MAYA AKIMOTO
Study on the gene expression of a persistent organophosphorus flame retardant-degrading enzyme in Sphingobium sp. TCM1
Shouji TAKAHASHI, Yoshio KERA, Katsumasa ABE
The flame retardant, tris(2-chloroethyl) phosphate (TCEP), is recently regarded as a potentially toxic and persistent environmental contaminant. We have previously isolated a TCEP-degrading bacterium, Sphingobium sp. strain TCM1, and further identified a TCEP-degrading enzyme (haloalkylphosphorus hydrolase, HAD) in this bacterium. HAD activity in the bacterial cells, however, decreased in a medium containing a high concentration of inorganic phosphate (Pi).
The genes whose expression is regulated by environmental Pi concentration belong to a gene family Pho regulon. Seven protein factors, PstSCAB and PhoURB, are involved in the regulation in bacteria. In E. coli, pstSCAB gene mutation leads to constitutive expression of Pho regulon. Therefore, if had gene belongs to Pho regulon, it would be possible to create a strain whose HAD activity is not repressed at a high Pi concentration. In this study, we aimed to show that had gene belongs to Pho regulon and to create a strain constitutively expressing Pho regulon.
I first tried to obtain a Pho regulon constitutive expression mutant using glycerol-2-phosphate as the sole carbon source in the presence of a high concentration of Pi. I obtained some strains that can grow on the medium, but they did not show constitutive expression of alkaline phosphatase, a member of Pho regulon.
I next examined to generate a pst gene deletion and a phoB gene mutants to create a strain constitutively expressing had gene and to confirm had gene belongs to Pho regulon, respectively. I obtained a pst gene partial deletion mutant but not the phoB mutant. I now characterize the pst gene mutants.
I also carried out had gene promoter deletion assay to identify Pi response region. This analysis showed that the 39-bp region between -37 and -75 bp upstream of had gene plays a major role in both the expression and the Pi regulation. In this region, we found a Pho box-like sequence.
To analyze the binding of PhoB to the Pho box-like sequence, we purified His-tagged TCM1 PhoB protein expressed in E. coli and confirmed its autophosphorylation ability. The EMSA analysis using the purified PhoB and an oligonucleotide having the Pho box-like sequence showed no PhoB binding to the oligonucleotide. However, it is necessary to optimize the binding conditions, because the binding is affected by various factors.
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