Yoko Asai

Transposon mutagenesis of coryneform bacteria

The Corynebacterium glutamicum insertion sequence IS31831 was used to construct two artificial transposons: Tn31831 and miniTn31831. The transposition vectors were based on a gram-negative replication origin and do not replicate in coryneform bacteria. Strain Brevibacterium flavum MJ233C was mutagenized by miniTn31831 at an efficiency of 4.3 x 104 mutants per microgram DNA. Transposon insertions occurred at different locations on the chromosome and produced a variety of mutants. Auxotrophs could be recovered at a frequency of approximately 0.2%. Transposition of IS31831 derivatives led not only to simple insertion, but also to cointegrate formation (5%). No multiple insertions were observed. Chromosomal loci of B. flavum corresponding to auxotrophic and pigmentation mutants could be rescued in Escherichia coli, demonstrating that these transposable elements are useful genetic tools for studying the biology of coryneform bacteria.

Cloning and sequencing of thesecY homolog from Coryneform bacteria

A conserved domain of the secY genes from Bacillus subtilis, Mycoplasma capricolum and Escherichia coli was used to design degenerate oligodeoxyribonucleotides. These synthetic DNA sequences were used to screen a lambda library of Brevibacterium flavum MJ233. A 1.5-kb KpnI fragment of a recombinant lambda phage containing the secY homology from Br. flavum MJ233 was subsequently subcloned into plasmid pUC118. The complete nucleotide (nt) sequence of the cloned fragment indicated that the deduced gene product of the Br. flavum secY homolog is composed of 440 amino acids (aa) with a deduced M(r) of 47,871. Comparison of this aa sequence to the corresponding sequences from E. coli and B. subtilis revealed a high degree of conservation, and suggested that the Br. flavum secY homolog is a membrane protein containing ten transmembrane segments. In addition, we could identify, downstream from secY, a putative coding sequence of the enzyme adenylate kinase. This gene organization is identical to that observed in the B. subtilis genome.