Detection of penicillin-binding proteins immunologically related to penicillin-binding protein 5 of Enterococcus hirae ATCC 9790 in Enterococcus faecium and Enterococcus faecalis

Penicillin-binding protein (PBP) 5 of Enterococcus hirae ATCC 9790 belongs to the class of the high-molecular mass, low-affinity PBPs which have been correlated with penicillin resistance in most Enterococcus species. Polyclonal antibodies were raised against PBP 5 and used to detect immunologically related membrane proteins in E. faecium and E. faecalis strains. Several strains of both species were found to have a membrane protein of similar molecular mass to E. hirae PBP 5 which reacted with the antibodies. Some E. faecium strains did not react with antibodies but their derivatives with increased penicillin minimal inhibitory concentrations did. In some E. faecalis strains the lack of a PBP 5-related protein was associated with failure to select stable penicillin-resistant derivatives.     

Role of class A penicillin-binding proteins in PBP5-mediated beta-lactam resistance in Enterococcus faecalis

Peptidoglycan polymerization complexes contain multimodular penicillin-binding proteins (PBP) of classes A and B that associate a conserved C-terminal transpeptidase module to an N-terminal glycosyltransferase or morphogenesis module, respectively. In Enterococcus faecalis, class B PBP5 mediates intrinsic resistance to the cephalosporin class of beta-lactam antibiotics, such as ceftriaxone. To identify the glycosyltransferase partner(s) of PBP5, combinations of deletions were introduced in all three class A PBP genes of E. faecalis JH2-2 (ponA, pbpF, and pbpZ). Among mutants with single or double deletions, only JH2-2 DeltaponA DeltapbpF was susceptible to ceftriaxone. Ceftriaxone resistance was restored by heterologous expression of pbpF from Enterococcus faecium but not by mgt encoding the monofunctional glycosyltransferase of Staphylococcus aureus. Thus, PBP5 partners essential for peptidoglycan polymerization in the presence of beta-lactams formed a subset of the class A PBPs of E. faecalis, and heterospecific complementation was observed with an ortholog from E. faecium. Site-directed mutagenesis of pbpF confirmed that the catalytic serine residue of the transpeptidase module was not required for resistance. None of the three class A PBP genes was essential for viability, although deletion of the three genes led to an increase in the generation time and to a decrease in peptidoglycan cross-linking. As the E. faecalis chromosome does not contain any additional glycosyltransferase-related genes, these observations indicate that glycan chain polymerization in the triple mutant is performed by a novel type of glycosyltransferase. The latter enzyme was not inhibited by moenomycin, since deletion of the three class A PBP genes led to high-level resistance to this glycosyltransferase inhibitor.     

Paradoxical effect of inserting, in Enterococcus faecalis penicillin-binding protein 5, an amino acid box responsible for low affinity for penicillin in Enterococcus faecium

Penicillin-binding proteins 5 (PBP5s) of enterococci are structurally and immunologically related proteins that are characterized by their low affinity for penicillin. For this reason, they are mainly involved in penicillin resistance, due essentially to their ability to take over the function of all other PBPs already bound and inhibited by the beta-lactam. It has been demonstrated that penicillin resistance in enterococci is acquired either by overproduction of PBP5 or by the presence of specific amino acid sequences in the protein that further decrease the affinity for penicillin. In particular, a specific amino acid box (ANNGA) previously identified in Enterococcus faecium is responsible for the high penicillin resistance displayed by this species. Here, we describe the insertion of the PBP5 amino acid box ANNGA in Enterococcus faecalis, an enterococcal species usually more sensitive to penicillin, by site-directed mutagenesis. Mutagenized PBP5 was re-introduced into a pbp5 mutant of E. faecalis obtained by insertion of transposon Tn916. Data indicate that this amino acid box brings about no reduction in penicillin sensitivity in the recipient E. faecalis strain, but, paradoxically, dramatically lowers the penicillin minimal inhibitory concentration caused by the native PBP5. We deduce that, although enterococcal PBP5s are a family of closely related proteins as far as biological function is concerned, differences exist in their three-dimensional structure that affect penicillin affinity.     

Synthesis of mosaic peptidoglycan cross-bridges by hybrid peptidoglycan assembly pathways in gram-positive bacteria

The peptidoglycan cross-bridges of Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium consist of the sequences Gly(5), l-Ala(2), and d-Asx, respectively. Expression of the fmhB, femA, and femB genes of S. aureus in E. faecalis led to the production of peptidoglycan precursors substituted by mosaic side chains that were efficiently used by the penicillin-binding proteins for cross-bridge formation. The Fem transferases were specific for incorporation of glycyl residues at defined positions of the side chains in the absence of any additional S. aureus factors such as tRNAs used for amino acid activation. The PBPs of E. faecalis displayed a broad substrate specificity because mosaic side chains containing from 1 to 5 residues and Gly instead of l-Ala at the N-terminal position were used for peptidoglycan cross-linking. Low affinity PBP2a of S. aureus conferred beta-lactam resistance in E. faecalis and E. faecium, thereby indicating that there was no barrier to heterospecific expression of resistance caused by variations in the structure of peptidoglycan precursors. Thus, conservation of the structure of the peptidoglycan cross-bridges in members of the same species reflects the high specificity of the enzymes for side chain synthesis, although this is not essential for the activity of the PBPs.     

Cloning, sequencing and expression in Escherichia coli of the low-affinity penicillin binding protein of Enterococcus faecalis

Abstract Low-affinity penicillin binding proteins are particular membrane proteins, in several Gram-positive bacteria, which are involved in β-lactam antibiotic resistance. The structural gene for the low-affinity penicillin binding protein 5 (PBP5) of Enterococcus faecalis was cloned and sequenced. From the sequence of the 3378 bp, a 2040 bp coding region was identified. From biochemical analysis it emerges that E. faecalis PBP5 is a type II membrane protein with an uncleaved N-terminal and is composed of 679 amino acids with a molecular weight of 74055. This protein showed 48 and 33% of identity with Enterococcus hirae PBP5 and Staphylococcus aureus PBP2a, both low-affinity PBPs involved in β-lactam resistance. Anti-PBP5 antibodies cross-reacted with a membrane protein present in other species of enterococci, but the entire gene fragment cloned hybridized only with DNAs of E. faecalis strains, thus suggesting that genes coding for low-affinity PBPs of enterococci are not stictly homologous. In this experiment digoxigenin-labelled E. faecalis DNA was used.     

Studies on the mechanism of intrinsic resistance to beta-lactam antibiotics in group D streptococci

Six penicillin-binding proteins (PBPs) were detected in clinical isolates of each one of three group D streptococci: Streptococcus bovis, S. faecalis and S. faecium. When examined in whole organisms, the PBPs of S. faecium, the most penicillin-resistant species of group D streptococci, generally had lower affinities for the antibiotic than those of S. faecalis (intermediate penicillin resistance), which in turn were of lower affinity than those of S. bovis (penicillin-sensitive). On the other hand, no quantitative correlation could be established between the binding of penicillin to any one PBP or group of PBPs, and the penicillin MIC value for the corresponding micro-organism. Examination of the amounts of antibiotic bound and the rates of binding to PBPs of equal numbers of protoplasts and whole bacteria of S. faecalis and S. faecium, indicated that there was no permeability barrier to benzylpenicillin in the cell walls of these species. The lower antibacterial effectiveness of cephalothin compared with ampicillin in group D streptococci was paralleled by the higher concentrations of cephalothin needed in competition assays to inhibit the lower molecular size PBPs of these bacteria.     

Identification of a streptococcal penicillin-binding protein that reacts very slowly with penicillin.

Penicillin-binding protein (PBP) 5 of Streptococcus faecium ATCC 9790 has an unusually low affinity for penicillin (50% binding occurred at a penicillin level of 8 micrograms/ml after 60 min of incubation, and the protein only became labeled after 20 min of incubation with high concentrations of radioactive penicillin). PBPs with similar properties are carried by strains of Streptococcus durans, Streptococcus faecalis, and Streptococcus lactis but not by strains of groups A, B, C, and G streptococci or Streptococcus pneumoniae. The strains carrying the slow-reacting PBP demonstrated a sensitivity to penicillin that was several hundred times lower than that of strains not carrying it. Spontaneous mutants with minimal inhibitory concentrations of penicillin of 20, 40, and 80 micrograms/ml were isolated from S. faecium ATCC 9790. They all showed a dramatic increase in the amount of slow-reacting PBP produced. Mutants with increased penicillin resistance were also isolated from wild-type strains of S. durans, S. faecalis, and S. faecium. All of them carried a greater amount of the slow-reacting PBP than that carried by the parent. Finally, it was found that resistant S. faecium ATCC 9790 mutants grew normally in the presence of penicillin concentrations that were far above that saturating all PBPs except PBP 5. Cell growth was, on the contrary, inhibited by a penicillin concentration that saturated the slow-reacting PBP by 90%. This penicillin dose was equal to the minimal inhibitory concentration.     

Interaction of beta-lactam antibiotics with penicillin-binding proteins from Bacillus megaterium

The binding properties of 25 beta-lactam antibiotics to Bacillus megaterium membranes have been studied. The affinities of the antibiotics for the penicillin-binding proteins (PBPs) are also reported. We found that PBP 4 has the highest affinity for nearly all the antibiotics studied whereas PBP 5 has the lowest affinity. Both PBP 4 and PBP 5 appear to be dispensable for the maintenance of bacterial growth and survival and appear to be DD-carboxypeptidases. Only the beta-lactam cefmetazol bound preferentially to PBP 5 and has been used to study the inhibition of DD-carboxypeptidase. Comparative studies with beta-lactam that simultaneously result in (a) binding to PBPs 1 and 3, (b) inhibition of cell growth and (c) lysis, stressed the importance of PBPs 1 and 3 for cell growth and survival.     

Population biology of Gram-positive pathogens: high-risk clones for dissemination of antibiotic resistance

Infections caused by multiresistant Gram-positive bacteria represent a major health burden in the community as well as in hospitalized patients. Staphylococcus aureus, Enterococcus faecalis and Enterococcus faecium are well-known pathogens of hospitalized patients, frequently linked with resistance against multiple antibiotics, compromising effective therapy. Streptococcus pneumoniae and Streptococcus pyogenes are important pathogens in the community and S. aureus has recently emerged as an important community-acquired pathogen. Population genetic studies reveal that recombination prevails as a driving force of genetic diversity in E. faecium, E. faecalis, S. pneumoniae and S. pyogenes, and thus, these species are weakly clonal. Although recombination has a relatively modest role driving the genetic variation of the core genome of S. aureus, the horizontal acquisition of resistance and virulence genes plays a key role in the emergence of new clinically relevant clones in this species. In this review, we discuss the population genetics of E. faecium, E. faecalis, S. pneumoniae, S. pyogenes and S. aureus. Knowledge of the population structure of these pathogens is not only highly relevant for (molecular) epidemiological research but also for identifying the genetic variation that underlies changes in clinical behaviour, to improve our understanding of the pathogenic behaviour of particular clones and to identify novel targets for vaccines or immunotherapy.     

湖北地区部分医院肠球菌耐药性监测及相关因素分析

目的：了解湖北地区2000年度肠球菌的分离情况及耐药状况,并对抗感染用药进行探索,指导临床合理用药。方法：对湖北地区15所三甲医院各类临床标本中的538株肠球菌采用API细菌鉴定系统或Vitek全自动细菌鉴定系统进行分离鉴定,用纸片扩散法进行药敏试验,并以WHO细菌耐药监测网提供的WHONET4软件分析系统对试验数据进行分析处理。结果：本试验共分离肠球菌13个种别538株,其中大多数是粪肠球菌408株,占75.8%,屎肠球菌54株,占10.0%;坚忍肠球菌24株,占4.46%;鸟肠球菌10株,占1.86%。酪黄肠球菌6株,占1.11%;母鸡肠球菌6株,占1.11%等。本研究结果表明肠球菌的主要感染部位为泌尿道31.4%和各种分泌物31.0%。同时从药敏结果可见屎肠球菌和坚忍肠球菌对大多数抗生素的耐药性高于粪肠球基本国策 ;并发现糖肽类抗生素耐药菌株比例尚不高;庆大霉素呈高水平耐药球菌株比例则较高;对青霉素和氨卞西林耐药,本研究结果亦有反映,并且鸟肠球菌的耐药性高于粪肠球菌近三倍。结论：本年度分离的肠球菌占全年总分离菌的第六位,说明我国目前由肠球菌引起的感染所占比例不高,但仍应引起临床的高度重视,并进行动态的监测。加强对肠球菌特别是VRR耐药性的监测是非常必要的,泌尿道肠球菌感染的抗生素中呋喃妥因的耐药率较四环素和喹诺酮类药物为低,故仍不失为治疗该类泌感的首选药物,菌株差别与地区有关,由于肠球菌属中的不同种对抗生素的敏感性不同。因此种的鉴定是对该地区医院感染暴发流行,选择治疗方案的重要工具。     

Replacement of the essential penicillin-binding protein 5 by high-molecular mass PBPs may explain vancomycin-β-lactam synergy in low-level vancomycin-resistant Enterococcus faecium D366

The mechanism of synergy between vancomycin and penicillin, as well as other beta-lactam antibiotics, was examined in a penicillin-resistant E. faecium (D366) expressing an inducible low-level resistance to vancomycin. It was demonstrated that penicillin per se was not able to reduce the inducible expression of the 39.5-kDa protein (VANB) or the carboxypeptidase activity which are involved in the mechanism of vancomycin resistance of this strain. Assays of competition between 3H-benzylpenicillin and diverse beta-lactam antibiotics suggested as the most likely explanation of the synergy that, once vancomycin resistance has been induced, the high-molecular mass penicillin-binding proteins (PBPs), and possibly PBP1 in particular, which have a high affinity for beta-lactam antibiotics, take over the role of the low-affinity PBP5 which is, in the non-induced strain, responsible for beta-lactam resistance.     

Drug resistance of 100 clinical strains of Enterococcus spp

The aim of this study was to evaluate the drug susceptibility of 100 Enterococcus spp. strains isolated from patients hospitalized in State Clinical Hospital No 1 in Warsaw. All strains were identified (API 20 STREP) and their susceptibility to antibiotics was tested (ATB STREP) in automatic ATB system. Additionally, PYRase activity, beta-lactamase production (in nitrocefin test), MICs for vancomycin and teicoplanin (E test), HLAR--high level aminoglycoside resistance and susceptibility to vancomycin, teicoplanin, piperacillin and piperacillin/tazobactam (disc diffusion method) were determined. E. faecalis ATCC 29212 was used as the control strain. Fifty E. faecalis, 45 E. faecium, 2 E. casseliflavus, 2 E. durans and 1 E. avium strain were cultured. All strains were PYRase-positive and beta-lactamase-negative. Ten isolates demonstrated intermediate susceptibility to vancomycin (6--E. faecalis and 4--E. faecium). One E. faecalis strain was intermediately susceptible to both glycopeptides. One E. casseliflavus strain showed low-level resistance to vancomycin, but this strain was susceptible to teicoplanin--phenotype Van C. HLAR strains were found among 31 E. faecalis and 40 E. faecium strains. 48 E. faecalis strains were susceptible to piperacillin and 49 to piperacillin/tazobactam. Whereas, 41 E. faecium were resistant to both these drugs. Thirty six per cent of isolates were resistant to penicillin and ampicillin, 73% to erythromycin, 87% to tetracycline, 89% to lincomycin and 56% to nitrofurantoin. Some discrepancies were noticed between the results of different methods applied for susceptibility testing--ATB system, E test and disc diffusion. These discrepancies concerned HLAR detection and susceptibility to glycopeptides determination. The best methods were: disc-diffusion for HLAR detection and E test for determination of resistance to vancomycin and teicoplanin. Increasing resistance to antimicrobial agents is observed in clinical Enterococcus spp. isolates cultured in our laboratory, especially in E. faecium strains. It is necessary to control the dissemination of multiresistant Enterococcus spp. strains in hospital wards.     

The microbiology of butyrate formation in the human colon

A physical map of the Enterococcus faecium ATCC19434 chromosome was constructed by NotI, I-CeuI and Sse8387I. The chromosome was a circular DNA of 2600 kb in size, and contained six rRNA operons (rrn). The locations and orientations of the six rrn operons and 24 different determinants were mapped. Genomes of three additional E. faecium strains were also analyzed by I-CeuI digestion, and the genome sizes were found to vary from 2550 to 2995 kb. We further investigated the genome sizes and number of rrn operons in four E. faecalis, one E. avium, and one E. durans strains. The genome sizes were larger than E. faecium: 3000-3250 kb in E. faecalis, 3445 kb in E. avium, and 3070 kb in E. durans. E. avium and E. durans contained six rrn operons as in E. faecium, but all the E. faecalis strains possessed four rrn operons.     

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.     

Antimicrobial resistance of Enterococcus species isolated from produce

The purpose of this study was to characterize the antibiotic resistance profiles of Enterococcus species isolated from fresh produce harvested in the southwestern United States. Among the 185 Enterococcus isolates obtained, 97 (52%) were Enterococcus faecium, 38 (21%) were Enterococcus faecalis, and 50 (27%) were other Enterococcus species. Of human clinical importance, E. faecium strains had a much higher prevalence of resistance to ciprofloxacin, tetracycline, and nitrofurantoin than E. faecalis. E. faecalis strains had a low prevalence of resistance to antibiotics used to treat E. faecalis infections of both clinical and of agricultural relevance, excluding its intrinsic resistance patterns. Thirty-four percent of the isolates had multiple-drug-resistance patterns, excluding intrinsic resistance. Data on the prevalence and types of antibiotic resistance in Enterococcus species isolated from fresh produce may be used to describe baseline antibiotic susceptibility profiles associated with Enterococcus spp. isolated from the environment. The data collected may also help elucidate the role of foods in the transmission of antibiotic-resistant strains to human populations.     

One or two low affinity penicillin-binding proteins may be responsible for the range of susceptibility of Enterococcus faecium to benzylpenicillin

Three benzylpenicillin-resistant, clinical isolates of Enterococcus faecium (MIC values 16-64 micrograms ml-1) contained six penicillin-binding proteins (PBPs), of which PBP5 was the most abundant and had the lowest affinity for the antibiotic. Four benzylpenicillin-susceptible strains (MIC values 0.031-0.5 microgram ml-1) were obtained as spontaneous derivatives from these above organisms. There were significant decreases in the amounts of PBP5 in each of the derivatives, with the concomitant appearance of a new, higher affinity PBP (5*) in three strains. Increased amounts of PBP5, with no changes in PBP5*, were found in several mutants with intermediate-level benzylpenicillin-resistance (MIC values 1-8 micrograms ml-1) selected from two of the susceptible strains. Examination of 18 other clinical isolates, with a wide range of susceptibilities to benzylpenicillin (MIC values 0.062-128 micrograms ml-1), showed that PBP5* was present in 13 strains, and PBP5 in all of them, but in differing amounts. The results concerning the relative amounts and relative affinities of PBPs 5* and 5 allowed the categorization of the various strains into six groups, within which organisms had somewhat similar susceptibilities to benzylpenicillin.     

Comparative study of vanA gene transfer from Enterococcus faecium to Enterococcus faecalis and to Enterococcus faecium in the intestine of mice

Vancomycin-resistant enterococci represent a large reservoir in animals because of the use of avoparcin as a growth promoter in Europe. These strains of animal origin enter the food chain and can either colonize the human gut or transfer their resistance genes to the human microbiota. In this study, we compared the transfer of vancomycin resistance from resistant animal Enterococcus faecium to sensitive human Enterococcus faecalis and E. faecium. We analysed these transfers in dibiotic mice and human faecal flora-associated mice. VanA transfer from animal E. faecium to human E. faecalis occurred in dibiotic mice. The transconjugants appeared rapidly and persisted at levels between 3.0 and 4.0 log10 colony-forming units g(-1) of faeces. In human faecal flora-associated mice, vanA gene transfer was not detected towards E. faecalis but was possible between E. faecium strains. Our experiments revealed the possibility of vanA transfer from animal E. faecium to human E. faecalis in vitro and in vivo in the intestine of dibiotic mice. However, intraspecies transfer of vanA gene seems more common than interspecies transfer among enterococci.     

Penicillin-binding proteins of clostridia

The penicillin-binding protein (PBP) profiles of 33Clostridium perfringens and sixClostridium species isolated from clinically significant infections were analyzed. Three new PBPs—PBPs 2B, 4B, and 5B (84, 70, and 49 kDa respectively)—and a high-molecular-weight PBP 6 (45 kDa) were demonstrated in theC. perfringens isolates. In addition to PBPs 1 and 2, PBPs 2B and 4B were seen to show low binding affinities for penicillin, although further studies are required to determine their possible roles in the development of penicillin resistance. The PBP profiles of theC. perfringens isolates were complex. Variations in apparent molecular weights (M _r s) of all PBPs, with the exception of PBP 5 and the presence or absence of PBPs 2, 3, and 4B, gave rise to nine different PBP patterns. The high-M _rPBPs 5 and 6, which exhibited high-penicillin-binding affinities, were with only one exception consistent within theC. perfringens isolates. These PBPs 5 and 6 of theC. perfringens isolates and independent PBPs found in the otherClostridium species studied indicate that PBP analysis may assist in the differentiation ofClostridium spacies.     

The ponA gene of Enterococcus faecalis JH2-2 codes for a low-affinity class A penicillin-binding protein

A soluble derivative of the Enterococcus faecalis JH2-2 class A PBP1 (*PBP1) was overproduced and purified. It exhibited a glycosyltransferase activity on the Escherichia coli 14C-labeled lipid II precursor. As a DD- peptidase, it could hydrolyze thiolester substrates with efficiencies similar to those of other class A penicillin-binding proteins (PBPs) and bind beta-lactams, but with k2/K (a parameter accounting for the acylation step efficiency) values characteristic of penicillin-resistant PBPs.     

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.