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 luxA B gene-cassette into multiple locations in the chromosome of a Pseudom onas fluorescens strain, thereby bringing the Ewe reporter genes under the control of resident promoters. To identify reporter bacteria resp onsive to nutritional stresses we isolated and characterized a collect ion of 23 gene fusions consistently displaying bioluminescence under n itrogen starvation and 12 phosphorus starvation inducible fusions. Bio luminescence of one group of mutants was induced after 4 to 6 h of sta rvation and was continuously expressed at a high level, whereas a seco nd group was induced earlier and the bioluminescence subsequently decl ined. Finally, a third group was induced later after 24 h of starvatio n. Four strains were selected for further study, namely, two Tn5-lux c ontaining strains which were induced by nitrogen starvation and two st rains induced by phosphorus starvation. Another two strains, carrying constitutively expressed lux fusions, were included as controls. An an alysis of biochemical characters, as well as LPS and protein compositi on, 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 wi ld-type under carbon and nitrogen starvation, whereas some of the stra ins were less resistant to phosphorus starvation. Expression of biolum inescence by the mutants during carbon, nitrogen and phosphorus starva tion was detectable even after 18 days and was not affected by high os molarity or low temperature.