Differential responses of Escherichia coli cells expressing cytoplasmic domain mutants of penicillin-binding protein 1b after impairment of penicillin-binding proteins 1a and 3

Abstract

Penicillin-binding protein 1b (PBP1b) is the major high-molecular-weight PBP in Escherichia coli. Although it is coded by a single gene, it is usually found as a mixture of three isoforms which vary with regard to the length of their N-terminal cytoplasmic tail. We show here that although the cytoplasmic tail seems to play no role in the dimerization of PBP1b, as was originally suspected, only the full-length protein is able to protect the cells against lysis when both PBP1a and PBP3 are inhibited by antibiotics. This suggests a specific role for the full-length PBP1b in the multienzyme peptidoglycan-synthesizing complex that cannot be fulfilled by either PBP1a or the shorter PBP1b proteins. Moreover, we have shown by alanine-stretch-scanning mutagenesis that (i) residues R(11) to G(13) are major determinants for correct translocation and folding of PBP1b and that (ii) the specific interactions involving the full-length PBP1b can be ascribed to the first six residues at the N-terminal end of the cytoplasmic domain. These results are discussed in terms of the interactions with other components of the murein-synthesizing complex.

FIG. 1

Amino-terminal sequences of the PBP1b mutants. Amino acid sequence of the cytoplasmic domain of PBP1bα coded by plasmid pPONB is shown (residues 1 to 64). The beginning of the sequences of the β and γ components are indicated by arrows. Amino acid sequences corresponding to the cytoplasmic domains of the PBP1b mutants (indicated on the right in parentheses) expressed by plasmids used in this study are aligned with the amino-terminal sequence of PBP1bα. Except for methionine, identical amino acids are shown by dashed lines and changed residues are indicated by their corresponding letters.

FIG. 2

α*, β*, γ, and δ forms of PBP1b. (A) Production of the α*, β*, γ, and δ forms of PBP1b. Membrane fractions from QCB1 (15 μg, lane 1) or QCB1 transformed with pPONB (10 μg, lane 2), pM46L (10 μg, lane 3), pR26ΔNt (15 μg, lane 4), pM46ΔNt (15 μg, lane 5), or pR63ΔNt (15 μg, lane 6) were analyzed by Western blotting using the antitag 12CA5 antibody. (B) Binding of 6-APA-FLU by the α*, β*, γ, and δ forms of PBP1b. Membrane fractions (100 μg) from QCB1 (lane 1) or QCB1 transformed with pPONB (lane 2), pM46L (lane 3), pR26ΔNt (lane 4), pM46ΔNt (lane 5), or pR63ΔNt (lane 6) and from MC6-RP1 (lane 7) were incubated with 6-APA-FLU and analyzed by Western blotting with an antifluorescein antibody. 1a, PBP1a mutant; 1b, PBP1b mutant.

FIG. 3

Stability of PBP1b dimers. Membrane fractions prepared from strain QCB1 (50 μg, lane 1) or QCB1 transformed with pPONB (25 μg, lane 2), pM46L (25 μg, lane 3), pR26ΔNt (50 μg, lane 4), pM46ΔNt (50 μg, lane 5), or pR63ΔNt (50 μg, lane 6) were incubated for 10 min in sample buffer with 5% β-mercaptoethanol at room temperature. Samples were subjected to SDS-PAGE and Western blotting with the anti-tag 12CA5 antibody. M, monomeric forms of PBP1b mutants; D, dimeric forms of PBP1b mutants.

FIG. 4

Effects of cephaloridine and aztreonam on the growth of strains expressing the α*, β*, and γ forms of PBP1b. When the OD₅₅₀ reached 0.1 (t = 0), cultures of E. coli MC6-RP1, QCB1 or QCB1 harboring pPONB, pM46L, pR26ΔNt, or pM46ΔNt growing exponentially at 37°C were divided into four subcultures, and antibiotics were added as described in Materials and Methods. Growth was monitored by measuring the OD₅₅₀ at 0, 30, 60, 90, 120, and 180 min. ⧫, Untreated control; ▴, cephaloridine (0.3 μg/ml); ■, aztreonam (0.5 μg/ml); ×, cephaloridine and aztreonam.

FIG. 5

ASS mutants of PBP1bα. (A) Production of the ASS mutants of PBP1bα. Membrane fractions from QCB1 (10 μg, lane 1) or QCB1 transformed with pM46L (10 μg, lane 2), pL46ASS1 (20 μg, lane 3), pL46ASS2 (20 μg, lane 4), pL46ASS3 (10 μg, lane 5), pL46ASS4 (10 μg, lane 6), or pL46ASS5 (10 μg, lane 7) were analyzed by Western blotting using the anti-tag 12CA5 antibody. (B) Binding of 6-APA-FLU by the ASS mutants. Membrane fractions (100 μg) from QCB1 (lane 1) or QCB1 transformed with pM46L (lane2), pL46ASS1 (lane 3), PL46SS2 (lane 4), pL46ASS3 (lane 5), pL46ASS4 (lane 6), or pL46ASS5 (lane 7) and from MC6-RP1 (lane 8) were incubated with 6-APA-FLU and analyzed by Western blotting with an antifluorescein antibody. 1a, PBP1a mutant; 1b, PBP1b mutant.

FIG. 6

Effects of cephaloridine and aztreonam on the growth of strains expressing the ASS mutants of PBP1bα. When OD₅₅₀ reached 0.1 (t = 0), cultures of E. coli QCB1 harboring pM46L, pL46ASS1, pL46ASS2, pL46ASS3, pL46ASS4, or pL46ASS5 growing exponentially at 37°C were divided into four subcultures, and antibiotics were added as described in Materials and Methods. Growth was monitored by measuring the OD₅₅₀ at 0, 30, 60, 90, 120, and 180 min. ⧫, Untreated control; ▴, cephaloridine (0.3 μg/ml); ■, aztreonam (0.5 μg/ml); ×, cephaloridine and aztreonam.