Genetic engineering of a radiation-resistant bacterium for biodegradation of ixed wastes. 1998 annual progress report
'Because of their tolerance to very high levels of ionizing radiation, members of the genus Deinococcus have received considerable attention over the past years. The type species of the genus, Deinococcus radiodurans, has been studied extensively in several labs. Although researchers are only beginning to understand the mechanisms by which this Gram-positive bacterium is able to repair massive DNA damage after radiation dosages as high as 5 Mrad, it has become evident that its recombination machinery has several unique characteristics (1--4). The aim of the present studies is to engineer D. radiodurans into a detoxifier for bioremediation of complex waste mixtures, containing heavy metals, halo-organics and radionuclides, making use of its ability to be biologically active in environments where they will be exposed to high levels of radiation. For that purpose, the authors aim to clone and express several broad spectrum oxygenases and heavy metal resistance determinants, and test survival and activities of these strains in artificial mixtures of contaminants, designed to simulate DOE mixed waste streams. This report summarizes work after 0.5 year of a 3-year project. The initial studies have focused on the development of an insertional expression system for D. radiodurans R1. This effort has involved two parts, namely: (1) promoter analysis, and (2) development of insertion systems. Several studies have shown that the expression signals used by D. radiodurans differ considerably from those found in other bacteria. Although D. radiodurans contains a typical eubacterial RNA polymerase core enzyme (based on TBLASTN searches on the genome sequence), Escherichia coli promoters are not recognized in D. radiodurans and vice versa (5). To expand the basic understanding of the requirements for transcription, and to optimize expression of (heterologous) genes, they will follow two strategies. First, a promoter-probe vector is being developed for the selection of promoter sequences from the D. radiodurans R1 genome. This system, which uses either lacZ or gfp as a reporter for expression, is based on single-copy replacement recombination (DCO) in either the thyA or dfrA (folA) gene. From numerous studies in both Gram-positive and Gram-negative organisms it is known that mutations in these genes, encoding thymidilate synthase and dihydrofolate reductase, respectively, render the host resistant to trimethoprim (e.g., 6). This obviates the need of an efficiently expressed antibiotic resistance marker for the initial selection of transformants. This system will then be used for the construction of a shotgun-library of promoter fragments and subsequent screening of D. radiodurans transformants for expression of b-galactosidase or fluorescence. The second strategy involves primer extension studies of a number of genes which are expected to be transcribed at a substantial level. This will enable us to map transcription start sites and identify possible -35 and -10 sequences.'
- Research Organization:
- Univ. of Washington, Seattle, WA (US)
- Sponsoring Organization:
- USDOE Office of Environmental Management (EM), Office of Science and Risk Policy
- OSTI ID:
- 13705
- Report Number(s):
- EMSP-60150-98; ON: DE00013705
- Country of Publication:
- United States
- Language:
- English
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