Escherichia coli Mutants Lacking All Possible Combinations of Eight Penicillin Binding Proteins: Viability, Characteristics, and Implications for Peptidoglycan Synthesis

Abstract

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 biological roles. To better understand the contributions these proteins make to the physiology ofEscherichia coli, we constructed 192 mutants from which eight 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 gene an internal coding sequence was removed and replaced with a kanamycin resistance cassette flanked by two ressites from plasmid RP4. Deletion of individual genes was accomplished by transferring each interrupted gene onto the chromosome of E. coli via λ phage transduction and selecting for kanamycin-resistant recombinants. Afterwards, the kanamycin resistance cassette 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 deleted and replaced by an 8-bpres site. These kanamycin-sensitive mutants were used as recipients in further rounds of replacement mutagenesis, resulting in a set of strains lacking from one to seven PBPs. In addition, thedacD gene was deleted from two septuple mutants, creating strains lacking eight genes. The only deletion combinations not produced were those lacking both PBPs 1a and 1b because such a combination is lethal. Surprisingly, all other 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 inactivated by the β-lactams mecillinam and aztreonam, respectively, several mutants did not lyse but continued to grow as enlarged spheres, so that one mutant synthesized osmotically resistant peptidoglycan when only 2 of 12 PBPs (PBPs 1b and 1c) remained active. These results have important implications for current models of peptidoglycan biosynthesis, for understanding the evolution of the bacterial sacculus, and for interpreting results derived 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 metabolism.

Role of penicillin-binding protein 1b in competitive stationary-phase survival ofEscherichia coli

“…Removal of Cam R resistance cassettes was achieved by transformation of each strain with a temperature-sensitive plasmid expressing FLP recombinase (Datsenko & Wanner, 2000). For complementation experiments, PBP mutant SF2341 (mrcB) was transformed with plasmid pSAD426-11, which expresses mrcB, or the plasmid pSAD444-1, which carries a mutant variant of mrcB (Denome et al, 1999).…”

“…Two of these proteins, PBP2 (mrdA) and PBP3 (ftsI), are essential for normal growth (Spratt, 1975) and are not further characterized in this study. Although the physiological functions of PBPs 1a, 1b, and 5 are fairly well understood (Denome et al, 1999;Nelson & Young, 2000), the biological roles of the remaining PBPs are not fully determined, even though most of their specific in vitro biochemical activities have been elucidated. Briefly, PBPs 1a (mrcA) and 1b (mrcB) are transglycosylases and transpeptidases (Ghuysen & Dive, 1994); PBP1c is a close homolog to PBPs 1a and 1b but has not been completely characterized (Schiffer & Holtje, 1999); PBPs 4, 5, 6, and DacD are carboxypeptidases (Matsuhashi et al, 1979;Amanuma & Strominger, 1980;Korat et al, 1991;Baquero et al, 1996); PBPs 4 and 7 are endopeptidases (Korat et al, 1991;Romeis & Holtje, 1994); AmpC is a b-lactamase, and AmpH binds many b-lactams, though it has not been shown to possess demonstrable b-lactamase activity (Henderson et al, 1997).…”

“…Although E. coli maintains 12 PBP genes, many of the gene products have overlapping biochemical functions and seem to be nonessential for exponential phase growth. In fact, an E. coli mutant lacking eight of 12 PBPs is viable (Denome et al, 1999). Perhaps individual PBPs play more important roles in natural environments, where cells compete for limited carbon and energy sources, encounter antimicrobial agents, and are subjected to a variety of other stresses.…”

A D, D‐carboxypeptidase is required for Vibrio cholerae halotolerance

“…Helicobacter pylori, Caulobacter crescentus) (Ghosh et al, 2008) do not contain obvious DD-CPase homologues. In E. coli, individual deletion of most LWM PBP genes does not impair cell growth or alter cell morphology (Denome et al, 1999). However, the absence of penicillin-binding protein 5 (PBP5; encoded by dacA), E. coli's principal DD-CPase, does lead to subtle changes in cell shape under some conditions, although it does not impair cell proliferation (Nelson and Young, 2000).…”

Moderate deacylation efficiency of DacD explains its ability to partially restore beta-lactam resistance inEscherichia coliPBP5 mutant

“…coli CS109 (K12 variant) (Denome et al, 1999) was used for sDacD gene amplification. Unless otherwise specified, enzymes for molecular analysis were purchased from New England Biolabs (Ipswich, MA) and other reagents from Sigma-Aldrich, (St. Louis, MO).…”