Hydroxylation of 2-octaprenylphenol in Escherichia coli K 12

A membrane-bound pathway for the biosynthesis of ubiquinone 8 (UQ8) in $\text{Escherichia coli}$ has been identified from the analysis of the precursors accumulated by mutants blocked in the pathway. Ubiquinone 8 (UQ8) deficient mutant which accumulate 2 octaprenylphenol (2-OPP) allowed to show that two components are involved in the hydroxylating system of this compound: one membranous, is cytochrome $\text{o}$ and the second cytoplasmic, is an NADPH cytochrome $\text{c}$ reductase.     

The argECBH-bearing EcoRI segment of the Escherichia coli chromosome

The transducing phage λd$\text{arg}\text{14}$, carrying a portion of the $\text{E. coli}$ chromosome including $\text{argECBH}$, is derived from the heat-inducible, lysis-defective strain λy199, which has the $\text{b}\text{519}$ and $\text{b}\text{515}$ deletions. Cleavage of λy199 DNA by $\text{Eco}$RI endonuclease, followed by agarose slab gel electrophoresis, results in bands corresponding to the known C, D, E, and F segments of λ, and a segment A′ (A plus B minus $\text{b}\text{519}$ minus $\text{b}\text{515}$, the cleavage site between A and B being eliminated). Cleavage of λd$\text{arg}\text{14}$ DNA by $\text{Eco}$RI yields the expected D, E, and F segments of λ and four other segments, termed 14-1 through 14-4, whose length is 17.5, 6.2, 3.0, and 2.0 kilobases, respectively, as determined by electron microscopy and corroborated by electrophoretic mobility. Heteroduplex analysis shows that the $\text{E. coli argECBH}$ cluster is on the 14-1 segment.     

Protein synthesis during a nutritional shift-up in Escherichia coli

The rate of synthesis of ribosomal protein increased immediately following a nutritional shift-up in $\text{Escherichia coli}$; while the rate of synthesis of elongation factors did not increase until 5–10 minutes had elapsed. The relative rates of synthesis of EFG and EFTs were constant at all times following shift-up. This constancy was not maintained between elongation factors and ribosomal protein early during shift-up. These data suggest that ribosomal and elongation factor proteins are not co-ordinately synthesized and argue against the postulate that their genes are part of one polycistron.     

Uncoupler and anaerobic resistant transport of phosphate in Escherichia coli

Active transport of inorganic phosphate into whole cells of a strain (AB3311) derived from $\text{Escherichia coli}$ K12 was found to be partially resistant to 50 μM carbonyl cyanide $\text{m}$-chlorophenyl hydrazone (CCCP), a powerful uncoupler of oxidative phosphorylation. The presence of 10 mM dithiothreitol (DTT) before the addition of CCCP completely prevented the inhibition of phosphate uptake caused by the uncoupler. The addition of DTT to the CCCP-inhibited system restored phosphate uptake to the control rate even when added 5 min after the phosphate transport assay was started. This uncoupler resistant transport is insensitive to anaerobiosis, or the addition of 10 mM KCN which reduces oxygen consumption to less than 1% that of aerobic controls. Additional studies of transport in a mutant (CBT302) deficient in membranebound Ca²⁺-, Mg²⁺-ATPase activity also demonstrated the retention of appreciable inorganic phosphate uptake under anaerobic conditions.     

Ribonuclease F,a putative processing endoribonuclease from Escherichia coli

A new endoribonuclease activity, RNase F, was partially purified from $\text{Escherichia coli}$ cells. This activity can cleave a precursor RNA molecule (of Species 1), isolated from T4 infected cells, in a specific site. This activity is different from the other three know processing endoribonucleases of $\text{E. coli}$ RNase III, RNase E and RNase P.     

Effect of trypsinization on thiamine transport in Escherichia coli Crookes

Trypsin treatment of intact $\text{Escherichia coli}$ Crookes cells is a probe for analyzing the functioning of protein sites in thiamine transport. These sites are cryptic since cells with different levels of thiamine transport activity respond differently to trypsin treatment. Mild trypsin treatment of cells with normal transport activity enhanced the velocity of uptake and decreased the capacity; more rigorous treatment reduced both parameters. Cells with low activity showed greatly increased rates of uptake and capacities under all but the mildest treatment conditions. These observations are consistent with a trypsin unmasking of thiamine transport sites in low activity cells and a destruction of the sites in higher activity cells.     

Outer membrane of Escherichia coli K-12: Tsx mutants (resistant to bacteriophage T6 and colicin K) lack an outer membrane protein

$\text{Tsx}$ mutants of $\text{Escherichia coli}$ are fully resistant to a set of T6-like bacteriophage and are resistant to colicin K. We demonstrate that these mutants are missing an outer membrane protein (the tsx-protein) of molecular weight 32,000 as measured by SDS-polyacrylamide gel electrophoresis. $\text{Tsx}$ mutants are receptor mutants which are unable to absorb either the bacteriophages or the colicin and the loss of receptor function can be demonstrated using outer membrane preparations.We suggest that the tsx-protein is the receptor for both the bacteriophage and colicin.     

DNA-dependent in vitro synthesis of Escherichia coli ribosomal protein L10 and the formation of an L10L12 complex

The $\text{in vitro}$ synthesis of ribosomal protein L10 has been demonstrated using λrif^d18 DNA as template. The L10 synthesized $\text{in vitro}$ forms a complex with ribosomal protein L12 and the L10 in this complex can be immunoprecipitated with L12 antiserum.     

Defective 30S ribosomal particles in a polyamine auxotroph of Escherichia coli

Polypeptide synthesis directed by poly(U) or MS 2 phage RNA is several fold more active in cell-free systems prepared from polyamine supplemented bacteria than in extracts of polyamine depleted cells. This effect depends on the presence of defective 30S ribosomal subunits in the starved bacteria. It is concluded that polyamines play a role in the normal biosynthesis, maturation and/or assembly of the small ribosomal subparticles.     

Isolation of relaxed-control mutants of Escherichia coli K-12 which are sensitive to glucose starvation

Mutants of $\text{Escherichia coli}$ K-12 which are sensitive to glucose starvation were isolated by an enrichment procedure using thymine starvation to select for nongrowing cells. Eleven independent isolates were obtained by this method. The mutants are also sensitive to glycerol starvation and to a lesser extent to nitrogen or amino acid starvation. The mutants are more sensitive than the parental strain to inhibitors of protein synthesis but not inhibitors of RNA or DNA synthesis. [³H]-leucine incorporation experiments indicate that protein synthesis is blocked in the mutants during recovery from glucose starvation or chloramphenicol inhibition. Incorporation of [³H]uridine in amino acid-starved cells demonstrates that the mutants are partially relaxed for control of RNA synthesis. Physiological and genetic experiments indicate that these mutants are different from previously isolated relaxed-control mutants.     

In vivo radiation-induced thymine residue release from E. coli DNA

Cells of $\text{E. coli}$ C thy^?321 are examined for thymine residue release from DNA following gamma-irradiation from 5 to 15 krad. Experimental conditions are designed to inhibit enzyme activity that might promote base residue release. Enzyme action is restricted in order to assess the physicochemical action of radiation on cellular DNA, and to this end irradiation is done under O₂, N₂, and N₂O saturating conditions. Both thymine and thymidine release from bacterial DNA are detected and quantitated, and three oxygen effects are noted in comparing yields of these products. No difference in effect is observed between N₂ and N₂O gassing conditions, suggesting that the hydroxyl radical has little effect on thymine or thymidine release from irradiated DNA $\text{in vivo}$.     

Structure of the capsular polysaccharide (K6-antigen) from Escherichia coli LP 1092

The linkage pattern of the K6-antigen was investigated using material from the urinary pathogen, $\text{Escherichia coli}$ LP 1092. The polysaccharide consists of ribose and 3-deoxy-$\text{D}\text{-}\text{manno}$-2-octulosonate (KDO) in a ratio of 2:1. Colorimetric procedures, Smith degradation, methylation analysis, and nuclear magnetic resonance spectroscopy were applied to the whole polysaccharide and to a trisaccharide “repeating unit” obtained by mild-acid catalyzed hydrolysis. Together, the data are compatible only with a branched chain structure …$\text{3Rib}\text{f}\text{β1→7KDO}\text{p}\text{β2→3Rib}\text{f}\text{β}$…

     

The release of endonuclease I from Escherichia coli by a new cold shock procedure

A new cold shock procedure has been developed for releasing large quantities of endonuclease I from $\text{E. coli}$, which neither involves EDTA-lysozyme treatment nor osmotic shock. Treatment of cells with ice-cold 0.1M Tris-0.2M KCl buffer, pH 7.4 results in the release of endonuclease I into the medium. Although the loss of endonuclease I from the cells is a rapid process, its recovery in the shock fluid is gradual and approaches maximum in about 90 minutes. Certain divalent metal ions such as Mg⁺⁺ and Mn⁺⁺ strongly inhibit the release of endonuclease I. The cold shock procedure is rather selective and the mechanism of the release of endonuclease I is different from that of osmotic shock procedure.     

The structure of the Escherichia coli pyruvate dehydrogenase complex is probably not unique

Measurements of apparent diffusion coefficients of the pyruvate dehydrogenase complex from $\text{E. coli}$, with the use of the active enzyme centrifugation method at different angular velocities, show that the complex is heterogeneous although the heterogeneity is limited. Conditions of extraction and conditions of centrifugation in which the complex would show a monodisperse behaviour have not been found.     

The interaction of phosphonate and homophosphonate analogues of 3-deoxy-D-arabino heptulosonate 7-phosphate with 3-dehydroquinate synthetase from Escherichia coli

Phosphonate and homophosphonate analogues of 3-deoxy-D-$\text{arabino}$ heptulosonate 7-phosphate and D-$\text{gluco}$ heptulosonate 7-phosphate behave as competitive inhibitors of 3-dehydroquinate synthetase. Phosphonates have better affinities than homophosphonates and protect efficiently the enzyme against thermal denaturation. No evidence has been obtained for 5-keto phosphonate intermediate formation in the interaction of such analogues with 3-dehydroquinate synthetase and NAD⁺.     

An endodextranase inhibitor from batch cultures of Streptococcus,mutans

An inhibitor of $\text{Streptococcus}$,$\text{mutans}$ endodextranase was detected in proteins prepared from batch cultures of $\text{S.}$,$\text{mutans}$ strains representing serotypes $\text{a}$ through $\text{g}$. Affinity chromatography of strain 6715-49 proteins, which apparently were free of endodextranase activity, yielded an active endodextranase and, in a separate peak, the endodextranase inhibitor. The presence of the inhibitor in culture fluids accounts for the absence of endodextranase activity in batch-grown cultures of $\text{S.}$,$\text{mutans}$ known to produce this enzyme.     

OKY-1581: A selective inhibitor of thromboxane synthesis invivo and invitro

OKY-1581 is an effective inhibitor of thromboxane synthesis $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$. The generation of thromboxane B₂ (TxB₂), prostaglandin E (PGE) and prostaglandin F (PGF) was measured following clotting and during platelet aggregation induced by collagen. The presence of OKY 1581 either $\text{in}$$\text{vivo}$ or $\text{in}$$\text{vitro}$ caused a reduction in TxB₂ generation during clotting and platelet aggregation with a concomitant increase in PGE and PGF. The effect could be observed two hours after oral or subcutaneous administration of 5 to 100 mg per rabbit and lasted for 24 to 48 hours. The reduction in TxB₂ was not accompanied by an inhibition of clotting or platelet aggregation. OKY-1581 appears to be a suitable agent for studying the role of TxB₂ in atherosclerosis.     

Metabolites of mating pheromone,rhodotorucine A, by a cells of Rhodosporidiumtoruloides

Rhodotorucine $\text{A}$ which induces mating tube formation of $\text{a}$ cells in $\text{Rhodosporidium}\text{toruloides}$ is metabolized rapidly by $\text{a}$ cells. By use of labeled rhodotorucine $\text{A}$, the degradation was found to be proteolytic. Two peptide fragments Tyr-Pro-Glu-Ile-Ser-Trp-Thr-Arg and Asn-Gly-Cys(S-farnesyl) were identified as the metabolites. Proteolysis of the pheromone mainly occurred on the cell surface. Culture filtrate of $\text{a}$ cells at log phase did not metabolize rhodotorucine $\text{A}$.     

Inactivation of Escherichia coli succinic thiokinase by selective oxidation of thiol groups by permanganate

Succinic thiokinase from $\text{Escherichia coli}$ was rapidly inactivated by permanganate ion at 25° and 0°. On the basis of the cysteic acid content of hydrolysates of treated protein, oxidation of 3 sulfhydryl groups appeared to effect total loss of thiokinase activity. However, titration of the same protein samples revealed that 4 important sulfhydryl groups (a fraction of which was possibly in disulfide form) were more likely oxidized during the inactivation process. Significant protection of the enzyme against permanganate inactivation was obtained by the following additions: ATP-Mg²⁺ and succinate (51%); desulfo-CoA alone (53%); and ATP-Mg²⁺, succinate, and desulfo-CoA (93%). No protection was observed when either inorganic phosphate or arsenate was added.