Incorporation of [2-3H]glycerol into cell surface components of Bacillus subtilis 168 and thermosensitive mutants affected in wall teichoic acid synthesis: effect of tunicamycin

Institut de génétique et de biologie microbiennes, Rue César-Roux 19, CH-1005 Lausanne, Switzerland 1 Author for correspondence: Harold M. Pooley. Tel: +41 21 3206075. Fax: +41 21 3206078. e-mail: Harold.Pooley{at}igbm.unil.ch A method is described for measuring the synthesis of poly(glycerol phosphate) [poly(groP)], the major wall teichoic acid (WTA), lipoteichoic acid (LTA) and phospholipid (P-lipid), through fractionation of [2- 3 H]glycerol ([2- 3 H]gro)-labelled Bacillus subtilis cells. When cultures of certain temperature-sensitive mutants defective in one of several tag genes, encoding enzymes involved in WTA synthesis, were transferred to the restrictive temperature, the synthesis of WTA underwent a specific, immediate, block, while that of LTA or P-lipid proceeded unimpeded. These results, in addition to confirming the role of tag genes, demonstrated, reciprocally, the specificity of the fractionation procedure used to distinguish label in WTA from that in LTA or P-lipid. Results of analysis of other, less severely affected, tag -deficient mutants, as well as of another genetically unrelated mutant developing comparable morphological phenotypes in non-permissive conditions, are discussed in relation to a possible mechanism generating the latter phenotype. Fractionation of B. subtilis 168 cells labelled either with [2- 3 H]gro or with [1- 14 C] N -acetylglucosamine, to which tunicamycin was added at 0·5 µg ml -1 (the MIC) revealed a specific and marked inhibition of poly(groP) as well as of poly(3- O -ß-D-glucopyranosyl- N -acetylgalactosamine 1-phosphate), the minor WTA. However, for 60 min at least, the syntheses of PG, LTA and P-lipid were barely affected. Keywords: poly(glycerol phosphate), tunicamycin, teichoic acid, thermosensitive tag mutants, phospholipid Abbreviations: GlcNAc, N -acetylglucosamine; [2- 3 H]gro, [2- 3 H]glycerol; LTA, lipoteichoic acid; ND; nephelometric density; PG, peptidoglycan; P-lipid, phospholipid; poly(GlcGalNAc 1-P), poly(3- O -ß-D-glucopyranosyl- N -acetylgalactosamine 1-phosphate); poly(groP), poly(glycerol phosphate); Ts, temperature sensitive; TUN, tunicamycin; WTA, wall teichoic acid