Use of CDP-glycerol as an alternate acceptor for the teichoic acid polymerase reveals that membrane association regulates polymer length

Abstract

The study of bacterial extracellular polysaccharide biosynthesis is hampered by the fact that these molecules are synthesized on membrane-resident carrier lipids. To get around this problem, a practical solution has been to synthesize soluble lipid analogs and study the biosynthetic enzymes using a soluble system. This has been done for the Bacillus subtilis teichoic acid polymerase, TagF, although several aspects of catalysis were inconsistent with the results obtained with reconstituted membrane systems or physiological observations. In this work we explored the acceptor substrate promiscuity and polymer length disregulation that appear to be characteristic of TagF activity away from biological membranes. Using isotope labeling, steady-state kinetics, and chemical lability studies, we demonstrated that the enzyme can synthesize poly(glycerol phosphate) teichoic acid using the elongation substrate CDP-glycerol as an acceptor. This suggests that substrate specificity is relaxed in the region distal to the glycerol phosphate moiety in the acceptor molecule under these conditions. Polymer synthesis proceeded at a rate (27 min(-1)) comparable to that in the reconstituted membrane system after a distinct lag period which likely represented slower initiation on the unnatural CDP-glycerol acceptor. We confirmed that polymer length became disregulated in the soluble system as the polymers synthesized on CDP-glycerol acceptors were much larger than the polymers synthesized on the membrane or previously found attached to bacterial cell walls. Finally, polymer synthesis on protease-treated membranes suggested that proper length regulation is retained in the absence of accessory proteins and provided evidence that such regulation is conferred through proper association of the polymerase with the membrane.

FIG. 1.

Structure and synthesis of teichoic acid in B. subtilis 168. (A) Chemical structure of poly(glycerol phosphate) teichoic acid attached to peptidoglycan. Gray, peptidoglycan; blue, GlcNAc; red, ManNAc; green, glycerol phosphate. (B) Teichoic acid biosynthetic pathway. The solid arrows indicate steps in the pathway that are catalyzed by the identified enzymes. The dashed arrows indicate the steps at which various nucleotide activated precursors are involved. For an explanation of the colors see above.

FIG. 2.

Synthesis of a large glycerol phosphate-containing product in the absence of membrane. TagF (100 nM) was incubated in the presence of 500 μM CDP-[¹⁴C]glycerol. The reaction was quenched after 23 h by addition of urea to 4 M. Reaction products were analyzed by gel filtration chromatography using an HPLC equipped for in-line scintillation counting. The results for zero time (solid line) and 23 h (dotted line) are shown. Formation of a high-molecular-weight product in the absence of membrane was observed (asterisk).

FIG. 3.

Analysis of the membrane-free reaction product. (A) Membrane-free reaction products were subjected to denaturing proteolysis in the presence of 20 μg/ml trypsin for 60 min and were analyzed by gel filtration chromatography (Waters ProteinPak 300SW). (Inset) Purified TagF (10 μg) was subjected to identical proteolysis for 0, 10, 25, and 60 min in the presence or absence of CDP-glycerol (2 mM). A sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel of the products of proteolysis is shown. The positions of molecular mass standards (in kDa) are indicated on the left. The molecular mass of TagF is 90 kDa. (B) Purified membrane-free reaction products were treated in the presence of 1 M HCl for 3 h at 100°C and analyzed by gel filtration chromatography (Waters Ultrahydrogel 120). The peaks at 15 and 20 min coeluted with glycerol phosphate and glycerol standards, respectively.

FIG. 4.

CDP-glycerol functions as the acceptor in the absence of membrane. (A) TagF (200 nM) was incubated with 150 μM [α-³²P]CDP-glycerol for 48 h. The reaction product was purified and analyzed by gel filtration chromatography using an HPLC system equipped for in-line scintillation counting. (Inset) Predicted structure of the reaction product, showing the position of ³²P. (B) Purified reaction product was treated in the presence of 0.5 M NaOH for 25 min at 37°C. Neutralized samples were analyzed by gel filtration chromatography as described above for panel A. The presence of base-labile [α-³²P]CMP in the membrane-free polymers suggested that CDP-glycerol was used as the acceptor for polymerization.

FIG. 5.

Dependence of membrane-free activity on time and enzyme concentration. TagF protein at concentrations of 50 (•), 75 (○), 100 (▾), and 125 nM (▿) was incubated in the presence of 6 mM CDP-glycerol for 120, 180, and 240 min. The reactions were quenched by addition of urea to 4 M, and the products were analyzed by paired ion chromatography. The production of CMP was assessed as described in Materials and Methods and plotted against time. (Inset) Steady-state velocity plotted against the concentration of TagF. The slope of the line represents the turnover of the enzyme under experimental conditions (27 min⁻¹) and is very similar to that previously obtained for membrane-associated TagF (34).

FIG. 6.

Analysis of polymers synthesized on native and protease-treated membranes. (A) TagF (50 nM) was incubated in the presence of 750 μM CDP-[¹⁴C]glycerol and 20 mg protein/ml native B. subtilis membranes. (B) Inverted B. subtilis membranes were treated with proteinase K as described in Materials and Methods and subsequently used as an acceptor for TagF. The results obtained for membranes treated with 1,000 μg/ml proteinase K are shown. After 24 h, each reaction was quenched by addition of urea to 4 M, membranes were separated by ultracentrifugation, and the synthesized polymers were extracted by treatment in 0.5 M NaOH for 25 min at 37°C. Extracted polymers were analyzed by gel filtration chromatography. (Inset) Western blot analysis of the membrane preparations treated with 0, 250, 500, or 1,000 μg/ml proteinase K. Lane TX contained membranes treated with 1,000 μg/ml proteinase K in the presence of 3% Triton X-100.