Regulation of the isofunctional genes ubiD and ubiX of the ubiquinone biosynthetic pathway of Escherichia coli.

The expressions of the isofunctional genes ubiD and ubiX of the ubiquinone biosynthetic pathway of Escherichia coli were compared under a variety of growth conditions and in several genetic backgrounds. LacZ operon fusions were constructed and were inserted in single copies into strain MC4100 and into its fnr, arcA or hemA carrying derivatives. During aerobic growth the expressions of both ubiD and ubiX depended on the carbon source: succinate>glycerol>glucose. Mutations in fnr, arcA or hemA increased the expressions of both genes. During anaerobic growth in LB medium glucose strongly inhibited the expression of ubiD but not of ubiX.

Identification of the ubiD gene on the Escherichia coli chromosome.

The open reading frame at 86.7 min on the Escherichia coli chromosome, "yigC," complemented a ubiD mutant strain, AN66, indicating that yigC is the ubiD gene. The gene product, a 497-amino-acid-residue protein, showed extensive homology to the UPF 00096 family of proteins in the Swiss-Prot database.

Low ubiquinone content in Escherichia coli causes thiol hypersensitivity.

Thiol hypersensitivity in a mutant of Escherichia coli (IS16) was reversed by complementation with a plasmid that carried the ubiX gene. The mutant had low ubiquinone content. Complementation elevated the ubiquinone level and eliminated thiol hypersensitivity. Analysis of chromosomal ubiX genes indicated that both parent and mutant strains were ubiX mutants. The low ubiquinone content of IS16 was possibly caused by a ubiD ubiX genotype. A ubiA mutant also exhibited thiol hypersensitivity. Neither IS16 nor the ubiA mutant strain could produce alkaline phosphatase (in contrast to their parent strains) after 2 h of induction, thus showing Dsb- phenotypes. The phenomena of thiol hypersensitivity and low ubiquinone content may be linked by their connections to the periplasmic disulfide bond redox machinery.

Expression of the heme biosynthetic pathway genes hemCD, hemH, hemM, and hemA of Escherichia coli.

Little is known about the control of latter steps of heme biosynthesis in Escherichia coli. In this study we examined the transcriptional regulation of genes that encode two intermediate heme pathway enzymes, porphobilinogen deaminase (hemC) and uroporphyrinogen III cosynthase (hemD), and the final enzyme of the pathway, ferrochelatase (hemH). We also reexamined the regulation of hemA and the gene located immediately upstream of hemA, hemM. The regulatory regions of hemC, hemH, hemA and hemM were fused to lacZ. The resultant operon fusions were inserted into the E. coli chromosome in single copy and expression monitored under conditions of oxygen and heme limitation. Expression of hemM appeared constitutive under the conditions tested here. In contrast, expression of hemCD, hemH and hemA were shown to be mildly regulated in response to heme availability. Thus, transcription of four of the nine genes of the E. coli heme pathway appears to be only mildly regulated in response to heme limitation.

Enzymatic basis of thiol-stimulated secretion of porphyrins by Escherichia coli.

1-Thioglycerol (TG) stimulates the synthesis of porphyrin in aerobically growing Escherichia coli. Here the levels of delta-aminolevulinate biosynthetic enzymes in untreated and TG-treated E. coli THU and PUC2 (a mutant of THU which overproduces porphyrins in the presence of thiols) cells were determined. TG treatment elevated the activity of glutamyl-tRNA reductase in both strains. The increased activity was not caused by activation of preexisting enzymes by thiols or by oxidizing agents but was dependent on new protein synthesis.

Interactions of triply phosphorylated human beta-casein: fluorescence spectroscopy and light-scattering studies of conformation and self-association.

Structural changes of triply phosphorylated human beta-casein, caused by shifts in temperature between 5 and 40 degrees C, were studied using intrinsic and extrinsic fluorescence, fluorescence polarization, turbidity, and light scattering measurements. Intrinsic fluorescence declined between 5 and 20 degrees C then rose between 25 and 40 degrees C, indicative of a shift of the tryptophan fluor toward a more nonpolar environment. The fluorescence of the extrinsic probe, 8-anilino-1-naphthalene-sulfonic acid (ANS), increased only slightly between 5 and 25 degrees C, and then more sharply between 25 and 40 degrees C, suggesting a change in conformation leading to a change in either the dissociation constant, Kd, or the number of ANS binding sites, N. The presence of Ca+2 ions did not significantly alter the pattern of changes of intrinsic and extrinsic fluorescence with changing temperature. For ANS binding, values of Kd and N were calculated by two different procedures, each based upon different assumptions. The results point to increased exposure of hydrophobic surfaces with increased temperature, strongly supportive of conformational changes. Although more opportunity for hydrophobic interaction leads to increased protein-protein association, turbidity and light-scattering also suggest ion bridge formation between protein molecules. A comparison of the primary sequences of beta-caseins from six species reveal residues that are common in all species examined and thus are pivotal in protein folding and conformation, intermolecular hydrophobic interactions and ion bridge formation with Ca+2 and inorganic phosphate.

Thiol-sensitive genes of Escherichia coli.

The effect of 1-thioglycerol on the expression of genes of Escherichia coli was investigated. Pulse-labeled proteins from aerobically growing, 1-thioglycerol-treated E. coli were separated by two-dimensional gel electrophoresis, and their radioactivities were compared with those of identical proteins from nontreated cells. The first 10 min of exposure to thiol stimulated the synthesis of 10% of the observed proteins and inhibited the production of 16% of the proteins. After 30 min of growth with thiol, the synthesis of 44% of the observed proteins was inhibited and synthesis of 18% of the proteins was stimulated. In general, the expression of genes of carbohydrate metabolism, amino acid metabolism, and protein biosynthesis were inhibited, while nucleic acid synthetic and repair gene expressions showed mixed responses. Synthesis of transport proteins was not affected. Transient stimulation of oxidative-stress proteins and sustained stimulation of the expressions of trxB, ompA, and ompB genes and those of several unidentified gene products were also observed. Whether these complex responses merely reflect adjustments by cellular subsystems to a suddenly reducing environment or whether they are manifestations of a reductive-stress regulon will have to await genetic analysis of this phenomenon.

Thiol-sensitive promoters of Escherichia coli.

Mu dX(lac) insertion mutants of Escherichia coli CSH50 in which the expression of the lacZ gene was sensitive to the presence of exogenous 1-thioglycerol or dithiothreitol were isolated. Both stimulatory and inhibitory mutants were found. The existence of several thiol-sensitive promoters suggests that exogenous thiols may provoke global stress responses in E. coli.

Allium sativum (garlic) inhibits lipid synthesis by Candida albicans.

The effect of aqueous garlic extract on the macromolecular synthesis of Candida albicans was studied. Protein and nucleic acid syntheses were inhibited to the same extent as growth, but lipid synthesis was completely arrested. Blockage of lipid synthesis is likely an important component of the anticandidal activity of garlic.

Depression of adenosylmethionine content of Escherichia coli by thioglycerol.

The mechanism of growth inhibition of Escherichia coli by thioglycerol was probed. The extent of growth inhibition depended on the ratio of thioglycerol to initial cell concentration. Exogenously added methionine and several methionine analogues reversed the inhibition of 2 to 40 mM thioglycerol. Exposure to thioglycerol elevated the intracellular concentration of methionine, but the level of S-adenosylmethionine was depressed. Thioglycerol was methylated in vivo to 3-methylthio-1,2-propanediol.

Inhibition of respiration of Escherichia coli by thioglycerol.

Anaerobic growth on glucose significantly protected Escherichia coli from growth inhibition by thioglycerol. Methionine and anaerobiosis completely overcame growth inhibition by 2 to 90 mM thioglycerol. The respiration of aerobically growing cells was partially inhibited by 20 to 90 mM thioglycerol.

Inhibition of Escherichia coli by thioglycerol.

Thioglycerol inhibits the growth of various Escherichia coli strains and other microorganisms, both gram positive and gram negative. The susceptibility of organisms varies. The bactericidal action of this substance is not continuous and stops after an initial burst. At subbactericidal concentrations synthesis of ribonucleic acid is the most strongly affected. This is not due to interference with nucleoside biosynthesis or to direct inhibition of ribonucleic acid polymerase by thioglycerol.

Stability of Escherichia coli membrane proteins during chloramphenicol treatment.

It is shown that there is no degradation of previously formed inner and outer membrane proteins of Escherichia coli during 1 h of chloramphenicol treatment.

Inhibition of ribonucleic acid synthesis by nalidixic acid in Escherichia coli.

The effect of low concentrations of nalidixic acid on ribonucleic acid (RNA) synthesis in Escherichia coli was examined. It was observed that RNA synthesis in exponentially growing cells was not significantly affected, in harmony with previous studies. However, RNA synthesis was markedly depressed by nalidixic acid during starvation for an amino acid or during chloramphenicol treatment. This effect was not caused by increased killing or inhibition of nucleoside triphosphate synthesis by nalidixic acid. The pattern of radioactive uracil incorporation into transfer RNA or ribosomes was not changed by the drug. The sensitivity of RNA synthesis to nalidixic acid in the absence of protein production may be useful in probing the amino acid control of RNA synthesis.

Stimulation of unbalanced ribonucleic acid synthesis in Escherichia coli by methanol.

Addition of methanol to a stringent strain of Escherichia coli, starving of methionine, stimulates unbalanced ribonucleic acid (RNA) synthesis. The newly formed RNA and ribonucleoprotein species sediment between 4S and 30S. As a result of methanol treatment, cells become permeable to actinomycin D. Damage to cellular membrane appears to influence the control for RNA synthesis.