2002 Fiscal Year Final Research Report Summary
Studies on the transcriptional regulatory system for sensing of environmental pollutants
| Project/Area Number |
12460043
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | Nagaoka University of Technology |
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Principal Investigator |
FUKUDA Masao Nagaoka University of Technology, Bioengineering, professor, 工学部, 教授 (20134512)
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| Co-Investigator(Kenkyū-buntansha) |
MASAI Eiji Nagaoka University of Technology, Bioengineering, associate professor, 工学部, 助教授 (20272867)
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| Project Period (FY) |
2000 – 2002
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| Keywords | environmental pollution / sensor / polychlorinated biphenyl / transcriptional regulation / two component system / biphenyl / Rhodococcus |
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| Research Abstract |
We characterized the gene regulation system by BphS1/BphT1 and BphS2/T2, which respond to the aromatic compounds and activate the transcription of biphenyl degradation genes in Rhodococcus sp. strain RHA1.
1. The substrate specificities of BphS1 and BphS2, which have high similarities each other, were investigated. Both BphSs responded to a wide range of aromatic compounds. The substrate specificities of them were alike, except for the case of biphenyl. Towards biphenyl, only BphS1 responded. The construction and analysis of BphS1-BphS2 hybrid sensor kinase revealed that the N terminus region of BphS (to amino acid number 405) is responsible for the response towards biphenyl.
2. The signal transduction system between BphS and BphT were investigated. Mutants of both BphSs were constructed by the substitution of His 1411 to Arg. His 1411 is a amino acid residue which corresponds to the conserved His which act as phosporylation site among many sensor kinases. Both mutants were expressed as soluble proteins, but lost their activities, which showed their importance in the phospho-relay to the BphTs. We also observed the cross-talk between BphS1 and BphT2, and BphS2 and BphT1, respectively.
3. The upregulation activity of BphSTs towards various promoters of biphenyl degradation genes (etbA1, ebdA1, etbD1, and bphA4-2) were investigated. The results showed that all the promoters mentioned above were regulated by BphST, and this suggested that these promoters have consensus sequences for BphT binding. The transcriptional start points of these promoters were determined by the primer extension analysis, and the results revealed a consensus motif that exists around -35 of each transcriptional start point. The overexpression and the purification of BphT protein were also performed by using E. coli as a host.
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