1887

Abstract

Glutamate racemase (MurI) provides -glutamate, a key building block in the peptidoglycan of the bacterial cell wall. Besides having a crucial role in cell wall biosynthesis, MurI proteins from some bacteria have been shown to act as an inhibitor of DNA gyrase. and MurI exhibit these dual characteristics. Here, we show that the two activities of MurI are unlinked and independent of each other. The racemization function of MurI is not essential for its gyrase-inhibitory property. MurI–DNA gyrase interaction influences gyrase activity but has no effect on the racemization activity of MurI. Overexpression of MurI provides resistance to the action of ciprofloxacin, suggesting the importance of the interaction in gyrase modulation. We propose that the moonlighting activity of MurI has evolved more recently than its racemase function, to play a transient yet important role in gyrase modulation.

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2008-09-01
2019-11-22
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Multiple sequence alignment showing highly conserved racemase motifs and the two conserved cysteine residues of MurI. The sequences used for the alignment are as follows: 1, ; 2, ; 3, ; 4, ; 5, ; 6, sp.; 7, ; 8, sp.; 9, ; 10, ; 11, . The signature motifs are highlighted by a bold dashed line. The schematic diagram of MurI shows the two highly conserved signature motifs along with the putative catalytic cysteine residues. [ PDF] (269 kb) Expression profile of MurI from pJAM2 constructs in . mc 155 cells harbouring either pJAM2 vector or pJAM2-mtmurI constructs (encoding wild-type, WT, or C75SC185S double mutant, DM, forms of MurI) were grown in Middlebrook 7H9 medium at 37°C to mid-exponential phase, induced with 2% acetamide and grown for a further 6 h. The protein expression profiles were checked by 12% SDS-PAGE. Lanes: 1, protein molecular mass markers; 2, cell extract from cells harbouring vector; 3 and 4, uninduced and induced cell extracts, respectively, from transformants harbouring pJAM2-mtmurI (WT); 5 and 6, uninduced and induced cell extracts, respectively, from transformants harbouring pJAM2-mtmurI (DM). [ PDF] (637 kb) The nucleoid status upon overexpression of MurI resembles that of novobiocin-treated cells. Comparison of nucleoid structures in mc 155 cells harbouring the pJAM2 vector or pJAM2-mtmurI (WT) or pJAM2-mtmurI (DM) constructs, induced with 2% acetamide. Novobiocin-treated cells were used as positive control showing the relaxed diffused nucleoids for cells upon treatment with this gyrase inhibitor. Left panels, phase-contrast images; middle panels, fluorescent images showing the DAPI-stained nucleoid; right panels, overlay of the two images. Scale bars, 5 μm. [ PDF] (436 kb)

PDF

Multiple sequence alignment showing highly conserved racemase motifs and the two conserved cysteine residues of MurI. The sequences used for the alignment are as follows: 1, ; 2, ; 3, ; 4, ; 5, ; 6, sp.; 7, ; 8, sp.; 9, ; 10, ; 11, . The signature motifs are highlighted by a bold dashed line. The schematic diagram of MurI shows the two highly conserved signature motifs along with the putative catalytic cysteine residues. [ PDF] (269 kb) Expression profile of MurI from pJAM2 constructs in . mc 155 cells harbouring either pJAM2 vector or pJAM2-mtmurI constructs (encoding wild-type, WT, or C75SC185S double mutant, DM, forms of MurI) were grown in Middlebrook 7H9 medium at 37°C to mid-exponential phase, induced with 2% acetamide and grown for a further 6 h. The protein expression profiles were checked by 12% SDS-PAGE. Lanes: 1, protein molecular mass markers; 2, cell extract from cells harbouring vector; 3 and 4, uninduced and induced cell extracts, respectively, from transformants harbouring pJAM2-mtmurI (WT); 5 and 6, uninduced and induced cell extracts, respectively, from transformants harbouring pJAM2-mtmurI (DM). [ PDF] (637 kb) The nucleoid status upon overexpression of MurI resembles that of novobiocin-treated cells. Comparison of nucleoid structures in mc 155 cells harbouring the pJAM2 vector or pJAM2-mtmurI (WT) or pJAM2-mtmurI (DM) constructs, induced with 2% acetamide. Novobiocin-treated cells were used as positive control showing the relaxed diffused nucleoids for cells upon treatment with this gyrase inhibitor. Left panels, phase-contrast images; middle panels, fluorescent images showing the DAPI-stained nucleoid; right panels, overlay of the two images. Scale bars, 5 μm. [ PDF] (436 kb)

PDF

Multiple sequence alignment showing highly conserved racemase motifs and the two conserved cysteine residues of MurI. The sequences used for the alignment are as follows: 1, ; 2, ; 3, ; 4, ; 5, ; 6, sp.; 7, ; 8, sp.; 9, ; 10, ; 11, . The signature motifs are highlighted by a bold dashed line. The schematic diagram of MurI shows the two highly conserved signature motifs along with the putative catalytic cysteine residues. [ PDF] (269 kb) Expression profile of MurI from pJAM2 constructs in . mc 155 cells harbouring either pJAM2 vector or pJAM2-mtmurI constructs (encoding wild-type, WT, or C75SC185S double mutant, DM, forms of MurI) were grown in Middlebrook 7H9 medium at 37°C to mid-exponential phase, induced with 2% acetamide and grown for a further 6 h. The protein expression profiles were checked by 12% SDS-PAGE. Lanes: 1, protein molecular mass markers; 2, cell extract from cells harbouring vector; 3 and 4, uninduced and induced cell extracts, respectively, from transformants harbouring pJAM2-mtmurI (WT); 5 and 6, uninduced and induced cell extracts, respectively, from transformants harbouring pJAM2-mtmurI (DM). [ PDF] (637 kb) The nucleoid status upon overexpression of MurI resembles that of novobiocin-treated cells. Comparison of nucleoid structures in mc 155 cells harbouring the pJAM2 vector or pJAM2-mtmurI (WT) or pJAM2-mtmurI (DM) constructs, induced with 2% acetamide. Novobiocin-treated cells were used as positive control showing the relaxed diffused nucleoids for cells upon treatment with this gyrase inhibitor. Left panels, phase-contrast images; middle panels, fluorescent images showing the DAPI-stained nucleoid; right panels, overlay of the two images. Scale bars, 5 μm. [ PDF] (436 kb)

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