Reconstitution of denatured E. coli alkaline phosphatase with E. coli ribosome.

E. coli alkaline phosphatase was denatured by physical/chemical means. In vitro reconstitution of this denatured enzyme was assisted by 70S E. coli ribosome, as shown by the recovery of its catalytic competence. Almost total recovery of activity of the totally inactivated enzyme was obtained in presence of equimolar concentration of 70S ribosome at 50 degrees C.     

Lysophospholipase of Escherichia coli.

A lysophospholipase from Escherichia coli cells was purified about 1,500-fold to near homogeneity by extraction with Tris-HCl buffer, streptomycin treatment, (NH4)2SO4 fractionation, column chromatographies on Sephadex G-200, DEAE-cellulose and hydroxylapatite-cellulose, and polyacrylamide gel electrophoresis. The final preparation had a molecular weight of 39,500 plus or minus 500. The enzyme hydrolyzes 1-acylglycerylphosphorylethanolamine, 2-acylglycerylphosphorylethanoiamine, and 1-acylglycerylphosphorylglycerol, but does not attack diacylphospholipids with long chain fatty acids, such as phosphatidylethanolamine and phosphatidylglycerol. The enzyme does not show any esterase activity against p-nitrophenyl acetate or palmitate. Although it does not hydrolyze triacylglycerol or diacylglycerol, it hydrolyzes 1-acylglycerol at almost the same rate as 1-acyl-sn-glycerol-3-phosphorylethanolamine. Results indicated that the acyl-hydrolyzing activities toward monoacyl-glycerylphosphorylethanolamine and monoacylglycerol belong to the same enzyme. In general, acidic and nonionic detergents inhibited the reaction. This lysophospholipase preparation hydrolyzes the monomolecular and micellar forms of lysophospholipids as well as of monoacylglycerol. The monomolecular and micellar forms of Triton X-100 both inhibited the hydrolyses of lysophospholipids and monoacylglycerol.     

Morphogenesis of Escherichia coli.

Morphogenesis of the rod-shaped Escherichia coli is determined by controlled growth of an exoskeleton made of murein (peptidoglycan). Recent insights in the growth strategy of the stress-bearing murein sacculus has contributed to our understanding of how the required concerted action of murein polymerizing and hydrolyzing enzymes is achieved. The proteins involved are coordinated by the formation of multienzyme complexes. In this review, we summarize the recent results on murein structure and metabolism. On the basis of these findings, we present a model that explains maintenance of the specific rod shape of E. coli.     

Mistranslation in E. coli.

Flagellin, the protomeric subunit of bacterial flagella, contains no cysteine. We have detected the incorporation of trace quantities of 35S-cysteine into flagellin, highly purified and then resolved by SDS polyacrylamide gel electrophoresis, to measure mistranslation in vivo. Under normal conditions, this value is about 6 X 10(-4) pmoles cysteine per pmole flagellin. This value is greatly increased during growth in low concentrations of streptomycin and neomycin, antibiotics which are known to stimulate misreading in vitro. Of the specific types of misreading which streptomycin stimulates in vitro, only misreading of the CGU and CGC arginine codons could give rise to illegitimate incorporation of cysteine. In agreement, partial arginine starvation increases the incorporation of 35S-cysteine into flagellin in a relA- mutant, with or without streptomycin, but has no such effect in its isogenic relA+ partner- Assuming from these results that 35S-cysteine incorporation into flagellin reflects misreading of CGU/C coda, we deduce a misreading probability per codon in the range of 10(-4).     

gltBDF operon of Escherichia coli.

A 2.0-kilobase DNA fragment carrying antibiotic resistance markers was inserted into the gltB gene of Escherichia coli previously cloned in a multicopy plasmid. Replacement of the chromosomal gltB+ gene by the gltB225::omega mutation led to cells unable to synthesize glutamate synthase, utilize growth rate-limiting nitrogen sources, or derepress their glutamine synthetase. The existence of a gltBDF operon encoding the large (gltB) and small (gltD) subunits of glutamate synthase and a regulatory peptide (gltF) at 69 min of the E. coli linkage map was deduced from complementation analysis. A plasmid carrying the entire gltB+D+F+ operon complemented cells for all three of the mutant phenotypes associated with the polar gltB225::omega mutation in the chromosome. By contrast, plasmids carrying gltB+ only complemented cells for glutamate synthase activity. A major tricistronic mRNA molecule was detected from Northern (RNA blot) DNA-RNA hybridization experiments with DNA probes containing single genes of the operon. A 30,200-dalton polypeptide was identified as the gltF product, the lack of which was responsible for the inability of cells to use nitrogen-limiting sources associated with gltB225::omega.     

icdB mutants of Escherichia coli.

icdB mutations map at 16 min, lead to the specific loss of citrate synthase, and are complemented by a prophage containing a gltA+ gene. Thus, they are allelic with gltA.     

Peptidase-deficient mutants of Escherichia coli.

Mutant derivatives of Escherichia coli K-12 deficient in several peptidases have been obtained. Mutants lacking a naphthylamidase, peptidase N, were isolated by screening for colonies unable to hydrolyze L-alanine beta-naphthylamide. Other mutants were isolated using positive selections for resistance to valine peptides. Mutants lacking peptidase A, a broad-specificity aminopeptidase, were obtained by selection for resistance to L-valyl-L-leucine amide. Mutants lacking a dipeptidase, peptidase D, were isolated from a pepN pepA strain by selection for resistance to L-valyl-glycine. Starting with a pepN pepA pepD strain, selection for resistance to L-valyl-glycyl-glycine or several other valine peptides produced mutants deficient in another aminopeptidase, peptidase B. Mutants resistant to L-valyl-L-proline lack peptidase Q, an activity capable of rapid hydrolysis of X-proline dipeptides. Using these selection procedures, a strain (CM89) lacking five different peptidases has been isolated. Although still sensitive to valine, this strain is resistant to a variety of valine di- and tripeptides. The ability of this strain to use peptides as sources of amino acids is much more restricted than that of wild-type E. coli strains. Strains containing only one of the five peptidases missing in CM89 have been constructed by transduction. The peptide utilization profiles of these strains show that each of the five peptidases can function during growth in the catabolism of peptides.     

PfkA locus of Escherichia coli.

pfkA was know, on the basis of three mutants, as the likely locus of phosphofructokinase in Escherichia coli, and the unlinked pfkB1 mutation suppressed these mutations by restoring some enzyme activity (Morrissey and Fraenkel, 1972). We now report a new search for the complete inactivation of pfkA (e.g., by deletion or amber mutation), done to assess whether the pfkB1 suppression is by an independent enzyme, phosphofructokinase activity 2 (Fraenkel, Kotlarz, and Buc, 1973). Ten new phosphofructokinase mutants all were at pfkA, rather than at pfkB or pfkC. One of them (pfkA9) gave temperature-sensitive reverants with heat-labile enzyme. Another (pfkA11) proved genetically to be a nonsense mutation, but showed no restored activity when suppressed by supF. However, even unsuppressed it was found to contain an enzyme related to phosphofructokinase activity 1 kinetically (more allosteric), physically (almot identical subunit), and antigenically. All the pfkA mutants apparently contained cross-reacting material to activity 1. All (including pfkA11) were suppressed by the pfkB1 mutation. Several results support the idea that pfkA is the structural gene for the main phosphofructokinase of E. coli (activity 1), but that there is some restriction to its complete inactivation.     

Thiol-sensitive genes of Escherichia coli.

The effect of 1-thioglycerol on the expression of genes of Escherichia coli was investigated. Pulse-labeled proteins from aerobically growing, 1-thioglycerol-treated E. coli were separated by two-dimensional gel electrophoresis, and their radioactivities were compared with those of identical proteins from nontreated cells. The first 10 min of exposure to thiol stimulated the synthesis of 10% of the observed proteins and inhibited the production of 16% of the proteins. After 30 min of growth with thiol, the synthesis of 44% of the observed proteins was inhibited and synthesis of 18% of the proteins was stimulated. In general, the expression of genes of carbohydrate metabolism, amino acid metabolism, and protein biosynthesis were inhibited, while nucleic acid synthetic and repair gene expressions showed mixed responses. Synthesis of transport proteins was not affected. Transient stimulation of oxidative-stress proteins and sustained stimulation of the expressions of trxB, ompA, and ompB genes and those of several unidentified gene products were also observed. Whether these complex responses merely reflect adjustments by cellular subsystems to a suddenly reducing environment or whether they are manifestations of a reductive-stress regulon will have to await genetic analysis of this phenomenon.     

alpha-Hemolysin of E. coli]

The activity of alpha-hemolysin increased at the log growth phase in the culture of E. coli P678 Hly+ hemolytic strain; this activity diminished with the change into the stationary phase, and then fell sharply. Replacement of the culture medium in the stationary growth phase by fresh one led to restoration of the hemolytic activity of the culture. The culture fluid separated from the cells at the stationary growth phase produced an inhibitory action on the alpha-hemolysin Ca ions activated and stabilized the alpha-hemolysin. Sodium citrate and sucrose served as hemolysis inhibitors. The action of alpha-hemolysin was maximal against human erythrocytes at pH 6.5. Hemolytic activity was characterized in time by a distinct lag-phase and the phase of the greatest rate of reaction. The duration of the lag-phase and also the rate of hemolysis depended on the concentration of alpha-hemolysin (with the increase of the hemolysin concentration lag-phase was shortened and the reaction was accelerated). There proved to be a linear relationship between the amount of erythrocytes taken into the reaction and the rate of hemoglobin release, and also there was noted a temperature activation of the hemolytic reaction.     

Expression of Escherichia coli pabA.

Escherichia coli pabA encodes the glutamine amidotransferase subunit of p-aminobenzoate synthase. p-Aminobenzoate synthase catalyzes the conversion of chorismate and glutamine to 4-amino-4-deoxychorismate, which is then converted to p-aminobenzoate by a 4-amino-4-deoxychorismate lyase. The 5'-terminal segment of pabA was previously shown to be transcribed from two different promoters, one near the pabA coding sequence (P1) and one preceding fic (P2). However, a pabA-lacZ translational fusion was expressed only from the mRNA originating at P1. We have determined that expression of a pabA-lacZ chromosomal fusion is not changed by p-aminobenzoate limitation, growth rate, catabolite repression, overexpression of either p-aminobenzoate synthase subunit, or gene dosage of pabA and pabB. The lack of pabA expression from P2 appears to be the result of a stable secondary structure in the intergenic space preceding pabA that sequesters the pabA ribosome binding site. Disruption of the secondary structure by mutation allowed expression of pabA from P2, as did translation of ribosomes into the fic-pabA intergenic region.