Characterization of a Bacillus subtilis thermosensitive teichoic acid-deficient mutant: gene mnaA (yvyH) encodes the UDP-N-acetylglucosamine 2-epimerase - PubMed (original) (raw)

Comparative Study

Characterization of a Bacillus subtilis thermosensitive teichoic acid-deficient mutant: gene mnaA (yvyH) encodes the UDP-N-acetylglucosamine 2-epimerase

Blazenka Soldo et al. J Bacteriol. 2002 Aug.

Abstract

The Bacillus subtilis thermosensitive mutant ts-21 bears two C-G-->T-A transitions in the mnaA gene. At the nonpermissive temperature it is characterized by coccoid cell morphology and reduced cell wall phosphate content. MnaA converts UDP-N-acetylglucosamine into UDP-N-acetylmannosamine, a precursor of the teichoic acid linkage unit.

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Figures

FIG. 1.

Correction of the strain ts-21 mutations by subclones of the mnaA-gtaB region. Plasmids correcting or not correcting the thermosensitive phenotype are indicated by + or −, respectively. The map corresponds to the region previously designated orfX-gtaB (27). Plasmids p6309, p6311, p6312, p6328, and p6344 were previously described (27). pBS635 was obtained by cloning into pBAD-TOPO vector (Invitrogen) the PCR product extending from nucleotide 639 of lytR to nucleotide 43 of gtaB and generated on strain 168 (5) DNA.

FIG. 2.

Cell morphology and cell wall phosphate content of mutant L6571 (ts-21) and its thermoresistant derivatives. Cells were grown for 20 h on LB agar plates at 30 and 47°C. Cell wall phosphate was determined for cells grown at 47°C. Residues at positions 69 and 374 in MnaA of each investigated strain are indicated. WT, wild type.

FIG. 3.

Alignment of the B. subtilis MnaA domains comprising Thr-69 and Pro-374 with their counterparts from different bacteria. All listed proteins are 43 to 64% identical to B. subtilis MnaA. The B. subtilis MnaA Thr-69 and Pro-374, as well as their equivalents in MnaA homologs, are boxed. Conserved residues are in boldface.

FIG. 4.

Coomassie blue-stained gel of the MnaA-His6 fusion protein obtained following overexpression in E. coli TOP10(pBS629). Cells were induced with 0.2% _l_-arabinose and grown for 5 h. Purified protein is indicated by the arrowhead. At a higher protein loading, a few minor bands could be detected. Molecular weight markers are in thousands.

FIG. 5.

MnaA-His6 assay. High-pressure liquid chromatography analysis was performed on trifluoroacetic acid hydrolysis products of ManNAc (dotted line), purified recombinant MnaA-His6 protein with UDP-GlcNAc without incubation (dashed line), and purified recombinant MnaA-His6 protein with UDP-GlcNAc incubated for 2 h (solid line). Under experimental conditions used, the equilibrium of interconversion between UDP-GlcNAc and UDP-ManNAc is reached in less than 10 min (data not presented). Peaks 1 to 5 are indicated.

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Characterization of a Bacillus subtilis thermosensitive teichoic acid-deficient mutant: gene mnaA (yvyH) encodes the UDP-N-acetylglucosamine 2-epimerase - PubMed (original) (raw)