Penicillin binding protein 2 is dispensable in Escherichia coli when ppGpp synthesis is induced.

Abstract

Mecillinam, a beta‐lactam antibiotic which specifically inactivates penicillin binding protein 2 (PBP2) in Escherichia coli, prevents lateral cell wall elongation, inducing spherical morphology and cell death. Two mecillinam resistant mutants, lov‐1 and lovB, both able to dispense entirely with PBP2, are shown here to be affected in the aminoacyl‐tRNA synthetase genes argS and alaS, respectively. Although the argS and alaS mutants grow slowly, we show that there is no correlation between mecillinam resistance and either growth rate or translation speed. A role of the ribosomes in mecillinam sensitivity, suggested by our earlier report that the lov‐1 mutation is suppressed by certain rpsL(StrR) alleles affecting ribosomal protein S12, is supported by the present observation that a pseudo‐streptomycin dependent mutant is mecillinam resistant in the presence of streptomycin. The argS and alaS mutants have high pools of the nucleotide ppGpp (effector of the stringent response) and the mecillinam resistance of both mutations is suppressed by a relA mutation, inactivating the ribosome‐associated ppGpp synthetase and preventing ppGpp synthesis in response to aminoacyl‐tRNA starvation. Furthermore, a ptacrelA' multicopy plasmid makes a wild type strain mecillinam resistant. The effect of ppGpp is probably mediated by RNA polymerase, since sublethal doses of the polymerase inhibitor rifampicin suppress mecillinam resistance in argS, alaS and ptacrelA'‐bearing strains. We conclude that ppGpp regulates the transcription of a gene whose product is involved in mecillinam sensitivity, possibly as part of a chain of interacting elements which coordinate ribosomal activity with that of the PBPs.

A mutant cysteinyl-tRNA synthetase affecting timing of chromosomal replication initiation in B. subtilis and conferring resistance to a protein kinase C inhibitor.

“…Interestingly, the spnA495 mutation was shown by recombinational analysis to map in a 400 bp region (S.Casaregola et al, manuscript in preparation) which corresponds to the catalytic part of the cysteinyl-tRNA synthetase (CysRS). This region binds the acceptor stem of the tRNA as defined for the E. coli glutamyltRNA synthetase by Rould et al (1989) Vinella et al (1992) recently described mutations in alanyl and arginyl-tRNA synthetases in E. coli which confer resistance to mecillinam, an inhibitor of lateral cell wall growth. It was proposed that these mutants, which produce higher levels of ppGpp, a product of the Stringent Response, are defective in the co-ordination of cell surface growth and mass increase.…”

Biochemistry and Comparative Genomics of SxxK Superfamily Acyltransferases Offer a Clue to the Mycobacterial Paradox: Presence of Penicillin-Susceptible Target Proteins versus Lack of Efficiency of Penicillin as Therapeutic Agent

“…As a corollary, peptide segments other than the conserved motifs must serve as recognition sites specifying the morphogenetic apparatus to which Eco2 and Eco3 attach. Cell division and viability in the absence of Eco2 but in the presence of Eco3 are restored by increasing the pool of ppGpp or the level of FtsQAZ (115,238). Hence Eco2, but not Eco3, is dispensable in particular genetic backgrounds.…”

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

“…We therefore compared the concentration of FtsZ protein in a wild-type strain with that in two mecillinam-resistant derivatives, one carrying the argS201 mutation and the other bearing the plasmid pGC401, which has a p lac -relAЈ construction and permits IPTG induction of ppGpp synthetase activity; the latter strain, cultivated at 6 × 10 ¹5 M IPTG, has a twofold increase in its ppGpp pool and is resistant to mecillinam (Joseleau-Petit et al, 1994;Vinella et al, 1992). The concentration of FtsZ protein was determined in exponential-phase cultures of these three strains, using quantitative immunoblots as described above.…”