AmpN-AmpG Operon Is Essential for Expression of L1 and L2 β-Lactamases in Stenotrophomonas maltophilia

ABSTRACT AmpG is an inner membrane permease which transports products of murein sacculus degradation from the periplasm into the cytosol in Gram-negative bacteria. This process is linked to induction of the chromosomal ampC beta-lactamase gene in some members of the Enterobacteriaceae and in Pseudomonas aeruginosa. In this study, the ampG homologue of Stenotrophomonas maltophilia KJ was analyzed. The ampG homologue and its upstream ampN gene form an operon and are cotranscribed under the control of the promoter P ampN. Expression from P ampN was found to be independent of β-lactam exposure and ampN and ampG products. A ΔampN allele exerted a polar effect on the expression of ampG and resulted in a phenotype of null β-lactamase inducibility. Complementation assays elucidated that an intact ampN-ampG operon is essential for β-lactamase induction. Consistent with ampG of Escherichia coli, the ampN-ampG operon of S. maltophilia did not exhibit a gene dosage effect on β-lactamase expression. The AmpG permease of E. coli could complement the β-lactamase inducibility of ampN or ampG mutants of S. maltophilia, indicating that both species have the same precursor of activator ligand(s) for β-lactamase induction.

Restricted Mobility of Cell Surface Proteins in the Polar Regions of Escherichia coli

ABSTRACT The polar regions of the Escherichia coli murein sacculus are metabolically inert and stable in time. Because the sacculus and the outer membrane are tightly associated, we investigated whether polar inert murein could restrict the mobility of other cell envelope elements. Cells were covalently labeled with a fluorescent reagent, chased in dye-free medium, and observed by microscopy. Fluorescent material was more efficiently retained at the cell poles than at any other location. The boundary between high and low fluorescence intensity areas was rather sharp. Labeled material consisted mostly of cell envelope proteins, among them the free and murein-bound forms of Braun's lipoprotein. Our results indicate that the mobility of at least some cell envelope proteins is restrained at regions in correspondence with underlying areas of inert murein.

On the Architecture of the Gram-Negative Bacterial Murein Sacculus

ABSTRACT The peptidoglycan network of the murein sacculus must be porous so that nutrients, waste products, and secreted proteins can pass through. Using Escherichia coli and Pseudomonas aeruginosa as a baseline for gram-negative sacculi, the hole size distribution in the peptidoglycan network has been modeled by computer simulation to deduce the network's properties. By requiring that the distribution of glycan chain lengths predicted by the model be in accord with the distribution observed, we conclude that the holes are slits running essentially perpendicular to the local axis of the glycan chains (i.e., the slits run along the long axis of the cell). This result is in accord with previous permeability measurements of Beveridge and Jack and Demchik and Koch. We outline possible advantages that might accrue to the bacterium via this architecture and suggest ways in which such defect structures might be detected. Certainly, large molecules do penetrate the peptidoglycan layer of gram-negative bacteria, and the small slits that we suggest might be made larger by the bacterium.

Membrane-Bound Lytic Endotransglycosylase inEscherichia coli

ABSTRACT The gene for a novel endotype membrane-bound lytic transglycosylase, emtA, was mapped at 26.7 min of theE. coli chromosome. EmtA is a lipoprotein with an apparent molecular mass of 22 kDa. Overexpression of the emtA gene did not result in bacteriolysis in vivo, but the enzyme was shown to hydrolyze glycan strands isolated from murein by amidase treatment. The formation of tetra- and hexasaccharides, but no disaccharides, reflects the endospecificity of the enzyme. The products are characterized by the presence of 1,6-anhydromuramic acid, indicating a lytic transglycosylase reaction mechanism. EmtA may function as a formatting enzyme that trims the nascent murein strands produced by the murein synthesis machinery into proper sizes, or it may be involved in the formation of tightly controlled minor holes in the murein sacculus to facilitate the export of bulky compounds across the murein barrier.

Growth of the Stress-Bearing and Shape-Maintaining Murein Sacculus of Escherichia coli

SUMMARY To withstand the high intracellular pressure, the cell wall of most bacteria is stabilized by a unique cross-linked biopolymer called murein or peptidoglycan. It is made of glycan strands [poly-(GlcNAc-MurNAc)], which are linked by short peptides to form a covalently closed net. Completely surrounding the cell, the murein represents a kind of bacterial exoskeleton known as the murein sacculus. Not only does the sacculus endow bacteria with mechanical stability, but in addition it maintains the specific shape of the cell. Enlargement and division of the murein sacculus is a prerequisite for growth of the bacterium. Two groups of enzymes, hydrolases and synthases, have to cooperate to allow the insertion of new subunits into the murein net. The action of these enzymes must be well coordinated to guarantee growth of the stress-bearing sacculus without risking bacteriolysis. Protein-protein interaction studies suggest that this is accomplished by the formation of a multienzyme complex, a murein-synthesizing machinery combining murein hydrolases and synthases. Enlargement of both the multilayered murein of gram-positive and the thin, single-layered murein of gram-negative bacteria seems to follow an inside-to-outside growth strategy. New material is hooked in a relaxed state underneath the stress-bearing sacculus before it becomes inserted upon cleavage of covalent bonds in the layer(s) under tension. A model is presented that postulates that maintenance of bacterial shape is achieved by the enzyme complex copying the preexisting murein sacculus that plays the role of a template.