Overproduction of Inactive Variants of the Murein Synthase PBP1B Causes Lysis in Escherichia coli

Abstract

Penicillin-binding protein 1B (PBP1B) of Escherichia coli is a bifunctional murein synthase containing both a transpeptidase domain and a transglycosylase domain. The protein is present in three forms (␣, ␤, and ␥) which differ in the length of their N-terminal cytoplasmic region. Expression plasmids allowing the production of native PBP1B or of PBP1B variants with an inactive transpeptidase or transglycosylase domain or both were constructed. The inactive domains contained a single amino acid exchange in an essential active-site residue. Overproduction of the inactive PBP1B variants, but not of the active proteins, caused lysis of wild-type cells. The cells became tolerant to lysis by inactive PBP1B at a pH of 5.0, which is similar to the known tolerance for penicillin-induced lysis under acid pH conditions. Lysis was also reduced in mutant strains lacking several murein hydrolases. In particular, a strain devoid of activity of all known lytic transglycosylases was virtually tolerant, indicating that mainly the lytic transglycosylases are responsible for the observed lysis effect. A possible structural interaction between PBP1B and murein hydrolases in vivo by the formation of a multienzyme complex is discussed.

Outer Membrane Vesicle Production by Escherichia coli Is Independent of Membrane Instability

“…Transpeptidases and transglycosylases are critical in growth and crosslinking of the peptidoglycan. This mutant is likely to produce an inactive PBP1b truncate, and, in fact, MK7E17 has integrity defects that resemble those observed in E. coli upon overexpression of inactive versions of PBP1b (18). Our SDS-PAGE analysis of MK7E17 supernatant material suggests that the bulges observed previously are probably composed primarily of OM (data not shown).…”

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

“…With time, the continued lytic activity would eventually lead to bacteriolysis. While the proposal presented above is highly speculative and other explanations may exist, it would also account for the lysis observed when inactive variants of PBP 1b, which is known to complex with PBP 3 and MltA among other proteins (see reference 1 and references therein), are overproduced in E. coli (28). Again, the excessive amounts of PBP 1b would not permit the formation of complete biosynthetic complexes but nonetheless would attract lytic transglycosylase(s) to eventually cause the autolysis.…”

Peptidoglycan Hydrolases of Escherichia coli

“…Furthermore, the simultaneous use of two ␤-lactams targeting different HMM PBPs has a synergistic effect that also causes rapid lysis, even though each drug is at a sublytic concentration (85). Yet another kind of interference with PG polymerization leading to lysis was reported when inactive variants of the polymerase PBP1b were overproduced in cells with a normal level of wildtype PBP1b (171).…”