Division Delay Induced in Escherichia coli by Near-Ultraviolet Radiation

Beams of near-ultraviolet radiation at several principal emission lines of a mercury arc were isolated with a grating monochromator and directed upon cell suspensions. During subsequent incubation at room temperature in Nutrient Broth, the population was studied by removing samples and obtaining cell numbers and cell size distributions with an electronic cell counter. Division delay without lethality was observed. The shapes of the dose-response curves for induction, the doses of near-ultraviolet radiation required, and the action spectrum for division delay were found to be similar to those for growth delay (in broth) and for photoprotection. These findings indicate that all three effects, division delay, growth delay, and photoprotection, are induced by a common type of critical event. Changes in cell size distribution in the culture during incubation in Nutrient Broth after near-ultraviolet irradiation are very similar for control and irradiated populations, although these changes occur at a much later time in the irradiated population. This indicates that, in Nutrient Broth, the population recovers completely from the inhibition of growth and division, thus justifying use of the term "delay," and suggesting that the damage is nongenetic.     

Escherichia coli Tryptophanase in the Enteric Environment

The activity of the enzyme tryptophanase in the enteric environment was investigated to elucidate the significance of the enzyme in the metabolism of Escherichia coli. The tryptophanase activity, tryptophan content, and indole concentration as well as the numbers of E. coli were determined in the intestinal and fecal contents of conventional, germ-free, and monocontaminated axenic laboratory mice. Increasing the tryptophan content of the diet of mice having a conventional microflora increased the tryptophanase activity of the enteric microflora by a factor of almost 2 but did not increase the numbers of E. coli either absolutely or relative to other facultative enteric coliforms. In the enteric environment, E. coli is responsible for very little tryptophanase activity, a fraction calculated to be less than 0.02%. The values for the experimental parameters were much the same in the contents of the cecum and in the fecal material.     

Radiation Inhibition of Amino Acid Uptake by Escherichia coli

The inhibition of macromolecular synthesis in Escherichia coli by ionizing radiation has been investigated. The survival of the ability to incorporate arginine, leucine, isoleucine, histidine, uracil, and glucose after various doses of gamma radiation, deuteron and alpha particle bombardment has been measured. All amino acids are incorporated by processes which show the same radiation sensitivity. The sensitivity of uracil corresponds to a volume which is roughly spherical, of radius about 160A, whereas the amino acids possess sensitive regions which are long and thin in character. The uptake of glucose is concerned with a smaller, roughly spherical unit. The possible identification of the radiation-sensitive targets with cellular constituents is discussed. The long thin character observed for amino acids suggests that the sensitive region affected by radiation is an unfolded form of a ribosome, or alternatively a long nucleic acid molecule. For uracil the sensitive region fits with a 70S ribosome, while for glucose a smaller particle would fit the data.     

Tryptophanase in sRNA control of the Escherichia coli cell cycle

The field of gene regulation underwent a major revolution with the discovery of small non-coding RNAs (sRNAs) and the various roles they play in organisms from bacteria to man. Escherichia coli has more than 60 sRNAs that are transcribed primarily from intergenic regions. They usually target the leader region of mRNAs and prevent their translation. Protein targets are relatively rare. In this issue of Molecular Microbiology, Chant and Summers provide an example of a totally unexpected protein target. They show that dimers of plasmid ColE1 make an sRNA that interacts directly with the enzyme tryptophanase and enhances its affinity for its substrate, tryptophan. A breakdown product, indole, then arrests cell division until the dimers are resolved to monomers. The monomerization helps to prevent plasmid loss. Targeting a catabolic enzyme to buy time for recombination is an amazing example of adaptation, which illustrates the power of a selfish element (a plasmid in this case) to exploit the host cell machinery to its advantage.     

Die Wirkung von Ultraschall auf Tryptophanase aus Escherichia coli

Zusammenfassung In der vorliegenden Arbeit wurde die Wirkung des Ultraschalls auf die Tryptophanase in Escherichia coli untersucht. Es konnte nachgewiesen werden, daß Ultraschall das Tryptophanase-Protein angreift, während das Coenzym Pyridoxalphosphat nicht verändert wird. Weiterhin wurde die Veränderung des Pyridoxalphosphats durch Ultraschall in vitro festgestellt.

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Summary The action of ultrasonic waves on the tryptophanase in Escherichia coli is described. The proteine of the tryptophanase is changed by ultrasonic waves, whereas the coenzyme pyridoxalphosphate is stable. In vitro pyridoxalphosphate is changed by ultrasonic waves.

     

Action Spectrum for Growth Delay Induced in Escherichia coli B/r by Far-Ultraviolet Radiation

An action spectrum for growth delay induced in Escherichia coli B/r by far-ultraviolet radiation (230 to 295 nm) was obtained. It resembles the action spectrum for killing obtained in the same experiments, indicating that the chromophore for growth delay is probably the same as the chromophore for killing. Another action spectrum for killing, obtained under conditions more suitable for chromophore identification, suggests that nucleic acid, either deoxyribonucleic acid or ribonucleic acid, is the chromophore for growth delay induced by far ultraviolet. Isoprenoid quinones, which seem to be important chromophores for growth delay induced by near-ultraviolet radiation (above 300 nm), appear to play a negligible role in growth delay induced by wavelengths below 300 nm.     

Bulge Formation in Escherichia coli Induced by l-Arabinose under Hypertonic Conditions with Sodium Chloride

A pseudotropine-forming tropinone reductase was extracted from root cultures of Hyoscyamus niger that produce the tropane alkaloids hyoscyamine and scopolamine. The enzyme stereospecifically reduces tropinone to pseudotropine, oxidizing NADPH. It has an approximate molecular weight of 84,000 and a pH optimum between 5.8 and 6.25. The Km value for tropinone is 35.1 μmol/l and for NADPH 21.1 μmol/l. Substrate specificity was tested for NADPH and several tropinone analogues.     

Inhibition of growth of Shiga toxin-producing Escherichia coli by nonpathogenic Escherichia coli

During routine quality control testing of diagnostic methods for Shiga toxin-producing Escherichia coli (STEC) using stool samples spiked with STEC, it was observed that the Shiga toxin could not be detected in 32 out of 82 samples tested. Strains of E. coli isolated from such stool samples were shown to be responsible for this inhibition. One particular isolate, named E. coli 1307, was intensively studied because of its highly effective inhibitory effect; this strain significantly reduced growth and Shiga toxin levels in coculture of several STEC strains regardless of serovar or Shiga toxin type. The probiotic E. coli Nissle 1917 inhibited growth and reduced Shiga toxin levels in STEC cultures to an extent similar to E. coli 1307, but commensal E. coli strains and several other known probiotic bacteria (enterococci, Bacillus sp., Lactobacillus acidophilus ) showed no, or only small, inhibitory effects. Escherichia coli 1307 lacks obvious fitness factors, such as aerobactin, yersiniabactin, microcins and a polysaccharide capsule, that are considered to promote the growth of pathogenic bacteria. We therefore propose strain E. coli 1307 as a candidate probiotic for use in the prevention and treatment of infections caused by STEC.     

Inhibition of Escherichia coli B by homoarginine

Homoarginine inhibits the growth of Escherichia coli B, but not of E. coli K-12. These two strains also differ in regard to repressibility of the arginine-forming enzymes. In K-12, arginine acts as a repressor whereas in B it does not. The latter difference is determined by different alleles of a regulator gene, arg R. In K-12 × B crosses, it was shown that the genetic determinant for homoarginine sensitivity is closely linked to or identical with arg R. Homoarginine-resistant mutants of B were isolated. The biochemical mechanism of homoarginine inhibition is not known. However, whether or not a strain is sensitive to homoarginine seems to depend on the intracellular level of arginine. In B this level is relatively low and inflexible as a result of the action of a repressor whose formation is determined by the B-specific allele of arg R.     

Inhibition of the growth of Escherichia coli by chlortetracycline

1. This study extends previous work concerned with the ribonucleic acid made by Escherichia coli during inhibition of protein synthesis by chlortetracycline. 2. The antibiotic caused an initial stimulation in the rate of RNA synthesis. 3. RNA made during inhibition was stable during continued incubation in the presence of the antibiotic although it was extensively degraded in resting cell suspensions. 4. Most of the RNA accumulated during chlortetracycline action was in particles that sedimented more slowly than ribosomes. During the recovery of cells from the effects of the antibiotic, accumulated RNA was apparently not degraded and ribosomes were synthesized from the RNA in the particles.     

Effect of Intracellular Concentration of Vitamin B6 on Tryptophanase Activity in Escherichia coli

The intracellular concentration of vitamin B6 (B6) of a wild type strain (WG1) of Escherichia coli B remained almost constant (0.25 to 0.35 nmol per mg cells) when different amounts of B6 were extracellularly added. However, B6-requiring mutants were strongly affected by extracellular B6.

Activities of tryptophanase in a B6-requiring mutant, strain WG3, grown under various conditions were measured. A clear correlation between intracellular B6 concentration and the ratio of holo-tryptophanase activity (i.e., holo-activity/total activity) was observed.

Tryptophanase of strain WG3 grown under B6-deficient conditions and the enzyme of strain WG1 were purified and their properties compared. The purified enzymes of both strains WG3 and WG1 showed similar characteristics, but a difference was observed in the antigen activities.     

Formation of ethylene by Escherichia coli.

Escherichia coli strain SPA O converts methionine to ethylene by an inducible enzyme system. L-Cysteine, L-homocysteine, methionine derivatives and the sulphur-containing analogues of L-methionine also act as precursors of ethylene. Ethylene is produced by cell suspensions only in the presence of air; cell-free preparations can produce ethylene aerobically and anaerobically, but the extent to which they do so depends on the mode of culture growth. Light stimulates ethylene production by cell suspensions and its presence is essential for production by cell-free preparations. The kinetics of ethylene biogenesis and its pH and temperature optima suggest that ethylene is a secondary metabolite.     

Inhibition by Phenylalanine of Thiazole Biosynthesis in Escherichia coli

Phenylalanine inhibited thiazole biosynthesis in a thiamine-regulatory mutant of Escherichia coli, and the inhibition was overcome by tyrosine.     

Inhibition of Thiamine Transport by Chloroethylthiamine in Escherichia coli

Chloroethylthiamine was found to inhibit an entrapment of thiamine as thiamine monophosphate by blocking thiamine monophosphokinase in the cytoplasm after thiamine was taken up by the cells of Escherichia coli.     

Inhibition of Escherichia coli Division by Protein X

We propose that protein X provides the connection between damage to Escherichia coli DNA and inhibition of septation and cell division. This connection is needed to guarantee that each new bacterium receives a complete DNA copy. We present several new experiments here which demonstrate that the degree to which septation is inhibited following damage to DNA is correlated with the amount of protein X that is produced. Rifampin selectively blocks protein X production. This drug was shown to allow cells whose DNA had been damaged by nalidixic acid to resume septation. Several mutants formed septa-less filaments and also produced protein X at 42 degrees C; rifampin both inhibited their production of protein X and permitted them to form septa and divide. Essentially complementary results were obtained with a dnaA mutant which at 42 degrees C stopped making DNA, did not produce protein X, and continued to divide; added bleomycin degraded DNA, induced protein X, and inhibited septation. These results, as well as previous observations, are all consistent with the proposal that protein X is produced as a consequence of DNA damage and is an inhibitor of septation. We suggest that septation could require binding of a single-stranded region of DNA to a septum site in the membrane. Protein X could block this binding by combining with the DNA. This control could provide an emergency mechanism in addition to the usually proposed coordination in which completion of DNA synthesis creates a positive effector for a terminal step of septation. Or it could be the sole coordinating mechanism, even under unperturbed growth conditions.     

Inhibition of Escherichia coli glutamine synthetase by phosphinothricin

The inhibition of Escherichia coli glutamine synthetase by phosphinothricin [2-amino-4-(methylphosphinyl)butanoic acid] has been studied. This amino acid was observed to function as an active site directed inhibitor exhibiting time-dependent inhibition of glutamine synthetase in the presence of ATP or adenylylimidodiphosphate (AMPPNP) but not adenylyl(β,γ-methylene) diphosphonate (AMPPCP). The inactivation was observed to be pseudo-first order. Phosphinothricin was also found to inhibit the enzyme reversibly under initial rate conditions and was competitive with respect to glutamate with K_1S = 18 ± 3 μm. The inactive enzyme inhibitor complex was found to contain approximately 11 molecules of ADP and of ³²P per dodecamer using [γ-³²P]ATP. Reactivation of the inactive enzyme complex was achieved by incubating the enzyme complex in 50 mm acetate (pH 4.4), 1 m KCl, and 0.40 m (NH₄)₂SO₄. ADP, phosphinothricin, and P_i were released upon reactivation.     

Protoplast Formation in Escherichia coli

A procedure for protoplast formation in Escherichia coli is described. Removal of the cell wall was confirmed by examination of cells in thin-section preparations.