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 of Escherichia 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 res sites from plasmid RP4. Deletion of individual genes was accomplished by transferring each interrupted gene onto the chromosome of E. coli via lambda 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-bp res 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, the dacD 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 beta-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.

FIG. 1

Cloned wild-type PBP genes and locations of internal deletions. Underneath the PBP name is listed the DNA fragment that was isolated and subcloned from the specified phage in the Kohara et al. genomic library (18). Each DNA fragment was subcloned into the vector pBCSK⁻ (Stratagene) except for PBP 4, which was cloned into pBCKS⁻, and AmpH, which was cloned into pBCSK⁺. The light line represents the cloned DNA fragment, and the dark arrow above the line represents the position, length, and direction of the reading frame for an individual PBP gene. The heavy rectangle represents the internal DNA fragment that was deleted from the coding sequence and replaced with a HindIII linker. Names of the plasmids carrying these genes and deletions are listed at the right. Except for PBPs 5 and 6, each construct is represented by two plasmids: the first plasmid carries the entire DNA fragment and includes the cloned wild-type gene; the second plasmid carries the DNA fragment from which the internal segment was deleted and replaced by a HindIII linker (designated by the “Δ” prefix). For PBPs 5 and 6, only the deletion plasmids are represented because the wild-type genes were not cloned (Materials and Methods). Letters denote restriction enzyme sites (A, ApaI; B, BamHI; Bb, BstBI; Bg, BglI; Bs, BstEII; D, BspDI, E, EcoRI; H, HindIII; K, KpnI; M, MluI; N, NdeI; P, PstI; RV, EcoRV; S, SalI; Sm, SmaI; St, StuI; T, BstXI; W, BsiWI; X, XhoI). A letter enclosed in parentheses denotes a site destroyed during construction of the plasmid; letters in parentheses at the extreme ends of the entire DNA fragment were sites in the cloning vector that were destroyed, sometimes including neighboring sites in the multiple cloning site (data not shown). Sites that define the ends of the internal deletions were destroyed and replaced by a HindIII linker. To create marked genes for transfer to the E. coli chromosome, a res-npt-res (kanamycin-resistance) gene cassette from pCK155 (Materials and Methods) was inserted into the HindIII site of each deletion plasmid.

FIG. 2

Schematic illustration of the steps by which multiple genes were deleted from the chromosome of E. coli. Kan^R, kanamycin resistance.

FIG. 3

Pedigree of the PBP deletion mutants. The chart reflects the order in which genes were deleted from each strain. The original parental background was E. coli CS109. Mutants from which a single gene was deleted have names with numbers in the teens; mutants from which two genes were deleted have names with numbers in the 200’s; mutants with three genes deleted are numbered in the 300’s, etc. In the chart, the strain number is given but the “CS” prefix for each strain name was omitted for clarity. Underneath the number of each strain is listed the PBP gene that was deleted from the preceding parent to create that strain. Therefore, to determine the total complement of genes that were deleted from a particular strain, simply move “upward,” following the arrows backward to the ultimate parent, CS109, noting which genes were deleted in that lineage. More strains are shown than the minimum 192 required to make all possible mutants because some combinations were constructed by deleting genes in different orders. The mutants are listed in numerical order in Table 1, which includes a list of the genes deleted from each strain.

FIG. 4

SDS-PAGE of ¹²⁵I-penicillin-X-labeled PBPs from selected mutants. PBPs were labeled, separated by SDS-PAGE, and visualized by autoradiography as described elsewhere (11). (A) E. coli mutants from which a single PBP gene was deleted (deleted PBPs in parentheses): lane 1, CS109 (wild-type parent); lane 2, CS9-19 (PBP 7), lane 3, CS11-2 (PBP 4); lane 4, CS12-7 (PBP 5); lane 5, CS13-2 (PBP 1a); lane 6, CS14-2 (AmpC); lane 7, CS15-1 (AmpH); lane 8, CS16-1 (PBP 1b); lane 9, CS17-1 (PBP 6). (B) Representative subset of E. coli mutants from which a progressive number of PBPs were deleted: lane 1, CS109 (wild-type parent); lane 2, CS17-1 (PBP 6); lane 3, CS211-2 (PBPs 5 and 6); lane 4, CS322-1 (PBPs 4, 5, and 6); lane 5, CS446-1 (PBPs 4, 5, 6, and 7); lane 6, CS531-3 (PBPs 4, 5, 6, and 7 and AmpH); lane 7, CS612-1 (PBPs 4, 5, 6, and 7, AmpH, and AmpC); lane 8, CS702-1 (PBPs 1b, 4, 5, 6, and 7, AmpH, and AmpC); lane 9, CS701-1 (PBPs 1a, 4, 5, 6, and 7, AmpH, and AmpC). Note that in Fig. 4B, in lanes 8 and 9, some very faint bands equal to or smaller in size than PBP 4 are artifacts due to spillover from an adjacent lane (not shown); other gels of the samples in lanes 8 and 9 showed no such bands.

FIG. 5

Lysis of PBP mutants by mecillinam. Cultures of E. coli mutants were grown in LB broth at 37°C, and the absorbance at 550 nm was measured. At time zero, mecillinam (10 μg/ml, final concentration) was added to each culture. Labels to the right of the curves list the PBPs that were deleted from the strain used to generate the data. (A) wt (wild type), CS109; 1b, CS16-1; 1b 5, CS224-2. (B) wt, CS109; 1b C (AmpC), CS227-1; 1b 5 C, CS309-1. (C) wt, CS109; 1b 6 7 C, CS436-2; 1b 5 6 7 C, CS526-1.

FIG. 6

Growth of PBP mutants after simultaneous addition of mecillinam and aztreonam. Cultures of E. coli were grown in LB broth at 37°C, and the absorbance at 550 nm was measured. At time zero, aztreonam and mecillinam (Azt+Mec) were added to give a 10-μg/ml final concentration of each. Absorbance values for mutants CS403-3 and CS801-4 remained stable to at least 200 min (data not shown). Circles, CS109 (parental wild type [wt]); squares, CS801-4 (PBPs 1a, 4, 5, 6, and 7, AmpH, AmpC, and DacD deleted); triangles, CS403-3 (PBPs 1a, 4, and 7 and AmpH deleted).

FIG. 7

Morphology of E. coli CS403-3 after simultaneous addition of mecillinam and aztreonam. E. coli CS403-3 was grown in LB broth at 37°C to an absorbance at 550 nm of 0.2, at which point both mecillinam and aztreonam were added to give a final concentration of 10 μg of each per ml. At the times indicated, samples were withdrawn for phase-contrast microscopy. All photographs were captured with a cold charge-coupled device camera at a magnification of ×1,000, and all images represent the same relative magnification so that direct comparisons of cell size can be made between time points. The size of an individual E. coli cell before addition of antibiotics was approximately 0.8 by 1.5 μm (see cells at time zero) and serves as a measure of the size of cells at other time points. Arrows indicate ghost cells that are emptied of cytoplasmic contents.