| Tributyltin compounds have been used on a global scale for many decades now, and thus have become global environmental contaminants. These compounds are highly toxic, and deleterious effects on numerous organisms have been demonstrated. Yet, little is known of the molecular mechanisms of tributyltin's extreme toxicity. To obtain a better understanding of such mechanisms, a luxAB gene-fusion library of Escherichia coli was screened for changes in gene expression upon cellular exposure to tributyltin. Two clones, designated TBT1 and TBT3, were thus found, both showing an increased light emission in the presence of added tributyltin. Northern blotting analyses confirmed a marked increase in the transcription of the tributyltin-responsive gene identified from each clone. These genes appear to play a protective role when cells are exposed to tributyltin at concentrations ≧ 10 mug/ml, with minimal-dose responses of 0.1 mug/l when grown on LB media. Speciation studies indicated TBT+ as the active chemical species in eliciting these responses. Mapping and sequencing of these tributyltin-responsive genes revealed that the luxAB reporter element had inserted within the uhpT gene in the TBT1 clone. This gene encodes a sugar-phosphate transporter protein, which has been shown to be up-regulated by external glucose-6-phosphate and 2-deoxyglucose-6-phosphate. On a kinetic level, the increased expression of uhpT by tributyltin closely mirrors that produced by 2-deoxyglucose-6-phosphate. In addition to tributyltin, TBT1 also responds to dibutyltin, monobutyltin, trimethyltin, triethyltin, tripropyltin, trifluoroacetic acid, and vanadium. Similar mapping and sequencing experiments revealed the luxAB reporter genes within the stpA gene in the TBT3 clone, but in an antisense orientation, such that they were not under the regulatory control of stpA. The lack of an appropriate open reading frame for this 140-nucleotide transcript (identified by Northern blotting analysis), coupled with the high degree of secondary structure of RNA from this chromosomal region, led to the proposal of this transcript as an sRNA. TBT3 also responds to trimethyltin, triethyltin, tripropyltin, triethylgermanium, and tributylsilane. These clones also have potential to serve as highly sensitive biosensors of biologically available contaminants in environmental samples. |
