Abstract
The cell wall of most Gram-positive bacteria contains equal amounts of peptidoglycan and the phosphate-rich glycopolymer wall teichoic acid (WTA). During phosphate-limited growth of the Gram-positive model organism Bacillus subtilis 168, WTA is lost from the cell wall in a response mediated by the PhoPR two-component system, which regulates genes involved in phosphate conservation and acquisition. It has been thought that WTA provides a phosphate source to sustain growth during starvation conditions; however, WTA degradative pathways have not been described for this or any condition of bacterial growth. Here, we uncover roles for the Bacillus subtilis PhoP regulon genes glpQ and phoD as encoding secreted phosphodiesterases that function in WTA metabolism during phosphate starvation. Unlike the parent 168 strain, ΔglpQ or ΔphoD mutants retained WTA and ceased growth upon phosphate limitation. Characterization of GlpQ and PhoD enzymatic activities, in addition to X-ray crystal structures of GlpQ, revealed distinct mechanisms of WTA depolymerization for the two enzymes; GlpQ catalyzes exolytic cleavage of individual monomer units, and PhoD catalyzes endo-hydrolysis at nonspecific sites throughout the polymer. The combination of these activities appears requisite for the utilization of WTA as a phosphate reserve. Phenotypic characterization of the ΔglpQ and ΔphoD mutants revealed altered cell morphologies and effects on autolytic activity and antibiotic susceptibilities that, unexpectedly, also occurred in phosphate-replete conditions. Our findings offer novel insight into the B. subtilis phosphate starvation response and implicate WTA hydrolase activity as a determinant of functional properties of the Gram-positive cell envelope.
Highlights
The cell wall of most Gram-positive bacteria contains equal amounts of peptidoglycan and the phosphate-rich glycopolymer wall teichoic acid (WTA)
In B. subtilis 168, synthesis is accomplished by sequential actions of the tag gene products (TagO, TagA, TagB, and TagF), producing WTA of 30 –50 glycerol phosphate (GroP) units, that is modified with ␣-glucose (TagE) and D-alanine and transferred (TagT, TagU, and TagV are implicated) to the 6Ј-hydroxyl on muramic acid in the glycan strands of PG [1]
Results glpQ and phoD Are Involved in WTA Metabolism during Phosphate Starvation of B. subtilis 168 —Cultures of B. subtilis strains disrupted in the phoPR locus become static upon depletion of Pi from growth media [28]
Summary
GlpQ and phoD Are Involved in WTA Metabolism during Phosphate Starvation of B. subtilis 168 —Cultures of B. subtilis strains disrupted in the phoPR locus become static upon depletion of Pi from growth media [28]. Phosphate-starved deletion mutants contained comparable amounts of cell wall uronic acid as the wild-type strain (Fig. 2C) and exhibited similar secreted APase activity (Fig. 2D), both indicative of functional PhoPR activity. We conclude from these findings that glpQ and phoD play a major role in the loss of WTA in phosphate-starved B. subtilis that is separate from genetic repression of WTA synthesis. Parent strain, indicating that the mutants were unable to utilize polymeric WTA in the hydrolysate, and growth was stimulated only by the WTA degradation products generated in vitro These results suggest that the activities catalyzed by GlpQ and PhoD enable the utilization of WTA as a phosphate reserve under limiting conditions. Kinetic parameters for hydrolysis of p-nitrophenyl phosphates and glycerol 3-phosphate by PhoD
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