PBP5 complementation of a PBP3 deficiency in Enterococcus hirae

Abstract

The low susceptibility of enterococci to beta-lactams is due to the activity of the low-affinity penicillin-binding protein 5 (PBP5). One important feature of PBP5 is its ability to substitute for most, if not all, penicillin-binding proteins when they are inhibited. That substitution activity was analyzed in Enterococcus hirae SL2, a mutant whose pbp5 gene was interrupted by the nisRK genes and whose PBP3 synthesis was submitted to nisin induction. Noninduced SL2 cells were unable to divide except when plasmid-borne pbp5 genes were present, provided that the PBP5 active site was functional. Potential protein-protein interaction sites of the PBP5 N-terminal module were mutagenized by site-directed mutagenesis. The T167-L184 region (designated site D) appeared to be an essential intramolecular site needed for the stability of the protein. Mutations made in the two globular domains present in the N-terminal module indicated that they were needed for the suppletive activity. The P197-N209 segment (site E) in one of these domains seemed to be particularly important, as single and double mutations reduced or almost completely abolished, respectively, the action of PBP5.

FIG. 1.

SDS-PAGE and fluorogram of the membrane-bound PBPs of different E. hirae strains. (A) Membranes were labeled for 60 min with 100 μM [¹⁴C]PenG. Lane 1, purified soluble PBP5; lane 2, R40; lane 3, ATCC 9790; lane 4, SL1. (B) Membranes (pretreated for 15 min with 10 μM nonradioactive PenG) were incubated for 60 min with 100 μM [¹⁴C]PenG for specific labeling of PBP5_Eh. Lane 1, purified soluble PBP5_Efm; lane 2, SL1/pbp5_Eh; lane 3, SL1/pbp5(K172N/R173N); lane 4, SL1/pbp5(K199Q/K203Q); lane 5, SL1/pbp5(K203Q); lane 6, SL1/pbp5(5K-S); lane 7, SL1/pbp5(R173N); lane 8, SL1/pbp5(K199Q).

FIG. 2.

SDS-PAGE and fluorogram of the 77-kDa membrane-bound PBP3 of nisin-induced SL1/pbp3i cells. Lane 1, noninduced cells; lanes 2 to 5, nisin-induced cells (10, 20, 40, and 80 ng/ml, respectively). Membranes (500 μg) were labeled for 10 min with 100 μM [¹⁴C]PenG. Part of the gel shows PBPs 2 to 4. Under these conditions, PBP2 was barely visible.

FIG. 3.

Growth curves of SL2 cells (cell counts) transformed with wild-type and mutated pbp5_Eh genes. Mean values of four to six independent cultures were used. Shown are induced (○) and noninduced (•) SL2, induced (□) and noninduced (▪) SL2/pbp5_Eh, noninduced SL2/pbp5(K199Q/K203Q) (×), SL2/pbp5(K203Q) (⋄), SL2/pbp5(5K-S) (⧫), and SL2/pbp5(K199Q) (▴).

FIG. 4.

SDS-PAGE and fluorogram of the membrane-bound PBP5_Eh of E. hirae SL2 cells expressing pbp5_Eh variants. (A) Membranes were labeled for 60 min with 100 μM [¹⁴C]PenG. Lane 1, noninduced SL2; lane 2, induced SL2; lane 3, noninduced SL2/pbp5_Eh; lane 4, induced SL2/pbp5_Eh. (B) Membranes were labeled for 10 min with 10 μM [¹⁴C]PenG. Lane 1, noninduced SL2/pbp5_Eh; lane 2, induced SL2/pbp5_Eh. Under these conditions, labeling of PBP5_Eh is generally low.

FIG. 5.

Model presenting the principal putative protein-protein interaction sites identified in the N-terminal module of the PBP5 structure extending from residues 39 to 353 (53). Conserved motifs of class B PBPs are shown as the thickened gray segments (roman numerals). The sequences and the positions of the predicted interaction sites are represented by letters and black segments. The numbers and positions of substituted amino acids are indicated by black circles. Nt and Ct, N and C termini.