Tn5530 from Burkholderia cepacia strain 2a encodes a chloride channel protein essential for the catabolism of 2,4-dichlorophenoxyacetic acid.
Chloride channel proteins (ClC) are found in living systems where they transport chloride ions across cell membranes. Recently, the structure/function of two prokaryotic ClC has been determined but little is known about the role of these proteins in the microbial metabolism of chlorinated compounds. Here we show that transposon Tn5530 from Burkholderia cepacia strain 2a encodes a ClC protein (BcClC) which is responsible for expelling Cl(-) ions generated during the catabolism of 2,4-dichlorophenoxyacetic acid (a chlorinated herbicide). We found that BcClC has the ability to transport Cl(-) ions across reconstituted proteoliposome membranes. We created two mutants in which the intrachannel glutamate residue of the protein, known to be responsible for opening and closing the channel (i.e. gating), was changed in order to create constitutively open and closed forms. We observed that cells carrying the closed-channel protein accumulated Cl(-) ions intracellularly leading to a decrease in intracellular pH, cell stasis and death. Further, we established that BcClC has the same gating mechanism as that reported for the ClC protein from Salmonella typhimurium. Our results show that the physiological role of ClC is to maintain cellular homeostasis which can be impaired by the catabolism of chlorinated compounds.
Sebastianelli
Antonio
A
Department of Biosciences, University of Kent, Giles Lane, Canterbury CT2 7NJ, UK.
Bruce
Ian J
IJ
D015084:2,4-Dichlorophenoxyacetic Acid / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016956:Burkholderia cepacia / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018118:Chloride Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004251:DNA Transposable Elements / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011510:Proteolipids / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D001426:Bacterial Proteins; D018118:Chloride Channels; D004251:DNA Transposable Elements; D011510:Proteolipids; C020485:proteoliposomes; D015084:2,4-Dichlorophenoxyacetic Acid