Alkaline phosphatase isozyme conversion by cell-free extract of Escherichia coli

Isozyme type 1 of alkaline phosphatase in Escherichia coli K-12 was converted to types 2 and 3 after incubation of type 1 isozyme with the supernatant of a sonicated cell-free extract prepared from the cells carrying the cloned iap+ gene on a multi-copy plasmid. By comparison, the lysate prepared from cells carrying the iap+ gene only on the chromosome showed much less isozyme-converting activity. The reaction was promoted by Mg2+ at concentrations of 10 to 50 mM. Protease inhibitors, antipain and leupeptin which inhibit the isozyme conversion in vivo, also inhibited the isozyme conversion in vitro. These results suggest that cells carrying the multiple copy iap+ plasmid overproduce a kind of proteolytic enzyme which removes the amino-terminal arginine residues from isozymes 1 and 2.     

Alkaline phosphatase activity of Escherichia coli starved in sterile lake water microcosms

Escherichia coli grown in high or low phosphate medium was inoculated into a lake water starvation medium. The viable count decreased at 37°C but not at the lower temperatures over 70 d. Alkaline phosphatase was monitored using a colorimetric assay with pNPP as the substrate. Derepression of the enzyme occurred in cultures starved for > 30 d in the lake water and within 5 d in lake water microcosms supplemented with carbon and nitrogen sources where there was rarely an increase in viable count. Chloramphenicol prevented the synthesis of alkaline phosphatase suggesting that, even under starvation conditions, de novo synthesis of the enzyme occurs.     

Ribonuclease sensitivity of Escherichia coli ribosomes

Santer, Melvin (Haverford College, Haverford, Pa.), and Josephine R. Smith. Ribonuclease sensitivity of Escherichia coli ribosomes. J. Bacteriol. 92:1099-1110. 1966.-The ribonucleic acid (RNA) contained in 70S ribosomes and in 50S and 30S subunits was hydrolyzed by pancreatic ribonuclease. A 7% amount of the RNA was removed from the 70S particle; at 10(-4)m magnesium concentration, a maximum of 24 and 30% of the RNA in the 50S and the 30S fractions, respectively, was removed by ribonuclease. At the two lower magnesium ion concentrations, 50S ribosomes did not lose any protein, whereas 30S ribosomes lost protein as a result of ribonuclease treatment. A number of proteins were removed from the 30S particles by ribonuclease, and these proteins were antigenically related to proteins present in 50S ribosomes. The differential effect of ribonuclease on 50S and 30S ribosomes suggested that they have structural dissimilarities.     

Hydrophobic interactions of Escherichia coli ribosomes

We have demonstrated the presence of hydrophobic sites on the surface of Escherichia coli ribosomes by means of hydrophobic chromatography on Octyl-Sepharose. Both 30-S and 50-S ribosomal subunits adsorb to Octyl-Sepharose at a low salt concentration, and can be eluted from it with a nonionic detergent without substantial changes in structure or activity. By testing a series of LiCl-derived ribosomal cores for their ability to adsorb to Octyl-Sepharose we have shown that the interaction of ribosomal particles with Octyl-Sepharose is dependent on the presence of certain ribosomal proteins; the core particles which lack these proteins do not bind to Octyl-Sepharose. The binding of a series of different ribosomal cores to nitrocellulose filters (Millipore) yielded the same pattern as was observed with Octyl-Sepharose, i.e. the more protein-depleted the particles, the less they were adsorbed. Thus, the adsorption of ribosomes to Millipore filters and to Octyl-Sepharose is presumably of the same hydrophobic nature.     

Escherichia coli ribosomes translate in vivo with variable rate.

The question of whether or not 'rare' codons are translated with the same rate as 'common' codons was investigated by measuring the translation time for two genes, lacI and bla, rich in rare codons, and comparing the results with the translation times measured on fus, tsf, tuf and rpsA which have very few rare codons. The rate of synthesis of the lac repressor was first measured with the up-promoter mutation lacIq1 present on the high copy number plasmid pBR322. In such a strain the average translation times for lacI and bla were 50% slower than the rate calculated from the translation time for the four ribosomal proteins. In a strain having lacIq1 on an F'lac episome this difference was much smaller, thus slow translation of genes rich in rare codons is exaggerated in strains with increased drain on the rare codon tRNAs. The data do not exclude that only a subset of the rare codons is translated more slowly. Translation times were also measured in cells growing in different media, and the translation chain growth rate was found to increase by approximately 40% going from acetate medium to a fully supplemented medium.