Inhibition of induction of adaptive response by o-vanillin in Escherichia coli B

Mutations induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were strongly enhanced in the presence of o-vanillin in E. coli B. The enhancement was also observed in uvrA, umuC, recA, polA, or alkB mutants. This effect was lower in an alkA mutant, but was restored in an alkA umuC double mutant. By contrast, the enhancing effect was almost blocked in an ada and ada umuC double mutant. It was necessary to add simultaneously MNNG and o-vanillin to the growth medium. Further investigations were conducted on the induction of ada and umuC genes using ada'-lacZ' and umuC'-lacZ' plasmids. o-Vanillin suppressed the induction of the ada gene by MNNG treatment, but not that of the umuC gene. In fact expression of the umuC gene was induced by lower concentrations of MNNG in the presence of o-vanillin. The results suggest that o-vanillin inhibits induction of the adaptive response, and consequently, the MNNG-induced mutation frequency is increased due to unrepaired O6-methylguanine.     

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 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.     

L-asparaginase synthesis by Escherichia coli B

We have studied the influence of strain of organism, temperature, and medium on the production of the antileukemic intracellular enzyme L-asparaginase by E. coli B grown in shaken flasks. Five strains of E. coli B exhibited wide differences in their capacities to synthesize the EC-2 form of L-asparaginase active against leukemia. For the most productive strain, when grown in a casein hydrolysate medium, maximal production of L-asparaginase occurred at 25°C. At this temperature, the organism required glycerol, glucose, or other mono-saccharides to synthesize L-asparaginase. Synthesis was stimulated when glycerol was used in place of glucose, but not in its presence. The effect of glycerol on L-asparaginase synthesis was most evident when the cells were grown at 37°C, rather than at 25°C. With 0.25% glucose, cells had a specific activity of 409 I.U./g; with glycerol cells had a specific activity of 553 I.U./g. At 25°C, both cell and L-asparaginase synthesis were increased by the use of 0.25% glycerol resulting in only a slight increase in specific activity of the cells. The addition of zinc, copper, manganese, iron, L-asparagine, L-glutamine, or L-aspartic acid had no effect on L-asparaginase synthesis in the casein hydrolysate medium. L-aspartic acid (10^?2 M) enhanced L-asparaginase synthesis in a synthetic medium that lacked these metals or L-asparagine, L-glutamine, or L-aspartic acid; cells grown under these conditions had a specific activity of 90 I.U./g. In the casein hydrolysate medium, cell morphology was correlated with temperature of incubation.     

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.     

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 Injured Escherichia coli by Several Selective Agents

A population of Escherichia coli ML30 cells was exposed to a quaternary ammonium compound, and injury to the cells was measured by a comparison of counts on Trypticase Soy Agar and Violet Red Bile Agar. Substantial injury could not be detected with a minimal medium. The ingredients of Violet Red Bile Agar were tested against damaged cells. The bile salts mixture alone in the medium prevented as many injured cells from growing as did any combination of the selective agents and inhibited as many injured bacteria as were inhibited by Violet Red Bile Agar itself. These dyes and salts were similarly assayed in minimal agar, and comparable results were obtained. Individual bile salts and other potential selective agents were added to the minimal medium, and the media were tested for inhibition of injured E. coli. Sodium deoxycholate was the bile salt most inhibitory to damaged E. coli cells.     

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 Escherichia coli Biodegradative Threonine Dehydratase by Pyruvate

Pyruvate inhibits Escherichia coli K-12 biodegradative threonine dehydratase activity by a mechanism distinct from product inhibition by alpha-ketobutyrate and catabolite inactivation by intermediary metabolites.     

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.     

Inhibition of Escherichia coli growth and respiration by polymyxin B covalently attached to agarose beads.

Polymyxin B was attached to agarose beads by stable covalent bonds and the antimicrobial activity of the immobilized peptide was examined. Polymyxin-agarose inhibited the growth of Escherichia coli and Pseudomonas aeruginosa, but not Bacillus subtilis. In addition, the respiration of E. coli, E. coli spheroplasts, and B. subtilis protoplasts was inhibited by immobilized polymyxin, whereas the respiration of B. subtilis was unaffected by polymyxin-agarose. The activity of polymyxin-agarose was not due to the release of free peptide from the derivative. These data indicate that polymyxin can inhibit the growth and respiration of gram-negative bacteria by interacting with the outer surface of these cells. It is proposed that perturbation of outer membrane structure by polymyxin-agarose indirectly affected the selective permeability of the inner membrane and inhibited respiration. The results of this study emphasize the importance of outer membrane structural integrity for the normal functions of gram-negative bacteria.     

Inhibition of Escherichia coli cytidine deaminase by a phosphapyrimidine nucleoside

The nature of the interaction between Escherichia coli cytidine deaminase and the phosphapyrimidine nucleoside 1 has been studied kinetically and spectrophotometrically. Compound 1 was designed as a transition-state analog, and is a potent, slow-binding inhibitor of cytidine deaminase (Ashley, G. W., and Bartlett, P. A. (1982) Biochem. Biophys. Res. Commun. 108, 1467-1474). We present evidence that the binding of 1 is reversible, with no covalent linkage between the enzyme and 1. At pH 6, the rate of recovery of enzyme activity from dissociation of the E X I complex is strongly dependent on the concentration of E X I, indicating that the inhibitor dissociates reversibly. UV difference spectroscopy reveals that the chromophore of 1 is unaltered on binding to the enzyme, thus eliminating the possibility of reversible, covalent modification of the enzyme. For the binding of the active beta-anomers of 1 to cytidine deaminase, the following kinetic parameters were determined at pH 6: kon = 8300 M-1 S-1, koff = 7.8 X 10(-6) S-1, Ki = 0.9 nM. We were also able to observe and characterize time-dependent inhibition of E. coli cytidine deaminase by tetrahydrouridine, 3. This interaction involves involves initial formation of a loose complex (KD = 1.2 microM), followed by isomerization in a slow step to give a more tightly bound complex (Ki = 0.24 microM) with forward and reverse rate constants kf = 3.81 min-1 and kr = 0.95 min-1, respectively.