Magnetic semiconductor: structural, magnetic, and conducting properties of the salts of the 6-oxoverdazyl radical cation with M(dmit)(2) anions (M = Ni, Zn, Pd, and Pt, dmit = 1,3-dithiol-2-thione-4,5-dithiolate).

Five kinds of (1:1), (1:3), and (2:1) salts of 3-[4-(diethylmethylammonio)phenyl]-1,5-diphenyl-6-oxoverdazyl radical cation [V](+) with M(dmit)(2) anions (M = Ni, Zn, Pd, and Pt, dmit = 1,3-dithiol-2-thione-4,5-dithiolate) ([V](+)[Ni(dmit)(2)](-) (1), [V](+)[Ni(dmit)(2)](3)(-) (2), [V](+)(2)[Zn(dmit)(2)](2-) (3), [V](+)(2)[Pd(dmit)(2)](2-) (4), and [V](+)(2)[Pt(dmit)(2)](2-) (5)) and an iodide salt of [V](+) ([V](+)[I](-) (6)) have been prepared, and the magnetic susceptibilities (chi(M) values) have been measured between 1.8 and 300 K. The chi(M) of the (1:1) Ni salt (1) can be well reproduced by the sum of the contributions from (i) a Curie-Weiss system with a Curie constant (C) of 0.376 K emu/mol and a negative Weiss constant (theta) of -1.5 K and (ii) the one-dimensional Heisenberg antiferromagnetic alternating chain system with 2J(A-B)/k(B) = -274 K (alternation parameter alpha = J(A-C)/J(A-B) = 0.2). The chi(M) of the (1:3) Ni salt (2) can be well explained by the two-term contributions from (i) the Curie-Weiss system with C = 0.376 K emu/mol and theta = -5.0 K and (ii) the dimer system with 2J/k(B) = -258 K. The magnetic properties of 1 and 2 were discussed based on the results obtained by crystal structure analysis and ESR measurements of 1 and 2. The chi(M) values of the (2:1) Zn, Pd, Pt salts 3, 4, and 5 and [V](+)[I](-) salt 6 follow the Curie-Weiss law with C = 0.723, 0.713, 0.712, and 0.342 K emu/mol and theta = -2.8, -3.1, -2.6, and +0.02 K, respectively, indicating that only the spins of the verdazyl radical cation contribute to the magnetic property of these salts. The salts 1, 3, and 5 are insulators. On the other hand, the conductivity (sigma) of the Ni salt 2 and Pd salt 4 at 20 degrees C was sigma = 8.9 x 10(-2) and 1.3 x 10(-4) S cm(-)(1) with an activation energy E(A) = 0.11 and 0.40 eV, respectively. The salts 2 and 4 are new molecular magnetic semiconductors.

Temperature-sensitive mutations in various genes of Escherichia coli K12 can be suppressed by the ssrA gene for 10Sa RNA (tmRNA).

An Escherichia coli strain with a deletion in the ssrA gene that encodes 10Sa RNA (tmRNA) was used to screen for temperature-sensitive (ts) mutants whose ts phenotypes were suppressible by introduction of the wild-type ssrA gene. Mutants in four different genes were isolated. Ts mutants of this type were also obtained in a screen for mutations in thyA, the structural gene for thymidylate synthase. The ThyA activity in crude extracts prepared from the ts mutants was temperature-sensitive. The presence of the ssrA gene caused an increase in the total amount of the temperature-sensitive enzyme expressed, rather than suppressing the ts activity of the enzyme itself. SsrA-DD, a mutant form of 10Sa RNA, suppressed the ts phenotype of a thyA mutant, suggesting that degradation of a tagged peptide was not required for suppression of the ts phenotype. Considering the fact that ssrA-suppressible mutants could be isolated as temperature-sensitive mutants with mutations in different genes, it seems evident that trans-translation can occur on mRNA that is not lacking its stop codon.

Abortive assembly of succinate-ubiquinone reductase (complex II) in a ferrochelatase-deficient mutant of Escherichia coli.

Heme molecules play important roles in electron transfer by redox proteins such as cytochromes. In addition, a structural role for heme in protein folding and the assembly of enzymes has been suggested. Previous results obtained using Escherichia coli hemA mutants, which are unable to synthesize 5-aminolevulinic acid, a precursor of porphyrins and hemes, have demonstrated a requirement for heme biosynthesis in the assembly of a functional succinate-ubiquinone reductase (SQR or complex II), which is a component of the aerobic respiratory chain. In the present study, in order to investigate the role of the heme in the assembly of E. coli SQR, we used a hemH (encodes ferrochelatase) mutant that lacks the ability to insert iron into the porphyrin ring. The hemH mutant failed to insert functional SQR into the cytoplasmic membrane, and the catalytic portion of SQR [the flavoprotein subunit (Fp) and the iron-sulfur protein subunit (Ip)] was localized in the cytoplasm of the cell. It is of interest to note that protoporphyrin IX accumulated in the mutant cells and inactivated the cytoplasmic succinate dehydrogenase (SDH) activity associated with the catalytic Fp-Ip complex. In contrast, SQR was assembled into the membrane of a heme-permeable hemH double mutant when hemin was present in the culture. Only a low level of SQR activity was found in the membrane when hemin was replaced by non-iron metalloporphyrins: Mn-, Co-, Ni-, Zn- and Cu-protoporphyrin IX, or protoporphyrin IX These results indicate that heme iron is indispensable for the functional assembly of SQR in the cytoplasmic membrane of E. coli, and provide a new insight into the biological role of heme in the molecular assembly of the multi-subunit enzyme complex.

Yeast [PSI+] "prions" that are crosstransmissible and susceptible beyond a species barrier through a quasi-prion state.

The yeast [PSI(+)] element represents an aggregated form of release factor Sup35p and is inherited by a prion mechanism. A "species barrier" prevents crosstransmission of the [PSI(+)] state between heterotypic Sup35p "prions." Kluyveromyces lactis and Yarrowia lipolytica Sup35 proteins, however, show interspecies [PSI(+)] transmissibility and susceptibility and a high spontaneous propagation rate. Cross-seeding was visualized by coaggregation of differential fluorescence probes fused to heterotypic Sup35 proteins. This coaggregation state, referred to as a "quasi-prion" state, can be stably maintained as a heritable [PSI(+)] element composed of heterologous Sup35 proteins. K. lactis Sup35p was capable of forming [PSI(+)] elements not only in S. cerevisiae but in K. lactis. These two Sup35 proteins contain unique multiple imperfect oligopeptide repeats responsible for crosstransmission and high spontaneous propagation of novel [PSI(+)] elements.

Amber mutations in ribosome recycling factors of Escherichia coli and Thermus thermophilus: evidence for C-terminal modulator element.

Ribosome recycling factor, referred to as RRF, is essential for bacterial growth because of its activity of decomposition of the post-termination complex of the ribosome after release of polypeptides. In this study, we isolated a conditionally lethal amber mutation, named frr-3, in the Escherichia coli RRF gene at amino acid position 161, showing that the truncation of the C-terminal 25 amino acids of RRF is lethal to E. coli. An RRF gene cloned from Thermus thermophilus, whose protein is 44% identical and 68% similar to E. coli RRF, failed to complement the frr-3(Am) allele. However, truncation of the C-terminal five amino acids conferred intergeneric complementation activity on T. thermophilus RRF, demonstrating the modulator activity of the C-terminal tail. Rapid purification of T. thermophilus RRF was achieved by T7-RNA polymerase-driven overexpression for crystallography.

Novel temperature-sensitive mutants of Escherichia coli that are unable to grow in the absence of wild-type tRNA6Leu.

Escherichia coli has only a single copy of a gene for tRNA6Leu (Y. Komine et al., J. Mol. Biol. 212:579-598, 1990). The anticodon of this tRNA is CAA (the wobble position C is modified to O2-methylcytidine), and it recognizes the codon UUG. Since UUG is also recognized by tRNA4Leu, which has UAA (the wobble position U is modified to 5-carboxymethylaminomethyl-O2-methyluridine) as its anticodon, tRNA6Leu is not essential for protein synthesis. The BT63 strain has a mutation in the anticodon of tRNA6Leu with a change from CAA to CUA, which results in the amber suppressor activity of this strain (supP, Su+6). We isolated 18 temperature-sensitive (ts) mutants of the BT63 strain whose temperature sensitivity was complemented by introduction of the wild-type gene for tRNA6Leu. These tRNA6Leu-requiring mutants were classified into two groups. The 10 group I mutants had a mutation in the miaA gene, whose product is involved in a modification of tRNAs that stabilizes codon-anticodon interactions. Overexpression of the gene for tRNA4Leu restored the growth of group I mutants at 42 degrees C. Replacement of the CUG codon with UUG reduced the efficiency of translation in group I mutants. These results suggest that unmodified tRNA4Leu poorly recognizes the UUG codon at 42 degreesC and that the wild-type tRNA6Leu is required for translation in order to maintain cell viability. The mutations in the six group II mutants were complemented by introduction of the gidA gene, which may be involved in cell division. The reduced efficiency of translation caused by replacement of the CUG codon with UUG was also observed in group II mutants. The mechanism of requirement for tRNA6Leu remains to be investigated.

Effects of starvation for heme on the synthesis of porphyrins in Escherichia coli.

A study is described of the regulation of porphyrin synthesis in Escherichia coli using a heme-permeable, hemH deletion mutant, designated VS212. This strain utilizes only exogenous hemin that is supplied in the medium and accumulates porphyrins since the final step in the synthesis of heme is genetically blocked. It is possible, therefore, to monitor the rate of synthesis of heme by examining the accumulation of porphyrins. Using this system, we found that the rate of production of porphyrins depended on the availability of heme. The lower the concentration of hemin in the medium, the higher the level of porphyrins that accumulated. We next examined the mechanism responsible for the activation of porphyrin synthesis upon starvation for heme. The main activation occurred at the step that leads to the synthesis of 5-aminolevulinic acid (ALA). Starvation for heme induced the expression of a hemA-lacZ fusion gene, as previously reported, but an activation pathway that is independent of the hemA promoter was also identified. We found that starvation for heme caused the stringent response, and such starvation promoted the synthesis of porphyrins without having any effect on the expression of the hemA-lacZ fusion gene. We suggest a model for the regulation of porphyrin synthesis whereby the synthesis of porphyrins is coordinated with that of proteins.

Control of the availability of exogenous 5-aminolevulinic acid in Escherichia coli.

We observed novel phenomena on the growth of a hemA deletion mutant (H500) of Escherichia coli: First, H500 cells grown in rich medium required an increased amount of exogenous 5-aminolevulinic acid (ALA) for normal growth, as compared with cells grown in poor medium. Second, H500 cells grown with an insufficient supply of ALA had a prolonged lag phase but relatively normal growth during the logarithmic phase. An increased requirement of exogenous ALA in rich media was due to an impermeability of the cells to ALA, resulting in a reduced ability to synthesize porphyrins. The latter could be also explained by change in permeability that was repressed under heme-deficient conditions.

Differential expression of two hemA mRNAs encoding glutamyl-tRNA reductase proteins in greening cucumber seedlings.

The first committed step of porphyrin synthesis in higher plants is the reduction of glutamyl-tRNA to glutamate 1-semialdehyde. This reaction is catalyzed by glutamyl-tRNA reductase, which is encoded by hemA genes. Two hemA cDNA clones (hemA1 and hemA2) were obtained from cucumber (Cucumis sativus) cotyledons by the PCR and cDNA library screening. They showed significant homology with published hemA sequences. Southern blot analysis of cucumber genomic DNA revealed that these genes are located at different loci and that there is another gene similar to the hemA genes. Accumulation of hemA1 mRNA was detected primarily in cotyledons and hypocotyls of greening cucumber seedlings, whereas that of hemA2 mRNA was detected in all tissues examined. Illumination of cucumber seedlings increased markedly the accumulation of hemA1 mRNA, but it did not induce remarkable changes in that of hemA2 mRNA. These findings suggest that hemA1 mRNA was accumulated in response to the demand of Chl synthesis in photosynthesizing tissues, whereas hemA2 mRNA was expressed in response to the demand of the synthesis of porphyrins other than chlorophylls.

Two hydrophobic subunits are essential for the heme b ligation and functional assembly of complex II (succinate-ubiquinone oxidoreductase) from Escherichia coli.

Complex II (succinate-ubiquinone oxidoreductase) from Escherichia coli is composed of four nonidentical subunits encoded by the sdhCDAB operon. Gene products of sdhC and sdhD are small hydrophobic subunits that anchor the hydrophilic catalytic subunits (flavoprotein and iron-sulfur protein) to the cytoplasmic membrane and are believed to be the components of cytochrome b556 in E. coli complex II. In the present study, to elucidate the role of two hydrophobic subunits in the heme b ligation and functional assembly of complex II, plasmids carrying portions of the sdh gene were constructed and introduced into E. coli MK3, which lacks succinate dehydrogenase and fumarate reductase activities. The expression of polypeptides with molecular masses of about 19 and 17 kDa was observed when sdhC and sdhD were introduced into MK3, respectively, indicating that sdhC encodes the large subunit (cybL) and sdhD the small subunit (cybS) of cytochrome b556. An increase in cytochrome b content was found in the membrane when sdhD was introduced, while the cytochrome b content did not change when sdhC was introduced. However, the cytochrome b expressed by the plasmid carrying sdhD differed from cytochrome b556 in its CO reactivity and red shift of the alpha absorption peak to 557.5 nm at 77 K. Neither hydrophobic subunit was able to bind the catalytic portion to the membrane, and only succinate dehydrogenase activity, not succinate-ubiquinone oxidoreductase activity, was found in the cytoplasmic fractions of the cells. In contrast, significantly higher amounts of cytochrome b556 were expressed in the membrane when sdhC and sdhD genes were both present, and the catalytic portion was found to be localized in the membrane with succinate-ubiquitnone oxidoreductase and succinate oxidase activities. These results strongly suggest that both hydrophobic subunits are required for heme insertion into cytochrome b556 and are essential for the functional assembly of E. coli complex II in the membrane. Accumulation of the catalytic portion in the cytoplasm was found when sdhCDAB was introduced into a heme synthesis mutant, suggesting the importance of heme in the assembly of E. coli complex II.

Partial inhibition of protein synthesis accelerates the synthesis of porphyrin in heme-deficient mutants of Escherichia coli.

Mutants of Escherichia coli defective in the HemA protein grow extremely poorly as the result of heme deficiency. A novel hemA mutant was identified whose rate of growth was dramatically enhanced by addition to the medium of low concentrations of translational inhibitors, such as chloramphenicol and tetracycline. This mutant (H110) carries mutation at position 314 in the hemA gene, which resulted in diminished activity of the encoded protein. Restoration of growth of H110 upon addition of the drugs mentioned above was due to activation of the synthesis of porphyrin. However, this activation was not characteristic exclusively of cells with this mutant hemA gene since it was also observed in a heme-deficient strain bearing the wild-type hemA gene. The activation did not depend on the promoter activity of the hemA gene, as indicated by studies with fusion genes. It appears that partial inhibition of protein synthesis via inhibition of peptidyltransferase can promote the synthesis of porphyrin by providing an increased supply of glutamyl-tRNA for porphyrin synthesis. Glutamyl-tRNA is the common substrate for peptidyltransferase and HemA.

Cloning and sequencing of a previously unidentified gene that is involved in the biosynthesis of heme in Escherichia coli.

We have isolated a number of porphyrin (Por)-synthesis mutants as light-resistant revertants of a light-sensitive strain delta visA (hemH) of Escherichia coli that accumulates protoPor IX in the cell. Among such mutants, we found a double mutant (H103) with mutations in hemA and in a new gene downstream of hemA. This new gene, designated hemK, was located at 27 min on the linkage map of the E. coli chromosome. By nucleotide (nt) sequencing, it was demonstrated that hemK forms part of the hemA-prfA-hemK operon and encodes 225 amino acids that show no significant homology to any protein in the standard databases. The mutant strain H103 formed small colonies and showed no catalase activity even in the presence of 5-aminolevulinic acid (ALA), indicating its inability to catalyze a step in the biosynthesis of heme from ALA. An extract of H103 cells has readily detectable ALA dehydratase and porphobilinogen deaminase activities. H103 cells carrying a plasmid that included only hemA as an insert accumulated protoPor and coproPor, but showed no sensitivity to light, a result that suggests that it may be deficient in protoporphyrinogen oxidase activity.

Cloning and identification of the hemG gene encoding protoporphyrinogen oxidase (PPO) of Escherichia coli K-12.

Cells of the VSR751 strain, which was previously isolated as a photoresistant revertant of the visA-deleted (hemH-deleted) strain of Escherichia coli K-12, accumulated uroporphyrin (uro), coproporphyrin (copro) and protoporphyrin IX (proto), but did not accumulate as much protoporphyrin as cells of the parental strain (hemH-deleted). Therefore, we concluded that strain VSR751 must be defective in protoporphyrinogen oxidase (PPO), the product of the hemG gene. By complementation analysis using VSR751, we isolated and identified this gene. The hemG gene is located at 86 mim on the E. coli chromosome, just upstream of the rrnA operon, and is transcribed clockwise in the same direction as the rrnA operon. This gene encodes a 181-amino acid protein with a calculated molecular mass of about 21 kDa. Sequence analysis revealed the presence of flavodoxin motif, suggesting tha a cofactor of this enzyme is flavin mononucleotide, which is consistent with the previous report that the mammalian PPO had the flavin cofactor.

Cloning and sequencing of the hemE gene encoding uroporphyrinogen III decarboxylase (UPD) from Escherichia coli K-12.

Among the photoresistant revertants of the visA-deleted (hemH-deleted) strain of Escherichia coli K-12, three mutants defective in the hemE gene encoding uroporphyrinogen III decarboxylase (UPD) were identified. Using one of the mutants, we cloned and sequenced the hemE of E. coli. We found an open reading frame of 353 codons, which encoded a predicted amino acid (aa) sequence that exhibited a high degree of homology over its entire length to the aa sequence of UPD from humans and other organisms. This hemE was located at 90.3 min near the hupA gene on the linkage map of the E. coli chromosome.