LUMINESCENCE-BASED SYSTEMS FOR DETECTION OF BACTERIA IN THE ENVIRONMENT

The development of techniques for detection and tracking of microorgan isms in natural environments has been accelerated by the requirement f or assessment of the risks associated with environmental release of ge netically engineered microbial inocula. Molecular marker systems are p articularly appropriate for such studies and luminescence-based marker s have the broadest range of applications, involving the introduction of prokaryotic (lux) or eukaryotic (luc) genes for the enzyme lucifera se. Lux or luc genes can be detected on the basis of unique DNA sequen ces by gene probing and PCR amplification, but the major advantage of luminescence-based systems is the ability to detect light emitted by m arked organisms or by luciferase activity in cell-free extracts. Lumin escent colonies can be detected by eye, providing distinction from col onies of indigenous organisms, and the sensitivity of plate counting c an be increased greatly by CCD imaging. Single cells or microcolonies of luminescent organisms can also be detected in environmental samples by CCD image-enhanced microscopy, facilitating study of their spatial distribution. The metabolic activity of luminescence-marked populatio ns can be quantified by luminometry and does not require extraction of cells or laboratory growth. Metabolic activity, and potential activit y, of marked organisms therefore can be measured during colonization o f soil particles and plant material in real time without disturbing th e colonization process. In comparison with traditional activity techni ques, luminometry provides significant increases in sensitivity, accur acy, and, most importantly, selectivity, as activity can be measured i n the presence of indigenous microbial communities. The sensitivity, s peed, and convenience of luminescence measurements make this a powerfu l technique that is being applied to the study of an increasingly wide range of ecological problems. These include microbial survival and re covery, microbial predation, plant pathogenicity, phylloplane and rhiz osphere colonization and reporting of gene expression in environmental samples.