Use of a site‐specific recombination‐based biosensor for detecting bioavailable toluene and related compounds on roots

Wiley - Tập 5 Số 4 - Trang 238-249 - 2003
N. Carol Casavant1, Dan R. Thompson1, Gwyn A. Beattie2, Gregory J. Phillips2, Larry J. Halverson1,2
1Department of Agronomy, and
2Department of Microbiology, Iowa State University, Ames, IA 50011–1010, USA.

Tóm tắt

SummaryWe constructed and characterized a plasmid‐based genetic system that reports the expression of a toluene‐responsive promoter (PtbuA1) by effecting an irreversible, heritable change in the biosensor cell. Expression of the reporter gene gfp is strongly repressed in the absence of expression from the PtbuA1 promoter, and high level gfp expression in the original cell and its progeny is mediated by the site‐specific recombination machinery of bacteriophage P22 to initiate removal of a repressor cassette. The reporter plasmid pTolLHB was functional in two soil saprophytes, Pseudomonas fluorescens A506 and Enterobacter cloacae JL1157, with the efficiency and sensitivity to low toluene concentrations being optimal in P. fluorescens A506. In culture, 80–100% of the A506 (pTolLHB) population expressed gfp following exposure to 0.2 µm toluene for one to three hours. Compared to the response of A506 containing a plasmid‐borne PtbuA1‐gfp fusion, the recombination‐based biosensor was more sensitive at detecting low toluene and trichloroethylene concentrations. An A506 (pTolLHB) inoculum, which had a background of 2.5% of the cells expressing gfp, was introduced onto barley roots in soil microcosms. If toluene was introduced into the microcosms, after 24 h, 72% of the A506 (pTolLHB) cells recovered from roots expressed gfp, indicating bioavailable toluene to rhizosphere bacteria. When toluene was not introduced, 16.5% of the A506 (pTolLHB) cells recovered from the roots expressed gfp, indicating that natural inducers of the PtbuA1 promoter were present in the barley rhizosphere. When introduced into rhizotrons containing barley plants and toluene vapours, the biosensor allowed localization of the availability of toluene along the seminal roots. In rhizotrons that were not exposed to toluene vapours, the biosensor exhibited high PtbuA1‐promoter activity in distinct regions along the seminal roots, indicating spatial heterogeneity plant‐ or rhizosphere microbial community‐derived inducers of the PtbuA1 promoter. This recombination‐based toluene biosensor thus was useful in identifying bacterial exposure to transient or low levels of toluene, or related compounds, directly in the environment.

Từ khóa


Tài liệu tham khảo

10.1021/es00049a001

10.1128/AEM.64.7.2730-2735.1998

Buckingham J., 1994, Dictionary of Natural Products

10.1128/jb.178.21.6327-6337.1996

10.1111/j.1365-2958.1995.mmi_18040671.x

10.1073/pnas.91.7.2634

10.1146/annurev.arplant.47.1.445

10.1128/AEM.68.7.3588-3596.2002

10.1094/MPMI-7-0440

10.1016/1360-1385(96)10019-4

10.1046/j.1462-2920.2000.00095.x

10.1073/pnas.76.4.1648

Gilbert E.S., 1997, Plant compounds that induce polychlorinated biphenyl biodegradation by Arthrobacter sp. strain B1B, Appl Environ Microbiol, 63, 1933, 10.1128/aem.63.5.1933-1938.1997

10.1094/MPMI.2001.14.10.1197

10.1128/AEM.64.12.5023-5026.1998

10.1094/Phyto-77-882

10.1094/MPMI.2000.13.11.1243

10.2134/jeq2000.00472425002900020029x

10.1128/jb.176.12.3749-3756.1994

Platt R., 2000, Veterinary Microbiology and Preventive Health

10.1006/plas.1999.1433

Sambrook J., 1989, Molecular Cloning: A Laboratory Manual.

SAS Institute I.(2001) JMP version 4.04. Cary NC SAS Institute.

10.1016/S0031-9422(00)81133-4

10.1146/annurev.arplant.52.1.407

10.1080/10643389609388492

10.1128/AEM.63.10.4053-4060.1997

10.1128/AEM.68.4.1962-1971.2002

10.1128/JB.184.3.760-770.2002

10.1111/j.1462-2920.2006.01080.x

10.1128/AEM.64.3.1006-1012.1998

Thông tin
Thông tin xuất bản

Wiley

Tập 5 Số 4

238-249

Thông tin tác giả