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.     

Effect of Cathepsin D Treatment on ATPase Activity of Rabbit Myofibril

The effect of cathepsin D and pepsin treatment on rabbit myofibril was studied by measuring the amount of proteolytic products and Mg-enhanced ATPase activity.

When myofibril was treated with cathepsin D at 3°C and pH 5.0 or 5.5, a little but detectable amount of nonprotein nitrogenous compounds was released. However, there was no change in ATPase activity of myofibril, though treated with cathepsin D of higher units than assumed to be in muscle.

When myofibril was treated with pepsin under the same condition as used above, there was an increase in KCl-concentration dependence of ATPase activity followed by a decrease in the maximal value of ATPase activity.

From the present results, it was concluded that cathepsin D might not take a main role on the post-mortem degradation of myofibril.     

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.     

A New Method for Estimation of the Mycelial Weight in Koji

A New method was devised for the estimation of the mycelial weight in rice-koji. In this method, the content of glucosamine in koji was used for the calculation of mycelial weight. The content of glucosamine in the mycelia of Aspergillus oryzae, koji, and rice was determined by a colorimetry after hydrolysis of these materials with sulfuric acid and purification of glucosamine fraction with a Dowex 50 W column. And the values of glucosamine were 114.5 mg/g in mycelia, 3.03 in the koji for amazake,* 1.34 in the koji for sake, and 0.0 in rice. The mycelial contents calculated from these data were 2.6% dry weight in amazake-koji and 1.2% in sake-koji.     

Distribution of Nuclease O Inhibitor among Aspergillus Species

Leucine dehydrogenase was inhibited by p-chioromercuribenzoate and HgCl₂, but not by 5,5′-dithiobis(2-nitrobenzoic acid), 4,4′-dithiopyridine and N-ethylmaleimide. Modification of sulfhydryl groups of the enzyme with p-chloromercuribenzoate and HgCl₂ was accompanied with a loss of the enzyme activity. The 6 reactive sulfhydryl groups per enzyme molecule play an essential role for catalysis. Approximately 12 sulfhydryl groups were titrated per molecule in the presence of 8 m urea: the enzyme contains 2 sulfhydryl groups per subunit, and one of them participates in the catalytic action. Fluorometric and gel filtration studies on binding of NADH to the enzyme revealed that the enzyme contains 6 coenzyme binding sites per molecule.

These results are compatible with the hexameric structure of leucine dehydrogenase composed of identical subunits, showing that each subunit has one catalytic site and one indispensable sulfhydryl group.     

Properties of Nucleoside Phosphorylase from Enterobacter aerogenes

The properties of uridine Phosphorylase (UPase) and purine nucleoside Phosphorylase (PNPase) at high temperature were investigated. Both enzymes were found to be distributed in a wide range of bacteria and were partially purified from Enterobacter aerogenes AJ 11125 by heat treatment, ammonium sulfate fractionation and column chromatographies onDEAE-cellulose and Sephadex G-150. The UPase was purified 109-fold, and it showed an optimum pH of 8.5 and optimum temperature of 65°C, and activity toward uridine, 2′-deoxyuridine, thymidine and uracil arabinoside but not cytidine. The Km values of UPase for uridine were 0.7 mm at 40°C and 1.8 mm at 60°C. The PNPase was purified 83-fold, and it showed an optimum pH of 6.8 and optimum temperature of 60°C, and significant activity toward purine arabinosides as well as purine ribosides. The Km values of PNPase for inosine were 0.8 mm at 40°C and 2.2 mm at 60°C.     

Studies on Proteolysis in Stored Muscle

Some physicochemical properties of the cathepsin D purified from the rabbit muscle (L. dorsi) were investigated.

The sedimentation coefficient (s_20,w) and the molecular weight determined from sedimentation equilibrium experiment was 3.83 S and 29,000~30,000, respectively.

The amino acid composition of the enzyme was determined with an automatic amino acid analyzer.

The proteolytic specificity of the enzyme was also investigated using the B-chain of oxidized beef insulin as the substrate. The cathepsin D cleaved the bonds Phe-Val, Ala-Leu, Leu-Tyr and Tyr-Leu. The specificity of the cathepsin D was fairly similar to that of the pepsin.     

Some Properties of Neutral Proteolytic System in Rabbit Skeletal Muscle

The properties of the neutral proteolytic activity concentrated in a fraction (F–1) separated from rabbit muscle homogenate were examined by measuring the effects of various reagents and metal ions, the time course of the proteolysis and Ca-stability. The obtained results have indicated that F–1 contains two types of neutral protease active on proteins, tentatively named Protease I and II, The former, which is activated by Ca²⁺ and Ca-labile, shows an explosive production of Cu-Folin phenol reagent positive materials at the early stage of incubation. The latter, which is Ca-stable, shows a large production of ninhydrin positive materials throughout the incubation time. The proteolysis by F–1 was similar to the autolysis of muscle homogenate in all the properties examined. Therefore, Proteases I and II were assumed to be main enzymes responsible for the muscle proteolysis at the neutral pH region. As there has been no factor denying their functioning in living muscle, it is probable that Proteases I and II take important parts in the muscle catabolism.     

De novo sequence analysis of cytochrome P450 1–3 genes expressed in ostrich liver with highest expression of CYP2G19

The cytochrome P450 (CYP) 1–3 families are involved in xenobiotic metabolism, and are expressed primarily in the liver. Ostriches (Struthio camelus) are members of Palaeognathae with the earliest divergence from other bird lineages. An understanding of genes coding for ostrich xenobiotic metabolizing enzyme contributes to knowledge regarding the xenobiotic metabolisms of other Palaeognathae birds. We investigated CYP1–3 genes expressed in female ostrich liver using a next-generation sequencer. We detected 10 CYP genes: CYP1A5, CYP2C23, CYP2C45, CYP2D49, CYP2G19, CYP2W2, CYP2AC1, CYP2AC2, CYP2AF1, and CYP3A37. We compared the gene expression levels of CYP1A5, CYP2C23, CYP2C45, CYP2D49, CYP2G19, CYP2AF1, and CYP3A37 in ostrich liver and determined that CYP2G19 exhibited the highest expression level. The mRNA expression level of CYP2G19 was approximately 2–10 times higher than those of other CYP genes. The other CYP genes displayed similar expression levels. Our results suggest that CYP2G19, which has not been a focus of previous bird studies, has an important role in ostrich xenobiotic metabolism.