Cadmium-regulated gene fusions in Pseudomonas fluorescens

To study the mechanisms soil bacteria use to cope with elevated concentrati ons of heavy metals in the environment, a mutagenesis with the lacZ-based r eporter gene transposon Tn5-B20 was performed. Random gene fusions in the g enome of the common soil bacterium Pseudomonas fluorescens strain ATCC 1352 5 were used to create a bank of 5000 P. fluorescens mutants. This mutant ba nk was screened for differential gene expression in the presence of the tox ic metal cadmium. Fourteen mutants were identified that responded with incr eased or reduced gene expression to the presence of cadmium. The mutants we re characterized with respect to their metal-dependent gene expression and their metal tolerance. Half the identified mutants reacted with differentia l gene expression specifically to the metal cadmium, whereas some of the ot her mutants also responded to elevated concentrations of copper and zinc io ns. One of the mutants, strain C8, also showed increased gene expression in the presence of the solvent ethanol, but otherwise no overlap between cadm ium-induced gene expression and general stress response was detected. Molec ular analysis of the corresponding genetic loci was performed using arbitra ry polymerase chain reaction (PCR), DNA sequencing and comparison of the de duced protein products with sequences deposited in genetic databases. Some of the genetic loci targeted by the transposon did not show any similaritie s to any known genes; thus, they may represent 'novel' loci. The hypothesis that genes that are differentially expressed in the presence of heavy meta ls play a role in metal tolerance was verified for one of the mutants. This mutant, strain C11, was hypersensitive to cadmium and zinc ions, In mutant C11, the transposon had inserted into a genetic region displaying similari ty to genes encoding the sensor/regulator protein pairs of two-component sy stems that regulate gene expression in metal-resistant bacteria, including czcRS of Ralstonia eutropha, czrRS of Pseudomonas aeruginosa and copRS of P seudomonas syringae. Although the P. fluorescens strain used in this study had not been isolated from a metal-rich environment, it nevertheless contai ned at least one genetic region enabling it to cope with elevated concentra tions of heavy metals.