A conditional-lethal mutant of Bacillus subtilis 168 with a thermosensitive glycerol-3-phosphate cytidylyltransferase, an enzyme specific for the synthesis of the major wall teichoic acid
Summary: A biochemical analysis was undertaken of thermosensitive mutants of Bacillus subtilis 168 harbouring mutations in several tag genes, involved in the synthesis of the major wall teichoic acid, poly(glycerol phosphate), poly(groP). Incorporation of a pulse of [2-3H]glycerol into whole cells, following shift to the restrictive growth temperature, was used to assess synthesis of this polymer and to seek evidence of accumulation of a specific precursor. The rate of incorporation into poly(groP) was strongly decreased in all mutants; glycerol uptake was diminished by 80% or more for a strain harbouring mutation tagB1 (formerly tag-1) and one bearing tagD11 (formerly tag-11). The pool of CDP-glycerol (CDP-gro), a specific precursor of poly(groP), was increased, relative to the wild-type, for all mutations except tagD11, where the pool of CDP-gro was reduced. Cytoplasmic extracts, assayed at the permissive temperature for glycerol-3-phosphate cytidylyltransferase (gro-PCT), the enzyme synthesizing CDP-gro, revealed wild-type activities for all mutations except tagD11. Gro-PCT activity in the latter strain was 100-fold lower and, unlike that in all other mutant strains, highly thermolabile. This thermosensitivity suggests that tagD encodes gro-PCT. The identification, in a gene encoding a poly(groP)-specific enzyme, of a mutation conferring a thermosensitive growth phenotype renders explicit the conclusion that synthesis of this teichoic acid is essential for the growth of B. subtilis.