Escherichia coli mutants lacking all possible combinations of eight penicillin binding proteins: Viability, characteristics, and implications for peptidoglycan synthesis

The penicillin binding proteins (PBPs) synthesize and remodel peptidoglycan , the structural component of the bacterial cell wall. Much is known about the biochemistry of these proteins, but little is known about their biologi cal roles. To better understand the contributions these proteins make to th e physiology of Escherichia coli, we constructed 192 mutants from which eig ht PBP genes were deleted in every possible combination. The genes encoding PBPs 1a, 1b, 4, 5, 6, and 7, AmpC, and AmpH were cloned, and from each gen e an internal coding sequence was removed and replaced with a kanamycin res istance cassette flanked by two res sites from plasmid RP4. Deletion of ind ividual genes aas accomplished by transferring each interrupted gene onto t he chromosome of E. coli via lambda phage transduction and selecting for ka namycin-resistant recombinants. Afterwards, the kanamycin resistance casset te was removed from each mutant strain by supplying ParA resolvase in trans , yielding a strain in which a long segment of the original PBP gene was de leted and replaced by an 8-bp res site. These kanamycin-sensitive mutants w ere used as recipients in further rounds of replacement mutagenesis, result ing in a set of strains lacking from one to seven PBPs. In addition, the da cD gene was deleted from two septuple mutants, creating strains lacking eig ht genes. The only deletion combinations not produced were those lacking bo th PBPs 1a and 1b because such a combination is lethal. Surprisingly, all o ther deletion mutants were viable even though, at the extreme, 8 of the 12 known PBPs had been eliminated. Furthermore, when both PBPs 2 and 3 were in activated by the beta-lactams mecillinam and aztreonam, respectively, sever al mutants did not lyse but continued to grow as enlarged spheres, so that one mutant synthesized osmotically resistant peptidoglycan when only 2 of 1 2 PBPs (PBPs 1b and 1c) remained active. These results have important impli cations for current models of peptidoglycan biosynthesis, for understanding the evolution of the bacterial sacculus, and for interpreting results deri ved by mutating unknown open reading frames in genome projects. In addition , members of the set of PBP mutants will provide excellent starting points for answering fundamental questions about other aspects of cell wall metabo lism.