Figures & data
Table 1. Strains and plasmids used in this study.
Table 2. Primers used in this study.
Fig. 1. pgcA and gtaB disruptants showed the same phenotype as the ugtP mutant.
Notes: (A) Detection of glucolipids of 168 (wild type) cells, PGCAd (pgcA::pMUTIN), GTABd (gtaB::pMUTIN T3), and YTB019 (ugtP::kan). Total lipids were extracted from 50 ml of cultures at log phase (OD530 ≈ 0.5). The total lipids were analyzed as described in MATERIALS AND METHODS. GPL: glucophospholipid; DGlcDG_bs: 3-[O-β-d-glucopyranosyl-(1→6)-O-β-d-glucopyranosyl]-1,2-diacylglycerol from B. subtilis; PG: phosphatidylglycerol; PE: phosphatidylethanolamine; Lys-PG: lysyl-phosphatidylglycerol. (B) Cell morphology of 168 (wild type) cell, PGCAd (pgcA::pMUTIN), GTABd (gtaB::pMUTIN T3), and YTB019 (ugtP::kan). Cells were grown in LB medium to an OD530 of 0.5. Cell morphology was observed as described in MATERIALS AND METHODS. The black bars indicate 10 μm.
Fig. 2. The abnormal morphology of the B. subtilis ugtP mutant was suppressed when heterologous MGlcDG synthase was expressed.
Notes: (A) Detection of glucolipids produced by heterologous glucosyltransferases. Total lipids of wild type cell (168), YTB019 (ugtP::kan), ugtP disruptant expressing almgs (MBS46), and both almgs and aldgs (MBS47) were extracted from 50 ml of cultures at log phase (OD530 ≈ 0.5). The total lipids were analyzed as described in MATERIALS AND METHODS. GPL: glucophospholipid; DGlcDG_bs: as described in the legend to Fig. A; MGlcDG_al: 3-O-α-d-glucopyranosyl-1,2-diacylglycerol produced by alMGS; DGlcDG_al: 3-[O-α-d-glucopyranosyl-(1→ 2)-O-α-d-glucopyranosyl]-1,2-diacylglycerol produced by alMGS and alDGS; CL: cardiolipin; PG: phosphatidylglycerol; PE: phosphatidylethanolamine; Lys-PG: lysyl-phosphatidylglycerol. (B) Cell morphology of wild type cell (168), YTB019 (ugtP::kan), ugtP mutant expressing almgs (MBS46), and both almgs and aldgs (MBS47). almgs and aldgs were expressed by addition of 1% d-xylose and 1 mM IPTG, respectively. Cells were grown in LB medium to an OD530 of approximately 0.5, then cell morphology was observed as described in MATERIALS AND METHODS. The black bars indicate 10 μm.
Fig. 3. Effect of heterologous glucolipids on activitiy of ECF sigmas in the B. subtilis ugtP mutant.
Notes: Cells were grown in LB medium to an OD530 of approximately 0.5. The subsequent lacZ assay was performed as described in MATERIAL AND METHODS. (A) LacZ activity of MBS51 (PsigM’-lacZ), MBS52 (PsigV-lacZ), and MBS53 (PsigX’-lacZ). Expression of almgs was regulated through the concentration of d-xylose (%) on inoculation. LacZ activities in MBS48 (PsigM’-lacZ), MBS49 (PsigV-lacZ), MBS50 (PsigX’-lacZ) are labeled WT, respectively. (B) LacZ activity of MBS54 (PsigM’-lacZ), MBS55 (PsigV-lacZ), and MBS56 (PsigX’-lacZ). The LacZ activities in MBS54 (PsigM’-lacZ), MBS55 (PsigV-lacZ), and MBS56 (PsigX’-lacZ) without inducers (d-xylose and IPTG) are shown as control. Expression of almgs was induced by 1% d-xylose, and that of aldgs was regulated via concentration of IPTG (μM) on inoculation.
Fig. 4. Effect of heterologous glucolipids production on activitiy of SigM and SigV in the B. subtilis wild type.
Notes: Cells were grown in LB medium to an OD530 of approximately 0.5. The subsequent lacZ assay was performed as described in MATERIALS AND METHODS. LacZ activities in MBS48 (PsigM’-lacZ) and MBS49 (PsigV-lacZ) are labeled WT, respectively. LacZ activity of MBS63 (PsigM’-lacZ), MBS64 (PsigV-lacZ) without inducers (d-xylose and IPTG) are shown as control. Expression of almgs was induced by 1% d-xylose, and that of aldgs was regulated via concentration of IPTG (μM) on inoculation.
Fig. 5. Effect of ugtPH18A expression and Mg2+ addition on activities of ECF sigmas in B. subtilis ugtP mutant.
Notes: Expression of ugtPH18A was regulated via IPTG concentration (μM) in MBS58 (PsigM’-lacZ), MBS59 (PsigV-lacZ), and MBS60 (PsigX’-lacZ). To examine the effect of Mg2+, 10 mM MgSO4 was added to the culture on inoculation. Cells were grown in LB medium to an OD530 of approximately 0.5. The LacZ assay was performed as described in MATERIALS AND METHODS.
Fig. 6. Effect of expression of heterologous glucolipids on activitiy of SigV and SigX in the B. subtilis without their anit-sigma factors.
Notes: Cells were grown in LB medium to an OD530 of approximately 0.5. LacZ activities in MBS49 (PsigV-lacZ) and MBS50 (PsigX’-lacZ) are labeled WT. To examine the effect of rsiV disruption, the PsigV-lacZ activities of MBS68, MBS70, MBS72, and MBS74 were measured. To examine the effect of rsiX disruption, the PsigX’-lacZ activities of MBS69, MBS71, MBS73, and MBS75 were measured. Expression of almgs was induced by 1% d-xylose, and that of aldgs was induced by 1 mM IPTG. The LacZ assay was performed as described in MATERIALS AND METHODS.
