Relative expression and stability of a chromosomally integrated and plasmid-borne marker gene fusion in environmentally competent bacteria.
A xylE-iceC transcriptional fusion was created by ligatinga 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, catechol2,3-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 10(7) Cit7m17 cells in the spectrophotometric assay and 10(3) 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 Cit7m17was 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 xylEin 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 taggedwith xylE for use in autecological studies.
Abebe
H M
HM
ManTech Environmental Technology Inc., USEPA Environmental Research Laboratory, 200 SW 35th St., Corvallis, OR 97333, USA.
Seidler
R J
RJ
Lindow
S E
SE
Short
K A
KA
Clark
E
E
King
R J
RJ
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D050557:Catechol 2,3-Dioxygenase; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D049308:Dioxygenases; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005819:Genetic Markers; D016254:Mutagenesis, Insertional; D010105:Oxygenases / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011549:Pseudomonas / Q000235:genetics*; D016958:Pseudomonas putida / Q000235:genetics; D011995:Recombination, Genetic; D015183:Restriction Mapping; D013053:Spectrophotometry; D014158:Transcription, Genetic
D001425:Bacterial Outer Membrane Proteins; D005819:Genetic Markers; C063113:ice nucleation protein; D010105:Oxygenases; D049308:Dioxygenases; D050557:Catechol 2,3-Dioxygenase