Abebe, H.M., Ramon J. Seidler, S.E. Lindow, K.A. Short, E. Clark, and R.J. King. 1997. Relative expression and stability of a chromosomally integrated and plasmid borne marker gene fusion in environmentally competent bacteria. Current Microbiology 34:71-78
A xylE-iceC transcriptional fusion was created by ligating a DNA fragment harboring the cloned xylE 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 the chromosome of Pseudomonas syringae Cit7 by homologous recombination. Both cis-merodiploid strain Cit7m17 and marker exchange strain Cit7h69 produced the XylE gene product, catechol 2,3-dioxygenase. Strain Cit7m17, in which XylE was influenced by transcription initiated by the amp promoter on pBR322, exhibited XylE activity in stationary phase at levels about 45 times higher than strain Cit7h69, permitting detection of 107 Cit7m17 cells in the spectrophotometric assay and 103 cells in HPLC measurements. The stability of xylE in both Cit7m17 and Cit7h69 was compared with maintenance of xylE in several plasmid-borne constructs in P. aeruginosa, Erwinia herbicola, and Escherichia coli. Only the xylE-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 xylE, 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 xylE 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 xylE for use in autecological studies are discussed. DGGS's based on subdivisions of the platonic solids, called Geodesic DGGS's, are then introduced. A number of existing and proposed Geodesic DGGS's are examined by looking at four design choices that must be made in constructing a Geodesic DGGS: the base platonic solid, the orientation of that solid relative to the earth's surface, the method of subdivision defined on a face of that solid, and a method for relating that planar subdivision to the corresponding spherical surface. Finally, an examination of these design choices leads us to the construction of the ISEA3H DGGS.