Overproduction of Penicillin-Binding Protein 2 and Its Inactive Variants Causes Morphological Changes and Lysis inEscherichia coli

Abstract

Penicillin-binding protein 2 (PBP 2) has long been known to be essential for rod-shaped morphology in gram-negative bacteria, including Escherichia coli and Pseudomonas aeruginosa. In the course of earlier studies with P. aeruginosa PBP 2, we observed that E. coli was sensitive to the overexpression of its gene, pbpA. In this study, we examined E. coli overproducing both P. aeruginosa and E. coli PBP 2. Growth of cells entered a stationary phase soon after induction of gene expression, and cells began to lyse upon prolonged incubation. Concomitant with the growth retardation, cells were observed to have changed morphologically from typical rods into enlarged spheres. Inactive derivatives of the PBP 2s were engineered, involving site-specific replacement of their catalytic Ser residues with Ala in their transpeptidase module. Overproduction of these inactive PBPs resulted in identical effects. Likewise, overproduction of PBP 2 derivatives possessing only their Nterminal non-penicillin-binding module (i.e., lacking their C-terminal transpeptidase module) produced similar effects. However, E. coli overproducing engineered derivatives of PBP 2 lacking their noncleavable, N-terminal signal sequence and membrane anchor were found to grow and divide at the same rate as control cells. The morphological effects and lysis were also eliminated entirely when overproduction of PBP 2 and variants was conducted with E. coli MHD79, a strain lacking six lytic transglycosylases. A possible interaction between the N-terminal domain of PBP 2 and lytic transglycosylases in vivo through the formation of multienzyme complexes is discussed.

A dynamically assembled cell wall synthesis machinery buffers cell growth

“…S4 A and B), cells gradually became wider and rounder over the course of several divisions (Fig. 3A), consistent with previous observations (11). Despite these morphological changes indicative of substantial PBP2 depletion, we detected no slowing of the growth rate, defined as the areal strain rate (1/A dA/dt), through two rounds of division (Fig.…”

“…In E. coli, PBP2 was previously observed to localize as dispersed puncta and at septa during division (9). PBP2 concentration affects cell size (10), and overexpression is lethal (11). Although MreB binds to the cytoplasmic face of the inner membrane, PBP2 is a transmembrane protein with a cytoplasmic N-terminus and a periplasmic domain required for transpeptidase activity.…”

Interaction of Penicillin-Binding Protein 2 with Soluble Lytic Transglycosylase B1 inPseudomonas aeruginosa

“…1) (18,19), suggesting that they may represent binding partners. To test this possibility, recombinant SltB1 was used as the ligand in affinity chromatography of P. aeruginosa preparations.…”

“…Cells of E. coli BL21 harboring pNBAC258-2 or pACKD16 were induced to overproduce P. aeruginosa SltB1-His 6 or sPBP2-His 6 , respectively, for purification as described previously (5,18). Membrane protein extraction from a 10-liter culture of P. aeruginosa PAO1 grown to mid-exponential phase (optical density at 600 nm, ϳ 0.6) was performed using a procedure adapted from Vollmer et al (34).…”

“…Consequently, each of the collected fractions were assayed specifically from the presence of PBPs by using both biotinylated ampicillin as described previously (18,19) and [phenyl-4(n)- 3 H]benzylpenicillin (25 Ci/mmol and 1 mCi/ml; TRK 779; GE Healthcare) according to the method described by Spratt (30,31) as modified by Preston et al (25). Prior to autoradiography of the latter, the gels were soaked in Amplify Scintillant (GE Healthcare) for 15 to 30 min and dried in vacuo on Whatman filter paper.…”