A mecillinam-sensitive peptidoglycan crosslinking reaction in Escherichia coli

The amidinopenicillin, mecillinam, induces the formation of spherical cells of Escherichia coli by inactivation of penicillin-binding protein 2 (PBP2). A mecillinam-sensitive peptidoglycan crosslinking reaction has been demonstrated in particulate membrane preparations from this organism. The activity was detected in membranes that contained elevated levels of PBP2 and in which crosslinking reactions due to all other PBPs had been inactivated with the cephamycin antibiotic, cefmetazole. The particulate membrane preparation catalyzed synthesis of peptidoglycan that was up to 20% crosslinked from nucleotide precursors. Crosslinkage of the peptidoglycan was inhibited 50% by 0.2 μg mecillinam per ml but was not inhibited by much higher concentrations of cephamycins, which have very low affinity for PBP2. The crosslinking reaction appears to be due to the transpeptidase activity of PBP2, which is implicated in the mechanism of cell shape determination, and is the killing target for mecillinam.     

Molecular divergence of a major peptidoglycan synthetase with transglycosylase - transpeptidase activities in Escherichia coli — Penicillin-binding protein 1Bs

Penicillin-binding protein 1Bs of Escherichia coli (Mr ca. 9 × 10⁴) gave three protein bands with slightly different mobilities on sodium dodecylsulfate — polyacrylamide gel electrophoresis. The enzymatic activities of each of these proteins were identified after renaturation of the proteins separated by electrophoresis. Each of them had two enzymatic activities of the last steps of synthesis of peptidoglycan from lipid-linked precursor, i. e., activity of transglycosylase, which extends the glycan chain, and activity of penicillin-sensitive transpeptidase, which crosslinks glycan chains with peptide cross-bridges. Trypsin treatment of each of the three proteins resulted in formation of a doublet of penicillin-binding proteins (Mr ca. 5 × 10⁴). The results strongly indicate that penicillin-binding protein 1Bs are bifunctional peptidoglycan synthetase proteins differing slightly in molecular structure.     

Novel S-Benzylisothiourea Compound That Induces Spherical Cells in Escherichia coli Probably by Acting on a Rod-shape-determining Protein(s) Other Than Penicillin-binding Protein 2

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

The Rcs phosphorelay is a cell envelope stress response activated by peptidoglycan stress and contributes to intrinsic antibiotic resistance

Gram-negative bacteria possess stress responses to maintain the integrity of the cell envelope. Stress sensors monitor outer membrane permeability, envelope protein folding, and energization of the inner membrane. The systems used by gram-negative bacteria to sense and combat stress resulting from disruption of the peptidoglycan layer are not well characterized. The peptidoglycan layer is a single molecule that completely surrounds the cell and ensures its structural integrity. During cell growth, new peptidoglycan subunits are incorporated into the peptidoglycan layer by a series of enzymes called the penicillin-binding proteins (PBPs). To explore how gram-negative bacteria respond to peptidoglycan stress, global gene expression analysis was used to identify Escherichia coli stress responses activated following inhibition of specific PBPs by the β-lactam antibiotics amdinocillin (mecillinam) and cefsulodin. Inhibition of PBPs with different roles in peptidoglycan synthesis has different consequences for cell morphology and viability, suggesting that not all perturbations to the peptidoglycan layer generate equivalent stresses. We demonstrate that inhibition of different PBPs resulted in both shared and unique stress responses. The regulation of capsular synthesis (Rcs) phosphorelay was activated by inhibition of all PBPs tested. Furthermore, we show that activation of the Rcs phosphorelay increased survival in the presence of these antibiotics, independently of capsule synthesis. Both activation of the phosphorelay and survival required signal transduction via the outer membrane lipoprotein RcsF and the response regulator RcsB. We propose that the Rcs pathway responds to peptidoglycan damage and contributes to the intrinsic resistance of E. coli to β-lactam antibiotics.     

Penicillin-binding proteins of pathogenic Escherichia coli strains

The penicillin-binding proteins of 11 pathogenic Escherichia coli strains, including enteropathogenic, enterotoxigenic, enteroinvasive, enteroaggregative, and enterohemorrhagic E. coli, were detected in gels following the labeling of isolated cell envelopes with [³H]benzylpenicillin. The electrophoretic profiles, sensitivities to and morphological changes induced by β-lactam antibiotics showed that the penicillin-binding proteins of most pathogenic E. coli possess structural and physiological functions similar to those of E. coli K12.     

Peptidoglycan of Pseudomonas aeruginosa

A practical method for preparing peptidoglycan from Ps. aeruginosa and E. coli was devised. After bacterial cells were dissolved in boiling 4% SDS solution, peptidoglycan was collected and washed with water by centrifugation. Peptidoglycan was treated further with pronase and lyophilized. The final preparation of peptidoglycan from Ps. aeruginosa appeared as a filmy coagulation in electron micrograph and its amino acid composition was determined as follows: Glu/Ala/A₂pm/Mur/GlcN (100/183/104/61/98). The lysozyme digest showed the same pattern as that of E. coli peptidoglycan. N-Terminal analysis suggested that about half of the peptide chains was interbridged by the peptide bond between Ala and A₂pm. The probable ratio of muropeptides in the peptidoglycan was estimated.     

The dynamic structure of the Escherichia coli cell envelope as probed by 15N nuclear magnetic resonance spectroscopy

Proton decoupled ¹⁵N NMR spectroscopy is shown to be a useful tool for probing the dynamic structure of the bacterial cell envelope. The proton decoupled ¹⁵N NMR spectra of Escherichia coli whole cells, cell envelopes and outer membranes were obtained and displayed resonances originating from protein side-chain groups, phosphatidylethanolamine, and peptidoglycan. Removal of phospholipids from the cell envelope resulted in a decrease in the motional freedom of peptidoglycan and cell envelope proteins. The mobility of the protein Arg side-chain groups is incresed in the absence of peptidoglycan. These data provide insights into the effect of supramolecular organization on the dynamic structure of the E. coli cell envelope.     

Modification of the Peptidoglycan of Escherichia coli in the Viable But Nonculturable State

The aim of this study was to analyse the chemical composition of peptidoglycan and the state of some of the enzymes involved in its metabolism in Escherichia coli KN126 in the viable but nonculturable (VBNC) state which is a survival strategy adopted by bacteria (including those of medical interest) when exposed to environmental stresses. When entering the VBNC state, E. coli cells miniaturised and became coccus-shaped. Analysis of peptidoglycan chemical composition, by separation in HPLC of muropeptides released by muramidase digestion of purified peptidoglycan, indicated a high degree of cross-linking, a threefold increase in unusual DAP–DAP cross-linking, an increase in muropeptides bearing covalently bound lipoprotein, and a shortening of the average length of glycan strands in comparison with dividing cells. Analysis of penicillin-binding proteins (PBPs), enzymes involved in the terminal stage of peptidoglycan assembly showed the disappearance of high-molecular-weight PBPs 1A, 1B, 2, and 3 in VBNC cells. Finally, VBNC cells displayed an autolytic capability which was far higher than that of exponentially growing cells. It is suggested that part of these alterations of peptidoglycan may be connected with the VBNC state. Received: 20 March 2001 / Accepted: 7 June 2001     

Identification of SPOR Domain Amino Acids Important for Septal Localization,Peptidoglycan Binding,and a Disulfide Bond in the Cell Division Protein FtsN

SPOR domains are about 75 amino acids long and probably bind septal peptidoglycan during cell division. We mutagenized 33 amino acids with surface-exposed side chains in the SPOR domain from an Escherichia coli cell division protein named FtsN. The mutant SPOR domains were fused to Tat-targeted green fluorescent protein (^TTGFP) and tested for septal localization in live E. coli cells. Lesions at the following 5 residues reduced septal localization by a factor of 3 or more: Q251, S254, W283, R285, and I313. All of these residues map to a β-sheet in the published solution structure of FtsN^SPOR. Three of the mutant proteins (Q251E, S254E, and R285A mutants) were purified and found to be defective in binding to peptidoglycan sacculi in a cosedimentation assay. These results match closely with results from a previous study of the SPOR domain from DamX, even though these two SPOR domains share <20% amino acid identity. Taken together, these findings support the proposal that SPOR domains localize by binding to septal peptidoglycan and imply that the binding site is associated with the β-sheet. We also show that FtsN^SPOR contains a disulfide bond between β-sheet residues C252 and C312. The disulfide bond contributes to protein stability, cell division, and peptidoglycan binding.     

How Bacteria Consume Their Own Exoskeletons (Turnover and Recycling of Cell Wall Peptidoglycan)

Summary: The phenomenon of peptidoglycan recycling is reviewed. Gram-negative bacteria such as Escherichia coli break down and reuse over 60% of the peptidoglycan of their side wall each generation. Recycling of newly made peptidoglycan during septum synthesis occurs at an even faster rate. Nine enzymes, one permease, and one periplasmic binding protein in E. coli that appear to have as their sole function the recovery of degradation products from peptidoglycan, thereby making them available for the cell to resynthesize more peptidoglycan or to use as an energy source, have been identified. It is shown that all of the amino acids and amino sugars of peptidoglycan are recycled. The discovery and properties of the individual proteins and the pathways involved are presented. In addition, the possible role of various peptidoglycan degradation products in the induction of β-lactamase is discussed.     

Plasma Membrane Damage Contributes to Antifungal Activity of Silicon Against Penicillium digitatum

Putative penicillin-binding proteins (PBPs) were identified in the genome of the Burkholderia cenocepacia strain J2315 based on homology to E. coli PBPs. The three sequences identified as homologs of E. coli PBP1a, BCAL2021, BCAL0274, and BCAM2632, were cloned and expressed as His₆-tagged fusion proteins in E. coli. The fusion proteins were isolated and shown to bind β-lactams, indicating these putative PBPs have penicillin-binding activity.     

Functional and Structural Characterization of PaeM,a Colicin M-like Bacteriocin Produced by Pseudomonas aeruginosa

Colicin M (ColM) is the only enzymatic colicin reported to date that inhibits cell wall peptidoglycan biosynthesis. It catalyzes the specific degradation of the lipid intermediates involved in this pathway, thereby provoking lysis of susceptible Escherichia coli cells. A gene encoding a homologue of ColM was detected within the exoU-containing genomic island A carried by certain pathogenic Pseudomonas aeruginosa strains. This bacteriocin (pyocin) that we have named PaeM was crystallized, and its structure with and without an Mg²⁺ ion bound was solved. In parallel, site-directed mutagenesis of conserved PaeM residues from the C-terminal domain was performed, confirming their essentiality for the protein activity both in vitro (lipid II-degrading activity) and in vivo (cytotoxicity against a susceptible P. aeruginosa strain). Although PaeM is structurally similar to ColM, the conformation of their active sites differs radically; in PaeM, residues essential for enzymatic activity and cytotoxicity converge toward a same pocket, whereas in ColM they are spread along a particularly elongated active site. We have also isolated a minimal domain corresponding to the C-terminal half of the PaeM protein and exhibiting a 70-fold higher enzymatic activity as compared with the full-length protein. This isolated domain of the PaeM bacteriocin was further shown to kill E. coli cells when addressed to the periplasm of these bacteria.     

Dynamic Polar Sequestration of Excess MurG May Regulate Enzymatic Function

Advances in bacterial cell biology have demonstrated the importance of protein localization for protein function. In general, proteins are thought to localize to the sites where they are active. Here we demonstrate that in Escherichia coli, MurG, the enzyme that mediates the last step in peptidoglycan subunit biosynthesis, becomes polarly localized when expressed at high cellular concentrations. MurG only becomes polarly localized at levels that saturate MurG''s cellular requirement for growth, and E. coli cells do not insert peptidoglycan at the cell poles, indicating that the polar MurG is not active. Fluorescence recovery after photobleaching (FRAP) and single-cell biochemistry experiments demonstrate that polar MurG is dynamic. Polar MurG foci are distinct from inclusion body aggregates, and polar MurG can be remobilized when MurG levels drop. These results suggest that polar MurG represents a temporary storage mechanism for excess protein that can later be remobilized into the active pool. We investigated and ruled out several candidate pathways for polar MurG localization, including peptidoglycan biosynthesis, the MreB cytoskeleton, and polar cardiolipin, as well as MurG enzymatic activity and lipid binding, suggesting that polar MurG is localized by a novel mechanism. Together, our results imply that inactive MurG is dynamically sequestered at the cell poles and that prokaryotes can thus utilize subcellular localization as a mechanism for negatively regulating enzymatic activity.Cells need ways to deal with having more of a specific protein than they need. Left unchecked, excess protein can be toxic to the cell and interfere with essential processes. In prokaryotes, a common mechanism for dealing with excess protein is degradation (30). Bacterial proteases can break down proteins, salvaging amino acids to produce new protein. This process costs time and energy, especially if the protein being degraded is essential and will need to be resynthesized later. Excess protein can also aggregate into insoluble inclusion bodies. In inclusion bodies, proteins are generally misfolded, and though in some cases these proteins can be refolded (24, 35), inclusion body proteins are not readily accessible for use by the cell (11). A potential alternative strategy for dealing with excess protein is to temporarily store the protein in an inactive form that can later be dynamically remobilized when needed. Here we propose that Escherichia coli uses subcellular localization of MurG to accomplish such dynamic storage.MurG is an essential, membrane-associated N-acetylglucosaminyl transferase involved in catalyzing the final step of peptidoglycan subunit biosynthesis (4, 21). In E. coli, the peptidoglycan cell wall determines both cell shape and growth rate (17). During growth and division, E. coli cells add new peptidoglycan both along the lateral cylindrical portion of the cell and at the division plane, but no new peptidoglycan is added at the cell poles (10). Previous efforts to study the localization of MurG have found that MurG localizes to the cell periphery and division plane in E. coli (25). In this study, we demonstrate that E. coli MurG also localizes to the cell poles in a concentration-dependent manner. We find that the polar MurG represents a dynamic pool of excess protein, suggesting that polar accumulation represents an accessible form of temporary storage.     

Molecular Dissection of Phage Endolysin: AN INTERDOMAIN INTERACTION CONFERS HOST SPECIFICITY IN LYSIN A OF MYCOBACTERIUM PHAGE D29*

Mycobacterium tuberculosis has always been recognized as one of the most successful pathogens. Bacteriophages that attack and kill mycobacteria offer an alternate mechanism for the curtailment of this bacterium. Upon infection, mycobacteriophages produce lysins that catalyze cell wall peptidoglycan hydrolysis and mycolic acid layer breakdown of the host resulting in bacterial cell rupture and virus release. The ability to lyse bacterial cells make lysins extremely significant. We report here a detailed molecular dissection of the function and regulation of mycobacteriophage D29 Lysin A. Several truncated versions of Lysin A were constructed, and their activities were analyzed by zymography and by expressing them in both Escherichia coli and Mycobacterium smegmatis. Our experiments establish that Lysin A harbors two catalytically active domains, both of which show E. coli cell lysis upon their expression exclusively in the periplasmic space. However, the expression of only one of these domains and the full-length Lysin A caused M. smegmatis cell lysis. Interestingly, full-length protein remained inactive in E. coli periplasm. Our data suggest that the inactivity is ensued by a C-terminal domain that interacts with the N-terminal domain. This interaction was affirmed by surface plasmon resonance. Our experiments also demonstrate that the C-terminal domain of Lysin A selectively binds to M. tuberculosis and M. smegmatis peptidoglycans. Our methodology of studying E. coli cell lysis by Lysin A and its truncations after expressing these proteins in the bacterial periplasm with the help of signal peptide paves the way for a large scale identification and analysis of such proteins obtained from other bacteriophages.     

Purification and biochemical characterization of Mur ligases from Staphylococcus aureus

The Mur ligases (MurC, MurD, MurE and MurF) catalyze the stepwise synthesis of the UDP-N-acetylmuramoyl-pentapeptide precursor of peptidoglycan. The murC, murD, murE and murF genes from Staphylococcus aureus, a major pathogen, were cloned and the corresponding proteins were overproduced in Escherichia coli and purified as His₆-tagged forms. Their biochemical properties were investigated and compared to those of the E. coli enzymes. Staphylococcal MurC accepted l-Ala, l-Ser and Gly as substrates, as the E. coli enzyme does, with a strong preference for l-Ala. S. aureus MurE was very specific for l-lysine and in particular did not accept meso-diaminopimelic acid as a substrate. This mirrors the E. coli MurE specificity, for which meso-diaminopimelic acid is the preferred substrate and l-lysine a very poor one. S. aureus MurF appeared less specific and accepted both forms (l-lysine and meso-diaminopimelic acid) of UDP-MurNAc-tripeptide, as the E. coli MurF does. The inverse and strict substrate specificities of the two MurE orthologues is thus responsible for the presence of exclusively meso-diaminopimelic acid and l-lysine at the third position of the peptide in the peptidoglycans of E. coli and S. aureus, respectively. The specific activities of the four Mur ligases were also determined in crude extracts of S. aureus and compared to cell requirements for peptidoglycan biosynthesis.     

Changes in peptidoglycan composition and penicillin-binding proteins in slowly growing Escherichia coli.

The composition of peptidoglycan of chemostat-grown cultures of Escherichia coli was investigated as a function of growth rate. As the generation time was lengthened from 0.8 to 13.8 h, there was a decrease in the major monomer (disaccharide tetrapeptide) and dimer (bis-disaccharide tetrapeptide), while disaccharide tripeptide moieties increased to greater than 50% of the total wall. The average chain length became much shorter; lipoprotein density tripled, and the number of unusual diaminopimelyl-diaminopimelic acid crossbridges increased fivefold. As cells grew more slowly, amounts of penicillin-binding proteins (PBPs) 1a-1b complex and 4 decreased, while amounts of PBPs 3 and the 5-6 complex increased. We propose that the chemical composition of E. coli cell walls changes with growth rate in a manner consistent with alterations in the activities of PBPs and cell shape.