Penicillin-binding protein 2 inactivation in Escherichia coli results in cell division inhibition, which is relieved by FtsZ overexpression

Abstract

Aminoacyl-tRNA synthetase mutants of Escherichia coli are resistant to amdinocillin (mecillinam), a P-lactam antibiotic which specifically binds penicillin-binding protein 2 (PBP2) and prevents cell wall elongation with concomitant cell death. The leuS(Ts) strain, in which leucyl-tRNA synthetase is temperature sensitive, was resistant to amdinocillin at 37°C because of an increased guanosine 5'-diphosphate 3'-diphosphate (ppGpp) pool resulting from partial induction of the stringent response, but it was sensitive to amdinocillin at 25°C. We constructed a leuS(Ts) A(rod4-pbpA)::KMr strain, in which the PBP2 structural gene is deleted. This strain grew as spherical cells at 37°C but was not viable at 25°C. After a shift from 37 to 25°C, the ppGpp pool decreased and cell division was inhibited; the cells slowly carried out a single division, increased considerably in volume, and gradually lost viability. The cell division inhibition was reversible when the ppGpp pool increased at high temperature, but reversion required de novo protein synthesis, possibly of septation proteins. The multicopy plasmid pZAQ, overproducing the septation proteins FtsZ, FtsA, and FtsQ, conferred amdinocillin resistance on a wild-type strain and suppressed the cell division inhibition in the leuS(Ts) A(rodA-pbpA)::KMr strain at 25°C. The plasmid pAQ, in which theftsZ gene is inactivated, did not confer amdinocillin resistance. These results lead us to hypothesize that the nucleotide ppGpp activatesftsZ expression and thus couples cell division to protein synthesis.

Analysis of the effect of ppGpp on the ftsQAZ operon in Escherichia coli

“…However, sixfold overproduction of FtsZ (6 × 10 ¹5 M IPTG) permitted essentially all cells to grow in the presence of mecillinam (Table 1). This corroborates our previous observation that pZAQ confers mecillinam resistance but pAQ does not (Vinella et al, 1993).…”

“…We originally observed that the multicopy plasmid pZAQ, which causes overproduction of FtsQ, FtsA and FtsZ from their natural promoters, conferred mecillinam resistance on its host, whereas pAQ, lacking a functional ftsZ gene, did not (Vinella et al, 1993). We wished to see whether amplification of FtsZ alone was sufficient to confer mecillinam resistance.…”

“…When added to a wild-type E. coli culture, the cells become spherical (Lund and Tybring, 1972), and, although the optical density increases for many generations, cell division stops rather quickly, at least in rich medium (Vinella et al, 1993). The loss of rod shape in the presence of mecillinam implies that PBP2 is required for lateral elongation of the peptidoglycan cell wall (Spratt, 1975).…”

“…We have shown that the division block caused by the inhibition or total loss of PBP2 can be relieved by overexpressing the cell division genes ftsQ, ftsA and ftsZ (Vinella et al, 1993), which belong to a complex operon (Yi et al, 1985). The FtsZ protein is known to form a ring at mid-cell (Bi and Lutkenhaus, 1991;Addinall et al, 1996;Lutkenhaus and Addinall, 1997) to which it attracts FtsA and other division proteins to form a large complex responsible for invaginating the membrane and synthesizing the septum.…”

“…We speculated that the mecillinam resistance observed in the presence of an increased ppGpp pool reflected a positive effect of this nucleotide on the transcription of the ftsQAZ operon (Vinella et al, 1993). This operon has five promoters (Fig.…”