DETECTING BIOAVAILABLE TOXIC METALS AND METALLOIDS FROM NATURAL WATER SAMPLES USING LUMINESCENT SENSOR BACTERIA S. M. TAURIAINEN*, M. P. J. VIRTA and M. T. KARP Department of Biotechnology, University of Turku, Turku, Finland (First received 1 April 1999; accepted in revised form 1 January 2000) AbstractÐWe have generated microbial sensors for analyzing the presence of various metals or metalloids by recombinant DNA technology. The strains are based on strictly regulated promoters controlling the expression of the ®re¯y luciferase gene in microbial cells. The regulator-reporter constructs are located in shuttle plasmids capable of replicating in gram-negative or -positive microbial organisms. The sensors developed are real-time indicators of metal responsive gene expression giving results in approximately 30 min, with optimal induction times ranging from 60 to 240 min. We describe here the performance of these metal sensing bacteria for the assessment of dierent water samples spiked with lead, arsenic, mercury or cadmium. We show that these bacteria are sensitive detectors of metal bioavailability, which is dicult or even impossible to measure by traditional analytical chemistry methods. All measurements were done using freeze-dried bacteria, which makes these sensors reagent- like and also easy to use in ®eld conditions. 7 2000 Elsevier Science Ltd. All rights reserved Key wordsÐluciferase, luc-gene, environment, cadmium, mercury, arsenite INTRODUCTION The methodology most often used today for metal or metalloid detection is atomic absorption spec- trometry (AAS). This and other methods of analyti- cal chemistry usually measure the total metal content of samples. They are not usually used to distinguish between dierent metal species, and they can not analyze the toxicity of metals. There are a variety of organisms used for the detection of non- speci®c toxicity, like Daphnia magna and Vibrio ®scheri (MicroTox 2 ), but these kinds of assays detect the total toxicity of samples. Bioavailability of metals or metalloids can be measured speci®cally by using engineered microbial cells designed for this purpose (Selifonova et al., 1993; Tauriainen et al., 1997; Virta et al., 1995). Living microbial cells can be used as sensitive sensors of environmental factors surrounding them. In fact, they are packages containing all the necess- ary compounds to carry out complex chemical reac- tions without the need of external additions. The use of recombinant DNA techniques for engineering microbial cells leads to interesting possibilities to generate speci®c microbial sensors. We have recently described several speci®c sensor bacteria for measuring bioavailable mercury (Virta et al., 1995), arsenite (Tauriainen et al., 1997, 1999), cad- mium and lead (Tauriainen et al., 1998). Sensors with analogous approaches have been also reported by other groups (Ramanathan et al., 1997; Scott et al., 1997; Selifonova et al., 1993; Tescione and Bel- fort, 1993). These sensor strains are based on the same concept: a metal responsive regulation unit regulates the expression of a sensitive reporter gene. A simpli®ed cartoon that shows how these sensor bacteria work is shown in Fig. 1. Metal responsive promoters can be obtained from metal resistant bacterial strains, in which resistance genes are usually located in plasmids and are precisely regu- lated. Many of these resistance mechanisms are well known and the respective genes have been cloned (for a recent review, see Silver, 1998). The ®re¯y (Photinus pyralis ) luciferase (Luc ) gene was used as a reporter in sensor strains con- structed. Fire¯y luciferase has become a widely used reporter since it was cloned in 1985 (deWet et al., 1985). Luciferase catalyzes the oxidation of the heterocyclic substrate D-luciferin in the presence of ATP producing visible light. The quanti®cation of light emission i.e. bioluminescence is one of the most sensitive means of detection and it can be measured from living cells with a liquid scintillation counter, a luminometer or even with X-ray ®lm. Wat. Res. Vol. 34, No. 10, pp. 2661±2666, 2000 7 2000 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/00/$ - see front matter 2661 www.elsevier.com/locate/watres PII: S0043-1354(00)00005-1 *Author to whom all correspondence should be addressed: Wallac OY, PO Box 10, FIN-20101 Turku, Finland. Tel.: +358-2-267-8698; fax: +358-2-267-8380; e-mail: sisko.tauriainen@wallac.®