Wall teichoic acid polymers are dispensable for cell viability in Bacillus subtilis

Abstract

An extensive literature has established that the synthesis of wall teichoic acid in Bacillus subtilis is essential for cell viability. Paradoxically, we have recently shown that wall teichoic acid biogenesis is dispensable in Staphylococcus aureus (M. A. D'Elia, M. P. Pereira, Y. S. Chung, W. Zhao, A. Chau, T. J. Kenney, M. C. Sulavik, T. A. Black, and E. D. Brown, J. Bacteriol. 188:4183-4189, 2006). A complex pattern of teichoic acid gene dispensability was seen in S. aureus where the first gene (tarO) was dispensable and later acting genes showed an indispensable phenotype. Here we show, for the first time, that wall teichoic acid synthesis is also dispensable in B. subtilis and that a similar gene dispensability pattern is seen where later acting enzymes display an essential phenotype, while the gene tagO, whose product catalyzes the first step in the pathway, could be deleted to yield viable mutants devoid of teichoic acid in the cell wall.

FIG. 1.

Growth of tagO deletion mutant. (A) Growth analysis was performed in LB for EB6, i.e., the wild-type B. subtilis (•), and the tagO deletion strain (EB1451) grown in the presence (▪) and absence (▾) of MgCl₂. Cultures were inoculated at a starting optical density at 600 nm (OD₆₀₀) of 0.001, and absorbance measurements were taken every 1 or 2 h. (B) Phase-contrast microscopy was performed on stationary-phase cultures of the (i) wild-type strain and the tagO deletion strain grown in the (ii) presence and (iii) absence of MgCl₂. Bar, 5 μm.

FIG. 2.

Ultrastructure of B. subtilis lacking wall teichoic acid. Strains of B. subtilis 168 were harvested at late log phase of growth and conventionally embedded in thin sections for examination with transmission electron microscopy as described previously (14). The (A) wild-type strain (EB6) along with the tagO deletion mutant (EB1451) in the (B) absence and (C) presence of MgCl₂ are depicted. Arrows highlight areas of thickened cell wall. Bar, 500 nm.

FIG. 3.

Testing tag gene dispensability in B. subtilis. (A) To address the dispensability of tagB, tagD and tagF donor strains were created containing deletions of tagO (marked with Erm^r) and one copy of tagB, tagD, or tagF (marked with Spec^r) (tagBDF) that contained a complementing copy of tagBDF at amyE (marked with Chl^r). Transformation into a recipient (wild-type) strain and selection on spectinomycin (Spec) (150 μg/ml) and xylose (2%) could allow for four possible outcomes (i to iv). (B) The outcome of this selection procedure performed to test the dispensability of tagD is depicted. In addition to showing Spec^r, all of the clones selected were also Erm^r and/or Chl^r. Erm, erythromycin; Chl, chloramphenicol.