Identification of Two Phosphate Starvation-induced Wall Teichoic Acid Hydrolases Provides First Insights into the Degradative Pathway of a Key Bacterial Cell Wall Component

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.

The distinct PhoPR mediated responses to phosphate limitation in Bacillus subtilis subspecies subtilis and spizizenii stem from differences in wall teichoic acid composition and metabolism

“…Together these changes reduce the cellular requirement for phosphate while maintaining the anionic character of the cell wall. Recently Myers and colleagues () reported that PhoPR regulon genes phoD and glpQ encode enzymes that cleave poly(glycerol phosphate) endo‐ and exo‐lytically respectively, thereby revealing a capability to recycle and reutilize the phosphorous content of cell wall material that is released during growth. Thus, WTA serves as a phosphorous store that can be mobilized under conditions of limitation (Myers et al ., ).…”

Phosphoglycerol-type wall- and lipoteichoic acids are enantiomeric polymers differentially cleaved by the stereospecific glycerophosphodiesterase GlpQ

“…GlpQ of B. subtilis and orthologs from other bacteria have been shown previously to specifically release Gro3P from GPC, glycerophosphoethanolamine, glycerophosphoglycerol, and bis(glycerophospho) glycerol. For the latter two substrates, K M and k cat values of 1.0 mM and 1275 min −1 , respectively 1.4 mM and 1517 min −1 , were determined for B. subtilis GlpQ (41,45,47). We confirmed the stereospecificity of recombinantly expressed, B. subtilis GlpQ for sn -glycero-3-phosphoryl substrates and determined the enzyme’s stability and catalytic optima, using sn -glycero-3-phosphocholine (GPC) as substrate (Supporting Information, Fig.…”

“…Besides synthesis, also the turnover of WTA and LTA needs to be differentially regulated, which so far has not been explored in much detail. Recently, the exo-acting sn -glycero-3-phosphate phosphodiesterase GlpQ, along with an endo-acting phosphodiesterase PhoD, has been implicated in the degradation of WTA during phosphate starvation (41). However, apart from WTA degradation during adaptation to phosphate starvation, turnover of WTA likely occurs also along with the turnover of PGN of the cell wall in B. subtilis and other Gram-positive bacteria (42–44).…”

“…Previous studies with the glycerophosphodiesterase GlpQ of B. subtilis as well as orthologous enzymes from Escherichia coli and S. aureus (amino acid sequence identities of 29 and 54 %, respectively) have revealed strict stereospecificity for glycerophosphodiesters harbouring sn -glycerol-3-phosphoryl groups, e.g. produced by phospholipases from membrane phospholipids (41,45–47). Accordingly, phosphatidylglycerol or lysophosphatidylglycerol, which harbour only free sn -glycerol-1-phosphoryl ends are not hydrolyzed by GlpQ and also bis(p-nitrophenyl)-phosphate, a chromogenic substrate for other phosphodiesterases, is not cleaved by GlpQ (45,46).…”

“…Intriguingly, LTA of S. aureus was found to be not a substrate of GlpQ, which however could be due to phosphoglycerol backbone modifications (47). In contrast, the enzyme shows broad substrate specificity with respect to the alcohol moiety and can hydrolyse a variety of different phospholipid head groups, such as glycerophosphocholine, glycerophosphoethanolamine, glycerophosphoglycerol, and bis(glycerophospho)glycerol (41,45,47).…”