Keiko Kohama

Genomic organization of the biotin biosynthetic genes of coryneform bacteria: Cloning and sequencing of the bioA-bioD genes from Brevibacterium flavum

Three coryneform bacteria, Brevibacterium flavum, Brevibacterium lactofermentum and Corynebacterium glutamicum have been shown to be able to convert 7-keto-8-aminopelargonic acid to biotin through a biotin synthetic pathway identical to that from Escherichia coli (Hatakeyama et al., DNA Sequence, in press, 1993). We report in this paper the cloning and sequencing of the biotin biosynthetic genes encoding the 7,8-diaminopelargonic acid aminotransferase (bioA) and the dethiobiotin synthetase (bioD) of B. flavum MJ233, by complementation of E. coli bioA and bioD mutants. Both bioA and bioD genes from B. flavum were located on a 4.0-kb SalI DNA fragment. Nucleotide sequence analysis of this fragment revealed that these genes consist of a 1272 bp and a 675 bp open reading frame, respectively. The deduced amino acid sequence of the 7,8-diaminopelargonic acid aminotransferase (BioA) is 51.3% and 31.9% identical to that of the E. coli and Bacillus spaericus bioA gene products, respectively. The deduced amino acid sequence of the dethiobiotin synthetase (BioD) is 25.9% and 32.7% identical to that of the E. coli and B. sphaericus bioD gene products, respectively. In addition, the genomic organization of the bioA, bioB and bioD genes in B. flavum has been shown to be different from that in E. coli and B. sphaericus.

Electrotransformation of intact cells of Brevibacterium flavum MJ-233

SummaryElectroporation allowed transformation of intact cells ofBrevibacterium flavum MJ-233. The two plasmids used for electroporation were pCRY2 (6.3 kilobases) and pCRY3 (8.2 kilobases). Both plasmids contain the chloramphenicol-resistance gene and the autonomous replication origin inB. flavum MJ-233. The efficiency of electrotransformation was optimal with cells harvested at the middle log phase of growth, and was imporved by the addition of 1.0U/ml of penicillin G to the culture medium. The optimum yield of transformants per μg DNA was 5×104 when the cell suspension was pulsed at a cell density of 1×1010/ml and at a DNA amount of 1.0μg.

Analysis of the biotin biosynthesis pathway in coryneform bacteria: Cloning and sequencing of the bioB gene from Brevibacterium flavum

The biotin biosynthetic pathway of three coryneform bacteria, Brevibacterium flavum, Brevibacterium lactofermentum, and Corynebacterium glutamicum were analysed by cross-feeding experiments using several Escherichia coli biotin-requiring mutants. The three strains of coryneform bacteria tested were able to convert 7-keto-8-aminopelargonic acid to biotin, through a biotin synthetic pathway identical to that from E. coli. The biotin biosynthetic gene, bioB, of B. flavum was cloned by phenotypic complementation of E. coli bioB mutants. The bioB gene was located on a 1.7 kb HindIII-SacI DNA fragment. Nucleotide sequence analysis of this fragment revealed that the bioB gene of B. flavum consists of a 1005 bp open reading frame. Its deduced amino acid sequence is 35.7% and 31.5% identical to that of the E. coli and Bacillus sphaericus bioB gene products, respectively. B. flavum mutants obtained by in vivo disruption of the bioB gene lost their ability to grow on minimal medium containing dethiobiotin, indicating that the bioB gene product is necessary for the conversion of dethiobiotin to biotin.

Cloning and Nucleotide Sequencing of The Poly(3-hydroxybutyrate) Depolymerase Gene From Pseudomonas pickettii

SUMMARY An extracellular poly (3-hydroxybutyrate) depolymerase (PHB depolymerase) producing bacterium, strain K1, was isolated from soil samples and identified as Pseudomonas pickettii. The PHB depolymerase gene was cloned from the genomic DNA of the strain K1 and the nucleotide sequence was determined. The 1960 bp Eco RI-Sma I DNA fragment containing the PHB depolymerase gene had an open reading frame of 1467 bp and the deduced amino acid sequence of this open reading frame was 492 residues long with a signal peptide of 30 amino acids.

Cloning and nucleotide sequencing of the tyrosine phenol lyase gene fromEscherichia intermedia

SummaryThe tyrosine phenol lyase (TPL) gene was cloned from the genomic DNA of aEscherichia intermedia strain and the nucleotide sequence of the TPL structural gene was determined. The 1801 bpHincll-Nrul DNA fragment containing the TPL gene had an open reading frame of 1368 bp and the deduced amino acid sequence was 456 residues long with a molecular weight of 51,441 daltons.