Growth of the Escherichia coli cell envelope

The growth pattern of the Escherichia coli envelope was studied by immunoelectron microscopy, using the outer membrane protein LamB specifically labelled by a double antibody gold particle technique. An operon fusion placing the lamB gene under lac promoter control permitted rapid turn-off of LamB synthesis. In the generation following turn-off no lamB-free regions appeared, strongly suggesting that bulk outer membrane material is not inserted in restricted growth zones.     

Aminopeptidase N from Escherichia coli. Unusual interactions with the cell surface.

The subcellular localization of aminopeptidase N (previously called aminoendopeptidase) has been investigated. This enzyme was found to be partially released (30-40%) by osmotic shock or by converting Escherichia coli K10 cells to spheroplasts. However, in all other E. coli strains (K12, B/r, MRE 600, ML 308) tested, this enzyme is not released at all by these procedures and thus behaves like a cytoplasmic enzyme. The crypticity of aminopeptidase N is surprisingly low, 75-85% of the enzyme activity is directly assayable in intact cells of any E. coli strain. Various inhibitors of transport systems do not interfer with this assay. Aminopeptidase activity could also be assayed in spheroplasts, even when an insolubilized substrate was used, which suggests a surface location of this enzyme. As well, N-ethylmaleimide (0.4 mM), under conditions which do not allow penetration in the cytoplasm, caused 70% inhibition of aminopeptidase N. Binding of 125I-labeled antiaminopeptidase N antibody to spheroplasts (from K12 strain) was used to assay the orientation of aminopeptidase N in the membrane. This enzyme is exposed on the outer surface of the cytoplasmic membrane. Confirmation of this orientation was obtained by comparing the accessibility of aminopeptidase, alkaline phosphatase and beta-galactosidase to fluorescamine in intact cells. Only 16% of the total beta-galactosidase was labeled with this fluorescent reagent whereas 44-45% of the aminopeptidase N and 59% of the alkaline phosphatase were labeled. Electron microscopic visualization of insolubilized reaction products of aminopeptidase N within the cells showed that these products are located at the poles of the cells. Neither mutant cells which were devoid of aminopeptidase N activity nor parental strains with the enzyme activity inhibited with phenylmercuric chloride contained the characteristic black caps. Thus, it appears that the periplasm is enlarged at the poles of the cells and that the reaction product is mainly located in these places. Investigation of the type of interactions of aminopeptidase N with the plasma membrane only revealed that aminopeptidase N has mainly an electrostatic interaction with the outer surface, probably mediated by magnesium ion bridges. Additional interactions are involved since disruption of the integrity of the cytoplasmic membrane is required to totally release this enzyme.     

High-sensitivity detection of newly induced LamB protein on the Escherichia coli cell surface.

The kinetics of the appearance at the cell surface of the outer membrane LamB protein after induction were determined by using specific antibodies and radioiodinated protein A as a probe. This was done in two different induction systems. First, LamB protein was induced in a wild-type strain by the simultaneous addition of cyclic AMP and maltose. Second, an operon fusion strain in which the lamB gene is expressed under lac promoter control was used; in this system, LamB protein can be induced by isopropyl-beta-D-thiogalactopyranoside. When uninduced cells were grown in glucose minimal medium, background expression of the lamB gene was found to be ca. 10-fold lower in lac-lamB cells than in wild-type cells. The level of LamB protein present in uninduced wild-type cells could, however, be reduced by supplementing the growth medium with Casamino Acids. After induction, the LamB protein appeared at the cell surface of both strains within a few minutes, and then the LamB level per cell increased linearly. The time lag in cell surface exposure of LamB protein differed slightly under both induction conditions: the LamB protein appeared at the surface of lac-lamB cells within 3 min of induction, whereas in wild-type cells it could not be detected earlier than after 4 to 5 min of induction.     

Going for baroque at the Escherichia coli K1 cell surface

Phase variation is usually thought of as the stochastic switching between alternatively expressed ('on') and unexpressed ('off') phenotypic states. However, coupling synthesis of a monotonous homopolysaccharide to a mechanism of random but incomplete chemical modification produces almost infinite structural variation. Potentially limitless variability implies that evolution can produce highly ornate or extravagant flourishes reminiscent of the baroque style. Here, we describe an analysis of capsular polysialic acid form variation in Escherichia coli K1, demonstrating that the large number of variant structures is controlled by a single contingency locus. The mechanism for generating maximum structural diversity from maximal genetic parsimony is conferred by a simple translational switch carried on a K1-specific prophage.     

Display of green fluorescent protein on Escherichia coli cell surface

In this study, expression of green fluorescence protein (GFP) on the external surface of Escherichia coli was achieved by construction of a fusion protein between Lpp-OmpA hybrid and a GFP variant, GFPmut2. The GFP was fused in frame to the carboxyl-terminus of Lpp-OmpA fusion previously shown to direct various other heterologous proteins to E. coli cell surface. Western blot analysis of membrane fractions identified the Lpp-OmpA-GFP fusion protein with the expected size (43 kDa). Immunofluorescence microscopy, immunoelectron microscopy, protease and extracellular pH sensitivity assays further confirmed that GFP is anchored on the outer membrane. The GFP displayed on the E. coli outer surface retained its fluorescence and was not susceptible to the indigenous outer membrane protease OmpT even though there are two putative OmpT proteolytic sites present in GFP. Optimization of the expression conditions was conducted using fluorometry, eliminating cumbersome immuno-labeling procedures. Surface-displayed GFP could be used in a variety of applications including screening of polypeptide libraries, development of live vaccines, construction of whole cell allosteric biosensors, and signal transduction studies.     

Bilinear cell growth of Escherichia coli.

Recent electron micrograph measurements of bacterial dimensions in exponentially growing cultures of Escherichia coli support a model of bilinear increase in cell surface area and volume, with a sharp doubling in growth rate at a discrete age during the cell cycle. The results also indicate coordinate regulation of increase of surface area and volume.     

Influence of surface cell structures on physicochemical properties of Escherichia coli

The partition of cells in a polyethylene glycol-dextran two phase system was used to compare the relative hydrophobicity of E. coli strains expressing different surface structures. The role of fimbriae and surface antigens on the behavior partition was investigated. The strains expressing PAP fimbriae and/or O-antigen showed a higher surface hydrophobicity than strains which express only type 1 fimbriae and/or R-antigen. No relation was found between K and H antigen and hydrophobicity measurements. The influence of surface structures on electrophoretic mobility has been evaluated. The polysaccharide capsules of AL 213 and AL 499 strains generated a high EPM. For non capsulated E. coli the EPM of rough strains (AL 46, 382) is higher than smooth strain (AL52).     

Growth kinetics of Colpoda steinii on Escherichia coli.

Colpoda steinii was grown in two-stage continuous cultures with Escherichia coli as prey species. The concentration of prey and the ciliate mean cell volume, dry weight, and number per milliliter were determined at known growth rates. Steady states were reached in the second-stage continuous cultures at all growth rates. Although changes occurred in mean cell size of the ciliates and in the number per milliliter at various growth rates, the yield of protozoan biomass per unit of prey consumed was constant at all growth rates. The data were compared with several equations proposed to describe the kinetics of protozoan growth as a function of prey density.     

Growth kinetics of Colpoda steinii on Escherichia coli.

Colpoda steinii was grown in two-stage continuous cultures with Escherichia coli as prey species. The concentration of prey and the ciliate mean cell volume, dry weight, and number per milliliter were determined at known growth rates. Steady states were reached in the second-stage continuous cultures at all growth rates. Although changes occurred in mean cell size of the ciliates and in the number per milliliter at various growth rates, the yield of protozoan biomass per unit of prey consumed was constant at all growth rates. The data were compared with several equations proposed to describe the kinetics of protozoan growth as a function of prey density.     

Immobilization of UDP-galactose 4-epimerase from Escherichia coli on the yeast cell surface

UDP-galactose 4-epimerase (EC 5.1.3.2, Gal E) from Escherichia coli catalyzes the reversible reaction between UDP-galactose and UDP-glucose. In this study, the Gal E gene from E. coli, coding UDP-galactose 4-epimerase, was cloned into pYD1 plasmid and then transformed into Saccharomyces cerevisiae EBY100 for expression of Gal E on the cell surface. Enzyme activity analyses with EBY100 cells showed that the enzyme displayed on the yeast cell surface was very active in the conversion between UDP-Glc and UDP-Gal. It took about 3 min to reach equilibrium from UDP-galactose to UDP-glucose.     

Lipid synthesis during the Escherichia coli cell cycle.

Lipid synthesis was examined in Escherichia coli cells at different stage of cell division. Exponentially growing cells were pulse-labeled with appropriate isotopes for 0.1 generation time, inactivated, and separated by size on a sucrose gradient. An abrupt increase in the rate of lipid synthesis occurred which was coincident with the initiation of cross walls. In contrast, the rate of protein synthesis during this same interval remained constant, resulting in an increased lipid/protein ratio in dividing cells. No changes in the composition of phospholipid head groups, fatty acids, or phospholipid molecular species were observed in cells at different stages of division. The observed increase in the rate of lipid synthesis may reflect a means by which the activities of membrane-associated enzymes are modulated during cross wall formation.     

Penicillin-binding site on the Escherichia coli cell envelope.

The binding of 35S-labeled penicillin to distinct penicillin-binding proteins (PBPs) of the "cell envelope" obtained from the sonication of Escherichia coli was studied at different pHs ranging from 4 to 11. At low pH, PBPs 1b, 1c, 2, and 3 demonstrated the greatest amount of binding. At high pH, these PBPs bound the least amount of penicillin. PBPs 1a and 5/6 exhibited the greatest amount of binding at pH 10 and the least amount at pH 4. With the exception of PBP 5/6, the effect of pH on the binding of penicillin was direct. Experiments distinguishing the effect of pH on penicillin binding by PBP 5/6 from its effect on beta-lactamase activity indicated that although substantial binding occurred at the lowest pH, the amount of binding increased with pH, reaching a maximum at pH 10. Based on earlier studies, it is proposed that the binding at high pH involves the formation of a covalent bond between the C-7 of penicillin and free epsilon amino groups of the PBPs. At pHs ranging from 4 to 8, position 1 of penicillin, occupied by sulfur, is considered to be the site that establishes a covalent bond with the sulfhydryl groups of PBP 5. The use of specific blockers of free epsilon amino groups or sulfhydryl groups indicated that wherever the presence of each had little or no effect on the binding of penicillin by PBP 5, the presence of both completely prevented binding. The specific blocker of the hydroxyl group of serine did not affect the binding of penicillin. These observations suggest that a molecule of penicillin forms simultaneous bonds between its S at position 1 and sulfhydryl groups of PBP 5 and between its C-7 and free epsilon amino groups of PBP 5.