A New Amino Acid Antibiotic KT–151, Produced by Streptomyces luteogriseus,Strain No. KT–151

From the results of taxonomic studies, Streptomyces sp. strain No. KT–151 isolated from a soil sample collected in Kumamoto City, was identified as a strain belonging to Streptomyces luteogriseus Schmitz, Deak, Crook and Hooper 1964. A new antibiotic, produced by this strain, was isolated as a leaflet crystal by ion-exchange chromatography and found to be an amino acid with the molecular formula, C₅H₁₂N₂O₂, and named antibiotic KT–151 (refered to as KT–151 hereinafter). The antibiotic showed antimicrobial activity against various Gram-positive and Gram-negative bacteria in a chemically defined medium but it was antagonized by several amino acids such as valine, leucine, isoleucine and threonine.     

Rapid Purification and Crystallization of Thermostable Malate Dehydrogenase Isolated from a Thermophilic Bacterium,Thermus flavus AT 62

A structural study of the water-soluble dextran made by Leuconostoc mesenteroides strain C (NRRL B-1298) was conducted by enzymic degradation and subsequent ¹³C-NMR analysis of the native dextran and its limit dextrins. The α-l,2-debranching enzyme removed almost all of the branched D-glucose residues, and gave a limit dextrin having a much longer sequence of the internal chain length (degree of linearity: n = 24.5 compared with the value of n = 3.3 for the native dextran). The degree of hydrolysis with debranching enzyme corresponded to the content of α-1,2-linkages determined by chemical methods, which suggested that most of the α-l,2-linkages in the dextran B-1298 constituted branch points of a single D-glucose residue. A synergistic increase of susceptibility of the dextran B-1299 was observed by simultaneous use of debranching enzyme and endodex-tranase. ¹³C-NMR spectral analysis indicated the similarity of structure of dextran B-1298 to that of B-1396, rather than that of B-1299. Occurrence of α-l,3-linkages in the limit dextrin was supported by a newly visualized chemical shift at 83.7 ppm.     

Benzoic Acid Derivatives Conjugated with Dicarboxylic Acids from Alfalfa (Medicago saliva)

Two compounds were isolated from alfalfa. Their structures were determined as benzoyl meso-tartaric acid (I) and benzoyl (S), (–)-malic acid (II) respectively. They were found in nature for the first time.     

Construction of New Shuttle Vectors for Acetbacter

Shuttle vector pMV301 was constructed by ligation of pMV102 found in A. aceti subsp. xylinum NBI 1002 to E. coli plasmid pACYC177. It is 6.0 kb in size, has unique restriction sites suitable for insertion of a foreign DNA fragment and confers ampicillin resistance to the Acetobacter host. This vector transforms A. aceti subsp. aceti 10-80S1 and industrial vinegar producer A. aceti subsp. xylinum NBI 1002 as well as E. coli. Various chimeric plasmids were also constructed by ligation of pMV102 to E. coli plasmids to examine the expression of drug resistance genes. In addition to the ampicillin resistance gene, resistance genes for kanamycin, chloramphenicol and tetracycline derived from E. coli plasmids were expressed in Acetobacter. Most of the constructed shuttle vectors were stably maintained in Acetobacter.     

Improved Transformation Method for Acetobacter with Plasmid DNA

An improved method for transformation of derivative strains of A. aceti subsp. aceti No. 1023 with plasmid DNA has been developed. Addition of polyethylene glycol or dimethylsulfoxide increased the transformation efficiency by a factor of about ten. In the presence of PEG 4,000, various transformation conditions were examined. Cells were also made transformation competent by treatment with other divalent cations than Ca²⁺ . The pH of the buffer did not affect the efficiency significantly. The growth phase influenced the efficiency. Mutants showing high competence were derived by treatment with N-methyl-N′-nitro-N-nitrosoguanidine. By the improved method using the highly transformable mutants, a transformation efficiency of approximately 105 transformants per γg plasmid DNA was achieved.     

Crystallization and Properties of N-Benzoylglycine Amidohydrolase from Pseudomonas putida

N-Benzoylgiycine amidohydrolase (hippurate hydrolase EC 3.5.1.32), which catalyzes the hydrolysis of hippuric acid to benzoic acid and glycine, was found in a cell-free extract of Pseudomonas putida C692-3 grown on a medium containing hippuric acid. The enzyme was purified from the extract by ammonium sulfate fractionation and column chromatographies on DEAE-cellulose, DEAE-Sephadex A-50, hydroxyapatite, and Sepharose CL-6B. The enzyme was finally crystallized. The crystalline enzyme was almost homogeneous on electrophoresis. The enzyme had a molecular weight of about 170,000 and consisted of four subunits identical in molecular weight (approximately 42,000). The enzyme hydrolyzed N-benzoylglycine most rapidly, and N-benzoyl-l-alanine and N-benzoyl-l-aminobutyric acid. The Km value for these substrates were 0.72 mm, 0.87 mm, and 0.87mm, respectively. The optimum pH of the enzyme reaction was 7.0 to 8.0 and the enzyme was stable from pH 6.0 to 8.0.     

Biological Activity and Mode of Action of a Specific Antiphage Substance,AFS

Purified AFS (anti-filamentous phage substance) produced by Streptomyces lavendulae AM–7a showed specific antiphage activity against the male specific, deoxyribonucleic acid-containing filamentous phages of Escherichia coli without any activity against other DNA-phages nor the male-specific ribonucleic acid-containing phages of E. coli. AFS brought about no inactivation of free particles of filamentous phage, fl, nor the receptor of the host cells for the phage, while it showed strong killing effect against the fl-infected host cells at the concentration below 0.01 μg/ml. Antiphage activity of AFS might be due to its highly specific killing effect only on the E. coli cells infected with the filamentous DNA phages, while it exerted no effect on the growth of the unifected E. coli nor other microorganisms. Killing by AFS seemed to require the energy metabolism of the phage-infected host cells. Macro-molecular synthesis and respiration of the infected host cells were inhibited soon after the addition of small amounts of AFS without any cell lysis.     

italic> of the Whole nif Genes of Klebsiella oxytoca,a Nitrogen Fixer in the Rhizosphere of Rice

We cloned in E. coli the whole 17 nif genes (nifQ-J) of Klebsiella oxytoca NG13 using pBR322 as a vector, and constructed a recombinant plasmid, pNOW25 (nif⁺, Ap^r, 42.6 kb). A non nif DNA fragment was deleted from the plasmid with XhoI, and a smaller plasmid, pNOK31 (nif+, Ap^r, 31.1 kb), was reconstructed.

We constructed the restriction map of the cloned nif genes. The map was the same as that of the K. pneumoniae M5a1 nif genes as to the EcoRI, HindIII, BamHI and XhoI sites, but differed considerably in the PstI, SalI and BglII sites.

E. coli KO60 containing pNOW25 or pNOK31 can grow on a N-free medium. The acetylene reduction activities of KO60 (pNOW25) and KO60 (pNOK31) were 280 nmol and 390 nmol/48 hr per 7 ml of N-free liquid medium, whereas the activity of K. oxytoca NG13 was 3800 nmol. Thus, the expressed activity of the nif system of K. oxytoca is rather low in E. coli even if the nif genes are cloned on a multicopy plasmid.     

Usefulness of Glucokinase from Bacillus stearothermophilus for the Enzymatic Measurement of Magnesium in Human Serum

(22R,23R,24S)-3α,5-Cyclo-22,23-diacetoxy-5a-ergostan-6-one (2b) is a new key intermediate of some naturally occurring brassinosteroids such as brassinolide (la), castasterone (lb), teasterone (lc) and typhasterol (Id). The cycloketone 2b was prepared in 10 steps via (22R,23R,24S)-6p- benzyloxy-3a,5-cyclo-22,23-dihydroxy-5a-ergostane (5) from stigmasterol. 2b was treated with a catalytic amount of /7-toluenesulfonic acid and sodium bromide to give an enone (7b), which was oxidized with osmium tetroxide and derived to give a 2a,3a-acetonide (8b). 8b was easily separated from its isomer by the use of silica gel column chromatography. 8b was oxidized with tri- fluoroperacetic acid and deacetylated to give la. 8b was deacetylated and deacetonized to give lb. 2b was treated with dilute sulfuric acid in acetic acid to give a 3/^-acetate (10). 10 was treated with sodium hydroxide to give lc. 2b was treated with hydrobromic acid to give a 3/i-bromide (12), which was treated with silver acetate to give a 3a-acetate (13). 13 was treated with sodium hydroxide to give Id.     

The Synthesis of Dimethyl Esters of dl-O,O′-Dimethylfukiic Acid and dl-O,O′-Dimethylepifukiic Acid

The synthesis of dimethyl esters of dl-O,O′-dimethylfukiic acid (11) and dl-O,O′-dimethylepifukiic acid (12) are described.