Taro Iizumi

Constitutive trichloroethylene degradation led by tac promoter chromosomally integrated upstream of phenol hydroxylase genes of Ralstonia sp. KN1 and its nucleotide sequence analysis

Ralstonia sp. KN1-10A is a strain capable of degrading trichloroethylene (TCE) constitutively due to the tac promoter (Ptac) integrated upstream of the phenol hydroxylase genes (phy) in its chromosome. The expression of Ptac was analyzed using luxAB of Vibrio harveyi as a reporter. After determining the nucleotide sequence of phyABCDE required for TCE degradation, a luxAB-encoding fragment was integrated downstream of phyE by homologous recombination in strain KN1-10A, obtaining strain KN1-10A-LX. In the same manner, the luxAB-encoding fragment was integrated into the chromosome of the wild-type strain, KN1. The resultant strain KN1-LX was used to analyze the gene expression caused by phenol induction. The expression induced by Ptac was compared to that by phenol induction. Although the level of luxAB expression led by Ptac was almost equal to that induced by phenol, the TCE degradation rate by the Ptac-carrying KN1-10A-LX was markedly slower than that by the phenol-induced KN1-LX. These results suggest that an important gene for TCE degradation was not transcribed by Ptac in KN1-10A-LX. The nucleotide sequence analysis showed the existence of a small gene, phyZ, upstream of phyA, and Ptac was found to be integrated into the middle of phyZ in KN1-10A-LX. The effect of phyZ on TCE degradation was examined by using recombinant strains expressing phyABCDE with or without phyZ in a plasmid. The coexistence of phyZ markedly accelerated TCE degradation. Through an exhaustive expression analysis, it was demonstrated that the chromosomal integration of Ptac was a very attractive method for high and stable production of phenol hydroxylase for TCE degradation.

Cloning and sequence analysis of an esterase gene from Pseudomonas sp. KWI-56.

The gene encoding an esterase from Pseudomonas sp. KWI-56 was cloned and sequenced. The nucleotide sequence contained an open reading frame encoding a polypeptide comprising 262 amino acids, whose molecular weight agreed well with the value obtained by SDS-PAGE. Comparison of the amino acid sequence with those of the other homologous enzymes suggested that Ser-92 and His-24l might be included in the catalytic triad.

Hyperproduction of Thermostable Lipase by Genetically Engineered Pseudomonas Species

One Pseudomonas species was isolated, and named KWI-56, which produced an extracellular thermostable lipase. More than 90% of the enzyme activity remained after 24 hours’ heat treatment at 60 °C [1].

Regulatory analysis of the Nitrosomonas europaea grpE-dnaK-dnaJ operon

The complete nucleotide sequences of the Nitrosomonas europaea grpE and dnaJ genes were determined. Transcriptional analysis showed that grpE was transcribed as polycistronic transcripts with the dnaK and dnaJ from a sigma(32)-dependent heat-inducible promoter located upstream of grpE. This promoter had significantly less activity than one located upstream of dnaK.