Identification and characterization of the regulatory elements of the inducible acetamidase operon from Mycobacterium smegmatis

Identification and characterization of the regulatory elements of the inducible acetamidase operon from Mycobacterium smegmatis Selvakumar Subbian and Sujatha Narayanan Abstract: The highly inducible acetamidase promoter from Mycobacterium smegmatis has been used as a tool in the study of mycobacterial genetics. The 4.2 kb acetamidase operon contains four putative open reading frames (ORFs) (amiC, amiA, amiD, and amiS) upstream of the 1.2 kb acetamidase ORF (amiE). In this article, using electrophoretic mobility shift assay and promoter probe analyses with a lacZ reporter system, we show the position of three putative operators within the acetamidase operon in M. smegmatis. Results from these studies reinforce previous findings about the involvement of multiple promoters in the regulation of acetamidase gene expression. Each of the identified operators are positioned up- stream of the respective promoter reported in previous studies. We also found that the crude cell lysate of M. smegmatis containing potential regulators, obtained from bacteria grown under inducing or noninducing conditions, binds to specific operators. The binding affinity of each operator with its cognate regulator is significantly different from the other. This supports not only the previous model of acetamidase gene regulation in M. smegmatis but also explains the role of these operators in controlling the expression of respective promoters under different growth conditions. Key words: gene regulation, acetamidase operon, Mycobacterium smegmatis promoter, electrophoretic mobility shift assay. Re´sume´ : Le promoteur hautement inductible de l’ace´tamidase de Mycobacterium smegmatis a e´te´ utilise´ comme outil dans une e´tude de la ge´ne´tique mycobacte´rienne. L’ope´ron de 4,2 kb de l’ace´tamidase contient quatre cadres de lecture ou- verts possibles (amiC, amiA, amiD et amiS) en amont d’un cadre de lecture ouvert de 1,2 kb (amiE). Dans cet article, graˆce a` des analyses de re´tention sur gel d’e´lectrophore`se et des analyses du promoteur dans un syste`me lacZ, nous avons situe´ trois ope´rateurs potentiels au sein de l’ope´ron de l’ace´tamidase chez M. smegmatis. Les re´sultats de ces e´tudes ap- puient nos re´sultats pre´ce´dents sur l’implication de multiples promoteurs dans la re´gulation de l’expression du ge`ne de l’ace´tamidase. Chacun des ope´rateurs identifie´s est situe´ en amont de son promoteur respectif identifie´ lors d’e´tudes pre´ce´- dentes. Nous avons aussi trouve´ qu’un extrait cellulaire brut de M. smegmatis contenant des re´gulateurs potentiels, obtenus de bacte´ries cultive´es sous des conditions inductibles et non-inductibles, lie les ope´rateurs spe´cifiques. L’affinite´ de la liai- son de chaque ope´rateur avec son re´gulateur propre diffe`re d’un a` l’autre. Ceci appuie non seulement le mode`le pre´ce´dent de la re´gulation de l’expression du ge`ne de l’ace´tamidase chez M. smegmatis mais explique aussi le roˆle de ces ope´rateurs dans le controˆle de l’expression ge´nique par ces promoteurs respectifs sous diffe´rentes conditions de croissance. Mots-cle´s : re´gulation ge´nique, ope´ron ace´tamidase, promoteur de Mycobacterium smegmatis, essai de re´tention sur gel d’e´lectrophore`se. [Traduit par la Re´daction] Introduction Amidase activity of Mycobacterium smegmatis has been shown to be inducible with distinct inducer and substrate specificities (Draper 1967). It has been reported that expres- sion of acetamidase, an aliphatic amidohydrolase, is induced more than 90-fold when the cells are grown in the presence of acetamide. However, in rich medium and (or) in the pres- ence of noninducing agents, such as succinate or glutamate, a low basal-level constitutive expression of acetamidase has been observed (Mahenthiralingam et al. 1993; Parish et al. 1997). The 1.2 kb acetamidase open reading frame (ORF) (amiE) and a 3 kb upstream sequence have been cloned and sequenced, and four putative ORFs (amiC, amiA, amiD, and amiS) have been identified upstream of amiE (Figs. 1A and 1B). The computed polypeptides for these ORFs have ho- mology with regulatory and transport proteins from other or- ganisms (Mahenthiralingam et al. 1993; Parish et al. 1997). The inducible nature of the acetamidase promoter from M. smegmatis has led to its use in the construction of novel expression vectors for mycobacteria (Parish and Stoker 1997; Triccas et al. 1998; Manabe et al. 1999; Dziadek et al. 2002). Previous studies have shown induction from the acetamidase promoter to occur at the transcriptional level, and a role for both positive and negative control elements Received 1 September 2006. Revision received 14 December 2006. Accepted 18 December 2006. Published on the NRC Research Press Web site at cjm.nrc.ca on 22 June 2007. S. Subbian1 and S. Narayanan.2 Department of Immunology, Tuberculosis Research Centre (ICMR), Mayor.V. Ramanathan Road, Chetput, Chennai 600 031, India. 1Present address: Department of Microbial and Molecular Pathogenesis, Reynolds Medical Building, Texas A&M University System Health Science Center, College Station, TX 77843, USA. 2Corresponding author (e-mail: [email protected]; [email protected]). 599 Can. J. Microbiol. 53: 599–606 (2007) doi:10.1139/W06-147 # 2007 NRC Canada has been suggested in the regulation of expression (Parish et al. 1997; Narayanan et al. 2000). In addition, a model was proposed involving promoters that were distinctly regulated under inducing and noninducing growth conditions. How- ever, experimental evidence of the presence of operators and their interaction with regulators in the regulation of acetamidase expression is lacking. Such studies are vital be- cause the interaction of regulatory factors with cognate oper- ators would explain the molecular mechanism involved in the differential expression of genes under changing environ- mental conditions. In this study, using the electrophoretic mobility shift assay (EMSA), we identified three putative operators (OP1, OP2, OP3) within the acetamidase operon and upstream of previ- ously reported promoters (Parish et al. 2001; Roberts et al. 2003). Whereas OP1 is engaged during the induction of the operon, OP2 is involved in suppression during noninducing conditions, and OP3 is involved in the basal constitutive ex- pression of the ORFs in the acetamidase operon. Specificity of the regulator–operator complexes were confirmed by cold-chase EMSA. Using a lacZ reporter system, we con- firmed the presence of promoters just downstream of OP1, OP2, and OP3. This finding complements previous observa- tions on the location and mode of action of various pro- moters within the acetamidase operon. Materials and methods Chemicals and enzymes All enzymes were purchased from Amersham Pharmacia Biotech (Piscataway, New Jersey) or New England Biolabs (Beverly, Massachusetts). All chemicals were purchased from Sigma-Aldrich (St. Louis, Missouri), unless otherwise noted. The gel shift assay system was obtained from Prom- ega (Madison, Wisconsin), and used in accordance with the manufacturer’s instructions. DNA was eluted from agarose gels using the GFX column purification kit from Amersham Pharmacia Biotech. Bacterial strains and growth conditions Bacterial strains used in this study are listed in Table 1. M. smegmatis mc2155 was grown at 37 8C, either in Luria– Bertani (LB) or in Kohn–Harris (K–H) minimal medium (Kohn and Harris 1941) supplemented with 2 g�L–1 of either acetamide (inducing conditions) or sodium succinate (nonin- ducing conditions) in a shaking incubator (220 r�min–1). Solid medium was made with 2% (m/v) agar. Growth me- dium was supplemented with kanamycin (50 mg�mL–1 for Escherichia coli; 25 mg�mL–1 for M. smegmatis mc2155) and X-gal (5-bromo-4-chloro-3-indolyl-b-D-galactopyrano- side) at a concentration of 40 mg�mL–1 wherever necessary. Fig. 1. (A) Organization of the acetamidase operon in Mycobacterium smegmatis. Different open reading frames (ORFs) in the acetamidase operon are represented by large arrows, which also show the direction of transcription of the ORF. The size of each ORF is shown in parentheses. The position of each ORF with respect to the operon is shown with broken lines above the operon. The small arrows marked as Pc, P1, P2, and P3 indicate the location and direction of the promoters identified in the acetamidase operon. The position of each promoter with respect to the operon is shown with broken lines below the promoters. (B) Regulatory elements of the acetamidase operon in M. smegmatis. The ORFs in the acetamidase operon are represented by large arrows, which also show the direction of transcription of the ORF. The location of operators OP1, OP2, and OP3 are marked with solid lines, and their positions are indicated with broken lines. The small arrows marked as Pc, P1, P2, and P3 indicate the location and direction of the promoters identified in the acetamidase operon. The double-headed arrows below the operon identify the region of the acetamidase operon (IA, IIA, IIB, IIIA, IIIB, and IV) used in the electro- phoretic mobility shift assay. The description and size of the fragments are given in Table 2. 600 Can. J. Microbiol. Vol. 53, 2007 # 2007 NRC Canada Transformation of E. coli and M. smegmatis mc2155 was performed as described elsewhere (Snapper et al. 1990; Chung and Miller 1993). The plasmid constructs used in this study are listed in Table 1. Preparation of cell-free crude extract of M. smegmatis mc2155 Mid-log-phase M. smegmatis mc2155 cultures were ino- culated into fresh K–H medium, supplemented with either acetamide or sodium succinate, and incubated for 18 h at 37 8C, with shaking at 200 r�min–1. All subsequent steps were carried out at 4 8C. The harvested cells were resus- pended in lysis buffer, containing 20 mmol�L–1 sodium phosphate (pH 7.2), 500 mmol�L–1 sodium chloride, 10% glycerol, and 2 mmol�L–1 phenylmethylsulfonyl fluoride. Bacterial suspensions were mixed with an equal volume of autoclaved 100-micron-sized glass beads, and beaten in a Mini Bead beater (BioSpec Products) in the presence of ice, with 30 s pulses for 3 min. The lysate was centrifuged at 10 000g for 20 min at 4 8C. Cleared supernatants were collected, and polyamine sulfate (5 mg per 100 mg of crude extract) was added to remove the nucleic acids. Protein con- centration in the final supernatant was estimated, using Lowry’s method (Lowry et al. 1951), and adjusted to a final concentration of 10 mg�mL–1 with autoclaved 1� PBS. Construction of recombinant plasmids Different fragments from the acetamidase operon (all de- rived from pAGAN1 (Parish et al. 1997)) of M. smegmatis were fused translationally to the lacZ reporter gene, and plasmids were constructed, as shown in Table 1. To con- struct plasmid pSS1, a 4.2 kb DNA fragment containing the acetamidase operon was obtained by restricting pAGAN1 with BamHI and inserting at the same site in the promoter probe vector pJEM13 (Timm et al. 1994). Plasmids pSS3 and pSS2 were derived from pSS1 after partial or full diges- tion, respectively, with BspMII, followed by self-ligation, such that pSS2 has a 2.1 kb insert and pSS3 has a 3 kb insert. Construct pSS5 was obtained from pSS1 after re- striction with ApaI as a 1.7 kb fragment. pSS21 and pSS22 were obtained as 357 and 266 bp fragments, re- spectively, using polymerase chain reaction (PCR) with specific primers (5’-CGGTGGTGAAGTCGATTCCC-3’ and 5’-CCATCCGAGACCTTCTCCGT-3’ for pSS21; and 5’-CGGTGGTGAAGTCGATTCCC-3’ and 5’-AAGATCAC- GGCCGCGTCTCC-3’ for pSS22), harboring overhanging sequences for BamHI and ScaI. After restriction digestion with BamHI and ScaI, the PCR products were ligated with pJEM13 digested with the same restriction enzymes. The in- tegrity of the cloned inserts and their proximity with respect to the lacZ reporter were confirmed by DNA sequencing and restriction analysis of the recombinants. All E. coli and my- cobacterial transformants were grown in K–H minimal me- dium supplemented with acetamide or succinate and were assessed for promoter activity with a b-galactosidase assay. b-Galactosidase assay A single bacterial colony from the LB agar plates, con- taining X-gal and the appropriate antibiotic, was inoculated into fresh LB medium with same antibiotic added, and grown at 37 8C for either 16 h (for E. coli) or 42 h (for M. smegmatis). Transformants harboring pJEM13 were in- cluded as negative controls. All the cultures were arrested when optical density at 600 nm (OD600) reached ~0.4–0.6. At this point, acetamide (inducing condition) or sodium suc- cinate (noninducing condition) was added, and the cultures were incubated for an additional 1 h (for E. coli) or 18 h (for M. smegmatis). Cultures were placed on ice to arrest growth, and the OD600 was measured. One hundred micro- litres of chloroform and 50 mL of 0.1% sodium lauryl sulfate were added to the bacterial pellet obtained from 2 mL of bacterial culture, and it was vortexed vigorously. The sus- pension of permeabilized cells (100 mL) was added to 900 mL of Z buffer (60 mmol�L–1 Na2HPO4, 40 mmol�L–1 NaH2PO4, 10 mmol�L–1 KCl, 1 mmol�L–1 MgSO4, and 50 mmol�L–1 2-mercaptoethanol) and 200 mL of 4 mg�mL–1 o-nitrophenyl-b-D-galactopyranoside (ONPG). Timed reac- tions were stopped with 500 mL of 1 mol�L–1 sodium bicar- bonate solution. Enzyme activity was calculated with OD420 and OD550 measurements, as discussed elsewhere (Miller Table 1. Bacterial strains and plasmids used in this study. Strain or plasmid Description Reference Strains Escherichia coli DH5a supE44, dlacU169 (y80 lacZ dM15), hsdR17, recA1, endA1, gyrA96 thi-1, relA1 Sambrook et al. 1989 Mycobacterium smegmatis mc2155 High-transformation mutant of M. smegmatis mc26 Snapper et al. 1990 Plasmids pJEM13 E. coli – mycobacteria shuttle, promoter probe vector with lacZ’ re- porter Timm et al. 1994 pSS1 4.2 kb acetamidase operon of M. smegmatis in pJEM13 This study pSS2 2.1 kb BamHI–BspMII fragment of acetamidase upstream region in pJEM13 This study pSS3 3.0 kb BamHI–BspMII fragment of acetamidase operon in pJEM13 This study pSS5 1.7 kb ApaI–BspMII fragment of acetamidase upstream region in pJEM13 This study pSS21 357 bp PCR fragment upstream to the ATG of acetamidase gene in pJEM13 This study pSS22 266 bp PCR fragment upstream to the ATG of acetamidase gene in pJEM13 This study Subbian and Narayanan 601 # 2007 NRC Canada 1972). Induction fold was calculated as the ratio of b-galac- tosidase activity (in Miller units) shown by a recombinant plasmid in induced and noninduced conditions. The experi- ments were repeated at least three times. EMSA For EMSA, seven DNA fragments were obtained from previously reported plasmids pAMI1-4 (Narayanan et al. 2000) after restriction digestion with appropriate restriction endonuclease to produce DNA fragments 200–800 bp in size (Fig. 1 and Table 2). The DNA fragments were purified using a GFX column purification kit, and their cohesive ends were modified with E. coli DNA polymerase I at 30 8C for 15 min in the presence of [g-32P]ATP (BARC, Trombay, India). The unincorporated nucleotides were removed by passing the la- beled reaction mixtures through a sephadex G 25-50 gel fil- tration column. The labeled fragment was diluted to a final concentration of 2 nmol�L–1. The binding reaction was set up in accordance with manufacturer’s instructions. In short, the conditions for the optimal binding of the regulators present in the crude extract of M. smegmatis with respective operator regions were found by incubating the labeled DNA fragments (2 nmol�L–1 each) with various concentrations of bacterial crude extracts obtained under either inducing or noninducing growth conditions in 1� binding buffer. For cold-chase EMSA, labeled DNA fragments (2 nmol�L–1 each) were incu- bated with a fixed concentration (30 mg) of crude extract of either inducing or noninducing conditions, along with 1, 10, 50, or 100� molar excess of the same but unlabeled DNA fragment. Reactions with labeled DNA fragments without crude extract were used as negative controls. For nonspecific competitor DNA control, unlabelled polydI/polydC (dI/dC) was included in the test reactions. The reaction mixtures were incubated at 25 8C for 15 min and were loaded directly onto a 6% native polyacrylamide gel and electrophoresed in 0.5� TBE buffer (50 mmol�L–1 Tris–borate (pH 8.0), 0.1 mmol�L–1 EDTA) at 100 V for 1.5 h. The gel was trans- ferred to a Whatman No. 3 sheet and vacuum dried at 70 8C for 1 h; bands were then visualized by autoradiography. Results Identification of promoters downstream of operators The organization of the acetamidase operon and the de- scription of different promoter constructs made are shown in Figs. 1A, 1B, and 2. To delineate the regulatory elements of the acetamidase operon, we first sought to characterize the promoters present in the operon. To do this, we used a 9.3 kb E. coli – mycobacteria shuttle promoter probe vector, pJEM13, that contained E. coli lacZ as the reporter gene. Different regions of the acetamidase operon obtained by re- striction digestion were ligated in-frame with the lacZ re- porter of pJEM13. All the six recombinant plasmids (pSS1, pSS2, pSS3, pSS5, pSS21, and pSS22) constructed were LacZ positive in both E. coli and M. smegmatis. However, the LacZ-positive E. coli transformants expressed LacZ to a Table 2. Description of the DNA frag- ments used in electrophoretic mobility shift assay studies. Fragment name Size (bp) Region covered in the acetamidase operon (bp) IA 498 40–538 IB 294 539–832 IIA 365 823–1188 IIB 457 1189–1646 IIIA 544 1627–2171 IIIB 266 2172–2438 IV 849 2418–3267 Fig. 2. Description of various deletion constructs made from the acetamidase operon to study promoter activity. The double-headed arrows represent the region of the acetamidase operon included in the respective plasmid. The numbers in each construct represent the region of the acetamidase operon included in the LacZ assay system. The broken lines correspond to the region missing in each promoter construct. The size of the insert and the name of the promoter construct are also shown. 602 Can. J. Microbiol. Vol. 53, 2007 # 2007 NRC Canada lesser extent than their M. smegmatis counterparts in the presence of acetamide (inducing conditions); however, under noninducing conditions, pSS1 and pSS3 expressed signifi- cantly higher levels of LacZ in E. coli (Fig. 3B). Among the various M. smegmatis transformants tested, the order of decreasing levels of LacZ expression were as follows: pSS2, pSS3, pSS21, pSS1, pSS22, and pSS5, respectively, under inducing conditions; and pSS21, pSS2, pSS22, pSS1, pSS3, and pSS5, respectively, under noninducing conditions (Fig. 3A). The induction fold (defined as the ratio of differ- ence in the level of expression from a cloned insert between inducing and noninducing growth conditions) was found to be highest for pSS3 (>100 fold), followed by pSS2 and pSS1 (Fig. 3A). It is interesting to note that the DNA frag- ment cloned in pSS5 is similar to that of pSS2, but there is a deletion of a 400 bp region from the 5’ end of pSS2. The loss of this region could be responsible for the 16-fold re- duction in LacZ expression between pSS5 and pSS2 in M. smegmatis. Identification of putative operator site in the acetamidase operon of M. smegmatis EMSA was used to identify the operator region(s) in- volved in the regulation of promoters of the acetamidase op- eron from M. smegmatis. Seven different radio-labeled fragments (IA, IB, IIA, IIB, IIIA, IIIB, and IV) from the en- tire acetamidase operon, each ~200–500 bp in size (Fig. 1B and Table 3), were incubated with the cell-free extract of M. smegmatis grown either in inducing or noninducing con- ditions in the EMSA. The bound products were analyzed on nondenaturing polyacrylamide gel electrophoresis. The spe- cificity of the protein–DNA complex formed was confirmed with competitive binding (cold chase), using increasing con- centrations of specific but unlabeled target DNA. Three of the seven tested fragments (IA, IIIA, and IIIB) showed a specific shift in their mobility with M. smegmatis crude ex- tracts in this assay (Fig. 4). The 478 bp DNA fragment IA (OP1) showed a specific shift only with bacterial extract from inducing conditions. The binding affinity of regulatory protein(s) with labeled IA required at least a 100� molar excess of unlabeled IA frag- ment to get rid of the complex formation (Fig. 5). A second 316 bp fragment, IIIA (OP2), showed specific binding with regulatory protein(s) in the bacterial crude extract from non- inducing conditions. This complex required a 100� molar excess of unlabeled IIIA fragment to eliminate the shift in the gel. The third 266 bp fragment IIIB (OP3) showed spe- cific binding of regulatory protein(s) from both inducing and noninducing crude extracts of M. smegmatis, and the protein–DNA complex thus formed required a 50� molar excess of unlabeled fragment to abolish the shift in mobility. There was no difference in the affinity for the regulatory protein(s) from induced and uninduced extracts to bind with this fragment (Fig. 5). EMSA reactions with the unlabelled Fig. 3. Levels of b-galactosidase expressed by various Mycobac- terium smegmatis (A) and Escherichia coli (B) transformants con- taining various promoter constructs from the acetamidase operon, as shown in Fig. 2. The transformants were grown either with acet- amide (inducing condition) or succinate (noninducing condition), as discussed in Materials and methods. For both E. coli and M. smeg- matis transformants, the level of b-galactosidase expressed by vec- tor pJEM13 was subtracted from the value obtained for the recombinants with the acetamidase operon. The experiment was re- peated at least three times. The values are plotted as mean ± SD. Table 3. Regulatory elements and their regions within the acetamidase operon of Mycobacterium smegmatis. Regulatory elements identified Region of acetamidase operon (bp) Reference Promoter P1/Pc 1291–1536 Parish et al. 2001 P2 1677–1986 P3 2458–3026 Operator OP1 40–538 This study OP2 1627–2171 OP3 2172–2438 Subbian and Narayanan 603 # 2007 NRC Canada nonspecific competitor DNA, dI/dC, did not show any change in migration of the shifted bands of the tested frag- ments (data not shown). Discussion The inducible nature of the acetamidase promoter renders it an important determinant in the construction of efficient expression systems for genetic manipulation studies in my- cobacteria (Parish and Stoker 1997; Triccas et al. 1998; Manabe et al. 1999; Dziadek et al. 2002). It has been shown that the regulation of acetamidase operon is at the level of transcription involving multiple promoters (Parish et al. 1997, 2001; Roberts et al. 2003). However, the molecular mechanism involved in the regulation of these promoters is not clearly understood. In this study, we provide experimen- tal evidence of the presence of potential operators within the acetamidase operon, which could be responsible for the dif- ferential expression of acetamidase promoters under differ- ent growth conditions. In this study, using an EMSA approach, we searched for potential operator regions within the acetamidase operon, and identified three DNA regions (OP1, OP2, and OP3) that showed specific shifts with regulatory protein(s) present in the crude cell lysates of M. smegmatis grown under either inducing or noninducing conditions. One of these operators, OP1, located upstream of amiA, showed specific binding with extracts from bacteria grown under inducing condi- tions. This shows the involvement of OP1 in induction dur- ing acetamidase catabolism. This finding is supported by promoter probe experiments, in which deletion of the region encompassing OP1 resulted in a significantly lower expres- sion of the lacZ reporter in pSS5 than in pSS2, which has an intact OP1. In addition, OP1 is located upstream from promoter P1 in the acetamidase operon reported by Parish et al. (2001). This promoter was shown to be responsible for the induction of acetamidase operon genes during the growth of bacilli in the presence of acetamide (Hutter and Dick 2000; Parish et al. 2001). Taken together, it is possible that promoter P1 is involved in the induction of the acetami- dase operon regulated by OP1. In previous studies, a promoter located upstream of amiD (P2) was found to be negatively regulated in the absence of the inducer, acetamide (Hutter and Dick 2000; Parish et al. Fig. 4. Electrophoretic mobility shift assay pattern of seven different radio-labeled fragments (IA, IB, IIA, IIB, IIIA, IIIB, and IV) from the acetamidase operon incubated with 10 mg of Mycobacterium smegmatis cell-free extract from bacteria grown in the presence of acetamide (inducing conditions, lane 1) or succinate (noninducing condition, lane 2). Labeled DNA fragments with reaction components alone serve as negative controls (lane 3). Arrows indicate a shift in mobility. Fig. 5. Cold-chase electrophoretic mobility shift assay (EMSA) to confirm the specificity of the operator–regulator complex. Each complex was treated with either no unlabelled (cold) DNA (lane 1) or 10� molar excess (lane 2), 50� molar excess (lane 3), and 100� molar excess (lane 4) of unlabelled DNA. Labeled probe incubated with EMSA components was included as a negative control (lane 5). Arrowheads indicate a shift in mobility. 604 Can. J. Microbiol. Vol. 53, 2007 # 2007 NRC Canada 2001). In our EMSA studies, we found an operator, OP2, up- stream of P2 that showed binding with the bacterial extract from uninduced growth conditions. On the basis of these studies, it is possible that OP2 is involved in the suppression of amiD. Because it has been suggested that AmiD itself is a regulatory protein, it might bind with OP2 to repress the acetamidase operon in the absence of acetamide. Alterna- tively, the binding of regulators at OP2 might negatively af- fect the expression of amiC by Pc, and the reduction of AmiC might suppress the overall repression of acetamidase operon, especially when the bacteria are grown in succinate. Finally, OP3, the third operator, might be involved in the constitutive expression of P3, the promoter located upstream of amiE, because this operator binds with proteins obtained from both induced and uninduced bacterial cultures. From promoter analysis studies using a LacZ reporter sys- tem, it is evident that most of the LacZ-positive recombi- nants expressed b-galactosidase to higher levels in mycobacteria than in E. coli. Also, the promoter in recombi- nant clone pSS2 (P1) expressed the reporter gene at higher levels during induced conditions. The 2 kb region cloned in this construct encompasses the transcription start point and extended –10 and –35 regions, which could be responsible for promoter activity (Narayanan et al. 2000). In addition to P1, we also confirmed the presence of the P3 promoter, lo- cated 70 bp upstream from the acetamidase coding gene, amiE, although this region does not contain putative –10, –35 sequences. This promoter expressed LacZ constitutively at lower levels than P1 in both E. coli and M. smegmatis, and was not influenced significantly by induction with acetamide. From the promoter analysis, it is clear that P1 was not effi- ciently recognized by E. coli, and P3 showed only a low-level constitutive expression in both hosts. This observation cau- tions the use of acetamidase promoters in heterologous hosts, such as E. coli. Many studies have reported the poor recogni- tion and expression of mycobacterial promoters in E. coli, where it has been documented that the conserved promoter el- ement, –10 and –35 regions, and (or) the intervening sequen- ces are responsible for the differential expression (Das Gupta et al. 1993; Kremer et al. 1995; Bashyam et al. 1996). Results from the LacZ reporter studies suggest that P1 is a highly inducible promoter of the acetamidase operon during induction. However, the region cloned in pSS2 and pSS3, which encompass P1, lacks some of the negative regulator(s) responsible for suppression under noninducing growth condi- tions. This might also be responsible for the low induction fold of pSS2 between inducing and noninducing conditions. Because expression of P3 was not significantly affected under either inducing or noninducing conditions, it might be a constitutive promoter operating independent of P1. Removal of a 468 bp region from the 5’ end of the acet- amidase operon (the C-terminal of AmiC) significantly re- duced reporter gene expression in pSS5 compared with pSS2. In previous studies, promoter P1 and a transcription start point were mapped nearly 1 kb downstream from this region. It is therefore possible that this region acts as an up- stream activating region for the acetamidase promoter. Also, this region forms the C terminal for a putative amide sensor protein, AmiC. This protein has homologies with the prod- ucts of nhhC and nhlC, which act as positive regulators in nitrogen metabolism in Rhodochrous rhodochrous J1 and also with the product of amiC from Pseudomonas aerugi- nosa (Komeda et al. 1996a, 1996b). The latter protein, along with another regulatory protein, AmiR, act as an amide sen- sor (Wilson et al. 1995). These homology results show not only the close association in the metabolism of nitrile com- pounds and amides but also a possible role for AmiC in the regulation of acetamidase expression in M. smegmatis. This finding is supported by this EMSA study, where part of this region encompassing OP1 showed a shift with induced cell extract from M. smegmatis. Hence, it is possible that this re- gion plays a positive regulatory role in the form of an amide sensor. Once induced with amides, the product of AmiC could bind with the amide, and this complex might exert a positive regulation by binding to OP1. The acetamidase op- eron also includes an ORF, amiS, that codes for a putative protein, similar to the membrane proteins of P. aeruginosa and R. rhodochrous involved in the transport of amides (Cussac et al. 1992; Wilson et al. 1995). Taken together, the putative ORFs present in the acetamidase operon of M. smegmatis that code for amide sensing (AmiC) and for amide transport (AmiS) could be expected to play key role, along with the upstream activating region, in regulating the induction of P1 through OP1. In summary, we have attempted to delineate the molecu- lar mechanism involved in the regulation of a highly induci- ble acetamidase operon from M. smegmatis. We report the identification of three operators (OP1, OP2, and OP3) in- volved in the regulation of the acetamidase operon. In addi- tion to confirming the presence of promoters P1 and P3 as previously reported for the acetamidase operon, we have provided evidence, through promoter probe studies, includ- ing LacZ reporter analysis and EMSA studies, that OP1 is located upstream of P1. This promoter, along with its cog- nate operator OP1, might control the induced expression of the acetamidase operon when bacteria are grown in the pres- ence of aliphatic amides. The EMSA results indicate the in- volvement of OP1, OP2, and OP3 in binding with regulators produced most likely from within the acetamidase operon. Because the binding is specific for a given condition and re- spective DNA fragment, fragments that contain the operator regions could be responsible for induction, suppression, and constitutive expression from the respective promoters. How- ever, studies on the nature of the regulators and their actual modes of interaction with respective operators are warranted for a complete understanding of the regulation of this op- eron. Acknowledgements We would like to thank Dr. Geetha Ramachandran for her help in the b-galactosidase assays, A.R. Senthilnathan for technical help, and Shanthi Sadasivan for secretarial help. S.S. was partly supported by a Senior Research Fellowship from the Council of Scientific and Industrial Research (CSIR), Government of India. References Bashyam, M.D., Kaushal, D., Dasgupta, S.K., and Tyagi, A.K. 1996. A study of mycobacterial transcriptional apparatus: identi- fication of novel features in promoter elements. J. Bacteriol. 178: 4847–4853. PMID:8759847. Subbian and Narayanan 605 # 2007 NRC Canada Chung, C.T., and Miller, R.H. 1993. Preparation and storage of competent Escherichia coli cells. Methods Enzymol. 218: 621– 627. PMID:8510550. Cussac, V., Ferrero, R.L., and Labigne, A. 1992. Expression of He- licobacter pylori urease genes in Escherichia coli grown under nitrogen-limiting conditions. J. Bacteriol. 174: 2466–2473. PMID:1313413. Das Gupta, S.K., Bashyam, M.D., and Tyagi, A.K. 1993. Cloning and assessment of mycobacterial promoters by using a plasmid shuttle vector. J. Bacteriol. 175: 5186–5192. PMID:8349558. Draper, P. 1967. The aliphatic acylamide amidohydrolase of Myco- bacterium smegmatis: its inducible nature and relation to acyl- transfer to hydroxylamine. J. Gen. Microbiol. 46: 111–123. PMID:6030461. Dziadek, J., Madiraju, M.V., Rutherford, S.A., Atkinson, M.A., and Rajagopalan, M. 2002. Physiological consequences associated with overproduction of Mycobacterium tuberculosis FtsZ in my- cobacterial hosts. Microbiology (Reading, U.K.), 148: 961–971. PMID:11932443. Hutter, B., and Dick, T. 2000. Analysis of the dormancy-inducible narK2 promoter in Mycobacterium bovis BCG. FEMS Micro- biol. Lett. 188: 141–146. doi:10.1111/j.1574-6968.2000. tb09185.x. PMID:10913697. Kohn, H., and Harris, J. 1941. On the mode of action of sulphona- mides I Action on E. coli. J. Pharmacol. Exp. Ther. 73: 343– 348. Komeda, H., Kobayashi, M., and Shimizu, S. 1996a. Characteriza- tion of the gene cluster of high-molecular-mass nitrile hydratase (H-NHase) induced by its reaction product in Rhodococcus rho- dochrous J1. Proc. Natl. Acad. Sci. U.S.A. 93: 4267–4272. doi:10.1073/pnas.93.9.4267. PMID:8633053. Komeda, H., Kobayashi, M., and Shimizu, S. 1996b. A novel gene cluster including the Rhodococcus rhodochrous J1 nhlBA genes encoding a low molecular mass nitrile hydratase (L-NHase) in- duced by its reaction product. J. Biol. Chem. 271: 15796– 15802. PMID:8662959. Kremer, L., Baulard, A., Estaquier, J., Content, J., Capron, A., and Locht, C. 1995. Analysis of the Mycobacterium tuberculosis 85A antigen promoter region. J. Bacteriol. 177: 642–653. PMID:7836298. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 265–275. PMID:14907713. Mahenthiralingam, E., Draper, P., Davis, E.O., and Colston, M.J. 1993. Cloning and sequencing of the gene which encodes the highly inducible acetamidase of Mycobacterium smegmatis. J. Gen. Microbiol. 139: 575–583. PMID:8473863. Manabe, Y.C., Chen, J.M., Ko, C.G., Chen, P., and Bishai, W.R. 1999. Conditional sigma factor expression, using the inducible acetamidase promoter, reveals that the Mycobacterium tubercu- losis sigF gene modulates expression of the 16-kilodalton alpha-crystallin homologue. J. Bacteriol. 181: 7629–7633. PMID:10601225. Miller, J.F. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Narayanan, S., Selvakumar, S., Aarati, R., Vasan, S.K., and Nar- ayanan, P.R. 2000. Transcriptional analysis of inducible aceta- midase gene of Mycobacterium smegmatis. FEMS Microbiol. Lett. 192: 263–268. doi:10.1111/j.1574-6968.2000.tb09392.x. PMID:11064205. Parish, T., and Stoker, N.G. 1997. Development and use of a con- ditional antisense mutagenesis system in mycobacteria. FEMS Microbiol. Lett. 154: 151–157. doi:10.1111/j.1574-6968.1997. tb12637.x. PMID:9297833. Parish, T., Mahenthiralingam, E., Draper, P., Davis, E.O., and Col- ston, M.J. 1997. Regulation of the inducible acetamidase gene of Mycobacterium smegmatis. Microbiology (Reading, U.K.), 143: 2267–2276. PMID:9245815. Parish, T., Turner, J., and Stoker, N.G. 2001. amiA is a negative regulator of acetamidase expression in Mycobacterium smegma- tis. BMC Microbiol. 1: 19. doi:10.1186/1471-2180-1-19. PMID:11570974. Roberts, G., Muttucumaru, D.G., and Parish, T. 2003. Control of the acetamidase gene of Mycobacterium smegmatis by multiple regulators. FEMS Microbiol. Lett. 221: 131–136. doi:10.1016/ S0378-1097(03)00177-0. PMID:12694921. Sambrook, J., Fritsch, E., and Maniatis, T. 1989. Molecular clon- ing: a laboratory manual. 2nd ed. Cold Spring Harbor Labora- tory Press, Cold Spring, Harbor, N.Y. Snapper, S.B., Melton, R.E., Mustafa, S., Kieser, T., and Jacobs, W.R., Jr. 1990. Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol. Mi- crobiol. 4: 1911–1919. doi:10.1111/j.1365-2958.1990.tb02040.x. PMID:2082148. Timm, J., Lim, E.M., and Gicquel, B. 1994. Escherichia coli – my- cobacteria shuttle vectors for operon and gene fusions to lacZ: the pJEM series. J. Bacteriol. 176: 6749–6753. PMID:7961429. Triccas, J.A., Parish, T., Britton, W.J., and Gicquel, B. 1998. An inducible expression system permitting the efficient purification of a recombinant antigen from Mycobacterium smegmatis. FEMS Microbiol. Lett. 167: 151–156. doi:10.1111/j.1574-6968. 1998.tb13221.x. PMID:9809415. Wilson, S.A., Williams, R.J., Pearl, L.H., and Drew, R.E. 1995. Identification of two new genes in the Pseudomonas aeruginosa amidase operon, encoding an ATPase (AmiB) and a putative in- tegral membrane protein (AmiS). J. Biol. Chem. 270: 18818– 18824. PMID:7642533. 606 Can. J. Microbiol. Vol. 53, 2007 # 2007 NRC Canada