Inhibition Of Peptidoglycan Synthesis Research Articles

Exploiting current understanding of antibiotic action for discovery of new drugs

The introduction of antibiotics for the chemotherapy of bacterial infections has been one of the most important medical achievements of the past 50 years. However, the emergence of bacterial resistance to antibiotics undermines the therapeutic utility of existing agents, creating a requirement for the discovery of new antibacterial drugs. Several drug discovery strategies have emerged, including incremental improvements to existing antibiotics by chemical manipulation and the search for novel drug targets based on genomic approaches. An alternative strategy seeks to exploit opportunities for drug discovery arising from an understanding of the mode of action of existing antibiotics. Thus biochemical pathways or processes inhibited by antibiotics already in clinical use may nevertheless contain key functions that represent unexploited targets for further drug discovery. A major benefit of employing pathways or processes that are already known to contain drug targets is that proof of principle for drug intervention is already established. This approach to drug discovery is illustrated by reviewing target sites for existing antibiotics and considering how this information might be applied for the discovery of new agents inhibiting peptidoglycan synthesis, tRNA synthesis, transcription and DNA replication

Synthesis and mode of action of hydrophobic derivatives of the glycopeptide antibiotic eremomycin and des-(N-methyl-D-leucyl)eremomycin against glycopeptide-sensitive and -resistant bacteria.

Des-(N-methyl-D-leucyl)eremomycin was obtained by Edman degradation of eremomycin. Derivatives with a hydrophobic substituent at the exterior of the molecule were then synthesized, and their antibacterial activities were compared with similar derivatives of eremomycin. Comparison of derivatives of eremomycin containing the n-decyl or p-(p-chlorophenyl)benzyl substituent in the eremosamine moiety (N') and n-decyl or p-(p-chlorophenyl)benzylamides with similar derivatives of eremomycin possessing the damaged peptide core (a defective binding pocket) showed that compounds of both types are almost equally active against glycopeptide-resistant strains of enterococci (GRE), whereas eremomycin derivatives are more active against staphylococci. Hydrophobic 7d-alkylaminomethylated derivatives of eremomycin (9, 10) demonstrated similar antibacterial properties. Since the basic mode of action of glycopeptide antibiotics involves binding to cell wall intermediates terminating in -D-Ala-D-Ala and this interaction is seriously decreased in the hexapeptide derivatives (lacking the critical N-methyl-D-leucine), we suggest that these hydrophobic derivatives may inhibit peptidoglycan synthesis in the absence of dipeptide binding. NMR binding experiments using Ac-D-Ala-D-Ala show that binding constants of these hexapeptide derivativies are decreased in comparison with the corresponding heptapeptides with intact binding pocket. This is in agreement with the decreased biological activity of the hexapeptide derivatives against vancomycin-sensitive strains in comparison with the activity of parent compounds. Binding to the lactate cell wall analogue Ac-D-Ala-D-Lac with decylamide of eremomycin 8 was not observed, demonstrating that the interaction with this target in GRE does not occur. While hydrophobic glycopeptide derivatives retain the ability to inhibit the synthesis of peptidoglycan in manner of natural glycopeptides, biochemical investigation supports the hypothesis that they inhibit the transglycosylase stage of bacterial peptidoglycan biosynthesis even in the absence of dipeptide or depsipeptide binding.

The identification and characterization of hydrazinyl urea-based antibacterial agents through combinatorial chemistry

An effort to identify novel inhibitors of peptidoglycan synthesis with antibacterial activity resulted in the discovery of a series of biaryl urea-based antibacterial agents through isolation of a by-product from a mixture-based combinatorial library of semi-carbazones and subsequent parallel synthesis efforts. The compounds were shown to possess broad spectrum antibacterial activity against gram-positive drug resistant pathogens, and showed apparent specificity for disruption of the bacterial cell wall biosynthesis pathway.

AddendumCorrelation between Increased Airway Responsiveness and Severity of Pulmonary Edema

The activity of existing antibiotics is diminishing due to the increasing number of resistant strains and by the increase of infections with naturally resistant microorganisms. New agents are urgently needed to meet this challenge and the molecular strategies adopted for the discovery of these compounds must focus on minimizing the emergence of future resistance to them. Novel compounds can be grouped on the basis of their mechanism of action: inhibitors of nucleic acid synthesis (fluoroquinolones), inhibitors of protein synthesis (ketolides, oxazolidinones, streptogramins, and glycylcyclines), inhibitors of peptidoglycan synthesis (β-lactams and glycopeptides), and agents interfering with membrane function (cationic peptides, and lipopeptides). Regarding the agents that are already in the research and development pipeline, only the oxazolidinones, the cationic peptides and the lipopeptide antibiotics can be truly considered as structurally novel inhibitors because the other agents are analogues of existing compounds that have been in use for many years.

Specific Binding of Nisin to the Peptidoglycan Precursor Lipid II Combines Pore Formation and Inhibition of Cell Wall Biosynthesis for Potent Antibiotic Activity

Unlike numerous pore-forming amphiphilic peptide antibiotics, the lantibiotic nisin is active in nanomolar concentrations, which results from its ability to use the lipid-bound cell wall precursor lipid II as a docking molecule for subsequent pore formation. Here we use genetically engineered nisin variants to identify the structural requirements for the interaction of the peptide with lipid II. Mutations affecting the conformation of the N-terminal part of nisin comprising rings A through C, e.g. [S3T]nisin, led to reduced binding and increased the peptide concentration necessary for pore formation. The binding constant for the S3T mutant was 0.043 x 10(7) m(-1) compared with 2 x 10(7) m(-1) for the wild-type peptide, and the minimum concentration for pore formation increased from the 1 nm to the 50 nm range. In contrast, peptides mutated in the flexible hinge region, e.g. [DeltaN20/DeltaM21]nisin, were completely inactive in the pore formation assay, but were reduced to some extent in their in vivo activity. We found the remaining in vivo activity to result from the unaltered capacity of the mutated peptide to bind to lipid II and thus to inhibit its incorporation into the peptidoglycan network. Therefore, through interaction with the membrane-bound cell wall precursor lipid II, nisin inhibits peptidoglycan synthesis and forms highly specific pores. The combination of two killing mechanisms in one molecule potentiates antibiotic activity and results in nanomolar MIC values, a strategy that may well be worth considering for the construction of novel antibiotics.

Antibacterial activity of synthetic analogues based on the disaccharide structure of moenomycin, an inhibitor of bacterial transglycosylase.

Moenomycin is a natural product glycolipid that inhibits the growth of a broad spectrum of Gram-positive bacteria. In Escherichia coli, moenomycin inhibits peptidoglycan synthesis at the transglycosylation stage, causes accumulation of cell-wall intermediates, and leads to lysis and cell death. However, unlike Esc. coli, where 5-6 log units of killing are observed, 0-2 log units of killing occurred when Gram-positive bacteria were treated with similar multiples of the MIC. In addition, bulk peptidoglycan synthesis in intact Gram-positive cells was resistant to the effects of moenomycin. In contrast, synthetic disaccharides based on the moenomycin disaccharide core structure were identified that were bactericidal to Gram-positive bacteria, inhibited cell-wall synthesis in intact cells, and were active on both sensitive and vancomycin-resistant enterococci. These disaccharide analogues do not inhibit the formation of N:-acetylglucosamine-ss-1, 4-MurNAc-pentapeptide-pyrophosphoryl-undecaprenol (lipid II), but do inhibit the polymerization of lipid II into peptidoglycan in Esc. coli. In addition, cell growth was required for bactericidal activity. The data indicate that synthetic disaccharide analogues of moenomycin inhibit cell-wall synthesis at the transglycosylation stage, and that their activity on Gram-positive bacteria differs from moenomycin due to differential targeting of the transglycosylation process. Inhibition of the transglycosylation process represents a promising approach to the design of new antibacterial agents active on drug-resistant bacteria.

'Intergenic' blr gene in Escherichia coli encodes a 41-residue membrane protein affecting intrinsic susceptibility to certain inhibitors of peptidoglycan synthesis.

In the annotation of genomic sequences, small open reading frames (ORFs) are often neglected, particularly if they have no homology to other ORFs or proteins. A mini-TnphoA insertion in a 602 bp 'intergenic' region of the Escherichia coli chromosome at genomic nucleotide 1702674 gave rise to a membrane-bound PhoA fusion protein and a two- to fourfold increase in the intrinsic susceptibility to a wide spectrum of beta-lactam antibiotics without affecting beta-lactamase activity or susceptibility to tetracycline, chloramphenicol, gentamicin or quinolones. Susceptibility was also increased to cycloserine and bacitracin, but not to fosfomycin or valinomycin; these drugs, like beta-lactams, inhibit peptidoglycan synthesis, although by different mechanisms. A clone bearing only 358 bp of this 'blr' region restored resistance to the parental level. Two amber mutations in the clone prevented such restoration and were counteracted by an amber suppressor, proving that the active species is a protein. The Blr protein has 41 amino acids, with a single predicted transmembrane helix, but no clear homology to any other protein. A transcriptional start exists 39 bp upstream from the translational start. The membrane location of Blr suggests that it may be part of an efflux pump or involved in murein metabolism. The results indicate that genes for other very small functional proteins may lie within 'intergenic' regions.

Genetic evidence that the bacteriophage phi X174 lysis protein inhibits cell wall synthesis.

Protein E, a 91-residue membrane protein of phiX174, causes lysis of the host in a growth-dependent manner reminiscent of cell wall antibiotics, suggesting E acts by inhibiting peptidoglycan synthesis. In a search for the cellular target of E, we previously have isolated recessive mutations in the host gene slyD (sensitivity to lysis) that block the lytic effects of E. The role of slyD, which encodes a FK506 binding protein-type peptidyl-prolyl cis-trans isomerase, is not fully understood. However, E mutants referred to as Epos (plates on slyD) lack a slyD requirement, indicating that slyD is not crucial for lysis. To identify the gene encoding the cellular target, we selected for survivors of Epos. In this study, we describe the isolation of dominant mutations in the essential host gene mraY that result in a general lysis-defective phenotype. mraY encodes translocase I, which catalyzes the formation of the first lipid-linked intermediate in cell wall biosynthesis. The isolation of these lysis-defective mutants supports a model in which translocase I is the cellular target of E and that inhibition of cell wall synthesis is the mechanism of lysis.

Les glycopeptides

Glycopeptides. Objectives: To review pharmacology, pharmacokinetic and therapeutic use of glycopeptides in intensive care units. Data sources: Extraction from Medline® database of French and English articles on glycopeptides and search along with major review articles. Data selection: The collected articles were reviewed and selected according to their quality and originality. The more recent data were selected. Data synthesis: Glycopeptides are bactericidal antibiotics which are only active against Gram positive species acting by inhibiting peptidoglycan synthesis. They had been in clinical use for almost 30 years without high-level resistance underlining. For ten years, there have been disturbing reports of first, resistance to vancomycin in enterococcal species and more recently in strains of Staphylococcus aureus by complex and large mechanisms of action. This new resistances may lead to a therapeutic impasse and a fatal issue for infected patients. The only response to this situation is the respect of prescription rules and the careful use of antibiotics. Conclusion: Considering their spectrum, glycopeptides are an antibiotic family which importance is fundamental to treat infected patients of intensive care units. Staff members of intensive care units are responsible for their good use.

Chlorobiphenyl-desleucyl-vancomycin inhibits the transglycosylation process required for peptidoglycan synthesis in bacteria in the absence of dipeptide binding

Novel glycopeptide analogs are known that have activity on vancomycin resistant enterococci despite the fact that the primary site for drug interaction, d-ala- d-ala, is replaced with d-ala- d-lactate. The mechanism of action of these compounds may involve dimerization and/or membrane binding, thus enhancing interaction with d-ala- d-lactate, or a direct interaction with the transglycosylase enzymes involved in peptidoglycan polymerization. We evaluated the ability of vancomycin (V), desleucyl-vancomycin (desleucyl-V), chlorobiphenyl-vancomycin (CBP-V), and chlorobiphenyl-desleucyl-vancomycin (CBP-desleucyl-V) to inhibit (a) peptidoglycan synthesis in vitro using UDP-muramyl-pentapeptide and UDP-muramyl-tetrapeptide substrates and (b) growth and peptidoglycan synthesis in vancomycin resistant enterococci. Compared to V or CBP-V, CBP-desleucyl-V retained equivalent potency in these assays, whereas desleucyl-V was inactive. In addition, CBP-desleucyl-V caused accumulation of N-acetylglucosamine-β-1,4-MurNAc-pentapeptide-pyrophosphoryl-undecaprenol (lipid II). These data show that CBP-desleucyl-V inhibits peptidoglycan synthesis at the transglycosylation stage in the absence of binding to dipeptide.

Identification of an antigen localized to an apparent septum within dividing chlamydiae.

The process of chlamydial cell division has not been thoroughly investigated. The lack of detectable peptidoglycan and the absence of an FtsZ homolog within chlamydiae suggest an unusual mechanism for the division process. Our laboratory has identified an antigen (SEP antigen) localized to a ring-like structure at the apparent septum within dividing chlamydial reticulate bodies (RB). Antisera directed against SEP show similar patterns of antigen distribution in Chlamydia trachomatis and Chlamydia psittaci RB. In contrast to localization in RB, SEP in elementary bodies appears diffuse and irregular, suggesting that the distribution of the antigen is developmental-stage specific. Treatment of chlamydiae with inhibitors of peptidoglycan synthesis or culture of chlamydiae in medium lacking tryptophan leads to the formation of nondividing, aberrant RB. Staining of aberrant RB with anti-SEP reveals a marked redistribution of the antigen. Within C. trachomatis-infected cells, ampicillin treatment leads to high levels of SEP accumulation at the periphery of aberrant RB, while in C. psittaci, treatment causes SEP to localize to distinct punctate sites within the bacteria. Aberrancy produced via tryptophan depletion results in a different pattern of SEP distribution. In either case, the reversal of aberrant formation results in the production of normal RB and a redistribution of SEP to the apparent plane of bacterial division. Collectively these studies identify a unique chlamydial-genus-common and developmental-stage-specific antigen that may be associated with RB division.

Chlorobiphenyl-desleucyl-vancomycin inhibits the transglycosylation process required for peptidoglycan synthesis in bacteria in the absence of dipeptide binding.

Novel glycopeptide analogs are known that have activity on vancomycin resistant enterococci despite the fact that the primary site for drug interaction, D-ala-D-ala, is replaced with D-ala-D-lactate. The mechanism of action of these compounds may involve dimerization and/or membrane binding, thus enhancing interaction with D-ala-D-lactate, or a direct interaction with the transglycosylase enzymes involved in peptidoglycan polymerization. We evaluated the ability of vancomycin (V), desleucyl-vancomycin (desleucyl-V), chlorobiphenyl-vancomycin (CBP-V), and chlorobiphenyl-desleucyl-vancomycin (CBP-desleucyl-V) to inhibit (a) peptidoglycan synthesis in vitro using UDP-muramyl-pentapeptide and UDP-muramyl-tetrapeptide substrates and (b) growth and peptidoglycan synthesis in vancomycin resistant enterococci. Compared to V or CBP-V, CBP-desleucyl-V retained equivalent potency in these assays, whereas desleucyl-V was inactive. In addition, CBP-desleucyl-V caused accumulation of N-acetylglucosamine-beta-1, 4-MurNAc-pentapeptide-pyrophosphoryl-undecaprenol (lipid II). These data show that CBP-desleucyl-V inhibits peptidoglycan synthesis at the transglycosylation stage in the absence of binding to dipeptide.

The lantibiotic mersacidin inhibits peptidoglycan synthesis by targeting lipid II.

The lantibiotic mersacidin exerts its bactericidal action by inhibition of peptidoglycan biosynthesis. It interferes with the membrane-associated transglycosylation reaction; during this step the ultimate monomeric peptidoglycan precursor, undecaprenyl-pyrophosphoryl-MurNAc-(pentapeptide)-GlcNAc (lipid II) is converted into polymeric nascent peptidoglycan. In the present study we demonstrate that the molecular basis of this inhibition is the interaction of mersacidin with lipid II. The adsorption of [14C]mersacidin to growing cells, as well as to isolated membranes capable of in vitro peptidoglycan synthesis, was strictly dependent on the availability of lipid II, and antibiotic inhibitors of lipid II formation strongly interfered with this binding. Direct evidence for the interaction was provided by studies with isolated lipid II. [14C]mersacidin associated tightly with [14C]lipid II micelles; the complex was stable even in the presence of 1% sodium dodecyl sulfate. Furthermore, the addition of isolated lipid II to the culture broth efficiently antagonized the bactericidal activity of mersacidin. In contrast to the glycopeptide antibiotics, complex formation does not involve the C-terminal D-alanyl-D-alanine moiety of the lipid intermediate. Thus, the interaction of mersacidin with lipid II apparently occurs via a binding site which is not targeted by any antibiotic currently in use.

A vancomycin-inducible lacZ reporter system in Bacillus subtilis: induction by antibiotics that inhibit cell wall synthesis and by lysozyme.

We have constructed a Bacillus subtilis strain in which expression of a vanH::lacZ gene fusion is regulated by VanR and VanS of Enterococcus faecium. This construct allows a nonpathogenic bacterial strain to be used as a model system for studying regulation of vancomycin resistance. Antibiotics and enzymes that affect cell wall biosynthesis and stability were tested for the ability to induce lacZ expression. As a result, fosfomycin and D-cycloserine were added to the group of peptidoglycan synthesis inhibitors shown to induce expression from the vanH promoter. Induction by cell wall hydrolytic enzymes, as well as by antibiotics whose actions may lead to the accumulation of chemically different peptidoglycan precursors, raises the possibility that models that postulate induction by peptidoglycan [correction of peptidodoglycan] precursors are wrong.

Permeability of mycobacterial cell envelopes to sterols: Peptidoglycan as the diffusion barrier

AbstractVancomycin, an inhibitor of peptidoglycan synthesis, depressed the growth of Mycobacterium sp. NRRL B 3805 and MB 3683, but not the β‐sitosterol side chain degradation and androstane derivatives accumulation. As a result, the specific activity (products formed/cell weight unit × h) increased threefold in the peptidoglycan‐deficient cells, indicating faster crossing of the sterol through the cell water barrier.Cell wall preparations: crude cell wall (CCW), purified cell wall (PCW), and peptidoglycan — enriched PCW — residue (PEPCW) were obtained and analysed in order to find a relationship between the vancomycin — induced chemical changes and the permeation rate of the sterol.The amounts of CCW, PCW and PEPCW, produced from 8 g lyophilised control cells were 445, 170 and 28 mg respectively. The respective figures were 176, 61, and 4.8 mg for vancomycin — treated cells. In addition to the lower content of the rigid layer, a distinct shift in the molar ratios of the peptidoglycan constituents: alanine, glutamic, diaminopimelic and muramic acids, and glucosamine was observed under the action of the murein inhibitor. The most significant change was that of muramic acid: diaminopimelic acid molar ratio, the compounds which are markers of glycan strands and tetrapeptides, respectively. In control cells it was approximately 1:1, and increased to 1.34–1.43: 1 in the compared preparation, which indicated a marked decrease in the tetrapeptide moieties crosslinking the main glycan strands. Together with the general lower content of murein, this modification may be responsible for the enhanced sterol permeation through the cell wall.

Direct correlation between overproduction of guanosine 3',5'-bispyrophosphate (ppGpp) and penicillin tolerance in Escherichia coli.

The penicillin tolerance exhibited by amino acid-deprived Escherichia coli has been previously proposed to be a consequence of the stringent response. Evidence indicating that penicillin tolerance is directly attributable to guanosine 3',5'-bispyrophosphate (ppGpp) overproduction and not to some other effect of amino acid deprivation is now presented. Accumulation of ppGpp in the absence of amino acid deprivation was achieved by the controlled overexpression of the cloned relA gene, which encodes ppGpp synthetase I. The overproduction of ppGpp resulted in the inhibition of both peptidoglycan and phospholipid synthesis and in penicillin tolerance. The minimum concentration of ppGpp required to establish these phenomena was determined to be 870 pmol per mg (dry weight) of cells. This represented about 70% of the maximum level of ppGpp accumulated during the stringent response. Penicillin tolerance and the inhibition of peptidoglycan synthesis were both suppressed when ppGpp accumulation was prevented by treatment with chloramphenicol, an inhibitor of ppGpp synthetase I activation. Glycerol-3-phosphate acyltransferase, the product of plsB, was recently identified as the main site of ppGpp inhibition in phospholipid synthesis (R. J. Health, S. Jackowski, and C. O. Rock, J. Biol. Chem. 269:26584-26590, 1994). The overexpression of the cloned plsB gene reversed the penicillin tolerance conferred by ppGpp accumulation. This result supports previous observations indicating that the membrane-associated events in peptidoglycan metabolism were dependent on ongoing phospholipid synthesis. Interestingly, treatment with beta-lactam antibiotics by itself induced ppGpp accumulation, but the maximum levels attained were insufficient to confer penicillin tolerance.

Beta-lactam-induced bacteriolysis of amino acid-deprived Escherichia coli is dependent on phospholipid synthesis

The penicillin tolerance of amino acid-deprived relA+ Escherichia coli is attributed to the stringent response; i.e., relaxation of the stringent response suppresses penicillin tolerance. The beta-lactam-induced lysis of amino acid-deprived bacteria resulting from relaxation of the stringent response was inhibited by cerulenin, or by glycerol deprivation in the case of a gpsA mutant (defective in the biosynthetic sn-glycerol 3-phosphate dehydrogenase). Therefore, beta-lactam-induced lysis of amino acid-deprived cells was dependent on phospholipid synthesis. The lysis process during amino acid deprivation can be experimentally dissociated into two stages designated the priming stage (during which the interaction between the beta-lactam and the penicillin-binding proteins occurs) and the beta-lactam-independent lysis induction stage. Both stages were shown to require phospholipid synthesis. It has been known for some time that the inhibition of phospholipid synthesis is among the plethora of physiological changes resulting from the stringent response. These results indicate that the inhibition of peptidoglycan synthesis and the penicillin tolerance associated with the stringent response are both secondary consequences of the inhibition of phospholipid synthesis.

Effect of inhibitors of cell envelope synthesis on β‐sitosterol side chain degradation by Mycobacterium sp. NRRL MB 3683

The role of the lipid bilayer and the peptidoglycan of the mycobacterial cell wall in the permeation of beta-sitosterol into the cell and its transformation to androst-1-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD) was studied. Specific inhibitors were used at concentrations affecting the biosynthesis of the assumed target structures, but causing only partial cell growth inhibition or exerting no effect on growth. m-Fluorophenylalanine and DL-norleucine which are known to disorganize the biosynthesis of amphipatic components of the outer layer of the lipid bilayer, used at concentrations 250 micrograms/ml and 400 micrograms/ml, respectively, increased the formation rate of AD+ADD from 0.3 (control) to 0.7 and 0.8 mg products/g dry weight/h. The disorganization of the underlying mycolyl-arabinogalactan structure by the action of the ethambutol at the concentration 40 micrograms/ml, at which the cell growth was apparently not affected, caused the decrease of the product formation from 135 mg/l to 70 mg/l. In the presence of isoniazid (350 micrograms/ml) only trace amounts of AD accumulated during 48 hours of transformation indicating much lower activity than that of the intact cells. The most effective among the tested inhibitors of peptidoglycan synthesis were glycine (15 mg/ml) and vancomycin (150 micrograms/ml) which enhanced the transformation activity of the treated cells nearly three times. Increased transformation rate was also obtained by the action of colistin at concentrations ranging from 10 micrograms/ml to 15 micrograms/ml.

Identification of the glmU gene encoding N-acetylglucosamine-1-phosphate uridyltransferase in Escherichia coli

The physiological properties of the EcoURF-1 open reading frame, which precedes the glmS gene at 84 min on the Escherichia coli chromosome (J. E. Walker, N. J. Gay, M. Saraste, and A. N. Eberle, Biochem. J. 224:799-815, 1984), were investigated. A thermosensitive conditional mutant in which the synthesis of the gene product was impaired at 43 degrees C was constructed. The inactivation of the gene in exponentially growing cells rapidly inhibited peptidoglycan synthesis. As a result, various alterations of cell shape were observed, and cell lysis finally occurred when the peptidoglycan content was 37% lower than that of normally growing cells. Analysis of the pools of peptidoglycan precursors revealed a large accumulation of N-acetylglucosamine-1-phosphate and the concomitant depletion of the pools of the seven peptidoglycan nucleotide precursors located downstream in the pathway, a result indicating that the mutational block was in the step leading from N-acetylglucosamine-1-phosphate and UTP to the formation of UDP-N-acetylglucosamine. In vitro assays showed that the overexpression of this gene in E. coli cells, directed by appropriate plasmids, led to a high overproduction (from 25- to 410-fold) of N-acetylglucosamine-1-phosphate uridyltransferase activity. This allowed us to purify this enzyme to homogeneity in only two chromatographic steps. The gene for this enzyme, which is essential for peptidoglycan and lipopolysaccharide biosyntheses, was designated glmU.

Effect of mecillinam on peptidoglycan synthesis during the division cycle of Salmonella typhimurium 2616

The effects of mecillinam, ampicillin and cephalexin on peptidoglycan synthesis in Salmonella typhimurium 2616 have been studied at equivalent concentrations or “isoactivities”. Using antibiotics at isoactivities allows a direct comparison of the biochemical effects of different antibiotics. When mecillinam was added at different times during the division cycle at a concentration that produced 50 % inhibition of peptidoglycan synthesis in an exponential culture over a short period of time, the inhibition of synthesis was greatest in the newborn cells and least in the dividing cells. Antibiotic competition experiments showed that mecillinam preferentially bound to penicillin-binding protein 2 in S. typhimurium 2616. High performance liquid chromatography analysis of the residual peptidoglycan synthesized in the presence of mecillinam showed an unexpected increase in pentapeptides and a significant increase in cross-linking. Other antibiotics added at equivalent activities did not show an increase in cross-linking.