FEMS Microbiology Ecology 14 (1994) 319-330 © 1994 Federation of European Microbiological Societies 0168-6496/94/$07.00 Published by Elsevier 319 FEMSEC 00544 Direct detection of rhizosphere-colonizing Pseudomonas sp. using an Escherichia coli rRNA promoter in a Tn7-1ux system M.V. Brennerova and D.E. Crowley * Department of Soil and Environmental Sciences, University of California, Riverside, California 92521, USA (Received 7 February 1994; revision received 21 May 1994; accepted 31 May 1994) Abstract: A promoterless Tn7-1ux system conferring bioluminescence was fused with an Escherichia coli rRNA gene promoter and compared with neo- or Iac-luxCDABE analogs after introduction in Pseudomonas cells. Fusion of the ribosomal promoter with IuxCDABE genes increased the bioluminescence of cells by approx. 100- to 1500-fold over the neo-lux system depending on the growth conditions and bacterial strain. When the cells were grown in suspension culture, light production and growth were strongly dependent on the nutrient composition of the medium. Root-colonizing competence was tested in nonsterile soil by autophoto- graphic detection of bacterial bioluminescence on plant roots. The lower detection limit of the autophotographic method for roots inoculated with Pseudomonas fluorescens 2-79 was 105 cfu g-1 fresh root weight. The new bioluminescence marker did not require addition of supplemental nutrients or the aldehyde substrate for the luciferase enzyme and provides a simple and highly sensitive detection method for long term in situ studies on the microbial ecology of specific bacterial strains. Key words: Autophotography; Microbial ecology; Pseudomonas; Rhizosphere; Tn7; Bioluminescence Introduction Bioluminescence is a versatile marker for tracking bacteria and studying their activity in the environment. Unlike antibiotic resistance mark- ers, bioluminescence provides a specific and highly sensitive method for in situ detection of bacteria after their release into different habitats. Because bioluminescence is dependent on avail- able energy within the cell, the relative metabolic * Corresponding author. Fax: 909-787-3993. activity of the marked population can be deter- mined quantitatively with nonextractive proce- dures [1,2]. A wide variety of broad host range vectors have been developed that employ the promoterless luciferase (Lux) encoding operon (lux) for use as a gene reporter [3-6]. Alterna- tively, when used as a marker to follow specific bacterial populations, the lux genes have been fused with inducible [3,7] or constitutively ex- pressed promoters [8-13] that permit the detec- tion of bacteria by either autophotographic or light-emission monitoring techniques. The luxCDABE genes encode the a and /3 SSDI 0168-6496(94)00027-T