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 mureinbound 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.The peptidoglycan (murein) sacculus of the bacterial cell wall plays a key role in bacterial morphogenesis (21,34,45,46,54). In gram-negative bacteria such as Escherichia coli, the sacculus occupies the periplasmic space between the cytoplasmic membrane (IM) and the outer membrane (OM) and interacts with both of them (24,34,35,44,45). Interaction with the OM is mediated by a number of proteins able to link murein to components of the OM. Among them, Braun's lipoprotein (Lpp) is remarkable as the only known protein that binds covalently to murein in E. coli (5, 6). Lpp is the most abundant protein in the cell envelope and coexists in free (60 to 70%) and murein-bound (30 to 40%) forms (4, 22). The attachment of Lpp to the OM presumably occurs through the insertion of the acyl chains into the inner phospholipid leaflet of the OM (4, 34, 57). Free Lpp molecules form trimers which may contribute to the strength of the murein-OM complex by additional noncovalent interactions (8,9,57). The sacculus and the OM also interact by means of noncovalently bound molecules such as the peptidoglycan-associated lipoprotein (PAL) and OmpA proteins. The binding between PAL and the sacculus is very strong and involves a murein-binding-specific sequence (26). PAL anchors to the OM primarily by the acylated N terminus, but direct protein-protein interactions with other OM proteins have been demonstrated (1, 26, 33). One of the major OM proteins is OmpA. It is an integral membrane protein with domains protruding from both the external and internal leaflets. On the outer side, it functions as a phage receptor, and on the internal side, it interacts directly with murein (18, 36). The interaction of OmpA with murein is strong enough to stabilize the cell envelope in the absence of Lpp (48,56). In addition to Lpp, PAL, and OmpA, a number of other proteins able to interact with the OM and the sacculus, such as OmpF and OmpC, may contribute to the total interaction as well (38).The turgor pressure of the cell forces a physical contact between the IM and the sacculus (23). However, no specific attaching elements have been demonstrated up to now. Interestingly the PAL-TOL complex might play such a role sinc...

Polar growth in the Alphaproteobacterial order Rhizobiales

“…D-cys labeling was performed as previously described (39) with modifications described in SI Materials and Methods. The TRSE staining protocol was modified to allow time-lapse microscopy of individual growing cells (21). Briefly, A. tumefaciens cells were grown to exponential phase and washed three times in 0.1 M NaHCO 3 , pH 8.3 buffer by centrifugation for 5 min at 5,000 × g. Washed cell pellets were resuspended in 200 μL of buffer and TRSE was added to a final concentration of 0.1 μg/mL.…”

The Selective Value of Bacterial Shape

“…This polar peptidoglycan is "inert" in that it is neither degraded and recycled nor diluted by addition of new material (43,52,171). Interestingly, this same stability extends to proteins and lipopolysaccharides in the outer membranes of the poles of gram-negative bacteria (51,91,235) and to proteins on the surfaces of gram-positive organisms (239). These inhomogeneities among lipids, proteins, and peptidoglycan indicate that bacterial poles differ from the rest of the cell, which makes them prime candidates for hosting specialized functional elements.…”

Bacterial Cell Wall Synthesis: New Insights from Localization Studies

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