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.     

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.     

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.     

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 $\text{in vivo}$.     

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.     

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.     

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.     

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.     

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.     

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.     

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⁺.     

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.     

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 $\text{A}$.     

Guanylate cyclase from Dictyosteliumdiscoideum

Guanylate cyclase from crude homogenates of vegetative $\text{Dictyostelium}$$\text{discoideum}$ has been characterized. It has a pH optimum of 8.0, temperature optimum of 25°C and requires 1 mM dithiothreitol for optimal activity. It strongly prefers Mn⁺⁺ to Mg⁺⁺ as divalent cation, requires Mn⁺⁺ in excess of GTP for detectable activity, and is inhibited by high Mn⁺⁺ concentrations. It has an apparent Km for GTP of approximately 517 μM at 1 mM excess Mn⁺⁺.The specific activity of guanylate cyclase in vegetative homogenates is 50–80 pmoles cGMP formed/min/mg protein. Most of the vegetative activity is found in the supernatant of a 100,000 x g spin (S100). The enzyme is relatively unstable. It loses 40% of its activity after 3 hours storage on ice. Enzyme activity was measured from cells that had been shaken in phosphate buffer for various times. It was found that the specific activity changed little for at least 8 hours. Cyclic AMP at 10^?4 M did not affect the guanylate cyclase activity from crude homogenates of vegetative or 6 hour phosphate-shaken cells.     

Studies on the mitogenic principle of the lipoprotein from the outer membrane of Escherichia coli

Lipoprotein, from the outer membrane of $\text{E. coli}$, and several derivatives were investigated for lymphocyte stimulating activity in different species. We could show that lipoprotein exhibits activity towards mouse and rat splenocytes and rabbit and bovine lymph node cells; human peripheral blood lymphocytes showed a weak but significant stimulation. Thymocytes of all species were also weakly activated. Altered molecular structures at the C-terminal end of lipoprotein had only small influence on activity, hydrolysis of N-terminal fatty acids abolished mitogenicity.     

Cloning and expression in Streptomyces lividans of a paromomycin phosphotransferase from Streptomyces rimosusforma paromomycinus

The paromomycin producing organism $\text{Streptomyces}$$\text{rimosus}$$\text{forma paromomycinus}$ is resistant to this antibiotic and contains a phosphotransferase which inactivates paromomycin. The gene encoding this enzyme has been inserted in the $\text{Streptomyces}$ vector pIJ702 and then cloned in $\text{Streptomyces}$$\text{lividans}$, selecting for paromomycin-resistance. Three plasmids have been isolated and one of them, pMJ1, contains a 2.2 kb insert with a single HindIII restriction site. Insertion of foreign DNA in this site blocks the expression of the phosphotransferase enzyme indicating that it is within the cloned gene. These findings provide a new dominant selective marker for $\text{Streptomyces}$ cloning vectors with the versatility of insertional inactivation.     

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 …$\text{3Rib}\text{f}\text{β1→7KDO}\text{p}\text{β2→3Rib}\text{f}\text{β}$…

     

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.     

A new purification procedure for Clostridium difficile enterotoxin

$\text{Clostridium difficile}$ produces two toxins, an enterotoxin and a cytotoxin. The enterotoxin was purified using fast methods (tangential flow filtration, fast protein liquid chromatography). The purified enterotoxin is composed of two subunits (A₁ = 41,500, A₂ = 16,000) and its pI is 3.5.