Conversion of the alpha component of penicillin-binding protein 1b to the beta component in Escherichia coli.

Among components alpha, beta, and gamma of penicillin-binding protein 1b, the alpha and gamma components were confirmed to represent the primary gene products by agreement of their N-terminal amino acid sequences with those predicted from the nucleotide sequence of the ponB (penicillin-binding protein 1b) gene with exclusion of the first methionine in each component. The generation of beta occurred primarily after cell disruption, and the simultaneous loss of alpha suggested the conversion of alpha to beta. The N-terminal amino acid sequence analyzed for beta showed that the conversion was due to the removal of 24 amino acids from the N terminus of alpha.     

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

The penicillin-binding proteins (PBPs) catalyze the synthesis and modification of bacterial cell wall peptidoglycan. Although the biochemical activities of these proteins have been determined in Escherichia coli, the physiological roles of many PBPs remain enigmatic. Previous studies have cast doubt on the individual importance of the majority of PBPs during log phase growth. We show here that PBP1b is vital for competitive survival of E. coli during extended stationary phase, but the other nine PBPs studied are dispensable. Loss of PBP1b leads to the stationary phase-specific competition defective phenotype and causes cells to become more sensitive to osmotic stress. Additionally, we present evidence that this protein, as well as AmpC, may assist in cellular resistance to beta-lactam antibiotics.     

Disulfide bridges are not involved in penicillin-binding protein 1b dimerization in Escherichia coli

PBP1b can be found as a dimer in Escherichia coli. Previous results suggested that dimerization involved the cysteine(s) in an intermolecular disulfide bond. We show that either deletion mutants or a mutant without cysteines is fully active and still binds penicillin and that the latter can also form dimers.     

Glycosyl transferase activity of the Escherichia coli penicillin-binding protein 1b: specificity profile for the substrate

The glycosyl transferase of the Escherichia coli bifunctional penicillin-binding protein (PBP) 1b catalyzes the assembly of lipid-transported N-acetylglucosaminyl-beta-1,4-N-acetylmuramoyl-L-Ala-gamma-D-Glu-meso-A2pm-D-Ala-D-Ala units (lipid II) into linear peptidoglycan chains. These units are linked, at C1 of N-acetylmuramic acid (MurNAc), to a C55 undecaprenyl pyrophosphate. In an in vitro assay, lipid II functions both as a glycosyl donor and as a glycosyl acceptor substrate. Using substrate analogues, it is suggested that the specificity of the enzyme for the glycosyl donor substrate differs from that for the acceptor. The donor substrate requires the presence of both N-acetylglucosamine (GlcNAc) and MurNAc and a reactive group on C1 of the MurNAc and does not absolutely require the lipid chain which can be replaced by uridine. The enzyme appears to prefer an acceptor substrate containing a polyprenyl pyrophosphate on C1 of the MurNAc sugar. The problem of glycan chain elongation that presumably proceeds by the repetitive addition of disaccharide peptide units at their reducing end is discussed.     

Variability in the posttranslational processing of penicillin-binding protein 1b among different strains of Escherichia coli

Screening of a number of unrelated strains of Escherichia coli confirms the existence of at least two patterns of molecular forms for penicillin-binding protein 1b in E. coli cell envelopes. Our data support that the beta-form of this protein is produced by posttranslational modification of the alpha-form and suggest that the absence of the beta-form in some strains is due to a strain-dependent variability in the alpha-form processing mechanism.     

Localization of penicillin-binding protein 1b in Escherichia coli: immunoelectron microscopy and immunotransfer studies.

We report the localization of penicillin-binding protein 1b (PBP 1b) in Escherichia coli KN126 and in an overproducing construct containing plasmid pHK231. We used PBP 1b-specific antiserum for the immunoelectron microscopy of ultrathin sections of whole cells and for immunoelectrophoresis of cytoplasm and isolated membrane fractions. We studied ultrathin sections of both glutaraldehyde-fixed cells that had been embedded after progressively lowering the temperature and cryofixed cells that had been freeze-substituted in Lowicryl K4M and HM20. Most of the PBP 1b-specific label was observed in the inner membrane (IM) and the adjacent cytoplasm, much less was observed in the outer membrane (OM); appreciable amounts were also seen in the bulk cytoplasm. Distribution and intensity of label were both temperature dependent: temperature shift-up to 37 degrees C, causing PBP 1b overproduction in the construct, showed a statistically highly significant increase in label of the IM, including a cytoplasmic zone (of at least 30 nm in depth) adjacent to the IM, a zone we termed the membrane-associated area. Concomitant with the temperature shift-up, a decrease in label density was observed in the bulk cytoplasm. Increased label was also found in IM-OM contact areas (zones of membrane adhesion). The periplasm did not show significant label. Western blotting (immunoblotting) revealed PBP 1b in most of the isolated membrane fractions; however, the highest label density was found in membrane fractions of intermediate density, supporting the suggestion of an increased concentration of PBP 1b in the membrane adhesion zones. In summarizing, we propose that PBP 1b is present in the membrane-associated area of the cytoplasm, from where proteins (such as PBP 1b or thioredoxin) gain access to their specific insertion sites in the envelope. The use of several methods of immunoelectron microscopy provided the first unequivocal evidence for localization of PBP 1b at membrane adhesion sites. Since such sites are specifically labeled with anti-PBP 1b serum, we hypothesize that they contain parts of the machinery for assembly and growth of the murein layer.     

Purification and sequencing of the active site tryptic peptide from penicillin-binding protein 1b of Escherichia coli

This paper reports the sequence of the active site peptide of penicillin-binding protein 1b from Escherichia coli. Purified penicillin-binding protein 1b was labeled with [14C]penicillin G, digested with trypsin, and partially purified by gel filtration. Upon further purification by high-pressure liquid chromatography, two radioactive peaks were observed, and the major peak, representing over 75% of the applied radioactivity, was submitted to amino acid analysis and sequencing. The sequence Ser-Ile-Gly-Ser-Leu-Ala-Lys was obtained. The active site nucleophile was identified by digesting the purified peptide with aminopeptidase M and separating the radioactive products on high-pressure liquid chromatography. Amino acid analysis confirmed that the serine residue in the middle of the sequence was covalently bonded to the [14C]penicilloyl moiety. A comparison of this sequence to active site sequences of other penicillin-binding proteins and beta-lactamases is presented.     

Interaction of monoclonal antibodies with the enzymatic domains of penicillin-binding protein 1b of Escherichia coli.

Monoclonal antibodies (MAbs) against four different antigenic determinants of penicillin-binding protein (PBP) 1b were used to study the transglycosylase and transpeptidase activities of PBP 1b. Enzyme kinetics in the presence of and without the MAbs were determined, and the synthesized murein was analyzed. Two MAbs against the transglycosylase domain of PBP 1b appeared to inhibit this reaction. One MAb inhibited only the transpeptidase reaction, and one inhibited both enzymatic activities of PBP 1b. The latter two MAbs bound to the transpeptidase domain of PBP 1b. The following major conclusions were deduced from the results. (i) Transpeptidation is the rate-limiting step of the reaction cascade, and it is dependent on the product of transglycosylation. (ii) PBP 1b has only one type of transpeptidase activity, i.e., a penta-tetra transpeptidase activity. (iii) PBP 1b is probably a globular protein which has two intimately associated enzymatic domains.     

Topographical and functional investigation of Escherichia coli penicillin-binding protein 1b by alanine stretch scanning mutagenesis.

Penicillin-binding proteins (PBPs) are the targets of beta-lactam antibiotics. We have used a systematic five-alanine substitution method (called ASS [alanine stretch scanning] mutagenesis) to investigate the functional or structural role of various stretches of amino acids in the PBP1b of Escherichia coli. To probe the specific activity of each variant, the antibiotic discs assay was used with strain QCB1 (delta ponB) in the presence of cefaloridine, which totally inhibits the complementing action of PBP1a. This in vivo test has been combined with a quick and efficient in vitro test of the penicillin-binding activity of each of these variants with fluorescent penicillin. This approach has enabled us to show an unexpected role of the N-terminal and C-terminal tails of PBP1b. Moreover, we have established the correct position in PBP1b of the SMN motif that, with the SXXK and the KTG motifs, constitutes the signature of the penicilloyl serine transferases family. Finally, we have shown that the transglycosylase and the transpeptidase domains are separated by an inert linker region, where substitutions and insertions can be made without hindering the in vivo and in vitro activity of the protein.     

Differential responses of Escherichia coli cells expressing cytoplasmic domain mutants of penicillin-binding protein 1b after impairment of penicillin-binding proteins 1a and 3

Penicillin-binding protein 1b (PBP1b) is the major high-molecular-weight PBP in Escherichia coli. Although it is coded by a single gene, it is usually found as a mixture of three isoforms which vary with regard to the length of their N-terminal cytoplasmic tail. We show here that although the cytoplasmic tail seems to play no role in the dimerization of PBP1b, as was originally suspected, only the full-length protein is able to protect the cells against lysis when both PBP1a and PBP3 are inhibited by antibiotics. This suggests a specific role for the full-length PBP1b in the multienzyme peptidoglycan-synthesizing complex that cannot be fulfilled by either PBP1a or the shorter PBP1b proteins. Moreover, we have shown by alanine-stretch-scanning mutagenesis that (i) residues R(11) to G(13) are major determinants for correct translocation and folding of PBP1b and that (ii) the specific interactions involving the full-length PBP1b can be ascribed to the first six residues at the N-terminal end of the cytoplasmic domain. These results are discussed in terms of the interactions with other components of the murein-synthesizing complex.     

Identification of the penicillin-binding active site of penicillin-binding protein 2 of Escherichia coli

We determined the active site of penicillin-binding protein (PBP) 2 of Escherichia coli. A water-soluble form of PBP 2, which was constructed by site-directed mutagenesis, was purified by affinity chromatography, labeled with dansyl-penicillin, and then digested with a combination of proteases. The amino acid composition of the labeled chymotryptic peptide purified by HPLC was identical with that of the amino acid sequence, Ala-Thr-Gln-Gly-Val-Tyr-Pro-Pro-Ala-Ser330-Thr-Val-Lys-Pro (residues 321-334) of PBP 2, which was deduced from the nucleotide sequence of the pbpA gene encoding PBP 2. This amino acid sequence was verified by sequencing the labeled tryptic peptide containing the labeled chymotryptic peptide region. A mutant PBP 2 (thiol-PBP 2), constructed by site-directed mutagenesis to replace Ser330 with Cys, lacked the penicillin-binding activity. These findings provided evidence that Ser330 near the middle of the primary structure of PBP 2 is the penicillin-binding active-site residue, as predicted previously on the basis of the sequence homology. Around this active site, the sequence Ser-Xaa-Xaa-Lys was observed, which is conserved in the active-site regions of all E. coli PBPs so far studied, class A and class C beta-lactamases, and D-Ala carboxypeptidases. The COOH-terminal amino acid of PBP 2 was identified as His633.     

Importance of the conserved residues in the peptidoglycan glycosyltransferase module of the class A penicillin-binding protein 1b of Escherichia coli

The peptidoglycan glycosyltransferase (GT) module of class A penicillin-binding proteins (PBPs) and monofunctional GTs catalyze glycan chain elongation of the bacterial cell wall. These enzymes belong to the GT51 family, are characterized by five conserved motifs, and have some fold similarity with the phage lambda lysozyme. In this work, we have systematically modified all the conserved amino acid residues of the GT module of Escherichia coli class A PBP1b by site-directed mutagenesis and determined their importance for the in vivo and in vitro activity and the thermostability of the protein. To get an insight into the GT active site of this paradigm enzyme, a model of PBP1b GT domain was constructed based on the available crystal structures (PDB codes 2OLV and 2OLU). The data show that in addition to the essential glutamate residues Glu233 of motif 1 and Glu290 of motif 3, the residues Phe237 and His240 of motif 1 and Gly264, Thr267, Gln271, and Lys274 of motif 2, all located in the catalytic cavity of the GT domain, are essential for the in vitro enzymatic activity of the PBP1b and for its in vivo functioning. Thus, the first three conserved motifs contain most of the residues that are required for the GT activity of the PBP1b. The residues Asp234, Phe237, His240, Thr267, and Gln271 are proposed to maintain the structure of the active site and the positioning of the catalytic Glu233.     

Catalytic mechanism of penicillin-binding protein 5 of Escherichia coli

Penicillin-binding proteins (PBPs) and beta-lactamases are members of large families of bacterial enzymes. These enzymes undergo acylation at a serine residue with their respective substrates as the first step in their catalytic events. Penicillin-binding protein 5 (PBP 5) of Escherichia coli is known to perform a dd-carboxypeptidase reaction on the bacterial peptidoglycan, the major constituent of the cell wall. The roles of the active site residues Lys47 and Lys213 in the catalytic machinery of PBP 5 have been explored. By a sequence of site-directed mutagenesis and chemical modification, we individually introduced gamma-thialysine at each of these positions. The pH dependence of kcat/Km and of kcat for the wild-type PBP 5 and for the two gamma-thialysine mutant variants at positions 47 and 213 were evaluated. The pH optimum for the enzyme was at 9.5-10.5. The ascending limb to the pH optimum is due to Lys47; hence, this residue exists in the free-base form for catalysis. The descending limb from the pH optimum is contributed to by both Lys213 and a water molecule coordinated to Lys47. These results have been interpreted as Lys47 playing a key role in proton-transfer events in the course of catalysis during both the acylation and deacylation events. However, the findings for Lys213 argue for a protonated state at the pH optimum. Lys213 serves as an electrostatic anchor for the substrate.     

Partial crypticity of penicillin-binding protein 1b in purified cell envelopes ofEscherichia coli

InEscherichia coli, penicillin-binding protein 1b (pbp 1b) is one of the critical proteins in the biosynthesis of the murein sacculus. In this communication we present evidence indicating that pbp 1b is unusually resistant to inactivation by n-butanol and that, under the standard conditions used in pbp-labeling experiments, a considerable fraction of the total pbp 1b in the cell envelope remains inaccessible to at least some -lactam antibiotics.     

Purification of penicillin-binding protein 2 of Escherichia coli.

A protein with a molecular weight of 60,000 (60K) constitutes approximately 20% of the envelope protein of Azotobacter vinelandii. This protein was removed from cells and purified from other proteins by a simple washing procedure that had no effect on cell viability. Anti-60K antiserum blocked azotophage A-22 adsorption and agglutinated both vegetative cells and cysts; ferritin-conjugated antibodies used in indirect labeling studies bound uniformly to the periphery of vegetative cells. We conclude that 60K is present on the outer surface of vegetative cells and cysts. The protein is similar to the surface protein alpha of Acinetobacter ssp. in molecular weight, reassociation characteristics, and high ratio of acidic to basic amino acids. We propose that 60K forms a layer external to the outer membrane of A. vinelandii.     

Membrane topology of penicillin-binding protein 3 of Escherichia coli

The beta-lactamase fusion vector, pJBS633, has been used to analyse the organization of penicillin-binding protein 3 (PBP3) in the cytoplasmic membrane of Escherichia coli. The fusion junctions in 84 in-frame fusions of the coding region of mature TEM beta-lactamase to random positions within the PBP3 gene were determined. Fusions of beta-lactamase to 61 different positions in PBP3 were obtained. Fusions to positions within the first 31 residues of PBP3 resulted in enzymatically active fusion proteins which could not protect single cells of E. coli from killing by ampicillin, indicating that the beta-lactamase moieties of these fusion proteins were not translocated to the periplasm. However, all fusions that contained greater than or equal to 36 residues of PBP3 provided single cells of E. coli with substantial levels of resistance to ampicillin, indicating that the beta-lactamase moieties of these fusion proteins were translocated to the periplasm. PBP3 therefore appeared to have a simple membrane topology with residues 36 to the carboxy-terminus exposed on the periplasmic side of the cytoplasmic membrane. This topology was confirmed by showing that PBP3 was protected from proteolytic digestion at the cytoplasmic side of the inner membrane but was completely digested by proteolytic attack from the periplasmic side. PBP3 was only inserted in the cytoplasmic membrane at its amino terminus since replacement of its putative lipoprotein signal peptide with a normal signal peptide resulted in a water-soluble, periplasmic form of the enzyme. The periplasmic form of PBP3 retained its penicillin-binding activity and appeared to be truly water-soluble since it fractionated, in the absence of detergents, with the expected molecular weight on Sephadex G-100 and was not retarded by hydrophobic interaction chromatography on Phenyl-Superose.     

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.     

Lipid modification of Escherichia coli penicillin-binding protein 3.

The primary structure of penicillin-binding protein 3 (PBP 3), an essential enzyme for cell division in Escherichia coli, was deduced from the nucleotide sequence of the ftsI gene (M. Nakamura, I. N. Maruyama, M. Soma, J. Kato, H. Suzuki, and Y. Hirota, Mol. Gen. Genet. 191:1-9, 1983). An amino acid sequence of Leu-26-Leu-Cys-Gly-Cys-30 was found near the amino terminus of the deduced sequence, showing a rather striking homology to the Leu-Leu-Ala-Gly-Cys consensus sequence for the modification and processing of precursors of the E. coli murein lipoprotein and other bacterial lipoproteins. As expected from this finding, PBP 3 was found to be modified with glycerol and fatty acids, although the lipid modification occurred only in a small fraction, accounting for less than 15% of the total PBP 3 molecules.     

Nucleotide sequence of the genes coding for alpha, beta and gamma subunits of the proton-translocating ATPase of Escherichia coli

A nucleotide sequence of 2328 base pairs comprising a portion of the gene cluster for the proton-translocating ATPase of $\text{E. coli}$ was determined. The sequence covers most of the gene for α subunit, the entire gene for γ subunit and the amino terminal portion of the gene for β subunit, along with the flanking regions of these genes. The amino acid sequences of these subunits deduced from the DNA sequences indicate that the α and γ subunits have 513 and 287 amino acid residues, respectively. A possible secondary structure for each subunit was estimated from the inferred primary structure. The intercistronic regions between the genes for α and γ and between γ and β are 49 and 26 base pairs, respectively. The significance of codon usage in these genes is discussed in correlation with their expression.