Kikuo Sen

Anaerobic growth and potential for amino acid production by nitrate respiration in Corynebacterium glutamicum

Oxygen limitation is a crucial problem in amino acid fermentation by Corynebacterium glutamicum. Toward this subject, our study was initiated by analysis of the oxygen-requiring properties of C. glutamicum, generally regarded as a strict aerobe. This organism formed colonies on agar plates up to relatively low oxygen concentrations (0.5% O2), while no visible colonies were formed in the absence of O2. However, in the presence of nitrate (

Identification of a second transcriptional start site for the insecticidal protein gene cryIVA of Bacillus thuringiensis subsp. israelensis

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Analysis of the sexual development-promoting region of Schizophyllum commune TRP1 gene

), the organism exhibited limited growth anaerobically with production of nitrite (

Expression of the two 130-kDa protein genes of Bacillus thuringiensis var. israelensis in Bacillus subtilis

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Effects of Bacillus thuringiensis var. israelensis 20-kDa protein on production of the Bti 130-kDa crystal protein in Escherichia coli.

), indicating that C. glutamicum can use nitrate as a final electron acceptor. Assays of cell extracts from aerobic and hypoxic cultures yielded comparable nitrate reductase activities, irrespective of nitrate levels. Genome analysis revealed a narK2GHJI cluster potentially relevant to nitrate reductase and transport. Disruptions of narG and narJ abolished the nitrate-dependent anaerobic growth with the loss of nitrate reductase activity. Disruption of the putative nitrate/nitrite antiporter gene narK2 did not affect the enzyme activity but impaired the anaerobic growth. These indicate that this locus is responsible for nitrate respiration. Agar piece assays using l-lysine- and l-arginine-producing strains showed that production of both amino acids occurred anaerobically by nitrate respiration, indicating the potential of C. glutamicum for anaerobic amino acid production.

Cloning and Nucleotide Sequences of the Two 130kDa Insecticidal Protein Genes of Bacillus thuringiensis var. israelensis

The nucleotide sequence of the cry11A gene from Bacillus thuringiensis subsp. israelensis strain HD522 was analyzed and the molecular characterization of Cry11A toxin was done. The 70-kDa Cry11A protoxin was processed in vitro into 36- and 32-kDa fragments by trypsin and into 34- and 32-kDa fragments by gut proteases from C. pipiens. These two processed fragments are associated together to form the heterodimer. The results of the binding assay with BBMV and the bioassay toward C. pipiens larvae suggested that the heterodimer was biologically as active as the non-digested Cry11A toxin and the intramolecular cleavage did not promote the insecticidal activity. These results suggested that a probable complex of the 36- or 34-kDa and 32-kDa fragments was also one of the possible active forms of Cry11A, and that the biological functions of Cry11A was not essentially affected by the intramolecular cleavage of the 70-kDa protein.