochrome P450, a mixed-function oxidase, catalyzes the incorporationof an oxygen atom from O₂ into a xenobiotic substrate, making the toxin more hydrophilic . If the product of the cytochrome P450-mediated reaction is not sufficiently hydrophilic but isthiol reactive, a subsequent reaction where the toxin is conjugatedto glutathione [GSH] occurs, an important process in the mammalian liver . In mammals, GS toxin conjugates are acted upon by a -glutamyl transpeptidase that cleaves the glutamic acid group from the GSH molecule followed by hydrolysis of the glycine moiety bya peptidase resulting in a cysteine toxin conjugate in a multiorgan process . This conjugate is acetylated in the kidney by an acetyl-coenzyme A-dependent N-acetyltransferase to form a mercapturic acid that is excreted [8].

Actinomycetes like M . tuberculosis do not produce GSH but instead synthesize a novel low-molecular-weight thiol, mycothiol [MSH] [13], a conjugate of N-acetyl-L-cysteine amide linked to thepsuedodisaccharide comprised of D-glucosamine [1-1] linkedto myo-inositol . Mycobacterium smegmatis mutants lacking MSHare more sensitive to oxidizing agents, electrophiles, and severalantibiotics [18, 21, 22], indicating that MSH-dependent detoxificationmechanisms exist in the mycobacterial cell . Indeed, a novelMSH-dependent detoxification mechanism where MSH reacts with a toxin, such as the fluorescent alkylating agent monobromobimane [mBBr], to form an MS toxin conjugate [designated MS-mB] which is acted upon by a mycothiol S-conjugate amidase [Mca] to yield a mercapturic acid [AcCyS-mB] and 1D-myo-inosityl 2-amino-2-deoxy-glucopyranoside[GlcN-Ins] [Fig . 1] was described by Newton et al . [14] . The substrates for this enzyme include the S conjugates of mBBr, iodoacetamide, and N-ethylmalemide [NEM] . Interestingly, one of the substrates for this enzyme is the S conjugate of cerulenin, an antibiotic from the actinomycete Cephalosporin ceruleans. Recently, the mycothiol S conjugate of rifamycin S, from which the semisynthetic antituberculous drug rifampin is derived,was demonstrated to be a substrate for Mca [25] . Maynes et al. [11] recently published the crystal structure of MshB, a homologof Mca that catalyzes the second step of MSH biosynthesis, cleavageof the amide bond in GlcNAc-Ins . They modeled the catalytic domain of Mca from this crystal structure and found that amino acid residues involved in binding the active-site zinc and in catalysis aligned perfectly, with the exception of Lys19 inMca in place of Ser20 in MshB.

 
A search of sequence databases revealed that homologs of Mcaare present in all other mycobacteria . Homologs of this genehave also been reported in several antibiotic biosynthetic operons,including the erythromycin biosynthetic operon . Furthermore,mercapturic acids of antibiotics have been found in the brothof some antibiotic-producing species [16] . For these reasons, we have postulated that this enzyme, in conjunction with mycothiol, may play a major role in detoxification of antibiotics . To better understand the role of Mca and MSH in xenobiotic detoxification, a targeted mutant of the mca gene in the saprophytic mycobacteria M . smegmatis was constructed . In this study we describe the generation and characterization of this M . smegmatis mca mutant.

 
Bacterial strains and culture conditions. Escherichia coli strain DH5 was used as the host strain forcloning experiments . E . coli was grown in Luria-Bertani brothand on Luria-Bertani solid media . M . smegmatis mc²155 was theparent wild-type strain used for construction of knockout mutants.M . smegmatis was grown in Middlebrook 7H9 broth [Difco] with0.05% Tween and was supplemented with either Middlebrook oleic acid-albumin-dextrose-catalase [OADC] supplement or 1% glucose.M . smegmatis was also grown on Middlebrook 7H10 solid medium[Difco] supplemented with OADC or 1% glucose . Ampicillin [100µg ml^–1 for E . coli], gentamicin [15 µg ml^–1 for E . coli and 10 µg ml^–1 for M . smegmatis], kanamycin[100 µg ml^–1 for E . coli and 25 µg ml^–1 for M . smegmatis], and hygromycin [100 µg ml^–1 forE . coli and 50 µg ml^–1 for M . smegmatis] were addedas needed . Complements of the mutant harboring the recombinantpALACE vector were grown on Middlebrook 7H10 solid medium supplementedwith OADC and 1% acetamide for induction of the cloned gene.

Molecular biology techniques. Genomic DNA was isolated from M . smegmatis cultures as describedby Hatfull and Jacobs [7]. M . smegmatis transformations werecarried out using a Bio-Rad Gene Pulser with mycobacterial cellsprepared as described by Snapper et al . [24] . Standard recombinantDNA techniques, such as restriction digestion, ligation, andtransformation, were carried out as described by Sambrook etal . [23] . Probes for Southern blotting were labeled with digoxigenin [DIG]-labeled deoxynucleoside triphosphates by using a RocheDIG labeling kit, and membranes were developed according tothe manufacturer's instructions [Roche Diagnostics] . The listof strains, plasmids, and oligonucleotides used in this studyare described in Table 1.

 
Bioinformatic analyses. The genomic sequence and the predicted genes were obtained forM . tuberculosis H37Rv [4] from the Tuberculist website [http://www.genolist.pasteur.fr/Tuberculist]. Sequence data for M . smegmatis were obtained from the Institute for Genomic Research [http://www.tigr.org] . BLAST searches wereperformed according to the methods of Altschul et al . [1] . PFAMdatabase information was obtained from http://www.sanger.ac.uk/Software/Pfam/,and PROSITE database information was obtained at http://www.expasy.ch/prosite.

Targeted mutagenesis of mca. A recombination cassette was constructed in order to disruptmca on the M . smegmatis chromosome . The M . smegmatis mca genefragment, spanning from 300 bp upstream to 40 bp downstream,was PCR amplified using primers Pgint1082I-5' and Pgint1082I-3',listed in Table 1 . The amplified gene was cloned into PCR cloningvector pCR 2.1 to yield pMK1082 . pMK1082 was digested with PmaCI,because there is a unique restriction site for this enzyme inthe middle of the M . smegmatis mca gene . A gentamicin resistancecassette was prepared by restriction digestion of pGINT vectorwith SacI . The appropriate SacI fragment containing the gentamicinresistance gene was agarose gel purified and treated with Klenowfragment of DNA polymerase to yield blunt ends . This fragmentwas ligated with pMK1082 restriction digested with PmaCI suchthat the resulting plasmid, pMR1082K, contained the gentamicinresistance gene disrupting the mca open reading frame . pMR1082Kalso has a kanamycin resistance marker in the pCR2.1 vectorbackbone that provides for negative selection. M . smegmatismc²155 was transformed with pMR1082K, and gentamicin-resistant,kanamycin-sensitive transformants were chosen for further analysis.In addition, M . smegmatis mc²155 was transformed with emptyvector pGINT to serve as a control for further characterizationstudies.

Sensitivity assays for antibiotics, alkylating agents, and oxidative agents. Three different methods were used for sensitivity assays . E-teststrips [AB Biodisk] were used to determine the MICs of isoniazid,rifampin, and vancomycin for the different strains [21] . Diskdiffusion assays were performed for vancomycin, cerulenin, lincomycin,erythromycin, rifampin, and streptomycin according to Rawatet al . [22] . In addition, the following alkylating agents andoxidants were tested: NEM, iodoacetamide, chlorodinitrobenzene[CDNB], mBBr, menadione, plumbagin, nitrofurantoin, cumene hydroperoxide,and hydrogen peroxide . For the preceding two methods, 7H10 solidmedia supplemented with 1% glucose were used, and these plateswere incubated for 48 to 72 h . The third method was the minimalbroth dilution assay, where the toxin or drug was serially dilutedin 1 ml of 7H9 medium supplemented with 1% glucose and cellswere added to each tube in the serial dilution, with an opticaldensity at 600 nm [OD₆₀₀] of 0.05 . After 2 to 3 days of incubationat 37°C the tubes were checked visually for growth . Allassays were performed in triplicate at least three times.

Determination of mycothiol levels and mycothiol amidase activity. Derivatization of cell extracts with mBBr and high-performanceliquid chromatography [HPLC] analysis of the derivatized samplesto determine the thiol content were performed essentially asdescribed earlier [2] . Control samples treated with NEM and then with mBBr were also analyzed . Results are reported as micromoles per gram of residual dry weight measured on the pellet obtainedfrom the 50% acetonitrile-water extraction.

Mca activity was tested essentially as described by Newton etal . [14] . Briefly, samples to be tested were pelleted and brought up in 200 µl of 25 mM HEPES, pH 7.5 . The cells were sonicated and centrifuged for 3 min at 14,000 x g . The cell extract proteinconcentration was measured using the Bio-Rad protein assay.A reaction volume of 50 µl consisted of 100 µg of protein sample in 25 mM HEPES [pH 7.5], 30 µM mycothiol-mBBr adduct [MS-mB], and 3 mM 2-mercaptoethanol . The samples were incubated for 30 min at 30°C, and then the reaction wasstopped by adding 50 µl of 40 mM methanesulfonic acid.The samples were subjected to HPLC analysis as described earlier[14].

Analysis of M . smegmatis mc²155 and Ami37 [mca::gent^r] treated with mBBr and rifamycin S in culture. Duplicate samples of 10 ml of late-log-phase M . smegmatis mc²155cells and Ami37, the mca knockout mutant, were incubated onice for 20 min . To the iced cells, 180 mM mBBr in acetonitrilewas added to a final concentration of 0.5 mM mBBr, and the cellswere incubated on ice for an additional 30 min . Excess 2-mercaptoethanol[1.0 mM] was added to scavenge unreacted mBBr and was allowedto react with the cells for 10 min on ice . The cells were thenharvested by centrifugation at 4°C, and the supernatantwas retained for analysis of thiol-mB derivatives in the medium.The cells were extracted with 50% acetonitrile in water . Afterincubation at 60°C for 10 min, the cells were acidifiedand centrifuged to remove cell debris . The supernatant was analyzedafter dilution with 10 mM methanesulfonic acid using the sameconditions described above for the MSH assay.

For rifamycin S treatment, four liters of M . smegmatis mc²155 and Ami37 was cultured to an OD₆₀₀ of 1 . The cells were then harvested and placed in 200 ml of ice-cold 7H9 Middlebrook medium containing 1% glucose . The cells were incubated with 1 mg of rifamycin S/ml for 1 h on ice and then were washed two timeswith ice-cold medium and resuspended in 20 ml of ice-cold medium.The cells were then diluted into 180 ml of 37°C medium andsampled at 1 min, 10 min, 60 min, 2 h, and 16 h . As describedby Steffek et al . [25], the cells were extracted and analyzedfor rifamycin S conjugates . Cellular rifamycin S conjugateswere quantified by HPLC at 315 nm, an isosbestic wavelengthfor the rifamycin S and rifamycin SV [RifSV] forms, using anapproximate extinction coefficient of 24,000 M^–1 cm^–1 and MS-RifSV standards.

Complementation of Ami37 with M . tuberculosis mca and M . smegmatis mca homolog. The M . tuberculosis mca gene was amplified from M . tuberculosisgenomic DNA and cloned into a replicative vector, pALACE, asdescribed earlier [22] to yield pM4 . The mca mutant was transformedwith pM4, and transformants were selected on 7H10 agar platessupplemented with OADC, 50 µg of hygromycin ml^–1,and 10 µg of gentamicin ml^–1 . To ascertain thatthe transformants contained the plasmid, the plasmids were rescued.Protein expression was checked on sodium dodecyl sulfate-polyacrylamidegel electrophoresis gels after induction of protein expressionby growth in 1% acetamide . One transformant, ami37palace, wasselected for further detailed study.

For complementation with the M . smegmatis mca gene, the pMK1082 plasmid described earlier and the pHINT vector were restriction digested with HindIII . The pHINT fragment was dephosphorylatedand then ligated to the pMK1082 fragment to create pHINTMK1082.Ami37 was transformed with pHINTMK1082, and the cells were selectedon 7H10 agar plates supplemented with OADC, 50 µg of hygromycinml^–1, and 10 µg of gentamicin ml^–1 . One transformant, ami37phint, was selected for further detailed study.

 
Bioinformatic analyses. M . tuberculosis mca is an 864-bp gene that codes for a proteinof 288 amino acids that has a molecular mass of 32.7 kDa anda pI of 5.1 . M . smegmatis mca is also an 864-bp gene that codesfor a 288-amino-acid protein . Fasta analysis of the two proteinsrevealed 78% similarity between 227 out of 288 of the aminoacids . Both genes have a PF02585 PFAM signature, which has beendescribed as a lmbE-like protein signature . The lmbE gene fromStreptomyces lincolnensis is a gene of unknown function presentin the lincomycin biosynthesis cluster [19].

Homologs of mca are present in all mycobacterial species sequenced so far, and homologs are also found in other antibiotic biosynthesis clusters, such as leinamycin biosynthesis genes of Streptomyces atroolivaceus, rifamycin biosynthesis genes of Amycolatopsis mediterranei, erythromycin biosynthesis genes of Streptomyces erythrae, and streptothricin biosynthesis genes of Streptomyces rochei . Three homologs are found in Streptomyces coelicolor, and two homologs are found in Streptomyces avermitilis . The M . tuberculosis genome also has two paralogs, Rv1170, which codes for MshB [3, 11, 15, 22], and Rv0323c, a gene that is present only in pathogenic mycobacteria and thus is absent in nonpathogenic M . smegmatis.

The mca gene is the first gene in an operon of three genes in M . tuberculosis . The second gene in the operon, Rv1083, 88 bpin length, encodes a 9.2-kDa protein of unknown function . Rv1083 has homology to the gene ML2390 [57% similarity in 59 out of 101 of the amino acids] from M . leprae . ML2390 has an N-terminal signal sequence and is thus possibly secreted . The last genein the operon, Rv1084, 673 bp in length, encodes a 71-kDa protein of unknown function . Interestingly, the Rv1084 gene containsa carboxypeptidase zinc binding domain [Prosite entry PS00133]and a thioredoxin conserved domain, COG1331 . The organizationof the operon in M . smegmatis is the same as that of M . tuberculosis.

Ami37 is a knockout mutant of mca. Genomic DNA, extracted from both the parental strain and putativemutants, was digested with BglII and was subjected to Southernhybridization using a DIG-labeled probe [Fig . 2B] . The probehybridized to a BglII fragment of approximately 6 kb in theparent strain [Fig. 2B, lane 3]; however, disruption of mcaby the gentamicin cassette introduced a second BglII site inthe mutant DNA [Fig . 2A] . Restriction digestion with BglII should result in two bands, one 2 kb in size and another approximately 5 kb in size . Two of the putative mutants, Ami37 [Fig . 2B, lane2] and Ami40 [Fig . 2B, lane 1], showed two bands of 2 and 5kb, indicating that the mca gene has been disrupted by the gentamicinresistance gene in these two clones . Mutant Ami37 was shownto have a MSH content equivalent to the parental strain [10± 2 µmol/g] and was chosen for further analysis.

 
Mutant Ami37 does not possess Mca activity. The mutant Ami37 was compared to the wild-type parental strainin terms of its ability to detoxify mBBr and to secrete mercapturicacids essentially as described previously [14] . Extracts ofthe mutant lack amidase activity with MS-mB [<0.0015 nmol/min/mg]and therefore do not produce the mercapturic acid product AcCys-mB.Under the same conditions, extracts of the parental strain exhibithigh activity with MS-mB [25 nmol/min/mg] . Thus, amidase activitytoward MS-mB is effectively eliminated in Ami37.

A close homolog of mca is mshB, which codes for MshB deacetylase,one of the enzymes involved in the biosynthesis of mycothiol[15] . This enzyme is known to possess Mca activity as well asGlcNAc-Ins deacetylase activity, albeit at a much lower levelthan that of Mca . To examine whether MshB activity is upregulatedin the mutant to compensate for loss of Mca, we assayed theparent and mutant strains for MshB amidase activity . The mBBradduct of Cys-GlcN-Ins [CySmB-GlcN-Ins] is known to be a very poor substrate for Mca [25] but is still the best known amidasesubstrate for MshB [G . Newton and R . C . Fahey, unpublished data].Assay of cellular extracts with CySmB-GlcN-Ins [0.1 mM] gave specific activities of 0.12 and 0.16 nmol min^–1 mg^–l for the parent and mutant strains, respectively . This indicatesthat only a small increase in MshB activity is observed in themca mutant and cannot significantly compensate for the lossof Mca activity.

Analysis of M . smegmatis mc²155 and Ami37 treated with mBBr in culture. Following an experiment which examined the fate of mycothioland mBBr in M . smegmatis cells treated with mBBr [14], we soughtto determine whether the mutant exhibited a phenotype in culturedifferent from that of the parent wild-type strain . When theparent strain mc²155 was treated with 0.5 mM mBBr in vivo for30 min on ice, both MS-mB and the product of the Mca reaction[AcCyS-mB] were found within the cells at a ratio of approximately1:3 . AcCyS-mB was present in the media at low levels [5% ofthe total intracellular content] [Table 2] . In contrast, inAmi37 only the conjugate MS-mB was detected intracellularly.There was also no trace of AcCys-SmB in media for the mutant,confirming that the mutant is unable to cleave the MS-mB conjugate[Table 2].

 
Analysis of M . smegmatis mc²155 and Ami37 treated with rifamycin S in culture. Steffek et al . [25] demonstrated that MSH could form a conjugatewith rifamycin S that served as a substrate for Mca . When M.smegmatis mc²155 was treated with rifamycin S on ice for 1 h,the cells washed with cold medium, and a cold suspension ofthe cells diluted into warm medium, initial analysis showedthat approximately 30% of the normal mycothiol content was presentas the MS-RifSV conjugate [Fig. 3] . MS-RifSV declined threeorders of magnitude to the limit of detection at 16 h [Fig.3] . The Ami37 mutant had a lesser amount of MS-RifSV initially,and it declined only threefold over 16 h . In the parent strain,the S conjugate of N-acetylcysteine with rifamycin S [AcCyS-RifSV], a mercapturic acid, was evident in the cells, demonstratingthe activity of the endogenous amidase on the MS-RifSV conjugate.No AcCyS-RifSV was found above the limit of detection [<0.02µmol/g] in Ami37, consistent with the disruption of themca gene . The loss of MS-RifSV from mc²155 could not be accountedfor in terms of products excreted into the medium, as analysisof the medium revealed maximal levels of AcCyS-RifSV and MS-RifSVequivalent to only 4 and 30%, respectively, of the MS-RifSVlost from the cells . Other components with similar UV absorptionwere noted in the HPLC analysis of both cells and medium andmay represent alternative metabolites . Ribosylation at the C-23hydroxyl has been identified as a mechanism for resistance torifampin in M . smegmatis [5, 20] and might also occur with rifamycinS, MS-RifSV, or AcCyS-RifSV.

 
Complementation of Ami37 with M . tuberculosis and M . smegmatis mca restores Mca activity. To confirm that the decrease in mycothiol was caused by thedisruption in mca, mca was reintroduced into Ami37 . The amidaseactivity for 100 µg of cell extracts was determined usingMS-mB as a substrate . In Fig. 4A, most of the MS-mB has beenconverted to the mercapturic acid AcCyS-mB in the parent strain,while in Ami37 cell extracts none of the substrate has beenconverted to mercapturic acid AcCyS-mB [Fig . 4B] . Introductionof M . tuberculosis mca epichromosomally or introduction of M. smegmatis mca integratively resulted in the complementationof the activity as both proteins are able to catalyze the conversionof MS-mB to AcCyS-mB [Fig . 4C and D, respectively], confirming that the M . tuberculosis and M . smegmatis proteins are functionallythe same.

 
Antibiotic sensitivity of Ami37. It has been demonstrated that Mca can react with a broad rangeof mycothiol S conjugates, including two different classes ofantibiotics, exemplified by cerulenin [14] and rifamycin S [25]. Both adducts are cleaved to produce a mercapturic acid and GlcN-Ins. Consequently, Ami37 was tested for sensitivity to ceruleninand rifampin, a derivative of rifamycin S that is used as an antituberculous drug . In the cerulenin and rifampin disk diffusion assay there was no difference in clearing between Ami37 andmc²155gint [data not shown] . For cerulenin, this result wasconfirmed with the minimal broth dilution assay where therewas no difference in the MIC between the two strains [Table3], indicating that the Mca-dependent detoxification pathwaydoes not play a major role in the detoxification of cerulenin,although cerulenin can form an S conjugate with mycothiol . TheMIC of rifampin for mc²155gint was fourfold higher than thatfor mc²155, as determined by both the minimal broth dilutionassay and E-test [Table 3].

 
Because Mca homologs have been identified in the antibiotic biosynthesis operons of S . erythrae [erythromycin biosynthesis] and S . lincolnensis [lincomycin biosynthesis] [16], the sensitivitiesof Ami37 and the parent strain to erythromycin and lincomycinwere also checked . In disk diffusion assays no significant differencewas found in the zone of clearing for erythromycin and lincomycinbetween Ami37 and mc²155gint [data not shown] . When minimalbroth dilution assay was performed, there was still no differencein the MIC of lincomycin for Ami37 and mc²155gint . In fact,both Ami37 and mc²155gint were able to grow in 90 µg oflincomycin/ml [Table 3] . The MIC of erythromycin was perhapstwofold higher for the parent strain as determined by the minimalbroth dilution assay and was sixfold higher as determined bythe E-test.

Recent studies have reported that mutants deficient in mycothiol are more sensitive to vancomycin and are more resistant to isoniazid [3, 10, 17, 18, 21] . To determine whether this sensitivity isalso dependent on Mca, the sensitivity of Ami37 to these twoantibiotics was checked by disk diffusion assay and E-test, followed by minimal broth dilution assay for vancomycin only. No difference in sensitivity was detected with the disk diffusion assay for vancomycin [data not shown], although a twofold difference in vancomycin MIC values between Ami37 and mc²155gint was detected in the E-test and minimal broth dilution assays [Table 3] . Aswith erythromycin, the MICs determined by minimal broth dilution assay were considerably higher than those of the E-test . For isoniazid there was no difference in zone of inhibition [datanot shown] or the MIC, indicating that Mca plays no role inthe resistance of mutants lacking mycothiol to isoniazid [Table 3].

Sensitivity of Ami37 and mc²155gint for a whole range of other antibiotics was checked by disk diffusion assays [data not shown]. Sensitivity to streptomycin, a second-line drug for M . tuberculosis treatment, was shown to increase in Ami37 . Complementation of the mutant with M . smegmatis mca and M . tuberculosis mca [Table 4] restored the parental strain sensitivity to streptomycin.The observed MIC of streptomycin was 10-fold higher for mc²155gintthan for Ami37 [Table 3] . As sensitivity to streptomycin formutants lacking MSH has not been previously reported and asMSH is a crucial part of the amidase-mediated detoxificationreaction, mutants in all steps of the mycothiol biosyntheticpathway were tested for sensitivity to streptomycin . As seenin Table 5, mutants disrupted in mycothiol biosynthetic geneswith the exception of the mshB mutant are also more sensitiveto streptomycin . The lack of difference in sensitivity betweenthe mshB mutant and the parent strain is not unexpected, becausewe have previously reported that the M . smegmatis mshB mutanthas 10% of the mycothiol content of the parent strain and isnot as sensitive as the other mutants to oxidants, toxins, andantibiotics [22].

 
Sensitivity of mca mutant and complement to toxins and oxidants. It was shown previously that sensitivity to iodoacetamide isMSH dependent, as all mutants in MSH biosynthesis are more sensitivethan the parent strain to iodoacetamide [21] . It has also been previously shown that mycothiol reacts with iodoacetamide to form a conjugate that is a substrate for Mca [14] . The relativeactivity of Mca for MS-acetamide compared to that of the MS-mBadduct is only 0.5% . Nevertheless, Ami37 is more sensitive than mc²155gint to iodoacetamide as determined by the disk inhibitionassay . Furthermore, this sensitivity is reversible upon complementation[Table 4] . Similarly, Ami37 is more sensitive to NEM [Table6], the MSH-NEM adduct being another substrate of Mca . The relativeactivity for MS-NEM is 2.1% compared to that of MS-mB, whichis greater than that of iodoacetamide but still substantiallyless than that reported for cerulenin . Ami37 is also more sensitiveto CDNB, a common substrate for GSH transferases, than mc²155gint[Table 6] . In contrast, there is no significant difference in sensitivity to mBBr, the substrate for which the highest amidase activity has been reported, as determined by the disk inhibition assay . Even in the minimal broth dilution assay, the mBBr MICfor Ami37 is 0.0063 µmol/ml, twofold less than 0.0125µmol/ml, the mBBr MIC for mc²155gint.

 
It was previously shown that mutants lacking mycothiol are more sensitive to redox cycling agents, menadione, plumbagin, and nitrofurantoin [21] . To investigate the role of Mca in the detoxificationof oxidants, sensitivity to oxidants was determined by the diskinhibition assay . In Table 5 it can be seen that Ami37 is moresensitive than mc²155gint to plumbagin and menadione . Such compoundsare known to form conjugates with thiols [6] . In contrast, thereis no difference in sensitivity to other oxidative stress inducers,such as hydrogen peroxide, cumene hydrogen peroxide, and nitrofurantoin [Table 6], that do not form conjugates with mycothiol.

 
The mca gene codes for an Mca that plays a key role in detoxification of electrophiles . Mca is a zinc metalloenzyme [25] and has beenshown to react with the mycothiol S conjugates of alkylatingagents and the antibiotics cerulenin and rifamycin S [14, 25].In this reaction, mycothiol reacts with toxins to form conjugatessimilar to glutathione toxin conjugates and the amidase cleavesan amide bond in the mycothiol moiety of the conjugate to releasea mercapturic acid and GlcN-Ins . In this report, we describethe generation and characterization of an mca-specific mutant,Ami37, disrupted in the mca gene of M . smegmatis . We show thatthe mca gene, either from M . smegmatis or M . tuberculosis, isable to restore the wild-type phenotype associated with themutation in M . smegmatis.

Mercapturic acids, products of the Mca-catalyzed reaction, have been found in fermentation broths of actinomycetes, such as granaticin A metabolite, Ws009A, seongomycin, cysfluoretin,and phenoxazinone metabolite [16] . It was also previously demonstratedthat mutants in the mycothiol biosynthetic pathway are moresensitive to antibiotics, toxins, and oxidants . This sensitivity of mutants lacking mycothiol may depend in part on the mycothiol-dependent amidase-catalyzed detoxification reaction [Fig . 1] . We consideredfirst the role of Mca in detoxification of antibiotics . If theamidase plays a role in the detoxification of an antibiotic, then the mca mutant should be more sensitive to this antibiotic.

We demonstrated that the mca mutant has enhanced sensitivity to streptomycin, a second-line drug employed in treatment for M . tuberculosis, as demonstrated by the increase in the zone of inhibition for Ami37 [Table 4] and the 10-fold decrease in the MIC for Ami37 compared to that for the control strain [Table 3] . Moreover, the complements ami37palace, where the M . tuberculosismca is overexpressed, and ami37phint, with the M . smegmatismca, exhibited reversion to wild-type levels of sensitivityto streptomycin . Furthermore, the mutants disrupted in the mycothiolbiosynthetic pathway, with the exception of the mshB mutant,are also more susceptible to streptomycin, as would be expectedif the detoxification was dependent on Mca activity [Table 5].Examination of the structure of streptomycin reveals the presenceof a thiol-reactive aldehyde that likely reacts with MSH toform a thiohemiacetal adduct . This reaction is reversible, andhow it could be involved in detoxification is not immediatelyobvious . Other metabolic modifications of the streptomycin moietymay be involved.

The sensitivity to several antibiotics in addition to streptomycin appears to depend upon Mca . It was previously reported thatmutants lacking mycothiol are sensitive to vancomycin, erythromycin,and rifampin [21] . Additionally, in the case of rifampin, the S conjugate of rifamycin, the parent antibiotic from which rifampin is derived, has been demonstrated to be a substrate for Mca[25] . No differences were seen in the zone of inhibition inthe disk diffusion assay for any of these antibiotics; thus,the MICs were checked by E-strips and by minimal broth dilutionassay for all these antibiotics . The MICs of rifampin, erythromycin,and vancomycin were fourfold higher, less than two fold higher,and twofold higher, respectively, for mc²155 than for Ami37[Table 3] . Thus, Mca cleavage of a MSH conjugate of these antibiotics may be important in the overall detoxification, but elucidation of the detailed chemistry involved will require further studies.

Examination of the M . smegmatis parent strain and mutant Ami37 treated with rifamycin S showed that a substantial quantityof the cellular MSH is converted to the MS-RifSV conjugate.In the parent strain, some of this is released to the mediumand some is cleaved by Mca to produce AcCyS-RifSV, which isalso found in the medium [Fig . 3 and 5] . However, a significantfraction of the cellular MS-RifSV conjugate produced in thecell subsequently disappeared and could not be accounted for in terms of cellular AcCyS-RifSV or identified secreted forms. HPLC analysis indicated that significant levels of additional rifamycin derivatives are produced in the cells and are releasedinto the medium [Fig . 5] . Prime candidates would be ribosylated forms of rifamycin S or its MSH-derived metabolites, because ribosylation is an established pathway for inactivation of the related antibiotic rifampin [5, 20] . Further studies are neededto elaborate the pathways involved.

 
Other antibiotics are not dependent upon Mca . Lincomycin alsohas a gene homologous to mca in its antibiotic biosynthesiscluster; however, Ami37 and mc²155gint were able to surviveand grow in 90 µg of lincomycin/ml [Table 3] . In addition, cerulenin, another antibiotic which forms an S conjugate with MSH that can serve as a substrate for Mca [14], produced nodifference in sensitivity as measured by the disk diffusionassay or the MIC as measured by the minimal broth dilution assaybetween Ami37 and mc²155gint [Table 3] . The lack of sensitivityof Ami37 to cerulenin and lincomycin suggests that formationof the mycothiol S conjugate suffices to detoxify this compoundand that accumulation of the S conjugate within the cell hadlittle or no adverse consequence.

Electrophiles severely damage biological molecules, such asDNA bases and protein sulfhydryl groups . The mutant disruptedin mca is more sensitive to NEM, iodoacetamide, and CDNB thanthe control strain [Table 5] . Because a number of natural products with structures related to the maleimide ring [e.g., maleimycin, showdomycin, and pencolide] are produced by bacteria and fungi, the MSH-dependent Mca detoxification system may protect thecell against electrophilic assault from such toxins . Moreover,in E . coli a GSH-dependent detoxification pathway has been described[12] where the GSH-NEM adduct produced in the cells serves asa substrate for an imidase which catalyzes the hydrolytic cleavageof the imide bond and converts the NEM to maleamic acid thatis secreted into the medium while the GSH is recycled . In contrast,in E . coli the GSH-CDNB adduct is excreted and the GSH failsto be recovered [9].

The results with the alkylating agent mBBr were unexpected.No difference in sensitivity was measured by disk diffusionassay [Table 6], and only a twofold decrease in the MIC of mBBr for Ami37 was determined [data not shown] . The mBBr adduct,MS-mB, is the best substrate known for the Mca enzyme . Indeed,even when M . smegmatis was incubated on ice with mBBr, the conjugateMS-mB formed spontaneously and the amide bond in this conjugatewas easily cleaved to yield AcCyS-mB, such that AcCyS-mB represented78% of the total of MS-mB and AcCyS-mB within the cell [Table2] . Moreover, the cells had already started to excrete AcCyS-mBinto the medium [Table 2] . Thus, while the MSH-Mca system is clearly involved in eliminating bimane from the cell, mycothiol itself and not Mca is the essential moiety in detoxifying mBBr.

Oxidants, such as plumbagin, a napthoquinone, and menadione,2-methyl-1,4-naphthoquinone, can rapidly form conjugates withthiols, such as mycothiol . Indeed, Zadzinski et al . [27] demonstratedthat incubation with 0.5 mM menadione results in a decreasein GSH concentration in yeast cells, as GS-menadione conjugatesare formed and exported . In a similar manner, menadione andplumbagin may form MS-menadione and MS-plumbagin conjugatesin mycobacteria that are cleaved by Mca to form mercapturicacids that are exported . Indeed, the disruption of mca resultsin an increase in sensitivity to these oxidants, as does thelack of mycothiol in mutants disrupted in the mycothiol biosyntheticpathway [21] . In contrast to plumbagin and menadione, Ami37is not sensitive to other oxidants, such as hydrogen peroxide,cumene hydrogen peroxide, and nitrofurantoin [Table 6], althoughsensitivity is exhibited by mutants lacking mycothiol [21].Thus, oxidants that can form stable S conjugates, such as plumbagin and menadione, may require Mca for detoxification, while the superoxide that they generate as redox cycling agents may be detoxified by other mycothiol-dependent or antioxidant mechanisms.

In conclusion, we have shown that an M . smegmatis Mca mutant is susceptible to several antibiotics, streptomycin in particular. The mutant is also more susceptible to electrophiles and oxidants that react with mycothiol directly . Thus, these findings support a major role for mycothiol and Mca in detoxification . The isolation of mercapturic acids and mycothiol adducts of these antibiotics and toxins will solidify the significance of this detoxification mechanism in mycobacteria.

 
This work was supported by grants to Y.A . from the TB Veterans Association and the Canadian Institute of Health Research, Institute of Infection and Immunity, MOP 64950, and grant MCB-0235705to R.C.F . from the National Science Foundation . Y.A . is a CanadianInstitute of Health Research and British Columbia Lung AssociationScholar.

We thank Mary Ko, Rayken Chow, and Teresa Koledin for technical assistance and the Institute for Genomic Research for providing access to M . smegmatis sequence data.

 
* Corresponding author . Mailing address: Department of Medicine, Division of Infectious Diseases, University of British Columbia, Vancouver, British Columbia V5Z 3J5, Canada . Phone: [604] 875-4329 . Fax: [604] 875-4013 . E-mail: yossi@interchange.ubc.ca .

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