Interaction of penicillin-binding protein 2 with soluble lytic transglycosylase B1 in Pseudomonas aeruginosa

Abstract

Soluble lytic transglycosylase B1 from Pseudomonas aeruginosa was coupled to Sepharose and used to immobilize interaction partners from membrane protein extracts. Penicillin-binding protein 2 (PBP2) was identified as a binding partner, suggesting that the two proteins function together in the biosynthesis of peptidoglycan. By use of an engineered truncated derivative, the N-terminal module of PBP2 was found to confer the binding properties.

FIG. 1.

Organization of the gene cluster harboring pbpA and modular structure of PBP2 from P. aeruginosa. (A) The cluster of genes at the pbpA (PBP2) locus on the P. aeruginosa chromosome includes dacA-dacC (dacA/C) and sltB1, encoding PBP5-PBP6 and SltB1, respectively. (B) The structure of PBP2 is comprised of an N-terminal transmembrane anchor (TM; residues 1 to 38), a non-PB module (residues 39 to 250), and a C-terminal PB module (residues 256 to 652) connected by a short linker sequence. Depicted within the PB module is the Ser-X-X-Lys consensus sequence involving Ser327 as the catalytic residue and site of penicillin binding.

FIG. 2.

PBP assays of fractions from affinity chromatography of membrane proteins on SltB-Sepharose. A P. aeruginosa PA01 membrane-protein extract (S) was applied to either SltB1-Sepharose or a control column of Tris-Sepharose in 10 mM Tris-HCl, 10 mM MgCl₂, 50 mM NaCl, and 0.05% Triton X-100. After the flowthrough fraction was collected, the resins were washed with the same buffer containing 150 mM NaCl (W) and then eluted with buffer containing 1 M NaCl (E₁) and 5 mM EGTA (E₂). Samples (30 μl) of the fractions were incubated with [³H]penicillin G (A) or biotinylated ampicillin (B) and analyzed by SDS-PAGE as described in Materials and Methods. The PBPs were visualized by autoradiography (A) or chemiluminescence (B). A sample of purified P. aeruginosa PBP2 (PBP2) was applied as a positive control, and the assignment of the other PBPs was based on apparent molecular masses.

FIG. 3.

Immunoblots of fractions from affinity chromatography of PBP2 applied to SltB1-Sepharose and Tris-Sepharose. Purified preparations of recombinant PBP2 (A) and the isolated non-PB module (B) were applied to either SltB1-Sepharose or a control Tris-Sepharose column. Conditions of the affinity chromatography and SDS-PAGE were as described in the legend to Fig. 2. Following electrophoresis, the recombinant proteins were detected by Western immunoblot analysis using an anti-six-His antibody as described in Materials and Methods. The positions of molecular mass markers (in kilodaltons) are indicated on the left, and the arrows denote the positions of PBP2 (top) and the non-PB module (bottom).

FIG. 4.

SPR analysis of interaction between P. aeruginosa SltB1 and PBP2. The soluble derivative of PBP2, sPBP2, in 10 mM HEPES, 150 mM NaCl, and 0.005% Tween 20 (pH 7.4) was applied to SltB1 immobilized on a CM5 sensor chip at a flow rate of 100 μl/min. The representative sensorgrams show the responses to injections of sPBP2 at concentrations of 230 nM (a), 460 nM (b), 920 nM (c), 1,840 nM (d), and 3,690 nM (e) using a Biacore 2000 spectrometer.