Onucleotide primers have been performed at the Center for D Technologies, University of Texas Health Science Center at San Antonio. Though the primers RVA, RVB have been designed to amplify the prime region of sapM and its upstream bp fragment (Frag I), primers RVC and RVD had been designed to amplify the area of sapM and its downstream bp fragment (Frag II). Also primers RvB and RVC have been engineered to have StuI web sites in them. Applying these primers and Mtb HRv D, we amplified fragments I and II in PCR and these fragments have been GS-4059 cloned into pCR. Vector (Invitrogen) to make plasmids pTBSAPM and pTBSAPM, respectively. The fragment II from plasmid pTBSAPM was released by cutting with StuI and BamHI and cloned in to the pTBSAPM cut with the same restriction enzymes. The resulting plasmid pTBSAPM features a D fragment, which has upstream and downstream regions of sapM but with substantial deletion in sapM coding area (Fig. S). The D fragment in pTBSAPM was released in the plasmid by digesting the plasmid with restriction enzymes HindIII and NotI along with the released fragment cloned into pNIL to make plasmid pTBSAPM. A bp PacI fragment, that contains sacB and lacZ genes as well as a gene for hygromycin resistance, was then isolated in the plasmid pGOAL and cloned into the PacI web site PubMed ID:http://jpet.aspetjournals.org/content/181/1/46 in the plasmid pTBSAPM. The resulting plasmid pTBSAPM was applied to create a markerless sapM mutant in Mtb DfbpA strain. Plasmid D from E. coli was isolated by utilizing a Qiaperp kit (Qiagen IncValencia, Calif.).the cuvettes was plated on H agar plates containing the antibiotic hygromycin ( mgmL) and Xgal ( mgmL). Transformants displaying blue color, resulting from single crossover event, were selected soon after 3 weeks of incubation at uC. Further screening on the transformants for the deletion of sapM area was performed by a twostep choice process. Very first, the blue colonies have been streaked onto H agar plates containing no antibiotics. Following growth, a loopfull of cells had been E-982 web resuspended into liquid medium, diluted serially to quite a few folds and plated onto H agar plates containing sucrose. Sucrose resistant colonies resulting from double crossover event were streaked onto plates with or devoid of hygromycin. Colonies showing no resistance to hygromycin have been filly streaked onto plates containing kamycin, because fbpA mutant is kamycin resistant. The D from these colonies had been further examined in Southern and PCR to confirm the deletion of sapM area.Complementation of DKO 
StrainTo complement the DKO strain with sapM gene, we constructed an integration plasmid carrying sapM gene as follows. Initially, we amplified the entire sapM gene and its upstream promoter region ( bp) by PCR working with primers RVA and RVEX (Table ) and Mtb HRv genomic D. The fragment was cloned in pCR. vector to result 
in plasmid pTBSAPMA. This plasmid was digested with KpnI and XbaI to release the fragment which was cloned in KpnI and XbaI digested pMVH, a derivative of pMV in which kamycin resistant marker is replaced with hygromycin marker, to have plasmid pMSAPMA. This plasmid was transformed into DKOC (DfbpADsapM double mutant) strain by electroporation. The colonies were selected in H hygromycin plates and the integration from the plasmid was confirmed by Southern blot (information not shown). The resulting strain was med as DKOcom.Southern and PCR AlysisTo confirm the deletion of sapM gene in Mtb DfbpA strain, we performed Southern alysis. We isolated chromosomal D from Mtb HRv, Mtb DfbpA and Mtb DfbpADsapM strains applying CTAB (cetyltrimeth.Onucleotide primers have been performed in the Center for D Technology, University of Texas Health Science Center at San Antonio. Though the primers RVA, RVB have been created to amplify the prime area of sapM and its upstream bp fragment (Frag I), primers RVC and RVD were developed to amplify the area of sapM and its downstream bp fragment (Frag II). Also primers RvB and RVC were engineered to possess StuI sites in them. Employing these primers and Mtb HRv D, we amplified fragments I and II in PCR and these fragments have been cloned into pCR. Vector (Invitrogen) to create plasmids pTBSAPM and pTBSAPM, respectively. The fragment II from plasmid pTBSAPM was released by cutting with StuI and BamHI and cloned in to the pTBSAPM reduce together with the same restriction enzymes. The resulting plasmid pTBSAPM has a D fragment, which has upstream and downstream regions of sapM but with substantial deletion in sapM coding region (Fig. S). The D fragment in pTBSAPM was released from the plasmid by digesting the plasmid with restriction enzymes HindIII and NotI and also the released fragment cloned into pNIL to create plasmid pTBSAPM. A bp PacI fragment, that consists of sacB and lacZ genes plus a gene for hygromycin resistance, was then isolated in the plasmid pGOAL and cloned in to the PacI web site PubMed ID:http://jpet.aspetjournals.org/content/181/1/46 in the plasmid pTBSAPM. The resulting plasmid pTBSAPM was utilised to generate a markerless sapM mutant in Mtb DfbpA strain. Plasmid D from E. coli was isolated by utilizing a Qiaperp kit (Qiagen IncValencia, Calif.).the cuvettes was plated on H agar plates containing the antibiotic hygromycin ( mgmL) and Xgal ( mgmL). Transformants showing blue colour, resulting from single crossover event, were chosen just after 3 weeks of incubation at uC. Further screening of the transformants for the deletion of sapM region was performed by a twostep selection technique. Initially, the blue colonies have been streaked onto H agar plates containing no antibiotics. Following development, a loopfull of cells had been resuspended into liquid medium, diluted serially to several folds and plated onto H agar plates containing sucrose. Sucrose resistant colonies resulting from double crossover occasion were streaked onto plates with or without hygromycin. Colonies showing no resistance to hygromycin were filly streaked onto plates containing kamycin, considering that fbpA mutant is kamycin resistant. The D from these colonies were additional examined in Southern and PCR to confirm the deletion of sapM region.Complementation of DKO StrainTo complement the DKO strain with sapM gene, we constructed an integration plasmid carrying sapM gene as follows. Initial, we amplified the whole sapM gene and its upstream promoter region ( bp) by PCR utilizing primers RVA and RVEX (Table ) and Mtb HRv genomic D. The fragment was cloned in pCR. vector to result in plasmid pTBSAPMA. This plasmid was digested with KpnI and XbaI to release the fragment which was cloned in KpnI and XbaI digested pMVH, a derivative of pMV in which kamycin resistant marker is replaced with hygromycin marker, to obtain plasmid pMSAPMA. This plasmid was transformed into DKOC (DfbpADsapM double mutant) strain by electroporation. The colonies had been selected in H hygromycin plates and also the integration of your plasmid was confirmed by Southern blot (information not shown). The resulting strain was med as DKOcom.Southern and PCR AlysisTo confirm the deletion of sapM gene in Mtb DfbpA strain, we performed Southern alysis. We isolated chromosomal D from Mtb HRv, Mtb DfbpA and Mtb DfbpADsapM strains utilizing CTAB (cetyltrimeth.