Assay for UDPglucose 6-dehydrogenase in phosphate-starved cells: gene tuaDof Bacillus subtilis 168 encodes the UDPglucose 6-dehydrogenase involved in teichuronic acid synthesis

A novel assay permitting the detection of UDPglucose 6-dehydrogenase activi ty in cell-free extracts obtained from phosphate-starved cultures of Bacill us subtilis is described. The critical step, the separation of phosphate-st arvation-induced exo-enzymes, phosphatases and phosphodiesterases from the cytoplasmic fraction containing the UDPglucose dehydrogenase, was achieved by protoplasting and removal of the periplasmic fraction by protoplast wash ing. Using this method, the following were unambiguously demonstrated: (i) the presence in the cytoplasm of an enzymic activity oxidizing UDPglucose t o UDPglucuronic acid, and (ii) that detection of the activity in whole-cell -free extracts is prevented by the presence of 'periplasmic' enzymes cataly sing the degradation of the sugar nucleotides. With this method, several B. subtilis 168 mutants unable to synthesize teichuronic acid were examined. Strains inactivated in gene tuaD, whose product shares homology with UDPglu cose 6-dehydrogenase and GDPmannose 6-dehydrogenase from other organisms, w ere shown to lack UDPglucose 6-dehydrogenase activity. Anion exchange chrom atography revealed that mutants deficient in tuaD lacked a cytoplasmic UDPg lucuronate pool.