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Y Kurusu

Publications and source records attributed to Y Kurusu.

27 records · Page 2Linked to original sources
PubMed1993

Identification and sequence determination of the acetohydroxy acid isomeroreductase gene from Brevibacterium flavum MJ233.

The enzyme acetohydroxy acid isomeroreductase (AHAIR) is the second enzyme in the parallel isoleucine-valine biosynthetic pathway. We previously reported the cloning and sequencing of the acetohydroxy acid synthase (AHAS) genes from Brevibacterium flavum MJ233. Analysis of the sequence downstream of the AHAS genes identified another open reading frame highly homologous at the amino acid level to the AHAIR gene from Escherichia coli (ilvC). We subcloned the B. flavum AHAIR gene on a 2.1 kb Bg/II-EcoRI fragment by complementation of an E. coli ilvC mutant. The nucleotide sequence of the B. flavum AHAIR gene consists of 338 codons (molecular weight of 36158). Comparison of the deduced protein sequence revealed a high degree of identity with the sequences of ilvC genes from other organisms. Disruption of the B. flavum ilvC gene by a kanamycin resistance cassette resulted in L-isoleucine and L-valine auxotrophy.

Alcohol Oxidoreductases↗
PubMed1993

Cloning and sequence determination of the acetohydroxy acid synthase genes from Brevibacterium flavum MJ233 by using the polymerase chain reaction.

Taking advantage of highly conserved domains present in the three acetohydroxy acid synthase (AHAS) isozymes from E. coli K-12, we designed degenerate oligonucleotides corresponding to these regions. These synthetic DNA sequences were used as primers in polymerase chain reactions in order to amplify DNA sequences from Brevibacterium flavum MJ233 chromosomal DNA. The polymerase chain reaction product was used as a probe to recover genomic fragments from a lambda library of B. flavum MJ233. A 5.8-kb EcoRI fragment hybridizing to the probe was isolated and amplification of this fragment in a B. flavum strain resulted in increased AHAS-specific activity. Sequence analysis revealed two open reading frames (ilvL and ilvS) highly homologous at the amino acid level to the corresponding domains of the three AHAS isozymes of E. coli K-12. Moreover, disruption of the putative ilvL gene by a kanamycin resistance cassette resulted in L-isoleucine and L-valine auxotrophy. These observations demonstrate that the cloned fragment encodes the AHAS gene of B. flavum MJ233.

Acetolactate Synthase↗
PubMed1991

Application of the tryptophanase promoter to high expression of the tryptophan synthase gene in Escherichia coli.

The application of an inducible regulation system using the tryptophanase operon promoter (TPase promoter; Ptna) was examined for its high expression of the tryptophan synthase (TS) gene in Escherichia coli. The main problem in the application of Ptna for industrial purposes is catabolite repression by glucose, since glucose is the most abundant carbon source. However, this problem could be avoided by changing glucose to an organic acid, such as succinate, fumarate, malate and acetate, in the course of cultivation after glucose initially added was completely consumed. Under these conditions, L-tryptophan was also used to induce tryptophan synthase. Thus, the specific activity of TS in E. coli strain no. 168 harbouring pBR322F-Ptna TS was increased 500-fold compared to that of the cultured host strain. About 1 mol L-tryptophan/l reaction mixture was formed from indole and L-serine at 37 degrees C for 3.5 h.

Carbon↗
PubMed1991

Identification of plasmid partition function in coryneform bacteria.

We have identified and characterized a partition function that is required for stable maintenance of plasmids in the coryneform bacteria Brevibacterium flavum MJ233 and Corynebacterium glutamicum ATCC 31831. This function is localized to a HindIII-NspV fragment (673 bp) adjacent to the replication region of the plasmid, named pBY503, from Brevibacterium stationis IFO 12144. The function was independent of copy number control and was not associated directly with plasmid replication functions. This fragment was able to stabilize the unstable plasmids in cis but not in trans.

Base Sequence↗
PubMed1990

Heat-inducible translational coupling in Bacillus subtilis.

Bacillus subtilis plasmid pGR71 is a promoter-probe shuttle vector derived from pUB110. The expression of the cat gene on pGR71 in B. subtilis requires the insertion of a Bacillus promoter and a ribosomal binding site (RBS) into the HindIII cloning site immediately upstream from the cat gene. A recombinant plasmid of pGR71, named pGR71-369, was obtained by a spontaneous deletion of a fragment containing most of the inserted HindIII fragment and the replication origin necessary for multiplication in Escherichia coli. The expression of the cat gene in B. subtilis cells carrying this plasmid was inducible by heat. Nucleotide sequence analysis of the upstream region of the cat gene, deletion analysis, and dot blot hybridization analysis of mRNA in various conditions revealed that the cat gene was expressed by heat-inducible translational coupling and that the regulatory region of heat inducibility was present in the upstream region of the cat gene.

Bacillus subtilis↗
PubMed1990

Electroporation-transformation system for coryneform bacteria by auxotrophic complementation.

We evaluated electroporation as an alternative system for genetic exchange for one of the coryneform bacteria, Brevibacterium flavum MJ233. The maximum number of transformants, 6 x 10(4) cells, was obtained when cells were cultured with Penicillin G (1 U/ml) and harvested at the middle-log phase. Electroporation was done using 12.5 kV/cm of pulse field strength, 1 x 10(10) cells, and 1 microgram of plasmid DNA. Other coryneform bacteria, Brevibacterium lactofermentum ATCC 13869, Corynebacterium glutamicum ATCC 31830, and B. stationis IFO 12144 were also transformed by electroporation. Electroporation has the advantage that intact cells can be used as host cells without the need for protoplast formation and regeneration. Moreover, minimal medium can be used, so auxotrophic complementation of the transformants is possible.

Brevibacterium↗
PubMedDate not supplied

Electrotransformation of intact cells of Brevibacterium flavum MJ-233.

Electroporation allowed transformation of intact cells of Brevibacterium flavum MJ-233. The two plasmids used for electroporation were pCRY2 (6.3 kilobase) and pCRY3 (8.2 kilobases). Both plasmids contain the chloramphenicol-resistance gene and the autonomous replication origin in B. flavum MJ-233. The efficiency of electrotransformation was optimal with cells harvested at the middle log phase of growth, and was improved by the addition of 1.0 U/ml of penicillin G to the culture medium. The optimum yield of transformants per micrograms DNA was 5 x 10(4) when the cell suspension was pulsed at a cell density of 1 x 10(10)/ml and at a DNA amount of 1.0 micrograms.

Brevibacterium↗
PubMedDate not supplied

Presence of mrr- and mcr-like restriction systems in coryneform bacteria.

Efficient transformation of Brevibacterium flavum MJ233C and Corynebacterium glutamicum ATCC 31831 (up to 5.0 x 10(7) transformants/microgram DNA) depends on the source of plasmid DNA. The transformation efficiencies of B. flavum MJ233C and C. glutamicum ATCC 31831 increased nearly 10(3)-fold when plasmid DNA was isolated from the recipient strain itself or from a damdcm Escherichia coli mutant, as compared with DNA passed through a modification-proficient E. coli strain. These results suggest the presence of a methyl-specific restriction system in certain strains of coryneform bacteria. In addition, electroporation conditions were optimized.

Brevibacterium↗