Construction of a mutant of Escherichia coli that has deletions of both the penicillin-binding protein 5 and 6 genes

A mutant of Escherichia coli has been constructed with deletions of the genes encoding penicillin-binding protein 5 (dacA) and penicillin-binding protein 6 (dacC). The construction of this mutant establishes that the complete loss of the two most abundant species of penicillin-binding proteins can be tolerated by E. coli. Moreover, the double deletion mutant had the same growth rate and morphology as an isogenic dacA+ dacC+ strain.     

Deletion of the penicillin-binding protein 5 gene of Escherichia coli.

A strain of Escherichia coli that has a deletion of the entire dacA gene has been constructed. The complete lack of penicillin-binding protein 5 in this strain establishes that the activity of this protein is not essential for the growth of E. coli.     

Molecular cloning of the gene of a penicillin-binding protein supposed to cause high resistance to beta-lactam antibiotics in Staphylococcus aureus.

A novel penicillin-binding protein, PBP-2' (Mr about 75,000), is known to be induced in excessively large amount by most beta-lactam compounds in cells of a clinically isolated strain of Staphylococcus aureus, TK784, that is highly resistant to beta-lactams and also most other antibiotics. This protein has very low affinities to most beta-lactam compounds and has been supposed to be the cause of the resistance of the cells to beta-lactams. A 14-kilobase DNA fragment was isolated from the cells that carried the gene encoding this penicillin-binding protein and also a genetically linked marker that is responsible for the resistance to tobramycin. This DNA was cloned on plasmid pACYC184 and was shown to cause both production of PBP-2' and resistance to tobramycin in Escherichia coli cells. However, the formation of PBP-2' in E. coli was only moderate and was independent of normal inducer beta-lactams. The PBP-2' formed in the E. coli cells showed slow kinetics of binding to beta-lactams similar to that of PBP-2' formed in the original S. aureus cells and gave a similar pattern of peptides to the latter when digested with the proteolytic V8 enzyme of S. aureus.     

Active-site and membrane topology of the DD-peptidase/penicillin-binding protein no. 6 of Enterococcus hirae (Streptococcus faecium) A.T.C.C. 9790.

The membrane-bound 43,000-Mr penicillin-binding protein no. 6 (PBP6) of Enterococcus hirae consists of a 30,000-Mr DD-peptidase/penicillin-binding domain and a approximately 130-residue C-terminal appendage. Removal of this appendage by trypsin proteolysis has no marked effect on the catalytic activity and penicillin-binding capacity of the PBP. Anchorage of the PBP in the membrane appears to be mediated by a short 15-20-residue stretch at the C-terminal end of the appendage. The sequence of the 50-residue N-terminal region of the PBP shows high degree of homology with the sequences of the corresponding regions of the PBPs5 of Escherichia coli and Bacillus subtilis. On this basis the active-site serine residue occurs at position 35 in the enterococcal PBP.     

Activity of penicillin-binding protein 3 from Escherichia coli.

The activity of penicillin-binding protein 3 of Escherichia coli has been studied both in vivo and in ether-permeabilized cells. The peptidoglycan transpeptidase activity of penicillin-binding protein 3 appears to use either nascent or exogenously added UDP-N-acetylmuramyl tripeptide-derived substrates as acceptors. By means of a defilamentation system which elicited the activity of penicillin-binding protein 3 in vivo, the structure of peptidoglycan made by this enzyme has been elucidated. This peptidoglycan, very probably of septal location, contained increased amounts of cross-linked peptidoglycan as well as a higher ratio of tripeptide-containing cross-linked subunits.     

A mutant of Escherichia coli defective in penicillin-binding protein 5 and lacking D-alanine carboxypeptidase IA.

A mutant of Escherichia coli defective in penicillin-binding protein 5 activity was isolated. The mutation (pfv) was shown to be located at 14.0 min on the E. coli chromosome map. Loss of penicillin-binding protein 5 in the pfv mutant was associated with the loss of D-alanine carboxypeptidase IA activity and increased sensitivity to beta-lactam antibiotics. We conclude that penicillin-binding protein 5 catalyzes the major D-alanine carboxypeptidase IA activity and that the enzyme activity, in vivo, protects E. coli cells from killing by low inhibitory concentrations of beta-lactam antibiotics.     

Identification of a new mutation in Escherichia coli that suppresses a pbpB (Ts) phenotype in the presence of penicillin-binding protein 1B

Analysis of the functional role of penicillin-binding protein 1B (PBP1B) of Escherichia coli led us to find a new mutation able to suppress thermosensitive growth of the pbpB2158(Ts) mutant strain, which harbors a thermosensitive PBP3 protein only in the presence of a ponB+ background. The mutation, originally isolated in a strain with a high dosage of PBP1B, could also suppress the pbpB(Ts) phenotype when a single copy of the ponB gene was introduced. These results clearly give further support to the implication of PPB1B in the septation process in Escherichia coli.     

An amino acid substitution that blocks the deacylation step in the enzyme mechanism of penicillin-binding protein 5 of Escherichia coli

A mutant of Escherichia coli has been described that produces an altered form of penicillin-binding protein 5 which still binds penicillin but is unable to catalyse the release of the bound penicilloyl moiety. We show that the mutation is caused by a single nucleotide transition that results in a change from glycine at residue 105 of the wild-type sequence of penicillin-binding protein 5 to aspartate in the mutant.     

Synthesis of penicillin-binding protein 6 by stationary-phase Escherichia coli.

The level of penicillin-binding protein 6, a D-alanine carboxypeptidase I, was found to be 2- to 10-fold higher in stationary-phase cells than in exponentially growing cells of Escherichia coli. This increase appeared to be due to de novo synthesis rather than to an unmasking of preexisting material. There was no comparable change in the amount of any of the other six penicillin-binding proteins.     

Nucleotide sequences of the pbpX genes encoding the penicillin-binding proteins 2x from Streptococcus pneumoniae R6 and a cefotaxime-resistant mutant, C506

Development of penicillin resistance in Streptococcus pneumoniae is due to successive mutations in penicillin-binding proteins (PBPs) which reduce their affinity for beta-lactam antibiotics. PBP2x is one of the high-Mr PBPs which appears to be altered both in resistant clinical isolates, and in cefotaxime-resistant laboratory mutants. In this study, we have sequenced a 2564 base-pair chromosomal fragment from the penicillin-sensitive S. pneumoniae strain R6, which contains the PBP2x gene. Within this fragment, a 2250 base-pair open reading frame was found which coded for a protein having an Mr of 82.35kD, a value which is in good agreement with the Mr of 80-85 kD measured by SDS-gel electrophoresis of the PBP2x protein itself. The N-terminal region resembled an unprocessed signal peptide and was followed by a hydrophobic sequence that may be responsible for membrane attachment of PBP2x. The corresponding nucleotide sequence of the PBP2x gene from C504, a cefotaxime-resistant laboratory mutant obtained after five selection steps, contained three nucleotide substitutions, causing three amino acid alterations within the beta-lactam binding domain of the PBP2x protein. Alterations affecting similar regions of Escherichia coli PBP3 and Neisseria gonorrhoeae PBP2 from beta-lactam-resistant strains are known. The penicillin-binding domain of PBP2x shows highest homology with these two PBPs and S. pneumoniae PBP2b. In contrast, the N-terminal extension of PBP2x has the highest homology with E. coli PBP2 and methicillin-resistant Staphylococcus aureus PBP2'. No significant homology was detected with PBP1a or PBP1b of Escherichia coli, or with the low-Mr PBPs.     

Lysis of Escherichia coli by beta-lactam antibiotics: deletion analysis of the role of penicillin-binding proteins 1A and 1B

Deletions of the ponA and ponB genes of Escherichia coli have been constructed in vitro and recombined into the chromosome to produce strains that completely lack penicillin-binding protein 1A or penicillin-binding protein 1B. In each case a DNA fragment internal to the gene was replaced by a fragment encoding an antibiotic resistance. The ponA and ponB deletions can therefore be readily introduced into other E. coli strains by P1 transduction of the antibiotic resistance. Although the complete absence of penicillin-binding protein 1A or penicillin-binding protein 1B was tolerated, the absence of both of these proteins was shown to result in bacterial lysis.     

Novel S-benzylisothiourea compound that induces spherical cells in Escherichia coli probably by acting on a rod-shape-determining protein(s) other than penicillin-binding protein 2

Random screening for inhibitors of chromosome partitioning in Escherichia coli was done by the anucleate cell blue assay. A novel S-benzylisothiourea derivative, S-(3,4-dichlorobenzyl)isothiourea, tentatively named A22, was found to induce spherical cells and spherical anucleate cells in E. coli. Mecillinam, a specific inhibitor of penicillin-binding protein 2, which induces spherical cells in E. coli, also caused anucleate cell production. Spherical cells induced by treatment with either A22 or mecillinam varied in size, and anucleate cells seemed to be more frequent among the smaller cells. These results suggest that loss of the rod shape in E. coli leads to asymmetric cell division that results in production of anucleate cells. No competition was observed even in the presence of a 10-fold excess A22 in an in vitro assay of 14C-penicillin G binding, but mecillinam specifically inhibited binding of 14C-penicillin G to penicillin-binding protein 2. Simultaneous treatment with mecillinam and cephalexin, a specific inhibitor of penicillin-binding protein 3, induced lysis of E. coli cells, but a combination of A22 and cephalexin did not. These results suggest that the target molecule(s) of A22 was not penicillin-binding protein 2. A22 may act on a rod-shape-determining protein(s) other than penicillin-binding protein 2, such as RodA or MreB.     

Reduced heat resistance of mutant spores after cloning and mutagenesis of the Bacillus subtilis gene encoding penicillin-binding protein 5.

Part of the gene encoding penicillin-binding protein 5 from Bacillus subtilis 168 was cloned in Escherichia coli with a synthetic oligonucleotide as a hybridization probe. The gene was designated dacA by analogy with E. coli. The nucleotide sequence was determined, and the predicted molecular mass was 45,594 daltons (412 amino acids). A comparison of the predicted amino acid sequence with that of the E. coli penicillin-binding protein 5 indicated that these enzymes showed about 25% identity. The B. subtilis dacA gene was mutated by integration of a plasmid into the structural gene by homologous recombination. A comparison of the mutant and control strains revealed that (i) the mutant lacked detectable penicillin-binding protein 5, (ii) the D-alanine carboxypeptidase activity of membranes isolated from the mutant was only 5% of that measured in membranes from the control strain, (iii) the mutant cells showed apparently normal morphology only during exponential growth, and after the end of exponential phase the cells became progressively shorter, (iv) the mutant sporulated normally except that the forespore occupied about two-thirds of the mother cell cytoplasm and, during its development, migrated towards the center of the mother cell, and (v) purified mutant spores were 10-fold less heat resistant but possessed normal refractility and morphology. Preliminary chemical analysis indicated that the structure of the cortex of the mutant was different.     

Production of thiol-penicillin-binding protein 3 of Escherichia coli using a two primer method of site-directed mutagenesis.

The active site serine residue of penicillin-binding protein 3 of Escherichia coli that is acylated by penicillin (Ser-307) has been converted to a cysteine residue using a simple and efficient two primer method of site-directed mutagenesis. The resulting thiol-penicillin-binding protein 3 was expressed under the control of the lacUV5 promoter in a high copy number plasmid. Constitutive expression of the thiol-enzyme (but not of the wild-type enzyme) was lethal, and the plasmid could only be maintained in E. coli strains that carried the lacIq mutation. Induction of the expression of the thiol-enzyme resulted in inhibition of cell division and the growth of the bacteria into very long filamentous cells. The inhibition of septation was probably due to interference of the function of the wild-type penicillin-binding protein 3 in cell division by the enzymatically inactive thiol-enzyme, and this implies that penicillin-binding protein 3 acts as part of a complex in vivo. We were unable to detect any acylation of the thiol-enzyme by penicillin, but it is not yet clear if this was because the thioester was not formed at an appreciable rate, or if it was formed but was too unstable to be detected by a modified penicillin-binding protein assay.     

dacD, an Escherichia coli gene encoding a novel penicillin-binding protein (PBP6b) with DD-carboxypeptidase activity.

In the course of a study of genes located at min 44 of the Escherichia coli genome, we identified an open reading frame with the capacity to encode a 43-kDa polypeptide whose predicted amino acid sequence is strikingly similar to those of the well-known DD-carboxipeptidases penicillin-binding proteins PBP5 and PBP6. The gene product was shown to bind [3H]benzylpenicillin and to have DD-carboxypeptidase activity on pentapeptide muropeptides in vivo. Therefore, we called the protein PBP6b and the gene dacD. As with other E. coli DD-carboxypeptidases, PBP6b is not essential for cell growth. A quadruple dacA dacB dacC dacD mutant was constructed and shown to grow as well as its isogenic wild-type strain, indicating that the loss of any known PBP-associated DD-carboxypeptidase activity is not deleterious for E. coli. We also identified the homologous gene of dacD in Salmonella typhimurium as one of the components of the previously described phsBCDEF gene cluster.     

Analysis of Neisseria gonorrhoeae peptidoglycan by reverse-phase, high-pressure liquid chromatography

The muramidase digest of peptidoglycan from Neisseria gonorrhoeae was isolated and analyzed by the use of a reverse-phase, high-pressure liquid chromatography system. As was found previously in the case of Escherichia coli, gonococci peptidoglycan is also composed of a greater number of muropeptides than can be resolved with thin-layer chromatography systems. Preliminary classification of the muropeptide components into subclasses based on O-acetyl modification and degree of cross-linkage was achieved. Examination of a penicillin-susceptible strain and a highly resistant strain with two penicillin-binding protein alterations synthesized distinctly different peptidoglycan structures, as revealed by this technique.     

A new beta-lactam-binding protein derived from penicillin-binding protein 3 of Escherichia coli.

In this paper we describe a new beta-lactam-binding protein from the cell envelope of Escherichia coli. It can be detected in cells grown at either 37 or 42 degrees C in medium containing glucose but not in cells grown at 30 degrees C. This novel component has an apparent molecular size that is 2.0 kilodaltons larger than that of penicillin-binding protein 3 and is derived from the latter through a divalent-cation-mediated process probably catalyzed by components located in the periplasmic space. The significance of this protein with regard to regulation of the amount of functional penicillin-binding protein 3 in the cell is discussed.     

Effects of furazlocillin, a beta-lactam antibiotic which binds selectively to penicillin-binding protein 3, on Escherichia coli mutants deficient in other penicillin-binding proteins.

Furazlocillin binds selectively to penicillin-binding protein 3 (PBP-3), prevents septation of Escherichia coli, and allows the cells to form long filaments without lysis. The effect of furazlocillin on the morphology, autolysis, and murein synthesis of E. coli mutants deficient in either PBP-1A, PBP-1Bs, or PBP-2 was studied. The results reveal that PBP-1A and PBP-1Bs functions are not equivalent since furazlocillin affects the morphology, autolysis, and murein synthesis of PBP1A- mutants quite differently from that of PBP-1Bs mutants. Different "PBP-2-" mutants were found to respond to furazlocillin in dramatically different ways: strain LS-1 cells formed elongated rods with a central bulge which eventually lysed, whereas SP6 cells formed stable "barbells" in which the two daughter cells were well separated but remained connected by a thick central region.     

Identification of the active site in penicillin-binding protein 3 of Escherichia coli.

We report the sequence of the active site tryptic peptide of penicillin-binding protein 3 from Escherichia coli. Purified penicillin-binding protein 3 was labeled with [14C]penicillin G and digested with trypsin, and the resulting radioactive peptides were isolated by a combination of gel filtration and high-pressure liquid chromatography. The major radioactive peak from high-pressure liquid chromatography was sequenced, and the peptide Thr-Ile-Thr-Asp-Val-Phe-Glu-Pro-Gly-Ser-Thr-Val-Lys, which comprises residues 298 to 310 in the amino acid sequence, was identified. This sequence is compared with the active site sequences from other penicillin-binding proteins and beta-lactamases.