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Keywords:

  • genetically modified microbial inocula;
  • luminescence-based microbial detection;
  • microbial activity in the soil;
  • Pseudomonas fluorescens

Abstract

Luminescent strains of Pseudomonas fluorescens 10586 were constructed in which luciferase production was constitutive by introduction of Vibrio fischeri luxABE genes on the chromosome and on a multicopy plasmid. Light production in liquid batch culture was directly proportional to biomass concentration during exponential growth and enabled detection by luminometry of 1.7 × 103 and 8.9 × 104 cells/ml for the plasmid and chromosomally marked strains, respectively. Luminescent colonies of both strains were detectable by eye, enabling viable cell enumeration on solid media against a background of non-luminescent strains. Following inoculation into sterile and non-sterile soil lower levels of detection were increased but detection of 8.1–59 × 103and 2.2–30 × 103 cells per g of soil was possible for plasmid and chromosomally marked strains. Maximum specific growth rate in liquid culture was unaffected by introduction of lux marker genes on the chromosome, but was reduced in the plasmid marked strain. The chromosomally encoded marker was stable in both liquid culture and in soil, but the plasmid was unstable during continuous subculturing in liquid medium and during growth in soil. The chromosomally encoded luminescence-marker system therefore provides a convenient, non-extractive technique for quantification of genetically modified soil microbial inocula.