Culturing of M. smegmatis and Preparation of Whole Cell Lysates
M. smegmatis strains were grown in 7H9TG (Middlebrook 7H9 broth supplemented with 0.2% glycerol and 0.05% Tween 80) at 37 °C, with shaking at 250 rpm, or on Middlebrook 7H10 agar. Cells were lysed by bead beating in buffer (10 mm Tris-Cl (pH 7.5), 100 mm NaCl, 10% glycerol, 5 μg/ml leupeptin, 5 μg/ml aprotinin, 5 μg/ml soybean trypsin inhibitor, 3 mm nicotinamide, and 1 μm trichostatin A). Nonidet P-40 and deoxycholate to final concentrations of 1% and 0.5%, respectively, were added to the lysate and mixing continued at 4 °C for 2 h, following which samples were centrifuged for 30 min at 4 °C at 13,000 × g. Supernatants were collected, and protein was estimated using a Micro BCA protein assay kit (Pierce).
Phylogenetic Analysis of the cNMP Binding Domain in Rv0998 and MSMEG_5458
The boundaries of the cNMP binding domain in MSMEG_5458 were identified by analysis in the Pfam database (pfam.sanger.ac.uk/). The sequence of the defined domain was used to identify orthologs in the SwissProt database using BLASTp. Individual full-length sequences of the hits thus obtained (>90) were then analyzed using Pfam to identify their cNMP binding domains. More than 156 domains were identified, because some proteins contained more than one cNMP binding domain in the full-length protein sequence. To this list of 156 sequences, the cNMP binding domain sequences of Rv0998, and the CRPs from M. tuberculosis
and Escherichia coli
were added, and all sequences were then aligned using ClustalW (EMBL). Sequences that were >80% identical were removed using JalView, and phylogenetic and molecular evolutionary analyses were conducted using MEGA version 4 (
- Tamura K.
- Dudley J.
- Nei M.
- Kumar S.
Cloning and Mutagenesis of MSMEG_5458 and Rv0998
The list of primers used for PCR and mutagenesis are provided in supplemental Table S1
. PCR was carried out on the genomic DNA of M. smegmatis
155 using MS5458FWD and MS5458RVS. The product was digested with BamHI and EcoRI and cloned into similarly digested pBKSII(+) to generate pBKS-MSMEG_5458. The clone was confirmed by sequencing (Macrogen, South Korea). A BamHI-EcoRV fragment from pBKS-MSMEG_5458 was cloned into BamHI-StuI-digested pPROEx-HTa to generate plasmid pPRO-MSMEG_5458. pPRO-MSMEG_5458 was digested with BamHI, end-filled, and digested with NotI, and the 1-kb fragment was cloned into SmaI-NotI-digested vector pGEX-6P-2 to generate pGEX-MSMEG_5458.
The Rv0998 gene was cloned using primers Rv0998FWD and Rv0998RVS. PCR was carried out on the genomic DNA of M. tuberculosis H37Rv, and the product was digested with EcoRI and XhoI and cloned into similarly digested pProEx-Htc to generate pPro-Rv0998. The clone was confirmed by sequencing (Macrogen). Plasmid pGEX-Rv0998 was generated by subcloning the EcoRI-XhoI fragment from pPRO-Rv0998 into similarly cut vector pGEX-6P-3.
Point mutations in MSMEG_5458
were generated by site-directed mutagenesis (
- Shenoy A.R.
- Visweswariah S.S.
). pBKS-MSMEG_5458 was used as the template for mutation of Arg-95 and Glu-234 using primers MS5458R95K and MS5458E234A, respectively, to generate pBKS-MSMEG_5458R95K
, respectively. Mutants were verified by sequencing (Macrogen). The BamHI-SnaBI fragment from pBKS-MSMEG_5458R95K
was cloned into similarly digested pPro-MSMEG_5458 to generate pPro-MSMEG_5458R95K
. The BamHI-KpnI fragment from pBKS-MSMEG_5458E234A
was cloned into similarly digested pProEx-Hta to generate pPro-MSMEG_5458 E234A
Expression and Purification of His-tagged Proteins
MSMEG_5458 or mutant proteins were expressed in the E. coli
strain on induction using 500 μm
isopropyl-β-thiogalactopyranoside. Cells were lysed by sonication in buffer containing 50 mm
Tris-Cl (pH 8.2), 5 mm
2-mercaptoethanol (2-ME), 100 mm
NaCl, 10% glycerol, 1 mm
benzamidine, and 2 mm
phenylmethylsulfonyl fluoride, followed by centrifugation at 30,000 × g
. The supernatant was interacted with nickel-nitrilotriacetic acid beads (Agarose Bead Technologies, Spain), and bound protein was eluted with buffer containing 200 mm
Tris-Cl (pH 8.2) buffer, 100 mm
NaCl, 5 mm
2-ME, 300 mm
imidazole, and 10% glycerol. Purified proteins were desalted into buffer containing 50 mm
Tris-Cl (pH 8.2), 5 mm
2-ME, 50 mm
NaCl, and 10% glycerol and stored in aliquots at −70 °C (supplemental Fig. S1a
Gel Filtration and Dynamic Light Scattering
Gel filtration was carried out in buffer containing 50 mm Tris-Cl (pH 8.2), 5 mm 2-ME, 50 mm NaCl, and 10% glycerol at 4 °C at a flow rate of 200 μl/min using a Superose 12 column and an AKTA fast protein liquid chromatography system (Amersham Biosciences). The column was calibrated using commercially available gel filtration standards (thyroglobulin (670 kDa), bovine γ-globulin (158 kDa), chicken ovalbumin (44 kDa), equine myoglobin (17 kDa), and vitamin B12 (1.35 kDa)).
Dynamic light scattering experiments were performed on a DynaPro Molecular Sizing Instrument (Protein Solutions). Samples of purified MSMEG_5458 in the same buffer used for gel filtration were analyzed at concentrations of ∼1 mg/ml at 25 °C. Several measurements were taken at 277 K and analyzed using DYNAMICS Version 3.3 software (Protein Solutions). Data collection times of 10 s were used in all cases for a minimum of 15 acquisitions.
Cyclic Nucleotide Binding Assays
Cyclic nucleotide binding assays were carried out in a final reaction volume of 100 μl containing 25 mm Tris-Cl (pH 7.5), 100 mm NaCl, 10% glycerol, purified protein, and [3H]cAMP (59 Ci/mmol, PerkinElmer Life Sciences) in presence and absence of unlabeled cAMP. Reactions were incubated at 37 °C for 1 h and then filtered through nitrocellulose filters (0.45 μm), which were then washed with 5 ml of ice-cold buffer (10 mm Tris-Cl and 150 mm NaCl). The filters were dried, and bound radioactivity was measured by scintillation counting in a liquid scintillation counter (PerkinElmer Life Sciences). Cyclic nucleotide displacement assays were carried out with 200 ng (∼50 nm) of purified protein using a fixed concentration of labeled [3H]cAMP and varying amounts of unlabeled cAMP or analogs. Data were analyzed using GraphPad Prism 5, and values shown represent the mean ± S.E. of triplicate assays.
Samples were electrophoresed on SDS-polyacrylamide gels (12% acrylamide, 1.2% bis-acrylamide) and transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore). MSMEG_5458 and USP polyclonal antibodies were generated in the laboratory and used at a dilution of 1:5,000. Acetyl-lysine antibody (Cell Signaling Technology, cat. no. 9441S) was used at a dilution of 1:2500. To show specificity of the immunoreactive bands, acetyl-lysine antibodies were incubated with acetylated bovine serum albumin for 1 h prior to addition to the Western blot. This would adsorb antibodies specific to acetylated lysine residues, thereby preventing their interaction with the proteins bound to the membrane. Bound antibody was detected by enhanced chemiluminescence (GE Healthcare).
GST Pulldown Assays
GST-MSMEG_5458 and GST-Rv0998 proteins were expressed in the E. coli cyc− SP850 strain upon induction using 500 μm isopropyl-β-thiogalactopyranoside. Cells were lysed by sonication in a buffer containing 50 mm Tris-Cl (pH 8.2), 5 mm 2-ME, 100 mm NaCl, 10% glycerol, 5 mm DTT, 5 mm EDTA, 1 mm benzamidine, and 2 mm phenylmethylsulfonyl fluoride, followed by centrifugation at 30,000 × g. The supernatant was interacted with glutathione-agarose beads (Sigma), and the matrix was washed thrice with lysis buffer, thrice with Wash I buffer containing 50 mm Tris-Cl pH 8.2, 5 mm DTT, 100 mm NaCl, 5 mm EDTA, and 0.1% Triton X-100, and thrice with Wash II buffer containing 50 mm Tris-Cl (pH 8.2), 5 mm DTT, 100 mm NaCl, 5 mm EDTA, and 10% glycerol. GST or GST fusion proteins bound to glutathione beads were interacted with 2 mg of cytosolic proteins from M. smegmatis/M. bovis Bacillus Calmette-Guérin at 4 °C for 1 h, and the beads were washed five times with 1 ml of buffer containing 150 mm NaCl, 100 mm Tris-Cl (pH 8.2), 10% glycerol, 2 mm phenylmethylsulfonyl fluoride, 5 mm 2-ME, and 1 mm benzamidine. Bound proteins were analyzed on a 12% polyacrylamide gel.
Protein bands were excised from the gel and washed with wash solution (50 mm ammonium bicarbonate/acetonitrile, 1:1, v/v) repeatedly to remove the stain. The gel pieces were dehydrated in acetonitrile and then dried in a centrifugal evaporator (Heto). The gel piece was then treated with 1.5 mg/ml DTT in 100 mm ammonium bicarbonate, and then alkylated with 10 mg/ml iodoacetamide in 100 mm ammonium bicarbonate. Pieces were washed with wash solution and dehydrated again. Pieces were then rehydrated in 50 mm ammonium bicarbonate with ∼200 ng of modified porcine trypsin (Promega, Madison, WI), and digestion was continued at 37 °C for 12 h. Extraction buffer (60% acetonitrile and 0.1% trifluoroacetic acid) was added to the gel pieces to extract the peptides. The extract was pooled and concentrated in centrifugal evaporator. Equal volumes of sample and 2,5-dihydroxybenzoic acid matrix (1 μl each) were mixed and spotted onto a MALDI plate (MTP 384 ground steel, target plate). The samples were analyzed by MALDI-TOF (Ultraflex TOF/TOF, Bruker Daltonics, Germany). Flex Control software was used to acquire data using 25 KvA Reflector mode, N2 Laser, 337 nm, and 50 Hz. Each spectrum was the sum of the ion intensities from 300 laser shots. Flex Analysis 2.0 was used to analyze the spectra. The peptide mass fingerprint data were used to identify proteins using MASCOT on an M. smegmatis Proteomics Database. Analysis allowed for a single missed cleavage with a variable modification of acetylation and fixed modification of carbamidomethylation of cysteine residues, with mass tolerance of 1 Da.
Cloning and Mutagenesis of MSMEG_4207
MSMEG_4207 was cloned using primers MSMEG_4207FWD and MSMEG_4207RVS. PCR was carried out on the genomic DNA of M. smegmatis mc2155, and the product was digested with BamHI and XhoI and cloned into similarly digested pBKSII(+) to generate pBKS-MSMEG_4207. The clone was confirmed by sequencing (Macrogen). The fragment BamHI-XhoI from pBKS-MSMEG_4207 was cloned into similarly digested pPROEx-HTb to generate plasmid pPRO-MSMEG_4207.
mutant was generated using mutagenic primers MSMEG_4207K104R1 and MSMEG_4207K104R2. Wild-type and mutant proteins were expressed in E. coli
BL21(DE3) and purified essentially as described for MSMEG_5458 (supplemental Fig. S1a
In Vitro Acetylation of USP
Assays were carried out in a 20-μl total reaction volume containing 25 mm Tris-Cl, 100 mm NaCl, 5 mm EDTA, 30 μm acetyl-CoA, MSMEG_5458/Rv0998, and 2 μg of MSMEG_4207(USP) as a substrate, in the presence and absence of cAMP. Reactions were incubated at 25 °C for 10 min, stopped by boiling in SDS sample buffer, and analyzed by Western blotting with acetyl-lysine antibody and enhanced chemiluminescence using ECL+ reagent (GE Healthcare).
For quantitation of immunoreactivity in a Western blot assay was used. Enzymatic assays were performed with varying concentrations of MSMEG_5458, acetyl-CoA, and USP as indicated, and incubated for the times indicated at 25 °C. Reactions were terminated by addition of SDS sample dye and boiled, and equal aliquots were loaded onto a gel for subsequent Western blot analysis using the acetyl-lysine antibody and enhanced chemiluminescence. Images were acquired with the FluorChem Q MultiImage III system (Alpha Innotech), and image intensities were quantified using the AlphaView Q version 3.03 software. Values obtained were analyzed by GraphPad Prism 5 using built-in equations for enzyme kinetics.
For measuring the rate of USP acetylation by MSMEG_5458, an enzyme-coupled assay was adopted. Acetyltransferase activity was continuously measured by using a UV-visible spectrophotometer (Varian Cary Bio 100). The assay reaction mixtures contained 0.2 mm NAD, 0.2 mm thiamine pyrophosphate, 5 mm MgCl2, 1 mm DTT, 2.4 mm α-ketoglutarate, 50 μm acetyl-CoA, 50 μm MSMEG_4207, 0.03 unit of α-ketoglutarate dehydrogenase, 190 nm MSMEG_5458, 100 mm sodium acetate, 50 mm Bis-Tris, and 50 mm Tris borate buffer, pH 7.5, in a total volume of 150 μl. All assay components except MSMEG_5458 and MSMEG_4207 were incubated at 25 °C for 5 min, and the reaction was initiated by the addition of MSMEG_4207 and MSMEG_5458. The rates were analyzed continuously for 2 min by measuring NADH production at 340 nm.
Generation of the ΔMSMEG_5458 Strain
A strain with a deletion of the MSMEG_5458
) was generated using the suicide vector approach (
). The region 5′ to MSMEG_5458
consisting of ∼850 bases upstream was amplified by PCR using primers UPKOMS5458FWD and UPKOMS5458RVS, and a fragment of ∼830 bases downstream of the acetyltransferase region of MSMEG_5458
was amplified using DOWNKOMS5458FWD and DOWNKOMS5458RVS primers. The 5′ amplicon was cloned into pBKSII vector using HindIII and NotI sites, and the 3′ amplicon was cloned into pBKSII(+) vector using NotI and BamHI sites to generate plasmids pBKS-MS5458–5′KO and pBKS-MS5458–3′KO. DNA sequencing confirmed the identity of the cloned sequences. The HindIII-NotI cut pBKS-MS5458–5′KO and NotI-BamHI cut pBKS-MS5458–3′KO inserts were ligated into BamHI-HindIII cut p2NIL plasmid with kanamycin selection marker (kindly provided by Prof. Neil Stoker) to generate p2NIL-MS5458–5′3′KO plasmid. The PacI fragment from pGOAL19 containing three markers (β-galactosidase, hygromycin resistance, and sucrose susceptibility) was cloned into p2NIL-MS5458-5′3′KO to generate plasmid p2NIL-MS5458–5′3′KO-PacI. Plasmid p2NIL-MS5458–5′3′KO-PacI (1 μg) was electroporated into M. smegmatis
155, and single crossovers and double crossovers were obtained essentially as described (
). Double crossovers were further tested by genomic PCR and Southern blotting to obtain the ΔMSMEG_5458
To construct the ΔMSMEG_5458 strain complemented with the MSMEG_5458 gene driven by its own promoter, a KpnI-MscI fragment from pBKS-MS5458–5′KO, and the MscI-XbaI fragment from pPRO-MSMEG_5458 were cloned into KpnI and XbaI digested vector pMH94h (a kind gift of W. R. Bishai, Johns Hopkins Centre for Tuberculosis Research) to generate pMH-MSMEG_5458C, which was electroporated in the ΔMSMEG_5458 strain. Positive integrants (ΔMSMEG_5458Comp) carrying the required insert were screened by colony PCR and validated by Southern hybridization. PCRs were carried out with primers MS5458FWD and MS5458RVS on genomic DNA prepared from the wild-type strain and the ΔMSMEG_5458 and ΔMSMEG_5458Comp strains to confirm the presence of the deletion in the ΔMSMEG_5458 strain and the complementation of MSMEG_5458.
Southern Blot Analysis
Southern blotting was carried out by electrophoretic separation of 2 μg of genomic DNA of wild-type, knock-out, and complement strains digested with EcoRI and AgeI and by transferring the DNA to Hybond nylon membranes (Amersham Biosciences). The probe was prepared from a 150-bp fragment amplified using primers MS5458FWD and UPKOMS5458RVS using pMH-MSMEG_5458C plasmid as a template. The probe was end-labeled with γ-32P-labeled ATP catalyzed by polynucleotide kinase (New England Biolabs). The blot was probed at 60 °C for 16 h and washed once in 2× SSC (0.3 mm NaCl and 30 mm sodium citrate) for 15 min at 60 °C prior to exposure to a PhosphorImager.
Immunoprecipitation of MSMEG_4207
Whole cell lysates were used for immunoprecipitation. For pre-clearing, the lysates were incubated with normal rabbit IgG for 1 h at 4 °C. Protein G beads were added to the lysates and incubated for an additional 30 min. The beads were removed by centrifugation, and the pre-cleared supernatant was interacted with MSMEG_4207 IgG overnight at 4 °C. Protein G beads were added to the lysates and incubated for an additional 2 h. The beads were pelleted at 4 °C and washed thrice with wash buffer (10 mm Tris-Cl (pH 7.5), 100 mm NaCl, 0.1% Triton X-100) and twice with TBS (10 mm Tris-Cl (pH 7.5), 100 mm NaCl). The beads were then boiled in SDS sample buffer and subsequently analyzed by SDS-PAGE and Western blotting.