Article Figures & Data
Figures
- Fig. 1.
Transcriptional analysis of the glvA, glvR, and glvC genes of B. subtilis, B. subtilis 168 cells were cultured in a rich sporulation medium (DSM) at 37°C for various periods (t0 means the time of onset of sporulation, and t−x andtx mean x hours before and after t0, respectively). mRNAs were prepared (see Materials and Methods) and subjected to Northern blot analysis. Ten micrograms of each RNA was separated on a 1% formaldehyde–agarose gel. Signals were detected with DIG-labeled RNA probes (panel A, probe A; panel B, probe R; panel C, probe C) specific to the glvA, glvR, and glvC mRNAs, respectively. The positions of mRNA signals and rRNAs are indicated by arrows on the left and right, respectively. A map of the three genes encoded by the glvoperon is shown below the panels.
- Fig. 2.
Growth of various mutants in MMM. (A) Open squares, 168 (wild type); open triangles, GLVAd (glvA::pMV1); filled triangles, GLVAd with 1 mM IPTG; open circles, MALLdd (malL::kan); open diamonds, MLGLVAd (glvA::pMV1malL::kan). (B) Open squares, 168 (wild type); open triangles, GLVRd (glvR::pMVR); filled triangles, GLVRd with IPTG; inverted triangles, GLVCd (glvC::pMV2).
- Fig. 3.
Effects of maltose and glucose on transcription of theglv operon. Maltose (2.5 mM) and/or 1% glucose was added toB. subtilis 168 cells at the beginning of growth in DSM. Northern blot analysis was carried out with probe A.
- Fig. 4.
Determination of the transcriptional start site by primer extension analysis. Total RNAs (40, 10, 10, and 10 μg) fromB. subtilis 168 cells cultured in DSM att0.5, DSM with 2.5 mM maltose [DSM (Mal)] att−1 and t0.5, and mSMM at t0.5, respectively, were used as RNA samples. Signals were detected with 32P-labeled primer V1-PEX. Dideoxy DNA sequencing reaction mixtures with the same primer were electrophoresed in parallel (lanes G, A, T, and C). The nucleotide sequence of the transcribed strand is given beside the sequence ladder and the arrow indicates the nucleotide at the transcriptional start site. A map of the glv operon and the nucleotide sequence of the upstream region of glv are shown below the primer extension analysis results.
- Fig. 5.
Northern blot analysis (A) and β-galactosidase activity (B) of the glvR-lacZ transcriptional fusion strain constructed in the B. subtilis chromosome. (A) Wild and GLVRd strains were grown at 37°C in DSM without and with 1 mM IPTG, respectively. RNAs prepared from cells were separated on a gel, and signals were detected with a DIG-labeled specific RNA probe (probe A). (B) Cell growth (A600) and β-galactosidase activity (units per A600) of theglvR-lacZ transcriptional fusion strain (GLVRd) are shown by open and filled symbols, respectively. Squares, B. subtilis168 (wild type); diamonds, GLVRd; circles, GLVRd with IPTG; triangles, GLVRd with IPTG plus maltose. A map of the insertionally inactivatedglv operon of GLVRd is shown at the top.
- Fig. 6.
β-Galactosidase activity of theglvR-lacZ transcriptional fusion strain (GLVR-PSP) with the intact glvR gene. Strain GLVR-PSP was grown in DSM with or without 1 mM IPTG and 2.5 mM maltose at 37°C. Growth (A600) and β-galactosidase activity (units per A600) are shown by open and filled symbols, respectively. Squares, B. subtilis 168 (wild type); diamonds, GLVR-PSP; circles, GLVR-PSP with IPTG; triangles, GLVR-PSP with IPTG plus maltose. A map of the glv operon of GLVR-PSP is shown at the top.
- Fig. 7.
β-Galactosidase activity of a PglvA-lacZ translational fusion localized at the amyE locus. The AMGLV strain having the PglvA-lacZ fusion at theamyE locus was transformed with a citrate-regulatedglvR plasmid, pHYCM2VR, and then β-galactosidase activity of the transformant cultured in DSM with or without 1% glucose and 2 mM citrate was measured. Glucose was added at 0 h and citrate was added at the time indicated by an arrow. Growth (A600) and β-galactosidase activity (units perA600) are shown by open and filled symbols, respectively. pHYCM2 is a control plasmid without theglvR gene. Squares, B. subtilis 168(pHYCM2); diamonds, AMGLV(pHYCM2); circles, AMGLV(pHYCM2VR); triangles, AMGLV(pHYCM2VR) with citrate; inverted triangles, AMGLV(pHYCM2VR) with citrate and glucose. A map of theamyE locus of AMGLV is shown at the top.
- Fig. 8.
The deduced cre and mutated cresequences of AMGLV and AMGLVCR, respectively (A), and β-galactosidase activity of AMGLVCR(pHYCM2VR) (B). AMGLVCR was constructed by changing the cre sequence to a mutated cre sequence upstream of the glvA-lacZ fusion at theamyE locus. β-Galactosidase activity of AMGLVCR(pHYCM2VR) cultured in DSM with or without 1% glucose and 2 mM citrate was measured. Glucose was added at 0 h and citrate was added at the time indicated by an arrow. Growth (A600) and β-galactosidase activity (units per A600) are shown by open and filled symbols, respectively. Squares, B. subtilis 168(pHYCM2); diamonds, AMGLVCR(pHYCM2); circles, AMGLVCR(pHYCM2VR); triangles, AMGLVCR(pHYCM2VR) with citrate; inverted triangles, AMGLVCR(pHYCM2VR) with citrate and glucose.
- Fig. 9.
Northern blot analysis of theccpA+ strain, AMCMVR, and the ccpAmutant, AMCMVRCC, in DSM with 2 mM citrate and 1% glucose. AMCMVR and AMCMVRCC contain PcitM-glvR at theamyE locus (top). Glucose was added att−4 and citrate was added att−2. Probe A was used for Northern blotting.
- Fig. 10.
Illustration of proposed mechanisms of PTS-dependent maltose transport and metabolism. Maltose is also incorporated into cells via an ABC transporter, whose system is explained with the PTS system in Discussion. Thin arrows indicate the metabolic pathway, and thick arrows and perpendicular ones indicate positive and negative controls, respectively. PEP, phosphoenolpyruvate; HTH, helix-turn-helix.
Tables
- Table 1.
Bacterial strains used in this study
Strain Relevant genotype Sourcea B. subtilis 168 trpC2 S. D. Ehrlich GLVAd trpC2 glvA::pMV1 This study GLVRd trpC2 glvR::pMVR This study GLVCd trpC2 glvC::pMV2 This study MALLdd trpC2 malL::kan This study MLGLVAd trpC2 malL::kan glvA::pMV1 This study GLVR-PSP trpC2 glvR::pMVRSD (spac-glvR glvC) This study AMGLV trpC2 amyE::(PglvA [−240 to +32]′-′lacZ cat)b pDHΔglv→168 AMGLVCR trpC2 amyE::(PglvA [−240 to +32 region carrying a CG-to-AT dinucleotide change at positions +6 and +7 relative to the glvA start point]′-′lacZ cat)b pDHΔglvCR→168 AMCMVR trpC2 amyE::(PcitM [−209 to +14]-glvR cat)c pDAFBCMVR→168 1A1 trpC2 ccpA::neo Y. Fujita AMCMVRCC trpC2 amyE::(PcitM [−209 to +14]-glvR cat)ccpA::neoc 1A1→AMCMVR E. coli JM109 recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi-1 Δ(lac-proAB)/F′ (traD36 proAB+laclqlacZ Δ M15) Takara C600 supE44 hsdR17 thi-1 thr-1 leuB6 lacY1 tonA21 Laboratory stock - Table 2.
Plasmids used in this study
Plasmid Relevant genotype Source or Reference pMUTIN2 bla erm lacZ lacI spac 33 pMUTIN4 bla erm lacZ lacI spac oid 33 pBluescript II SK(+) bla lacZ Stratagene pGEM-3Zf(+) bla lacZ Promega pUC119 bla lacZ Takara pDG782 bla kan BGSCa pHY300PLK bla tet Takara pMV1 pMUTIN2::ΔglvA This study pMVR pMUTIN2::ΔglvR This study pMV2 pMUTIN2::ΔglvC This study pMVR-SD pMUTIN4::ΔglvR (containingglvR SD sequence) This study pGV1 pGEM-3Zf(+)::ΔglvA This study pGVR pGEM-3Zf(+)::ΔglvR This study pGV2 pGEM-3Zf(+)::ΔglvC This study pUCMALL bla malL This study pUCMALLKm bla malL::kan This study pBCM2 pBluescript II SK(+) with a 242-bp citMpromoter region This study pHYCM2 pHY300PLK with a 223-bpcitM promoter region This study pDHAFBLZ bla amyE::(lacZ cat) 34 pHYCM2LZ pHY300PLK with a 223-bp citM promoter region and lacZ This study pBVR-SD bla glvR This study pHYCM2VR pHY300PLK with a 223-bpcitM promoter region and glvR This study pUCΔglv pUC119 with a 272-bp glvA promoter region This study pUCΔglvCR pUC119 with a 272-bpglvA promoter region (carrying a CG-to-AT dinucleotide change at positions +6 and +7 relative to the glvA start point) This study pDHΔglv pDHAFBLZ with a 272-bpglvA promoter region fused to lacZ This study pDHΔglvCR pDHAFBLZ with a 272-bp glvA promoter region fused to lacZ (carrying a CG-to-AT dinucleotide change at positions +6 and +7 relative to the glvA start point) This study pDHAFB bla amyE::cat lacI 34 pDHAFB2 bla amyE::cat This study pDAFBCMVR pDHAFB2 with a 242-bp citM promoter region and glvR This study ↵a BGSC, Bacillus Genetic Stock Center, The Ohio State University, Columbus.
- Table 3.
Primers used in this study
Primer Sequence (5′→3′) or sourcea Restriction site V1-EF gccgGAATTCCATTCTCAATCGTAATAGCG EcoRI V1-BR gcgcGGATCCTTCCCTACTCTGATGTGC BamHI VR-EF gccgGAATTCGAAGAACTGATCAATCAGC EcoRI VR-BR gcgcGGATCCTCTTCCGGCTGATCTTCC BamHI V2-HF gccgAAGCTTTCGTCGGTATCAGCACG HindIII V2-BR gcgcGGATCCGACCTCTTGATTCATGTCG BamHI VR-PSPE1 gccgGAATTCTATAATAGAAAGAAAATGGGG EcoRI VR-PSPB2 cgcGGATCCACTGTAACCGCTGAAACC BamHI GLVR-SDB gccgGGATCCAAATGGGGGGATCTGATAT BamHI GLVR-PROE2 gcgcGAATTCGCTTCCAAAGCGCTGAAT EcoRI CMUD-F4 gccgGAATTCTAAACGAACAGGACTGGG EcoRI CMUD-PH2B gcgcGGATCCGTCTTGCCTTTTTGCCATC BamHI GLV-UPF gccgGAATTCGGCATGTATCCGAATCG EcoRI GLV-UPR gccgGTCGACCTTCATATGACGACCTCC SalI GLV-creF ATAAATGGAATTGTAAAATTTATCAAGGAGGTCGTC GLV-creR GACGACCTCCTTGATAAATTTTACAATTCCATTTAT Mal-EF gccgGAATTCGTGGAAAGAAGCTGTCG EcoRI Mal-PB gccgCTGCAGCTAATGCCCATCACAGC PstI CCPA-F1 AGCGAGAGAAGCTAATGTAA CCPA-R2 GTGCGGCAGTTCGACGA V1-PEX AGAGCATGAGTACGATCC −21M13 Universal primer (Takara) M13RV Universal primer (Takara) PM-FK cggGGTACCGTGTGGAATTGTGAGCG KpnI PM-T7 TAATACGACTCACTATATAGTGTATCAACAAGCTGG ↵a The additional sequence (lowercase), restriction site (underlined), and the T7 promoter sequence (bold) are indicated. The PM-FK and PM-T7 primers were annealed to the outside of the multicloning site of pMUTIN derivatives.
