A bioluminescent derivative of Pseudomonas putida KT2440 for deliberate release into the environment

Recombinant derivatives of Pseudomonas putida strain KT2440 are of potential interest as microbial inoculants to be deliberately released for agricultural applications. To facilitate tracking of this strain and its derivatives after introduction into the environment, a mini-Tn5-'luxAB transposon was introduced into the chromosome of P. putida KT2440, yielding strain P. putida S1B1. Sequencing of the DNA region located upstream of the 'luxAB genes and similarity search with the P. putida KT2440 genome sequence, localized the transposon within a 3021-bp open reading frame (ORF), whose translated sequence showed significant similarity with the hypothetical YdiJ proteins from Escherichia coli and Haemophilus influenzae. A second ORF adjacent to and divergent from the ydiJ sequence was also found and showed significant homology with various LysR-type transcriptional activator proteins from several bacteria. Disruption of the ydiJ locus in P. putida S1B1 did not affect the survival of the strain in unvegetated or vegetated soils. Bioluminescent detection of P. putida S1B1 cells enriched in selective media directly from soil allowed detection of culturable cells in soil samples over a period of at least 8 months. The addition of the luxAB biomarker facilitates tracking in the root system of several plant species grown under sterile and non-sterile conditions. The correlation of the bioluminescent phenotype with the growth activity of P. putida S1B1 cells colonizing the root system of barley and corn plants was estimated by monitoring ribosomal contents using quantitative hybridization with fluorescence-labeled ribosomal RNA probes. A correlation between inoculum density, light output, and ribosomal contents was found for P. putida cells colonizing the root system of barley seedlings grown under sterile conditions. Although ribosomal contents, and therefore growth activity, of P. putida S1B1 cells extracted from the rhizosphere of corn plants grown in non-sterile soil were similar to those found in starved cells, the luminescent system permitted non-destructive in situ detection of the strain in the upper root system.