ISOLATION OF LUX REPORTER GENE FUSIONS IN PSEUDOMONAS-FLUORESCENS DF57 INDUCIBLE BY NITROGEN OR PHOSPHORUS STARVATION

We have used transposon Tn5 mutagenesis to insert a promoter-less luxAB gene-cassette into multiple locations in the chromosome of a Pseudomonas fluorescens strain, thereby bringing the Ewe reporter genes under the control of resident promoters. To identify reporter bacteria responsive to nutritional stresses we isolated and characterized a collection of 23 gene fusions consistently displaying bioluminescence under nitrogen starvation and 12 phosphorus starvation inducible fusions. Bioluminescence of one group of mutants was induced after 4 to 6 h of starvation and was continuously expressed at a high level, whereas a second group was induced earlier and the bioluminescence subsequently declined. Finally, a third group was induced later after 24 h of starvation. Four strains were selected for further study, namely, two Tn5-lux containing strains which were induced by nitrogen starvation and two strains induced by phosphorus starvation. Another two strains, carrying constitutively expressed lux fusions, were included as controls. An analysis of biochemical characters, as well as LPS and protein composition, did not reveal any discernible differences between the mutants and the wild-type strain. Survival experiments with the selected Tn5-lux containing strains showed that they all performed comparably to the wild-type under carbon and nitrogen starvation, whereas some of the strains were less resistant to phosphorus starvation. Expression of bioluminescence by the mutants during carbon, nitrogen and phosphorus starvation was detectable even after 18 days and was not affected by high osmolarity or low temperature.