Search

Ketocarotenoids are high-value natural pigments. The red diketocarotenoid astaxanthin particularly exhibits an extraordinary antioxidant activity, which raises its market demand for foods and nutraceuticals. We screened for ketocarotenoid-producing bacteria from both marine and freshwater environments. Phylogenetic analysis, based on 16S rRNA gene sequence, revealed 37 potential producers of ketocarotenoids that are related to α-proteobacteria, comprising 32 strains of Brevundimonas and 5 strains of Erythrobacter. Carotenoids analysis by HPLC-DAD and HPLC-MS revealed two groups; astaxanthin-producers (28 Brevundimonas strains) and adonixanthin-producers (Five Brevundimonas and 5 Erythrobacter strains). Strain FrW-Asx16 exhibited the highest carotenoid production (1060 µg g−1 dry cells with 16.6% astaxanthin). Strain FrW-Asx-5 producing 946.1 µg g−1 dry cells carotenoid exhibited the highest astaxanthin content (∼46%). The most intriguing result is the potential of producing natural colorants from freshwater bacterial isolates, and with high productivity and selectivity, suggesting a great promise for their application in food.

The conservon (cvn) of Streptomyces species encodes a putative membrane-associated signaling complex resembling the eukaryotic G-protein-coupled receptor (GPCR) system. The cvn is widely distributed in the genomes of Actinobacteria, indicating that it plays an important role in this group of bacterial species; however, the exact role of this regulatory system is hitherto poorly understood. In the present study, we generated null mutants for all 13 copies of the cvn operon distributed in the genome of Streptomyces coelicolor A3(2) and observed that the aerial mycelium formation and antibiotic production in a cvn1 mutant were markedly impaired. The cvn1 mutant formed aerial mycelium and produced actinorhodin and undecylprodigiosin at remarkably low levels on solid medium containing 1-2% glucose and at high levels on medium containing 6-10% glucose. The same phenotype as this was observed with a cvnA1 mutant. Transcriptional analyses revealed that the expression of sigU encoding a vegetative sigma factor was upregulated in the cvn1 mutant. Overexpression of rsuA encoding the σ(SigU) antagonist restored aerial mycelium formation and pigment production in the cvn1 mutant, suggesting that the developmental defect in the cvn1 mutant is based on the high expression level of sigU.

An ORF encoding the gene (nahA)-related carbohydrate hydrolase was found in chromosomal DNA of the symbiotic thermophile, Symbiobacterium thermophilum. BLASTX results indicated that the product of this structural gene is β-glucosidase or β-N-acetylhexosaminidase. To investigate details of the nahA gene product, cloning of the gene, overproduction of the gene product in Escherichia coli, and purification and characterization of the resulting protein were conducted. The nahA gene was amplified by PCR using fragmented chromosomal DNA of S. thermophilum as a template, sequenced, and then ligated into the BamHI and NdeI sites of plasmid pET-25b(+) to construct the expression vector pST-BNAH-A for overproduction of the gene product. Results of studies on the hydrolytic activity of cell-free extracts against pNP-β-Glc, pNP-β-GlcNAc and pNP-β-GalNAc, obtained by disruption of cultured E. coli cells harboring pST-BNAH-A, suggested that the nahA gene product was β-N-acetylhexosaminidase. Comparison of the amino acid sequence of the recombinant protein with those of other β-N-acetylhexosaminidases indicated that the β-N-acetylhexosaminidase of S. thermophilum is a member of the 3-glycoside hydrolase family. The recombinant enzyme was purified to homogeneity from cell-free extract in an overall yield of 24%. This purified β-N-acetylhexosaminidase possessed thermostability, was stable in alkaline solution, and exhibited greater hydrolytic activity against chitin oligosaccharides than against pNP-β-GlcNAc.

Apoptosis is morphologically related to premature mitosis, an aberrant form of mitosis. Staurosporine, a potent protein kinase inhibitor, induces not only apoptotic cell death in a wide variety of mammalian cells but also premature initiation of mitosis in hamster cells that are arrested in S phase by DNA synthesis inhibitors. Here we report on the biochemical differences between the two phenomena commonly caused by staurosporine. Rat 3Y1 fibroblasts that had been arrested in S phase with hydroxyurea underwent apoptosis by treatment with staurosporine, whereas S-phase-arrested CHO cells initiated mitosis prematurely when similarly treated with a low concentration of staurosporine. Chromosome condensation occurred in both apoptosis (3Y1) and premature mitosis (CHO). However, neither formation of mitotic spindles nor mitosis-specific phosphorylation of MPM-2 antigens was observed in apoptosis of 3Y1 cells, unlike premature mitosis of CHO cells. The p34cdc2kinase activated in normal and prematurely mitotic cells remained inactive in the apoptotic cells, probably because the active cyclin B/p34cdc2complex was almost absent in the S-phase-arrested 3Y1 cells. The absence of intracellular activation of p34cdc2in apoptosis was confirmed by immunohistochemical analyses using a specific antibody raised against Ser55-phosphorylated vimentin which is specifically phosphorylated by p34cdc2during M phase. Furthermore, phosphorylation of histones H1 and H3, which is associated with mitotic chromosome condensation, did not occur in the apoptotic cells. These results indicate that the two phenomena, staurosporine-induced apoptosis and premature mitosis, are different in their requirement for p34cdc2kinase activation and histone phosphorylation.

A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) is essential for aerial mycelium formation and streptomycin (Sm) production in Streptomyces griseus . A protein Ser/Thr kinase (AfsK), the product of the Streptomyces coelicolor A3(2) afsK gene, controlling secondary metabolism in this strain, reversed the aerial mycelium-negative phenotype of an A-factor-deficient mutant strain, S. griseus HH1, and induced sporulation without affecting A-factor productivity or Sm production. A mutant AfsK protein lacking kinase activity failed to induce aerial mycelium formation which indicates the importance of the kinase activity for suppression in S. griseus . These data suggest that a Ser/Thr kinase functionally similar to S. coelicolor A3(2) AfsK plays a regulatory role in aerial mycelium formation in S. griseus , either as a member in the A-factor regulatory network or independently of this network

Evidence is accumulating that demonstrates the key roles played by diffusible molecules in regulating cellular differentiation, even among prokaryotic microorganisms. This is exemplified by A-factor and its analogues, which act as autoregulators for morphological differentiation and secondary metabolism in Streptomyces. The identification of a specific receptor for A-factor and an A-factor-controlled promoter sequence in S. griseus indicate the close similarity of this system to eukaryotic hormonal control. The involvement of prokaryotic homologues of the eukaryotic Ser/Thr-kinases in the regulation of differentiation processes seems to be another characteristic feature of this group of bacteria. Recent evidence for the presence of these molecular signalling systems in Streptomyces is reviewed, along with the inherent implications.

A gene named sksl+was cloned as a suppressor of the K-252a-sensitivity phenotype of Schizosaccharomyces pombe (Sp) from a gene library of the parental Sp chromosomal DNA constructed with a multicopy vector pDB248'. The gene encoded a 308-amino-acid (aa) protein similar to the Caenorhabditis elegans F37A4.5 gene product and to the mouse and Drosophila Mov34 gene products. The sksl+ null mutants obtained by gene disruption were non-viable, indicating that sksl+ is essential for vegetative growth. The parental Sp strain carrying multiple copies of sksl+ showed distinct cross-resistance to staurosporine, thiabendazole and vanadate in addition to K-252a, although Sksl has no similarity in aa sequence to those of ATP-binding cassette (ABC)-type transporters. The multicopy plasmid containing sksl+conferred multidrug resistance (MDR), even in a mutant cell defective in pmdl+ encoding an ABC-type transporter. It is therefore unlikely that the function of pmdl+is involved in MDR conferred by sksl+. These results suggest that sksl+is a functionally novel MDR gene.

The serine protease (SSP) from a Gram-negative organism, Serratia marcescens, is excreted even from Escherichia coli. The protease is synthesized as a 1045-amino-acid precursor (preproSSP) composed of three functional portions, a typical NH2-terminal signal peptide of 27 amino acids, the mature protease part of 618 amino acids, and a large COOH-terminal part of 400 amino acids. After the mature part (Ala28 to Asp645) has been liberated into the medium, most of the COOH-terminal part (Phe717 to Phe1045) remains stably in the outer membrane. When a mutated gene encoding the prepro-SSP with deletion of the junction region (Ser646 to Gly716) between the mature protease and the processed COOH-terminal protein was expressed in E. coli, the SSP protein was not detected in any fractions of the cells nor in the medium, whereas a processed COOH-terminal protein was found in the outer membrane. However, when the outer membrane fraction prepared from the transformant exhibiting the junction region (Ser646 to Gly716) probably on the cell surface was co-cultured with the transformant, SSP was found in the medium. Furthermore, a significant portion of the SSP protein denatured with guanidine hydrochloride was correctly refolded in vitro into the active protease, only in the presence of the outer membrane preparation from the transformant exhibiting the junction region. These results suggest that the junction region is exposed outside the cells, and it plays a role for guiding the folding of SSP in the correct conformation.

Radicicol, an inhibitor of src or src-like kinases, causes hypophosphorylation and down-regulation of the RB retinoblastoma tumor suppressor protein in HL-60 human promyelocytic leukemia cells. Both of these changes in the RB protein typically are associated with myeloid or monocytic differentiation of these cells induced by retinoic acid or 1,25-dihydroxy vitamin D3. When added with either inducer, radicicol caused the typically induced myeloid or monocytic differentiation of these cells to be accelerated. By itself radicicol caused a transient inhibition of G1 to S transit, but did not cause phenotypic conversion. The down-regulation and dephosphorylation of RB by radicicol may thus facilitate cell differentiation.

The gene encoding an enzyme that catalyzes the hydroxylation at position 25 of vitamin D-3 was cloned from an actinomycete strain, Amycolata autotrophica, by use of a host-vector system of Streptomyces lividans. The amino acid sequence deduced from the nucleotide sequence revealed that this enzyme, tentatively named P-450VD25, contains several regions of strong similarity with amino acid sequences of cytochromes P-450 from a variety of organisms, primarily in the regions of an oxygen-binding site and a heme ligand pocket. Especially, P-450VD25 shows end-to-end similarity in amino acid sequence to P-450dNIR of Fusarium oxysporum and P-450SU2 of Streptomyces griseolus. The recombinant S. lividans strain containing the P-450VD25 gene on a multicopy plasmid converted vitamin D-3 in the medium into 25-hydroxyvitamin D-3 at a maximum yield of 10%.

Two macrolide-resistance determinants were cloned from a mycinamicin (Mm)-producing Micromonospora griseorubida strain in Streptomyces lividans and Streptomyces parvulus. One of the cloned genes, designated myrA, was cloned as a gene which conferred strong resistance to Mm and tyiosin (Ty), but not to erythromycin (Er) or josamycin (Jm) on S. lividans. Another gene, named myrB, was cloned as an ErRencoding gene which conferred MLS resistance (to macrolide, lincosamide and streptogramine B antibiotics) on S. parvulus. Both myrA and myrB were sequenced and the corresponding ORFs were determined. The deduced amino acid (aa) sequence of myrA showed no similarity to proteins in the available databases, suggesting that an unknown mechanism of macrolide resistance is exerted by the MyrA protein. The deduced aa sequence of myrB exhibited high similarity to 23S rRNA methyltransferases (MTases), such as ErmE and CarB, from a variety of microorganisms.

A DNA fragment of 42 kb encompassing one of the mycinamicin II (Mm)-resistance-encoding genes, myrB, from a Mm-producing strain, Micromonospora griseorubida, was cloned in Escherichia coli using the cosmid vector pJB8. Nucleotide sequencing of the neighboring region of myrB and a computer-aided analysis of the sequence predicted the presence of an open reading frame (ORF) with 254 amino acids which showed great similarity to the macrocin O-methyltransferase (tylF gene product) in tylosin (Ty)-producing Streptomyces fradiae. When a 1.0-kb AluI fragment containing the complete ORF was fused to the lacZ promoter in the correct orientation and expressed in E. coli, a mycinamicin III (MIII) O-methyltransferase (MOMT) activity was detected only upon induction with isopropyl-beta-D-thiogalactopyranoside (IPTG). All these data indicate that this ORF codes for the structural gene of MOMT, and it is designated mycF.

Mutagenesis of a fungus Mucor pusillus, a producer of an aspartic proteinase named Mucor pusillus rennin (MPR), was performed to obtain the mutated enzymes with decreased thermostability, which is desirable for practical use of the enzyme as a milk coagulant for cheese manufacturing. A fungal mutant strain which produced the mutant enzyme with distinctly reduced thermostability was isolated. Two different mutant alleles of the mpr gene, one with a single amino acid exchange of Ala101 for Thr and the other of Gly186 for Asp, were cloned out of this mutant strain. The mutated mpr genes were expressed in Saccharomyces cerevisiae under the control of the yeast GAL7 promoter to produce the active enzymes in extracellular medium. Both of the mutations, especially Gly186Asp, were confirmed to cause a marked decrease in thermostability of the enzyme. All mutants possessing exchanges of Gly186 for various amino acids by site-directed mutagenesis showed a decrease in thermostability, indicating involvement of this residue to maintain a conformation of the enzyme. A double mutant having the both exchanges, Ala101Thr and Gly186Asp, in a single molecule showed the lowest thermostability without decrease in the enzymatic activity as well as the relative ratio of clotting to proteolytic activity.

An ethanol-sensitive mutant strain of Acetobacter pasteurianus was isolated by chemical mutagenesis with N -methyl- N ′-nitro- N -nitrosoguanidine. The his1 gene of A. pasteurianus , which codes for histidinol phosphate aminotransferase (HPAT), was cloned on a multicopy plasmid pMV24 because of its ability to complement the ethanol-sensitive mutation. The mutant was found still to possess almost the same HPAT activity as the parental strain, which was increased to five times that of the basal activity by the introduction of a multicopy plasmid containing the his1 gene into the mutant. It was thus concluded that an increase in the HPAT activity led to the suppression of the ethanol-sensitive mutation.

Acetic acid resistance, which characterizes the genus Acetobacter , is conferred by three genes designated aarA , aarB , and raarC . The nucleotide sequence of aarC revealed that it coded for a protein of 492 amino acid residues. This gene product showed significant homology, with 44% identity, with the Neurospora crassa acu-8 gene product which is involved in acetate-utilization in this fungus. Consistent with this, an aarC -defective mutant, which was constructed by gene disruption with the cloned gene, showed an inability to assimilate acetic acid. Introduction of the aarC gene into the mutant restored both the resistance to acetic acid and the ability to assimilate acetic acid. These findings suggested that the aarC gene product was responsible for acetate assimilation and that the acetate-sensitive phenotype of the aarC mutant was caused by a defect in acetate assimilation.

Random mutagenesis of malate dehydrogenase from a thermophilic bacterium, Thermus flavus AT-62, had revealed that a Thr190----Ile replacement near the essential catalytic residue His187 caused marked modulation of the catalytic properties. For further exploration of a role of the residue at this position, this residue was substituted with each of the other amino acids by site-directed mutagenesis. Most of the mutations except for substitution with Ser caused increases in Km for oxaloacetate and increases Ki for oxaloacetate of 2-110 times. Substitution with His or Pro was characterized by the complete loss of substrate inhibition, along with a marked increase in Km for oxaloacetate. Kinetic analyses of the native and altered malate dehydrogenases at various pHs revealed that both Km and Ki for oxaloacetate decreased proportionally to the decrease in pH from 8.40 to 5.75, whereas kcat was nearly constant within the pH range. Apparent shifts of the optimum pH values toward acidity observed with most of the altered malate dehydrogenases were attributed to the increase in Ki, which facilitated the release from the substrate inhibition at a lower pH. Replacement of Thr310, a possible counterpart with which Thr190 forms a hydrogen bond, by Ile caused changes in the catalytic properties similar to those of the Thr190-substituted enzymes. These results suggest that not only the loss of the hydrogen bond between Thr190 and Thr310 but also properties of the residues introduced at position 190 cause modulation of the catalytic properties, probably through dislocation of the loop structure that contains the catalytic residue His187.

For investigation of an unknown open reading frame which is present upstream of the nitrile hydratase (NHase) gene from Rhodococcus sp. N-774, a longer DNA fragment covering the entire gene was cloned in Escherichia coli. Nucleotide sequencing and detailed subcloning experiments predicted a single open reading frame consisting of 521 amino acid residues of Mr 54,671. The amino acid sequence, especially its NH2-terminal portion, showed significant homology with those of indoleacetamide hydrolases from Pseudomonas savastanoi and Agrobacterium tumefaciens, and acetamidase from Aspergillus nidulans. The 521-amino acid coding region was therefore expressed by use of the E. coli lac promoter in E. coli, and was found to direct a considerable amidase activity. This amidase hydrolyzed propionamide efficiently, and also hydrolyzed, at a lower efficiency, acetamide, acrylamide and indoleacetamide. These data clearly show that the unknown open reading frame present upstream of the NHase coding region encodes an amidase. Because the TAG translational stop codon of the amidase is located only 75 base pairs apart from the ATG start codon of the alpha-subunit of NHase, these genes are probably translated in a polycistronic manner.

The amino acid sequences of cellulase from Bacillus subtilis (BSC) and that from an alkalophilic Bacillus sp. N-4 (NK1) show significant homology in most parts except for the C-terminal portions. Despite the high homology, the pH activity profiles of the two enzymes are quite different; BSC has its optimum pH at 6-6.5, whereas NK1 is active over a broad pH range from 6 to 10.5. In order to identify the structural features which determine such pH activity profiles, chimeric cellulases between BSC and NK1 were constructed using four restriction sites commonly present within the homologous coding sequences, and were produced in Escherichia coli. The chimeric cellulases showed various chromatographic behaviors, reflecting the origins of their C-terminal regions. The pH activity profiles of the chimeric enzymes in the alkaline range could be classified into either the BSC or NK1 type mainly depending on the origins of the fifth C-terminal regions. In the acidic range, the profile was determined only by the origin of the fourth enzyme region from the N terminus. Comparison of the kinetic parameters between pH 5 and 6 using p-nitrophenyl cellobioside as a substrate indicated that the fourth region is responsible for the pH-dependent change of the kcat value. Only a limited number of amino acids in the fourth region may affect on deprotonation of catalytic residues of the cellulases and modulate the catalytic activity in the acidic pH values.

The effect of staurosporine, a potent microbial inhibitor of protein kinases, on the cell cycle of cultured fibroblast cells was investigated. A low concentration of staurosporine (1–10 ng/ml) blocked the cell cycle of rat 3Y1 fibroblasts at the early G1 phase within 2 h after serum stimulation. On the other hand, a higher concentration of the drug (100 ng/ml) caused the specific G2 block. Both of these blocks were reversible. After release from the G2 block, highly synchronous transition to M phase was observed and both nuclear and cell divisions were completed within 180 min. This reversible G2 block showed a clear contrast to those by the other G2 arresters, trichostatin A and leptomycin B, which formed proliferative tetraploid cells after release by entering the cells into a new S phase without passage through M phase. The presence of trichostatin A or leptomycin B did not interfere with this synchronous progression through G2 M phases, suggesting that the arrest point of staurosporine was present in late G2 phase following those of trichostatin A and leptomycin B.

(R)-Trichostatin A (TSA) is a Streptomyces product which causes the induction of Friend cell differentiation and specific inhibition of the cell cycle of normal rat fibroblasts in the G1 and G2 phases at the very low concentrations. We found that TSA caused an accumulation of acetylated histone species in a variety of mammalian cell lines. Pulse-labeling experiments indicated that TSA markedly prolonged the in vivo half-life of the labile acetyl groups on histones in mouse mammary gland tumor cells, FM3A. The partially purified histone deacetylase from wild-type FM3A cells was effectively inhibited by TSA in a noncompetitive manner with Ki = 3.4 nM. A newly isolated mutant cell line of FM3A resistant to TSA did not show the accumulation of the acetylated histones in the presence of a higher concentration of TSA. The histone deacetylase preparation from the mutant showed decreased sensitivity to TSA (Ki = 31 nM, noncompetitive). These results clearly indicate that TSA is a potent and specific inhibitor of the histone deacetylase and that the in vivo effect of TSA on cell proliferation and differentiation can be attributed to the inhibition of the enzyme.

An antifungal antibiotic, leptomycin B (LMB), which induced cell elongation of fission yeast, Schizosaccharomyces pombe, was found to be a unique inhibitor of the cell cycle of mammalian and fission yeast cells. Proliferation of rat 3Y1 fibroblasts was reversibly blocked by LMB in both the G1 and G2 phases and the treated cells were presumably introduced into the resting state (GO). After removal of LMB, proliferative tetraploid cells were produced from the cells which had been arrested by LMB at the G2 phase, as a result of DNA replication without passage through the M phase. LMB also inhibited the proliferation of S. pombe in both the G1 and G2 phases. These results suggest that the molecular target of LMB is one of the components necessary for progression of both G1 and G2 in the eukaryotic cell cycle.

The tnaT gene of Symbiobacterium thermophilum encodes a protein homologous to sodium-dependent neurotransmitter transporters. Expression of the tnaT gene product in Escherichia coli conferred the ability to accumulate tryptophan from the medium and the ability to grow on tryptophan as a sole source of carbon. Transport was Na(+)-dependent and highly selective. The K(m) for tryptophan was approximately 145 nm, and tryptophan transport was unchanged in the presence of 100 microM concentrations of other amino acids. Tryptamine and serotonin were weak inhibitors with K(I) values of 200 and 440 microM, respectively. By using a T7 promoter-based system, TnaT with an N-terminal His(6) tag was expressed at high levels in the membrane and was purified to near-homogeneity in high yield.

A gene encoding an endo-type semi-alkaline cellulase was cloned from an alkalophilic Streptomyces strain in Streptomyces lividans, and its nucleotide sequence was determined. Downstream from the transcriptional start point, which was determined by high-resolution S1 mapping, an open reading frame of 388 amino acids (aa) was present. The N-terminal amino acid sequence of the mature enzyme determined by an Edman degradation procedure suggested that the cellulase had an extraordinarily long leader sequence of about 70 aa. Comparison with the leader sequences of endoglucosidase H from Streptomyces plicatus and the cellulase from Cellulomonas fimi suggested that the semi-alkaline cellulase was processed in two steps during maturation.

A Streptomyces bikiniensis DNA fragment complementing a deficiency in streptomycin (Sm) production was cloned on the plasmid vector pIJ385. Host strain S. bikiniensis SD1 used as the recipient in cloning displayed deficiency in biosynthesis of N -methyl- l -glucosamine, one of the moieties of Sm. The cloned fragment on the multicopy plasmid pIJ385 conferred sevenfold increase in Sm production in comparison with the wild-type parental strain. By subcloning, the region complementing the Sm deficiency of SD1 was narrowed to a 3.0-kb fragment.

K. oxytoca is a nitrogen-fixing bacterium living in the rhizosphere of rice plants. A strain with derepressed nitrogen fixing ability was constructed by insertion of a tetracycline-resistant (Tcr) gene into the coding sequence of the repressor gene (nifL) and another insertion of a synthetic promoter upstream from the activator gene (nifA) on the chromosome of K. oxytoca. The strain, R16, showed constitutive transcriptional activity of not only nifA gene but also nifB (iron-molybdenum cofactor) gene, and nitrogenase activity was derepressed to a level of 42% of that detected in the ammonia-less condition even in the presence of ammonia. R16, carrying a multicopy plasmid containing the constitutive nifA, acquired complete derepression from the control by ammonia and showed a high nitrogenase activity constitutively.

Chymosin, also known as rennin, a milk-clotting enzyme obtained from the stomach of calves, is used in the manufacture of cheese. The production of this enzyme by recombinant DNA technology is now becoming possible. A new source of this enzyme to replace or supplement the animal product or similar, naturally occurring fungal enzymes will be of great economic value.

Calf prochymosin produced in Escherichia coli cells harboring the expression plasmids was insoluble and formed large inclusion bodies, which were solubilized by 8 M urea. The conditions allowing correct refolding of denatured prochymosin were investigated. Dialysis at pH 10 in the presence of 500 mM NaCl was found to give the maximum renaturation, and subsequent acidic treatment for autocatalytic processing of refolded prochymosin allowed almost 100% recovery of chymosin.

Chemical modification with diethylpyrocarbonate (ethoxyformic anhydride) was examined to demonstrate the existence of an essential histidine residue at the NADPH-binding site of p-hydroxybenzoate hydroxylase (EC 1.14.13.2) from Pseudomonas desmolytica. Among some ligands, NADPH was noticeable in protecting the enzyme from the modification-caused inactivation. Although several amino acid residues were modified during the inactivation process, inhibition of the enzyme could be correlated with modification of a single histidine residue which was masked by addition of NADPH. The pK of the essential histidine residue was estimated to be 6.5-6.7. The Kd (Km) for NADPH of the inactivated enzyme was shown to have been increased greatly, although the Kd for substrate (p-hydroxybenzoate) was not changed.

To increase yields of calf prochymosin (PC) produced in Escherichia coli , PC cDNA was cloned in a plasmid vector under control of the trp promoter. The hybrid plasmid pCRSOl constructed for this purpose contains cDNA coding for PC (from the 5th Arg to the C-terminal Ile) fused to the N-terminal fragment of the trpE gene preceded by the trp promoter and attenuator region. E. coli C600 harboring this plasmid produces approx. 300000 molecules of PC per cell. This is about a tenfold increase above the amount obtained using lac UV5 promoter [Nishimori et al., Gene 19 (1982) 337-344]. A similar plasmid, pCR601, which contains the same coding sequence fused to the trp promoter and N-terminal fragment of the trpL gene, directs the production of PC at the same rate as pCR501. In pCR601 the trp attenuator is deleted. Another plasmid, pCR701, in which construction of a sequence coding for f Met-PC cDNA that was aided by chemical synthesis, was placed under direct control of the trp promoter, produced PC at a much lower rate. Extracts prepared from all these bacterial transformants in the presence of urea showed distinct milk-clotting activity after renaturation and processing.

The structures of fosfazinomycins A and B, phosphorus containing antibiotics, have been established by spectral evidence and degradation studies.

p-Hydroxybenzoate hydroxylase (EC 1.14.13.2) was rapidly inactivated by treatment with phenylglyoxal, by a process obeying pseudo-first order kinetics. The reaction with the reagent was also examined by amino acid analyses, radioactivity measurements, and spectrophotometric analyses. Results of these analyses were consistent with each other, which shows that the inactivation was due to modification of argiine residue(s). Addition of saturating amounts of p-hydroxybenzoate (or benzoate) during the treatment resulted in marked protection of the enzyme from the inactivation as well as a significant decrease in modification of arginine residues, while phenol showed no effect. Modification in the absence of p-hydroxybenzoate caused a spectral change in the flavin moiety of the enzyme similar to that due to the enzyme.substrate complex formation, and losses in both the overall activity and the substrate-binding ability accompanied the spectral change. On the other hand, such spectral change was not observed and the substrate-binding ability was retained even after the overall activity had decreased to a great extent when p-hydroxybenzoate as added during the modification treatment. These results suggest that phenylglyoxal (an analogue of the substrate) was incorporatd into the substrate-binding site and that an arginine residue is involved in the site, having an interaction with the carboxylate anion of the substrate.

The electron paramagnetic resonance(EPR) signals of Fusarium lipoxygenase were measured at liquid nitrogen temperature in the presence or absence of substrate, linoleic acid. The spin-state exchange of heme iron in Fusarium lipoxygenase from a low to high spin-state by the addition of linoleic acid was observed. The addition of linoleic acid to the enzyme at pH 9.0 gave rise to the appearance of EPR lines at g=5.92 and 3.58, while at pH 12.0, lines at g=6.12 and 3.41 were newly appeared. At the same time, the resonance at g=4.31 was increased both at pH 9.0 and 12.0 in the presence of linoleic acid.

Proliferation of rat 3Y1 cells was found to be specifically blocked by trichostatin A (TSA) at two distinct stages in the cell cycle. The first block occurred in the early G1 phase at least 9 h before the G 1 S boundary, whereas the second occurred during the G2 phase. When TSA-arrested cells at the G2 pahse were released from the inhibition, the cells with 4C DNA entered a new S phase without passage through the M phase, resulting in the formation of proliferative tetraploid cells. The removal of TSA induced a rapid transient increase in the transcription of c-fos and the cells required 15 h to enter the S phase after release. These results suggest that the cells arrested with TSA are quiescent (G0).

The mode of action of bacteriophage-induced lytic enzyme “LE95” was investigated. The LE95 hydrolyzed peptide portion in peptidoglycan of Ps. aeruginosa and E. coli . The exposed amino terminal amino acid was identified as glutamic acid by analysis of terminal amino acid by dinitrophenylation. This result suggested the LE95 hydrolyzed the peptide bond between L-alanine and D-glutamic acid in the peptidoglycan of Ps. aeruginosa and E. coli . The enzyme did not hydrolyze various peptides prepared from bacterial cell wall. This experimental result suggested that the glycan chain of peptidoglycan would be essential for the enzymic activity.

The structural changes in the cell membrane of λ-lysogenic Escherichia coli induced by colicin E2 were examined. The addition of colicin E2 made the cells susceptible to various detergents and the transport rate of o-nitrophenyl-β-D-galactoside into the colicin-treated cells was stimulated markedly by adding a low concentration of sodium dodecyl sulfate. The fluorescence intensity of 8-anilino-1-naphthalenesulfonate bound to the cells was markedly increased by adding colicin E2. Colicin E2 stimulated the incorporation of 32 P from prelabeled phosphatidylglycerol to cardiolipin. All these changes probably suggesting the structural alteration of the cell membrane were dependent on the presence of the rex gene of λ prophage in the cells.

The mechanism of alloisocitric acid fermentation by the fungus, Penicillium purpurogenum var. rubrisclerotium No. 1148, was studied using C 14 O 2 and citric acid-1,5-C 14 . By the aerobic incubation with glucose in the presence of C 14 O 2 , C 14 O 2 was incorporated into carbons 1 and 6 of alloisocitric acid in equal amounts. The pathway was assumed including the carboxylation of the C 3 compound to the C 4 -dicarboxylic acid, and the condensation of C 2 with the C 4 -dicarboxylic acid, which was randomized completely through the dicarboxylic acid shuttle. Isocitritase type condensation of C 4 with C 2 , Thunberg reaction, and the glyoxylic acid bypath seemed unlikely to be operative. The incorporation experiments of citric acid-1,5-C 14 showed that the labeling patterns of the residual citric acid and alloisocitric acid formed were completely the same. It suggested the presence of some direct conversion pathway from citric acid to alloisocitric acid without splitting of the carbon skeleton. From these with some other data, it was presumed that alloisocitric acid might be formed immediately from citric acid which was recycling through the tricarboxylic acid cycle. The presence of citric condensing enzyme and a few other enzymes of the tricarboxylic acid cycle was shown.

The mode of action of colicin E2 on ribosomes in Escherichia coli cells was investigated by zonal centrifugation analysis. Ribosome particles, both 50S and 30S, were degraded to smaller contents with the lapse of time by the action of colicin E2. Gradual reduction of S values of each particles could not be observed and degradative intermediates of possible RNA-protein complex were detected only at the position between 30S and 4S in the zonal centrifugation profile, which indicated the destruction of ribosome in burst-out attitude. 50S ribosome fraction influenced by colicin E2 contained both 23S and half-sized RNA. From these data, the mode of action of colicin E2 on ribosomes in E. coli was discussed.

1. Experiments were performed to observe the biochemical action of colicin E2 in the subcellular system, and purified colicin E2 was found to release DNA from the isolated DNA-membrane complex of Escherichia coli in the presence of ATP. 5 to 10 μg/ml of colicin E2 was sufficient to induce 50 % release. 2. No DNA degradation was observed during the release of DNA from the membrane complex by colicin E2. 3. The reaction was highly dependent on temperature and no reaction occurred below the critical temperature of 20°C. 4. ATP could be replaced by several nucleotide triphosphates, ADP and inorganic pyrophosphate. 5. No resistant and tolerant mutants to colicin E2 had been found whose DNA-membrane complexes showed resistance to the reaction of colicin E2in vitro. 6. Purified colicin E3 and K proteins did not release DNA from the complex, suggesting the presence of a highly specific interaction between colicin E2 protein and the membrane complex.

Crystals of the green, copper-containing enzyme nitrite reductase from Alcaligenes faecalis S-6 have been obtained. They are monoclinic, space group P2(1) with a = 78.0 A, b = 92.9 A, c = 78.5 A and beta = 111.8 degrees. The crystals diffract to better than 2 A and are suitable for high-resolution X-ray crystallography.