Teichoic acid is an essential polymer in Bacillus subtilis that is functionally distinct from teichuronic acid

Abstract

Wall teichoic acids are anionic, phosphate-rich polymers linked to the peptidoglycan of gram-positive bacteria. In Bacillus subtilis, the predominant wall teichoic acid types are poly(glycerol phosphate) in strain 168 and poly(ribitol phosphate) in strain W23, and they are synthesized by the tag and tar gene products, respectively. Growing evidence suggests that wall teichoic acids are essential in B. subtilis; however, it is widely believed that teichoic acids are dispensable under phosphate-limiting conditions. In the work reported here, we carefully studied the dispensability of teichoic acid under phosphate-limiting conditions by constructing three new mutants. These strains, having precise deletions in tagB, tagF, and tarD, were dependent on xylose-inducible complementation from a distal locus (amyE) for growth. The tarD deletion interrupted poly(ribitol phosphate) synthesis in B. subtilis and represents a unique deletion of a tar gene. When teichoic acid biosynthetic proteins were depleted, the mutants showed a coccoid morphology and cell wall thickening. The new wall teichoic acid biogenesis mutants generated in this work and a previously reported tagD mutant were not viable under phosphate-limiting conditions in the absence of complementation. Cell wall analysis of B. subtilis grown under phosphate-limited conditions showed that teichoic acid contributed approximately one-third of the wall anionic content. These data suggest that wall teichoic acid has an essential function in B. subtilis that cannot be replaced by teichuronic acid.

FIG. 1.

Chemical structures of the two types of predominant teichoic acid in B. subtilis. Poly(glycerol phosphate) is found in strain 168, and poly(ribitol phosphate) is found in strain W23. Covalent attachment of the polymer to peptidoglycan occurs via a highly conserved linkage unit consisting of a disaccharide and one to three glycerol phosphate monomers. Hydroxyl groups on the main chain are often glucosylated or d-alanylated. GroP, glycerol phosphate; RboP, ribitol phosphate; GlcNAc, N-acetylglucosamine; ManNAc, N-acetylmannosamine; Glc, glucose; MurNAc, N-acetylmuramic acid.

FIG. 2.

Xylose-dependent growth of tagB, tagF, and tarD deletion strains. (A) Analysis of growth of tag and tar deletion strains on solid medium. Parent strains EB313, EB521, and EB842 and deletion strains EB633, EB669, and EB856 were grown on solid LB medium containing chloramphenicol in the presence or absence of inducer (2% xylose). (B) Analysis of growth of tagB deletion strain in liquid medium. Strain EB521 was inoculated into LB medium containing chloramphenicol supplemented with 2% xylose (○), and EB633 was inoculated into LB medium containing chloramphenicol supplemented with 2% xylose (⋄), 0.06% xylose (□), 0.02% xylose (▪), 0.006% xylose (▿), or no xylose (▾). Growth was monitored by determining the optical density.

FIG. 3.

Xylose dependence of tag and tar gene deletion strains under phosphate-limited conditions. Parent strains EB124, EB313, EB521, and EB842 and deletion strains EB240, EB633, EB669, and EB856 were acclimatized to phosphate-limiting conditions (see Materials and Methods) and subsequently grown on solid PL medium supplemented with 2.5 mM phosphate (A) or 0.25 mM phosphate (B) in the presence and absence of an inducer (2% xylose).

FIG. 4.

Effects of phosphate and xylose levels on tagF deletion strain. EB669 and control strain EB312 were grown in liquid PL media in the absence of phosphate (□) or in the presence of 0.0313 mM phosphate (▪), 0.0625 mM phosphate (▿), 0.125 mM phosphate (▾), 0.25 mM phosphate (○), or 2.5 mM phosphate (•) and in the presence or absence of an inducer (2% xylose). Growth was monitored by determining the optical density. The experiment was performed in triplicate, and representative profiles are shown.