Reconstruction of Escherichia coli mrcA (PBP 1a) Mutants Lacking Multiple Combinations of Penicillin Binding Proteins

Abstract

Previously, we constructed a set of mutants from which eight penicillin binding protein (PBP) genes were deleted in 192 combinations from Escherichia coli (S. A. Denome, P. K. Elf, T. A. Henderson, D. E. Nelson, and K. D. Young, J. Bacteriol. 181:3981-3993, 1999). Although these mutants were constructed correctly as determined by restriction mapping and the absence of relevant protein products, we recently discovered by PCR mapping that strains from which mrcA (PBP 1a) was deleted were also missing two neighboring genes of unknown function (yrfE and yrfF). We created a new deletion mutation in mrcA and reconstructed 63 strains lacking PBP 1a and other PBP mutant combinations. The new mrcA mutants do not exhibit mucoidy, phage resistance, temperature sensitivity, growth rate defects, or antibiotic resistance, suggesting that these phenotypes require the loss of either yrfE or yrfF alone or in combination with the absence of multiple PBPs.Four high-molecular-weight penicillin binding proteins (PBPs) of Escherichia coli (PBPs 1a, 1b, 2, and 3) are responsible for synthesizing and assembling the peptidoglycan sacculus that forms the rigid bacterial cell wall (5, 6). However, E. coli also possesses at least seven low-molecular-weight PBPs (PBPs 4, 5, 6, and 7 and DacD, AmpC, and AmpH), the biological functions of which are either poorly characterized or completely unknown (2, 5, 6).To address this question of physiological function, we constructed a set of multiply mutated strains in which one to seven PBPs were deleted in every viable combination (2). At the time of construction, each strain was tested by restriction mapping and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to confirm that the correct genes and protein products had been deleted. Recently, we found we were unable to PCR amplify the mutated mrcA gene (encoding PBP 1a) from chromosomal preparations when using oligonucleotide primers hybridizing to sequences just upstream and downstream of the putative deletion endpoints. Primers further away from the mutated site did give an amplification product (data not shown), suggesting that a larger fragment had been deleted than was reported previously. DNA sequencing confirmed that one open reading frame (ORF) (yrfE) and the promoter and 5Ј end of a second open reading frame (yrfF) were deleted in addition to most of the mrcA gene (data not shown). The extent of the deletion is pictured schematically in Fig. 1B. Thus, every strain designated as ⌬mrcA in our previous publication (2) is actually a ⌬(mrcA-yrfE-yrfF) deletion. All other PBP gene deletions were correct as reported (data not shown).The data in Fig. 1 illustrates why the deletion was not correctly characterized earlier. When creating the original mrcA mutation we observed a single XhoI-BspDI DNA fragment, leading us to believe that one BspDI site existed in the cloned segment. However, after the mutants were constructed, the complete genomic sequence of E. coli (1) revealed there were three BspDI sites-two so close together t...

Contribution of Membrane-Binding and Enzymatic Domains of Penicillin Binding Protein 5 to Maintenance of Uniform Cellular Morphology of Escherichia coli

“…Because we recently discovered that strain CS701-1 was missing two non-PBP genes in addition to its documented PBP mutations (17), we introduced the plasmids into strain CS703-1, which is missing the same PBPs as CS701-1 but has no other mutations (17). CS703-1 exhibited the same morphological abnormalities as CS701-1, and the PBP 5/6 and PBP 5/DacD hybrid proteins reversed these effects (data not shown).…”

“…E. coli XL-1 Blue (recA endA hsdR supE thi recA gyrA relA lac) (Stratagene, La Jolla, Calif.) and E. coli DH5␣ (deoR recA endA hsdR supE thi gyrA relA) were used as hosts for constructing recombinant plasmids. Strains used in the morphological experiments were derived from CS109 (W1485 rpoS rph) (C. Schnaitman), as follows: CS604-2 (CS109 ⌬[mrcAyrfE-yrfF] ⌬dacB ⌬dacC ⌬pbpG ⌬ampC ⌬ampH); CS701-1 (CS109 ⌬[mrcA-yrfEyrfF] ⌬dacB ⌬dacA ⌬dacC ⌬pbpG ⌬ampC ⌬ampH); and CS703-1 (CS109 ⌬mrcA ⌬dacB ⌬dacA ⌬dacC ⌬pbpG ⌬ampC ⌬ampH) (5,17). PBP genes were expressed under the control of the arabinose promoter of pBAD18-CAM, provided by J. Beckwith (7).…”

Endopeptidase Penicillin-Binding Proteins 4 and 7 Play Auxiliary Roles in Determining Uniform Morphology ofEscherichia coli

“…a All strains were derived from E. coli CS109. Strain names with no alphabetical prefix are CS strains and have been described previously (4,18,23). b Numbers refer to the respective PBPs (e.g., 1a ϭ PBP 1a).…”

“…The amplified PCR products were transferred by electroporation into E. coli KM-32 (21), where they were incorporated into the chromosome via homologous recombination mediated by the phage lambda recombination system as described previously (18,21,22). Kanamycin-resistant colonies were selected, and colonies having the correct insertions were identified by diagnostic PCR of chromosomal DNA.…”

Elevated levels of σ S inhibit biofilm formation in Escherichia coli: a role for the Rcs phosphorelay

“…Phospho-RcsB (RcsB-P) then binds to DNA and affects the expression of an extensive Rcs regulon Ferrières & Clarke, 2003;Hagiwara et al, 2003;Takeda et al, 2001). In addition, three accessory proteins, RcsA, RcsF and YrfF (IgaA), participate in the modulation of the inputs and outputs of this phosphorelay Meberg et al, 2001;Stout et al, 1991;Tierrez & Garcia-del Portillo, 2004).…”