The effect of magnesium ion deprivation on the synthesis of mucopeptide and its precursors in Bacillus subtilis

1. Mg2+ or Mn2+ starvation causes suspensions of Bacillus subtilis strain W 23 to accumulate bound amino sugars that are soluble in trichloroacetic acid. 2. The presence of chloramphenicol or puromycin produces higher intracellular concentrations of amino sugars during Mg2+ starvation, but neither compound can stimulate the accumulation when Mg2+ is present. 3. The major component of the amino sugar fraction extracted from cells deprived of Mg2+ is a nucleotide containing uridine, phosphorus, N-acetylmuramic acid, alanine, glutamic acid and α∈-diaminopimelic acid in the molar proportions of 1:2:1:3:1:1. This compound represents at least 80% of the bound N-acetylhexosamine extracted by trichloroacetic acid. 4. Studies of the binding of this nucleotide with vancomycin support the proposal that it is the mucopeptide precursor UDP-N-acetylmuramyl-l-alanyl-d-glutaminyl- α∈-diaminopimelyl-d-alanyl-d-alanine. 5. A method is described for the isolation of this material labelled with [3H]α∈-diaminopimelic acid. 6. When Mg2+ is supplied to cells previously starved of Mg2+, the accumulated pool of amino sugars rapidly decreases. 7. The biosynthesis of mucopeptide is inhibited by 35–50% under conditions of Mg2+ starvation. The presence of EDTA increases this inhibition to 70%. The amount of N-acetylhexosamine that accumulates is balanced exactly by the associated fall in mucopeptide synthesis. 8. ‘Chase’ experiments show that the accumulated N-acetylhexosamine compound is utilized in mucopeptide synthesis.