Labeling pattern of major penicillin-binding proteins of Escherichia coli during the division cycle.

Escherichia coli cells were synchronized by the elutriation technique. The pattern of penicillin-binding proteins (PBPs) in synchronously growing cells was determined with an iodinated derivative of ampicillin in intact cells as well as in isolated membranes. This was done under nonsaturating conditions as well as under conditions in which the PBPs were saturated with [125I]ampicillin. No evidence was found for fluctuations in the PBP pattern: the PBPs seem to be present in a constant ratio throughout the division cycle. The E. coli cells exert their control on shape maintenance and cell wall growth apparently not on the level of concentration of PBPs in the cell but rather on activation of existing components.     

Overproduction of penicillin-binding protein 2 and its inactive variants causes morphological changes and lysis in Escherichia coli

Penicillin-binding protein 2 (PBP 2) has long been known to be essential for rod-shaped morphology in gram-negative bacteria, including Escherichia coli and Pseudomonas aeruginosa. In the course of earlier studies with P. aeruginosa PBP 2, we observed that E. coli was sensitive to the overexpression of its gene, pbpA. In this study, we examined E. coli overproducing both P. aeruginosa and E. coli PBP 2. Growth of cells entered a stationary phase soon after induction of gene expression, and cells began to lyse upon prolonged incubation. Concomitant with the growth retardation, cells were observed to have changed morphologically from typical rods into enlarged spheres. Inactive derivatives of the PBP 2s were engineered, involving site-specific replacement of their catalytic Ser residues with Ala in their transpeptidase module. Overproduction of these inactive PBPs resulted in identical effects. Likewise, overproduction of PBP 2 derivatives possessing only their N-terminal non-penicillin-binding module (i.e., lacking their C-terminal transpeptidase module) produced similar effects. However, E. coli overproducing engineered derivatives of PBP 2 lacking their noncleavable, N-terminal signal sequence and membrane anchor were found to grow and divide at the same rate as control cells. The morphological effects and lysis were also eliminated entirely when overproduction of PBP 2 and variants was conducted with E. coli MHD79, a strain lacking six lytic transglycosylases. A possible interaction between the N-terminal domain of PBP 2 and lytic transglycosylases in vivo through the formation of multienzyme complexes is discussed.     

Peptidoglycan synthetic activities in membranes of Escherichia coli caused by overproduction of penicillin-binding protein 2 and rodA protein

Penicillin-binding protein (PBP)-2 and the RodA protein are known to function in determining the rod shape of Escherichia coli cells. Peptidoglycan biosynthetic reactions that required these two proteins were demonstrated in the membrane fraction prepared from an E. coli strain that overproduced both of these two proteins and which lacked PBP-1B activity (the major peptidoglycan synthetase activity in the normal E. coli membranes). The cross-linked peptidoglycan was synthesized from UDP-N-acetylmuramylpentapeptide and UDP-N-acetylglucosamine in the presence of a high concentration of cefmetazole that inhibited all of PBPs except PBP-2. The peptidoglycan was synthesized via a lipid intermediate and showed up to 30% cross-linking. The cross-linking reaction was strongly inhibited by the amidinopenicillin, mecillinam, and by other beta-lactam antibiotics that have a high affinity for PBP-2, but not by beta-lactams that had very low affinity for PBP-2. The formation of peptidoglycan required the presence of high levels of both PBP-2 and the RodA protein in the membranes, but it is unclear which of the two proteins was primarily responsible for the extension of the glycan chains (transglycosylation). However, the sensitivity of the cross-linking reaction to specific beta-lactam antibiotics strongly suggested that it was catalyzed by PBP-2. The transglycosylase activity of the membranes was sensitive to enramycin and vancomycin and was unusual in being stimulated greatly by a high concentration of a chelating agent.     

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.     

Affinities of PBPs of enterococci to cefepime and ampicillin]

The beta-lactam resistance of genus Streptococcus has been explained by the low binding affinity of penicillin-binding proteins (PBPs) to the drug. This study was carried out to resolve the mechanisms of resistance to beta-lactam antibiotics in the species of genus Enterococcus by means of binding affinities of PBPs. Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium and Enterococcus avium were employed as assay microbes. Cefepime (CFPM) and ampicillin (ABPC) were used as representatives of cephems and penicillins, respectively. All the PBP fractions of S. pyogenes manifested high binding affinities to CFPM and ABPC, whereas PBPs 1 and 4 of E. faecalis showed high binding affinities to ABPC but not to CFPM. In E. faecium, PBPs of an exceptionally penicillin-susceptible strain manifested a high binding affinity to ABPC, but PBPs 5 and 6 showed low affinities to CFPM. beta-lactam resistant strains of E. faecium possessed PBPs 5 and 6 with low binding affinities to CFPM and ABPC. All the fractions of PBPs but PBP 1 in E. avium showed low binding affinities to CFPM. Although all the PBP fractions but PBPs 3 and 6 manifested high binding affinities to ABPC, PBPs 3 and 6 showed low binding affinities to ABPC. A strain of E. avium, which is susceptible to ABPC but moderately resistant to CFPM, lacked PBP 6. In conclusion, the resistance of E. avium to CFPM is based upon low binding affinities of the many fractions to this drug, and ABPC resistance is based upon PBPs 3 and 6 with low binding affinities to ABPC.     

Identification and cloning of the gene encoding penicillin-binding protein 7 of Escherichia coli.

Penicillin-binding protein (PBP) 7 of Escherichia coli is a poorly characterized member of the family of enzymes that synthesize and modify the bacterial cell wall. The approximate chromosomal position of the gene encoding this protein was determined by measuring the expression of PBPs during lytic infection of E. coli by each of the 476 miniset members of the Kohara lambda phage genomic library. Phages lambda 363 and lambda 364, encompassing the region from 47.7 to 48 min of the chromosome, overproduced PBP 7. One open reading frame, yohB, was present on both these phages and directed the expression of PBPs 7 and 8. The predicted amino acid sequence of PBP 7 contains the consensus motifs associated with other PBPs and has a potential site near the carboxyl terminus where proteolysis by the OmpT protein could occur, creating an appropriately sized PBP 8. The PBP 7 gene (renamed pbpG) was interrupted by insertion of a kanamycin resistance gene cassette and was moved to the chromosome of E. coli. No obvious growth defects were observed, suggesting that PBP 7 is not essential for growth under normal laboratory conditions.     

Expression and characterization of the ponA (ORF I) gene of Haemophilus influenzae: functional complementation in a heterologous system.

The coding sequence of the Haemophilus influenzae ORF I gene was amplified by PCR and cloned into different Escherichia coli expression vectors. The ORF I-encoded protein was approximately 90 kDa and bound 3H-benzyl-penicillin and 125I-cephradine. This high-molecular-weight penicillin-binding protein (PBP) was also shown to possess transglycosylase activity, indicating that the ORF I product is a bifunctional PBP. The ORF I protein was capable of maintaining the viability of E. coli delta ponA ponB::spcr cells in transcomplementation experiments, establishing the functional relevance of the significant amino acid homology seen between E. coli PBP 1A and 1B and the H. influenzae ORF I product. In addition, the physiological functioning of the H. influenzae ORF I (PBP 1A) product in a heterologous species established the ability of the enzyme not only to recognize the E. coli substrate but also to interact with heterologous cell division proteins. The affinity of the ORF I product for 3H-benzylpenicillin and 125I-cephradine, the MIC of beta-lactams for E. coli delta ponA ponB::spcr expressing the ORF I gene, and the amino acid alignment of the PBP 1 family of high-molecular-weight PBPs group the ORF I protein into the PBP 1A family of high-molecular-weight PBPs.     

Penicillin-binding proteins of Rhodopseudomonas sphaeroides and their membrane localization.

Cytoplasmic membranes (CM) prepared from both chemotrophic and phototrophic cells of Rhodopseudomonas sphaeroides possess penicillin-binding proteins (PBPs), as demonstrated by binding of [125]furazlocillin to isolated membranes, the subsequent separation of the constituent PBPs by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their detection by autoradiography. The major PBP present in CM from R. sphaeroides corresponds in molecular weight to PBP-5, the predominant PBP present in CM of Escherichia coli. In contrast, the outer membrane of R. sphaeroides shows only low-level furazlocillin-binding activity on a per milligram of protein basis compared with chemotrophic CM. The intracytoplasmic membrane (ICM) derived from phototrophic cells contains less than 5% of the furazlocillin-binding activity of the CM. Based on the specific localization of PBPs in the CM, it is possible to provide quantitative estimates of the extent of CM present in preparations of ICM. This method demonstrates that highly purified preparations of ICM contain less than 5% CM. Additionally, the assay for PBPs demonstrates that during ICM remodeling, which occurs upon a shift from phototrophic to chemotrophic growth, there is no significant insertion of PBPs into the ICM over the first two generations after a shift to chemotrophic growth.     

Contributions of PBP 5 and DD-carboxypeptidase penicillin binding proteins to maintenance of cell shape in Escherichia coli

Escherichia coli has 12 recognized penicillin binding proteins (PBPs), four of which (PBPs 4, 5, and 6 and DacD) have DD-carboxypeptidase activity. Although the enzymology of the DD-carboxypeptidases has been studied extensively, the in vivo functions of these proteins are poorly understood. To explain why E. coli maintains four independent loci encoding enzymes of considerable sequence identity and comparable in vitro activity, it has been proposed that the DD-carboxypeptidases may substitute for one another in vivo. We tested the validity of this equivalent substitution hypothesis by investigating the effects of these proteins on the aberrant morphology of DeltadacA mutants, which produce no PBP 5. Although cloned PBP 5 complemented the morphological phenotype of a DeltadacA mutant lacking a total of seven PBPs, controlled expression of PBP 4, PBP 6, or DacD did not. Also, a truncated PBP 5 protein lacking its amphipathic C-terminal membrane binding sequence did not reverse the morphological defects and was lethal at low levels of expression, implying that membrane anchoring is essential for the proper functioning of PBP 5. By examining a set of mutants from which multiple PBP genes were deleted, we found that significant morphological aberrations required the absence of at least three different PBPs. The greatest defects were observed in cells lacking, at minimum, PBPs 5 and 6 and one of the endopeptidases (either PBP 4 or PBP 7). The results further differentiate the roles of the low-molecular-weight PBPs, suggest a functional significance for the amphipathic membrane anchor of PBP 5 and, when combined with the recently determined crystal structure of PBP 5, suggest possible mechanisms by which these PBPs may contribute to maintenance of a uniform cell shape in E. coli.     

Effect of aminoglycosides on the binding of C14-benzylpenicillin by penicillin-binding proteins in Escherichia coli

It was shown that preincubation of E. coli intact cells with gentamicin and streptomycin induced a marked increase in binding of 14C-benzylpenicillin to its final targets in the membrane i.e. penicillin-binding proteins (PBP). The stimulating effect of the aminoglycosides was also confirmed in experiments with a membrane fraction isolated from the cells preincubated with the aminoglycosides. The PBPs of the cells preincubated with the aminoglycosides were studied with SDS-PAAG electrophoresis. It was revealed that under the action of the aminoglycosides the quantity of the labeled substance (intensity of the bands on the fluorograms) fixed by the low molecular PBPs i.e. D-alanine carboxypeptidases increased. Moreover, the composition of the high molecular less mobile PBPs (transpeptidases) changed. The data are discussed in regard to the peculiarities of the effect of the aminoglycosides on the cells (bactericidal action, membrane tropism). The effect of the aminoglycosides can influence (along with the others) the results of their combined use with beta-lactams.     

Comparative characteristics of penicillin-binding proteins of representatives of the genus Streptomyces

Penicillin-binding proteins (PBPs) in Strepomyces strains producing clavulanic acid and beta-lactamase and in Streptomyces strains not producing these compounds were studied comparatively. In S. clavuligerus, the organism producing clavulanic acid, there were detected 3 PBPs in the membrane fraction. S. griseus, the organism producing beta-lactamase, contained 6 PBP. In S. cacaoi and S. olivaceus, organisms producing neither beta-lactams nor beta-lactamase, there were detected 5 and 4 PBP, respectively. The set of the PBP in the organism producing clavulanic acid varied during fermentation. In a variant of S. clavuligerus isolated after protoplasting of the mycelial cells and their regeneration the content of the electrophoretically most mobile PBP lowered. The PBP of S. clavuligerus did no show any high affinity to other beta-lactams such as methicillin and ampicillin tested as competing agents of 14C-benzylpenicillin.     

Penicillin binding protein 5 affects cell diameter, contour, and morphology of Escherichia coli

Although general physiological functions have been ascribed to the high-molecular-weight penicillin binding proteins (PBPs) of Escherichia coli, the low-molecular-weight PBPs have no well-defined biological roles. When we examined the morphology of a set of E. coli mutants lacking multiple PBPs, we observed that strains expressing active PBP 5 produced cells of normal shape, while mutants lacking PBP 5 produced cells with altered diameters, contours, and topological features. These morphological effects were visible in untreated cells, but the defects were exacerbated in cells forced to filament by inactivation of PBP 3 or FtsZ. After filamentation, cellular diameter varied erratically along the length of individual filaments and many filaments exhibited extensive branching. Also, in general, the mean diameter of cells lacking PBP 5 was significantly increased compared to that of cells from isogenic strains expressing active PBP 5. Expression of cloned PBP 5 reversed the effects observed in DeltadacA mutants. Although deletion of PBP 5 was required for these phenotypes, the absence of additional PBPs magnified the effects. The greatest morphological alterations required that at least three PBPs in addition to PBP 5 be deleted from a single strain. In the extreme cases in which six or seven PBPs were deleted from a single mutant, cells and cell filaments expressing PBP 5 retained a normal morphology but cells and filaments lacking PBP 5 were aberrant. In no case did mutation of another PBP produce the same drastic morphological effects. We conclude that among the low-molecular-weight PBPs, PBP 5 plays a principle role in determining cell diameter, surface uniformity, and overall topology of the peptidoglycan sacculus.     

Contribution of membrane-binding and enzymatic domains of penicillin binding protein 5 to maintenance of uniform cellular morphology of Escherichia coli

Four low-molecular-weight penicillin binding proteins (LMW PBPs) of Escherichia coli are closely related and have similar DD-carboxypeptidase activities (PBPs 4, 5, and 6 and DacD). However, only one, PBP 5, has a demonstrated physiological function. In its absence, certain mutants of E. coli have altered diameters and lose their uniform outer contour, resulting in morphologically aberrant cells. To determine what differentiates the activities of these LMW PBPs, we constructed fusion proteins combining portions of PBP 5 with fragments of other DD-carboxypeptidases to see which hybrids restored normal morphology to a strain lacking PBP 5. Functional complementation occurred when truncated PBP 5 was combined with the terminal membrane anchor sequences of PBP 6 or DacD. However, complementation was not restored by the putative carboxy-terminal anchor of PBP 4 or by a transmembrane region of the osmosensor protein ProW, even though these hybrids were membrane bound. Site-directed mutagenesis of the carboxy terminus of PBP 5 indicated that complementation required a generalized amphipathic membrane anchor but that no specific residues in this region seemed to be required. A functional fusion protein was produced by combining the N-terminal enzymatic domain of PBP 5 with the C-terminal beta-sheet domain of PBP 6. In contrast, the opposite hybrid of PBP 6 to PBP 5 was not functional. The results suggest that the mode of PBP 5 membrane anchoring is important, that the mechanism entails more than a simple mechanical tethering of the enzyme to the outer face of the inner membrane, and that the physiological differences among the LMW PBPs arise from structural differences in the DD-carboxypeptidase enzymatic core.     

High-molecular-weight penicillin-binding proteins from membranes of bacilli

Mixtures of high-molecular-weight, cephalosporin-sensitive penicillin-binding proteins (PBPs) can be purified from Bacillus subtilis membranes by cephalosporin affinity chromatography (G. Kleppe and J. L. Strominger, J. Biol. Chem. 254:4856-4862, 1979). By appropriate modification of this technique, B. subtilis PBP 1 was purified to homogeneity, and a mixture of Bacillus stearothermophilus PBPs 1, 2, and 4 was isolated. [14C]penicillin-PBP complexes of high-molecular-weight PBPs purified from membranes of these two bacilli, after denaturation, were found to have chemical reactivities typical of the penicilloyl-serine derivative formed by D-alanine carboxypeptidase from B. stearothermophilus. Although enzymatic activity catalyzed by these and several other high-molecular-weight PBPs from gram-positive organisms has not been detected with cell wall-related substrates, a slow, enzymatic acylation of B. subtilis PBPs 1, 2ab, and 4 by [14C]-diacetyl-L-lysyl-D-alanyl-D-lactate was demonstrated. Further study is necessary to clarify the physiological relevance of the slow acylation by this analog of a natural cell wall biosynthetic intermediate.     

Identification of a penicillin-binding protein 3 homolog, PBP3x, in Pseudomonas aeruginosa: gene cloning and growth phase-dependent expression.

A homolog of Pseudomonas aeruginosa penicillin-binding protein 3 (PBP3), named PBP3x in this study, was identified by using degenerate primers based on conserved amino acid motifs in the high-molecular-weight PBPs. Analysis of the translated sequence of the pbpC gene encoding this PBP3x revealed that 41 and 48% of its amino acids were identical to those of Escherichia coli and P. aeruginosa PBP3s, respectively. The downstream sequence of pbpC encoded convergently transcribed homologs of the E. coli soxR gene and the Mycobacterium bovis adh gene. The pbpC gene product was expressed from the T7 promoter in E. coli and was exported to the cytoplasmic membrane of E. coli cells and could bind [3H] penicillin. By using a broad-host-range vector, pUCP27, the pbpC gene was expressed in P. aeruginosa PAO4089. [3H]penicillin-binding competition assays indicated that the pbpC gene product had lower affinities for several PBP3-targeted beta-lactam antibiotics than P. aeruginosa PBP3 did, and overexpression of the pbpC gene product had no effect on the susceptibility to the PBP3-targeted antibiotics tested. By gene replacement, a PBP3x-defective interposon mutant (strain HC132) was obtained and confirmed by Southern blot analysis. Inactivation of PBP3x caused no changes in the cell morphology or growth rate of exponentially growing cells, suggesting that pbpC was not required for cell viability under normal laboratory growth conditions. However, the upstream sequence of pbpC contained a potential sigma(s) recognition site, and pbpC gene expression appeared to be growth rate regulated. [3H]penicillin-binding assays indicated that PBP3 was mainly produced during exponential growth whereas PBP3x was produced in the stationary phase of growth.     

Direct quantitation of the number of individual penicillin-binding proteins per cell in Escherichia coli.

The penicillin-binding proteins (PBPs) are a set of enzymes that participate in the terminal stages of bacterial peptidoglycan assembly. As their name implies, these proteins also covalently bind and are inhibited by beta-lactam antibiotics. Although many studies have examined the relative binding affinities of a number of beta-lactam antibiotics, a surprisingly small number of studies have addressed the absolute numbers of each of the PBPs present in the bacterial cell. In the present study, the PBP values initially reported in Escherichia coli almost 20 years ago by B. G. Spratt (Eur. J. Biochem. 72:341-352, 1977) were refined. The individual PBPs from a known number of bacteria radiolabeled with [3H]benzylpenicillin were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The radioactive bands were located, excised, and quantitatively extracted from the gel slices. The radioactivity was measured by scintillation counting, and the absolute disintegrations per minute were calculated. From the specific activity of the labeled penicillin, the absolute disintegrations per minute, and the CFU per milliliter, a determination of the number of each of the PBPs per cell was made. The measurements were performed on multiple samples to place statistical limits on the numbers obtained. The values for the individual PBPs found in E. coli deviated in several ways from the previously reported observations. Of particular significance is the higher number of molecules of PBP 2 and 3 observed, since these PBPs are known to participate in cell morphogenesis. The PBP content in both rich Luria broth medium and M9 minimal medium was determined, with the slower-growing cells in minimal medium possessing fewer of the individual PBPs per cell.     

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.     

Topographical distribution of penicillin-binding proteins in the Escherichia coli membrane.

The penicillin-binding proteins (PBPs) found in the membranes of Escherichia coli X925 minicells (primarily cell ends or septa) were compared with those found in rod-shaped cells (primarily sidewalls) in an effort to determine whether certain PBPs are unevenly distributed over the bacterial cell membrane. The seven major PBPs of E. coli were all present in minicell membranes. PBP 1B was altered in minicells, however, appearing as two bands on sodium dodecyl sulfate-polyacrylamide gels rather than the usual three. PBP 2, which is needed for longitudinal growth of the cell but not for septum formation, was significantly reduced in minicell membranes. This observation is consistent with the fact that minicells contain very little sidewall material and raises the possibility that the specialized function of PBP 2 may be determined or regulated by its uneven topographical distribution in the membrane. None of the PBPs appeared to be selectively enriched in minicell membranes.     

Penicillin-binding protein 2x of Streptococcus pneumoniae. Expression in Escherichia coli and purification of a soluble enzymatically active derivative.

A 2.5-kb DNA fragment including the structural gene coding for the penicillin-binding protein 2x (PBP 2x) of Streptococcus pneumoniae has been cloned into the vector pJDC9 and expressed in Escherichia coli. Mapping of RNA polymerase binding sites by electron microscopy indicated that the pbpX promoter is well recognized by the E. coli enzyme. However, high-level expression occurred mainly under the control of the lac promoter upstream of the pJDC9 multiple cloning site. After induction with isopropyl beta-d-thiogalactopyranoside, PBP 2x was expressed as one of the major cellular proteins. PBP 2x produced in E. coli corresponded to the pneumococcal PBP 2x in terms of electrophoretic mobility, fractionation with the cytoplasmic membrane, and penicillin-binding capacity. Deletion of 30 hydrophobic N-terminal amino acid residues at positions 19-48 resulted in high-level expression of a cytoplasmic, soluble PBP 2x derivative (PBP 2x*) which still retained full beta-lactam-binding activity. A two-step procedure involving dye affinity chromatography was established for obtaining large amounts of highly purified enzymatically active PBP 2x*.     

Nucleotide sequence of the pbpA gene and characteristics of the deduced amino acid sequence of penicillin-binding protein 2 of Escherichia coli K12

We have determined the nucleotide sequence of the pbpA gene encoding penicillin-binding protein (PBP) 2 of Escherichia coli. The coding region for PBP 2 was 1899 base pairs in length and was preceded by a possible promoter sequence and two open reading frames. The primary structure of PBP 2, deduced from the nucleotide sequence, comprised 633 amino acid residues. The relative molecular mass was calculated to be 70867. The deduced sequence agreed with the NH2-terminal sequence of PBP 2 purified from membranes, suggesting that PBP 2 has no signal peptide. The hydropathy profile suggested that the NH2-terminal hydrophobic region (a stretch of 25 non-ionic amino acids) may anchor PBP 2 in the cytoplasmic membrane as an ectoprotein. There were nine homologous segments in the amino acid sequence of PBP 2 when compared with PBP 3 of E. coli. The active-site serine residue of PBP 2 was predicted to be Ser-330. Around this putative active-site serine residue was found the conserved sequence of Ser-Xaa-Xaa-Lys, which has been identified in all of the other E. coli PBPs so far studied (PBPs 1A, 1B, 3, 5 and 6) and class A and class C beta-lactamases. In the higher-molecular-mass PBPs 1A, 1B, 2 and 3, Ser-Xaa-Xaa-Lys-Pro was conserved. In the putative peptidoglycan transpeptidase domain there were six amino acid residues, which are common only in the PBPs of higher molecular mass.