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RELATIVE EXPRESSION AND STABILITY OF A CHROMOSOMALLY INTEGRATED AND PLASMID-BORNE MARKER GENE FUSION IN ENVIRONMENTALLY COMPETENT BACTERIA
Abebe, H. M., R. Seidler, S. E. Lindow, K. Short, E. Clark, AND R. J. King. RELATIVE EXPRESSION AND STABILITY OF A CHROMOSOMALLY INTEGRATED AND PLASMID-BORNE MARKER GENE FUSION IN ENVIRONMENTALLY COMPETENT BACTERIA. Current Microbiology 34(2):71-78, (1997).
A xyIE-iceC transcriptional fusion was created by ligating a DNA fragment harboring the cloned xyIE structural gene from the TOL plasmid of Pseudomonas putida mt-2 into the cloned iceC gene of Pseudomonas syringae Cit7. This fusion construct was integrated into chromosome of Pseudomonas syringae Cit7 by homologous recombination. Both cis-merodiploid strain Citm17 and marker exchange strain Cit7h69 produced the XyIE gene product, catechol 2,3-dioxygenase. Strain Citm17, in which XyIE was influenced by transcription initiated by the amp promoter on pBR322, exhibited XyIE activity in stationary phase at levels about 45 times higher than strain Cit7h69, permitting detection of 10 7 Cit7m17 cells in the spectrophotometric assay and 10 3 cells in HPLC measurements. The stability of xyIE in both Cit7m17 and Cit7h69 was compared with maintenance of xyIE in several plasmid-borne constructs in P.aeruginosa, Erwinia herbicola, and Escherichia coli. Only the xyIE-iceC fusion in the chromosome of Cit7h69 and Cit7m17 was stable in plate assays over the course of these studies. Even though strain Cit7h69 stably expressed xyIE, the low level of expression precludes its use in direct spectrophotometric or HPLC assays as a means for detecting cells in environmental samples. However, expression of xyIE in Cit7h69 is sufficient for identification of colonies harboring this marker gene which is useful in laboratory plate assays, and as a marker gene system for the detection of environmentally-competent strains chromosomally tagged with xyIE for use in autecological studies.
Record Details:Record Type: DOCUMENT (JOURNAL/PEER REVIEWED JOURNAL)
Organization:U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT