Mutator activity of a short Okazaki fragment mutant of Escherichia coli.

A mutant of Escherichia coli (sof) which was previously shown to have increased recombination frequency, to produce abnormally short "Okazaki fragments," and to be deficient in deoxyuridine triphosphatase has now been found also to possess mutator activity for several genes; point mutation rates and deletion rates are affected. The mutational stimulation effects are consistent with the hypothesis that incorporation of uracil into DNA is directly or indirectly responsible for the observed mutator activity.     

Crystallization of beta-galactosidase from Escherichia coli.

Two crystal forms of beta-galactosidase have been obtained from Escherichia coli. One crystal form is hexagonal space group P6222 or enantiomorph, with cell dimensions a = b = 154 A, c = 750 A. The second form is monoclinic, space group P21, with cell dimensions a = 107.9 A, b = 207.5 A, c = 509.9 A, beta = 94.7 degrees. The monoclinic form seems better suited to detailed structural analysis. The crystals are radiation-sensitive, but by using synchrotron radiation in conjunction with a long (400 mm) crystal-to-film distance it was possible to resolve the individual reflections. On the basis of crystal density measurements, there are four tetramers each of molecular weight 465,000 per asymmetric unit. The Patterson function strongly suggests that two of the tetramers are related to the other two by translation. The data are consistent with the tetramers having 222 point symmetry, but this is not proven.     

Purification and characterization of a mutant human platelet phospholipase A2 expressed in Escherichia coli. Cleavage of a fusion protein with cyanogen bromide.

Both methionine residues in phospholipase A2 (PLA2) from porcine pancreas have been replaced by leucines with retention of full enzymatic activity. The methionine-less mutant has been expressed as a Cro-LacZ fusion protein in Escherichia coli, from which a pro-PLA2 was liberated by chemical cleavage with CNBr. The general applicability of CNBr cleavage of proteins lacking methionine residue(s) was demonstrated by replacing the single Met8 in human platelet phospholipase A2 (HP-PLA2) by a leucine residue, and the introduction of a methionine at a position just preceding the HP-PLA2 sequence. This protein was expressed in E. coli as a 68-kDa Cro-LacZ fusion protein. CNBr cleavage liberated the HP-PLA2 fragment which was reoxidized in vitro. The [Met8----Leu]HP-PLA2 is monomeric in aqueous solutions, requires calcium ions in the millimolar range for enzymatic activity and has optimal activity around pH 8. p-Bromophenacyl bromide rapidly inactivates the enzyme with calcium ions having a protective effect. The highest specific activities, 2400 U/mg and 9300 U/mg, were found with pure micelles of 1,2-dioctanoyl-sn-glycero-3-phosphoglycol and with mixed micelles of taurodeoxycholate and 1,2-dioctanoyl-sn-glycero-3-phosphoglycol, respectively. In mixed micelles the activity on dioleoyl phospholipids decreases in the order phosphatidylglycerol greater than phosphatidylethanolamine much greater than phosphatidylcholine. The enzyme has low activity on monomeric 1,2-diheptanoyl-sn-glycero-3-phosphocholine as a substrate, but high activity on micelles with a distinct jump in activity at the critical micellar concentration. The binding of the HP-PLA2, porcine pancreatic PLA2 and PLA2 from Naja melanoleuca venom to lipid/water interfaces was determined with micellar solutions of the substrate analog n-hexadecylphosphocholine. The HP-PLA2 has a high apparent Kd (2 mM) compared to pancreatic (0.2 mM) and venom (0.03 mM) PLA2. In mixed micelles of taurodeoxycholate and 1,2-didodecanoyl-sn-glycero-3-phosphocholine, the competitive inhibition of HP-PLA2 by the R and S enantiomers of 2-tetradecanoylaminohexanol-1-phosphocholine, its phosphoglycol, and its phosphoethanolamine derivatives were tested. The S enantiomers are only weak inhibitors, whereas the R enantiomers are potent inhibitors. The inhibitory power depends on the nature of the polar head group and increases in the order phosphocholine much less than phosphoethanolamine less than phosphoglycol. The best inhibitor, (R)-2-tetradecanoylaminohexanol-1-phosphoglycol, binds 2200 times stronger than the substrate to the HP-PLA2 active site.     

Degradation of missense mutant beta-galactosidase proteins in Escherichia coli K-12.

Summary Ten out of 43 missense mutations in the lacZ gene of Escherichia coli gave rise to polypeptide chains that were degraded in vivo. While many of the mutants appeared to be fully or partially CRM^–, there appeared to be no obvious correlation between degradation, map position, altered subunit association and the half-life of the mutant proteins.     

Efflux of beta-galactosidase products from Escherichia coli.

Several different strains of Escherichia coli were grown on a variety of carbon sources under various growth conditions. Lactose was added (usually at mid-log phase), and the concentrations of the products of beta-galactosidase action on this sugar (galactose, glucose, and allolactose) were determined at various times thereafter in the total culture and in the medium. It was found that with each strain, with all carbon sources, and under all of the conditions studied, a very large proportion of the products were found in the medium. Control studies were carried out which showed that these results were not artifacts of the method of separating the cells from the medium. The results also did not arise from the secretion of beta-galactosidase into the medium, from the diffusion of substrates and products into and out of the cells due to leaks in the membrane, or from faults in the method of sugar analysis. In addition, the results showed that there were very high levels of products inside the cells under the conditions used and that the efflux of the products was rapid. The efflux might be energetically advantageous to the cell as well as being a means of storing excess products until needed.     

Stabilities of uncomplemented and complemented M15 beta-galactosidase (Escherichia coli) and the relationship to alpha-complementation.

M15 beta-galactosidase (Escherichia coli) is a mutant form of beta-galactosidase having residues 11-41 deleted. It is an inactive dimer but can be complemented to the active tetrameric form by the addition of a peptide containing the deleted residues. The activities of uncomplemented and complemented M15 beta-galactosidases decreased starting at 42 degrees C--uncomplemented over a narrow temperature range, complemented over a broad range. This is because uncomplemented protein is a simple dimer while complemented is a mix of interacting oligomers at high temperatures. The effects of added components on stability and alpha-complementation are best explained by binding effects on equilibria between native forms and forms susceptible to inactivation. Mg2+ stabilized complemented protein but destabilized uncomplemented protein (10x less Mg2+ was needed for complemented protein). Alpha-complementation increased somewhat at low Mg2+ but decreased at high Mg2+. These effects can be explained by differential Mg2+ binding to the native and susceptible forms. The enhancement of both stability and alpha-complementation by Na+ can be explained by preferential binding of Na+ to the native forms of both the uncomplemented and complemented proteins. Low 2-mercaptoethanol concentrations stabilized uncomplemented M15 beta-galactosidase, but high concentrations destabilized it. All concentrations destabilized complemented M15 beta-galactosidase. Alpha-complementation was enhanced by 2-mercaptoethanol. Thus, there is a correlation between stability of the uncomplemented protein and alpha-complementation at low 2-mercaptoethanol owing to interactions with native forms. The lack of correlation at higher 2-mercaptoethanol probably results from precipitation by 2-mercaptoethanol. In contrast to irreversible thermal inactivation, differences in reversible stability in urea were small. This suggests that quaternary structure and Mg2+ and Na+ sites are lost at low urea concentrations and are unimportant at the urea concentrations that result in reversible denaturation.     

Kinetic mechanism of phosphofructokinase-2 from Escherichia coli. A mutant enzyme with a different mechanism

The kinetic mechanisms of Escherichia coli phosphofructokinase-2 (Pfk-2) and of the mutant enzyme Pfk-2 were investigated. Initial velocity studies showed that both enzymes have a sequential kinetic mechanism, indicating that both substrates must bind to the enzyme before any products are released. For Pfk-2, the product inhibition kinetics was as follows: fructose-1,6-P2 was a competitive inhibitor versus fructose-6-P at two ATP concentrations (0.1 and 0.4 mM), and noncompetitive versus ATP. The other product inhibition patterns, ADP versus either ATP or fructose-6-P were noncompetitive. Dead-end inhibition studies with an ATP analogue, adenylyl imidodiphosphate, showed uncompetitive inhibition when fructose-6-P was the varied substrate. For Pfk-2, the product inhibition studies revealed that ADP was a competitive inhibitor versus ATP at two fructose-6-P concentrations (0.05 and 0.5 mM), and noncompetitive versus fructose-6-P. The other product, fructose-1, 6-P2, showed noncompetitive inhibition versus both substrates, ATP and fructose-6-P. Sorbitol-6-P, a dead-end inhibitor, exhibited competitive inhibition versus fructose-6-P and uncompetitive versus ATP. These results are in accordance with an Ordered Bi Bi reaction mechanism for both enzymes. In the case of Pfk-2, fructose-6-P would be the first substrate to bind to the enzyme, and fructose-1,6-P2 the last product to be released. For Pfk-2, ATP would be the first substrate to bind to the enzyme, and APD the last product to be released.     

Major proteins of the Escherichia coli outer cell envelope membrane. Sequence of the cyanogen bromide fragments of protein I from Escherichia coli B/r.

The sequence of the cyanogen bromide fragments of one of the major outer membrane proteins of E. coli B/r has been established with the aim of elucidating the primary structure of this protein. Separation of all fragments on one molecular sieve column was achieved upon citraconylation of these fragments. Overlapping peptides were obtained by digestion of the protein, or a cyanogen bromide fragment arising from incomplete cleavage, with trypsin or Staphylococcus aureus protease.     

Amino acid sequence of beta-galactosidase. VII. Isolation of the 24 cyanogen bromide peptides.

All of the 24 cyanogen bromide peptides of beta-galactosidase have been isolated in pure form. Of these 8 ranged in size from 2 to 5 residues and were purified by paper electrophoresis. The 16 large peptides, from 23 to 119 residues, were chromatographed at pH 5.0 on a carboxymethyl-cellulose column in 0.02 M ammonium acetate buffer containing 8 M urea. A number of peptides were obtained in pure from following Sephadex G-50 or G-75 gel filtration. Others were separated on sulfopropyl-Sephadex or diethyl-(2-hydroxylpropylaminoethyl)-Sephadex. There large peptides were obtained in over 50% yield and several others were obtained in more than 25% yield.     

Structure of N-terminal cyanogen bromide fragment of human plasma albumin.

The complete amino acid sequence of 87 residues of cyanogen bromide fragment CB1 (Asp), the N-terminal fragment of human plasma albumine molecule, has been established. The sequence was determined from the characterization of all tryptic peptides and of chymotryptic arginine-containing peptides in the fragment digested. Overlaps were obtained by tryptic and chymotryptic cleavage of the maleylated S-sulfo derivative of fragment CB1(Asp). Residue 34 is the only cysteine residue in the albumin molecule and it was determined in the form of S-carboxymethyl-cysteine. Edman and dansyl-Edman degradation were used for the sequential analysis.     

A cyanogen bromide fragment of S4 that specifically rebinds 16S RNA.

Escherichia coli ribosomal protein S4 was subjected to cyanogen bromide cleavage and was found to generate a complete cleavage product capable of rebinding 16S rRNA. This fragment, consisting of residues 1-103, was found to bind with an apparent association constant of 11 microM-1. This fragment was used in place of S4 in an in vitro reconstitution experiment. Although the particles formed had a protein composition not significantly different from reconstituted 30S ribosomal subunits, their sedimentation behavior was more like that of particles reconstituted without S4. These results indicate to us that, although residues 104-203 of S4 are involved in the assembly of the 30S ribosome, they are not necessary for the binding of S4 to 16S RNA. Taken with previous results, the domain of S4 involved in specific binding of 16S RNA can be confined to residues 47-103.     

Variable specific activity of Escherichia coli beta-galactosidase in bacterial cells

Escherichia coli lacZ is a frequently employed reporter gene for the monitoring of gene expression and recombinant protein production due the simple determination of beta-galactosidase activity in both qualitative and quantitative assays. In the absence of either total or recombinant protein synthesis, we observed a lack of correlation between protein amount and enzymatic activity in both engineered and native beta-galactosidases in Escherichia coli cells. A delayed fading of beta-galactosidase activity compared with the rapid degradation of intact protein suggests a progressive increase in enzyme-specific activity during the life of the protein. This intriguing event does not involve solubilization from major protein aggregates and it occurs both in vivo and in cell extracts, but not in solutions of purified protein. Possible explanations for this activation are examined in the context of the assisted protein folding network and proteolytic degradation of misfolded proteins.     

Threonine deaminase from Escherichia coli: feedback-hypersensitive enzyme from a genetic regulatory mutant.

A mutation, ilvA538, in the gene coding for the biosynthetic L-threonine deaminase of Escherichia coli K-12 has previously been demonstrated to have pleiotropic regulatory effects leading to low and invariant expression of some of the isoleucine-valine biosynthetic enzyme, and altered expression of the branched-chain aminoacyl-tRNA synthetases. Strain PS187, which carries the ilvA538 allele, has a partial growth requirement for L-isoleucine and is characterized by a sensitivity to growth inhibition by L-leucine. The experiments reported here demonstrate that the L-threonine deaminase produced by strain PS187 is hypersensitive to inhibition by the pathway end product L-isoleucine. In addition, L-leucine, which acts at relatively high concentrations in vitro as an inhibitor of L-threonine deaminase from the wild type, is a more potent inhibitor of the activity of the mutant enzyme. Forty-six derivatives of strain PS187 were isolated as spontaneous mutants resistant to the growth-inhibitory effects of L-leucine. Two of these, strains MSR14 and MSR16, produce an L-threonine deaminase that is more resistant than the wild type to L-isoleucine inhibition, and intermediate between the wild type and strain PS187 with respect to L-leucine inhibition. Strains MSR14 and MSR16 produce L-threonine deaminase and dihydroxyacid dehydrase, the ilvD gene product, at the low levels characteristic of the parent strain. Other L-leucine-resistant derivatives of strain PS187 produce higher levels of the feedback-hypersensitive L-threonine deaminase. Thus, the sensitivity to growth inhibition by L-leucine observed with strain PS187 appears to be related both to the hypersensitivity of L-threonine deaminase to inhibition of catalytic activity and to the low level of ilv gene expression. The results reported here indicated that L-threonine deaminase is structurally altered in strain PS187, and thus provide further support for the proposal that L-threonine deaminase participates as a genetic regulatory element for the expression of the branched-chain amino acid biosynthetic enzymes.