CHARACTERIZATION OF THE ESSENTIAL GENE GLMM ENCODING PHOSPHOGLUCOSAMINE MUTASE IN ESCHERICHIA-COLI

Two different approaches to identify the gene encoding the phosphogluc osamine mutase in Escherichia coli were used: (i) the purification to near homogeneity of this enzyme from a wild type strain and the determ ination of its N-terminal amino acid sequence; (ii) the search in data bases of an E. coli protein of unknown function showing sequence simi larities with other hexosephosphate mutase activities. Both investigat ions revealed the same open reading frame named yhbF located within th e leuU-dacB region at 69.5 min on the chromosome (Dallas, W. S., Dev, I. K., and Ray, P. H. (1993) J. Bacteriol, 175, 7743-7744). The predic ted 445-residue protein with a calculated mass of 47.5 kDa contained i n particular a short region GIVISASHNP with high similarity to the put ative active site of hexosephosphate mutases. In vitro assays showed t hat the overexpression of this gene in E. coil cells led to a signific ant overproduction (from 15- to 50-fold) of phosphoglucosamine mutase activity. A hexose 1,6-diphosphate-dependent phosphorylation of the en zyme, which probably involves the serine residue at position 102, is a pparently required for its catalytic action. As expected, the inactiva tion of this gene, which is essential for bacterial growth, led to the progressive depletion of the pools of precursors located downstream f rom glucosamine 1-phosphate in the pathway for peptidoglycan synthesis . This was followed by various alterations of cell shape and finally c ells were lysed when their peptidoglycan content decreased to a critic al value corresponding to about 60% of its normal level. The gene for this enzyme, which is essential for peptidoglycan and lipopolysacchari de biosyntheses, has been designated glmM.