Search

With use of polyribosomes isolated from Escherichia coli, biosynthesis of a specific lipoprotein of the E. coli outer membrane was investigated. The products of the cell-free system were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoprecipitation with anti-lipoportein serum, followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. It was found that one of the major products was the lipoprotein of the E. coli outer membrane. The lipoprotein was also found to be preferentially synthsized on smaller polyribosomes.

The existence of a free form of a specific lipoprotein of molecular weight 7,200 was examined in the envelopes of several gram-negative bacteria. When the envelope proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, distinct peaks were observed in Salmonella typhimurium, Serratia marcescens, and Pseudomonas aeruginosa at the same position as the free form of the lipoprotein of Escherichia coli. However, the peak was not observed in Proteus mirabilis. The protein at the peak in S. typhimurium was shown to contain little or no histidine as expected from the amino acid composition of the lipoprotein. Furthermore, antiserum against the highly purified lipoprotein from E. coli was shown to react with the proteins from S. typhimurium and S. marcescens and to form the specific immunoprecipitates. In contrast, the protein from P. aeruginosa did not react with the antiserum at all. Thus, it is concluded that S. typhimurium and S. marcescens have the free form of the lipoprotein in their envelopes as does E. coli. P. aeruginosa contains a protein of the same size as the lipoprotein, but it is not certain whether the protein is the same structural protein as the lipoprotein from E. coli. P. mirabilis may not have any free form of the lipoprotein, may have it in a very small amount, or may have a lipoprotein of different molecular weight serving the same function.

A mutant of Escherichia coli temperature-sensitive for deoxyribonucleic acid synthesis, dnaD, was found to have temperature-sensitive modification and restriction phenotypes. In contrast to the original observation by Carl (1970), the mutant could support the growth of lambda phage at 41 C. However, the lambda phages thus produced were able to form plaques with normal plating efficiency only on E. coli C, a restriction-less strain, but not on E. coli K. Since the lambda phages produced in the mutant at 30 C could form plaques equally well on both E. coli strains, it was concluded that the dnaD mutant has a temperature-sensitive modification phenotype. Furthermore, since the dnaD mutant allowed some growth of unmodified lambda.C phages at 41 C but less at 30 C, the mutant is also temperature sensitive in restriction. The relationship, if any, between temperature-sensitive deoxyribonucleic acid synthesis and temperature-sensitive modification-restriction in the dnaD mutant is not known. Similar experiments were done with three dnaC mutants and one dnaA mutant. Two dnaC mutants were found to have altered restriction phenotypes at 41 C, but none of the mutants were defective in modification.

From the complete amino-acid sequence of a lipoprotein from the outer membrane of E. coli, a three-dimensional molecular assembly model was constructed. It is proposed that the model provides a tubular hydrophilic channel through the outer membrane, which serves as a passive diffusion pore. An alpha-helix is constructed from the sequence, and six of them are arranged to form a superhelix with a hydrophilic interior and hydrophobic outer surface. The superhelical assembly is stabilized by seven ionic interactions between adjacent alpha-helices. Since the height of the assembly is 76 A, it could be inserted into the outer membrane and span the full 75-A thick membrane. The assembly is stabilized in the outer membrane not only by hydrophobic interaction between the surface of the assembly and the lipid bilayer, but also by three hydrocarbon chains of fatty acids linked to the amino-terminal end of the lipoprotein, which are flipped back along the assembly and inserted into the lipid bilayer of the outer membrane. Any two alpha-helices in an assembly are linked to the peptidoglycan at their carboxyl-terminal ends so that the outer membrane is anchored on the peptidoglycan layer. Six or more alpha-helices can form an assembly of this type. However, assuming that an assembly consists of six helices, there are 1.25 x 10(5) per cell hydrophilic channels of a diameter of 12.5 A and 35% of the cell surface is occupied by the assemblies.

A specific lipoprotein of the E. coli outer membrane has been synthesized in a cell-free system directed by purified messenger RNA. The mRNA for the lipoprotein was purified as 7S RNA about 250-fold from exponentially growing cells. Protein synthesis of the cell-free system was totally dependent upon the addition of the purified mRNA. The product of the cell-free system was identified as the specific lipoprotein by immunoprecipitation and by peptide mapping.

Some envelope proteins of Escherichia coli show variable behavior in acrylamide gel electrophoresis in 1% sodium dodecyl sulfate, depending upon the conditions of the solubilization. When solubilized in 1% sodium dodecyl sulfate at 70 C for 20 min, three distinct peaks (peaks 4, 6, and 7) are seen at molecular weights of 57,800, 44,300, and 38,400, respectively. However, when the envelope fractions are solubilized in 1% sodium dodecyl sulfate at 100 C for 5 min, or when they are treated with N, N-dimethylformamide at acidic pH before solubilization by our method, only a single peak at 48,000 molecular weight is observed in the molecular weight range mentioned above. That is, peaks 4 and 7 disappear and a new peak appears at the position overlapping with peak 6. Proteins isolated from peaks 4 and 7 show the similar molecular weight shifts to the new peak by the treatment at 100 C. No other peaks show any change by the heat treatment. The increase at the new peak is completely accounted for by the decrease at peaks 4 and 7, indicating that the new peak is composed of proteins from peaks 4, 6, and 7. However, it is concluded that these three peaks consist of distinctly different proteins for the following reasons: (i) they have different amino acid compositions, (ii) they show different solubilities in the nonionic detergent, Nonidet P-40, and as shown previously, (iii) peak 6 (protein Y) is related to deoxyribonucleic acid synthesis, and (iv) proteins in peaks 4, 6, and 7 have different resistance to proteolytic enzymes. Although the reasons for the anomalous molecular weight shifts of these peaks are not well understood at present, it is important to solubilize the E. coli envelope proteins by the standard method in order to investigate their properties and functions of the envelope proteins.

Synchronous cell division in an arginine auxotroph and a histidine auxotroph of Escherichia coli was obtained after starving for the required amino acid for 1 hr. However, cell division was not synchronized after starvation for 1 hr in another arginine auxotroph. This difference is proposed to depend on differences in the concentrations of polyamines in the cells. During amino acid starvation the ratio of putrescine concentration to spermidine concentration decreased in all strains, but it recovered afterward more rapidly in the third strain than in the other two. The cells divided when the ratio returned to normal in the Arg(-) mutants. Added putrescine permitted some of the cells of the first two mutants to divide sooner after amino acid starvation and thus eliminated synchrony. Spermidine added alone had no effect, but, when it was added together with putrescine, it restored synchronous division. Synchrony was established in the third mutant by adding spermidine after arginine starvation. Thus, both the variations in polyamine content and the effects of added polyamines suggest that the polyamines are essential in permitting cell division. We suggest that the molar ratio of putrescine to spermidine can be a critical factor for cell division. This effect of polyamines seems to be specific for cell division. Amino acid starvation does not induce delays in subsequent mass increase or deoxyribonucleic acid synthesis. Possible mechanisms of polyamine action are discussed.