Surveying biotransformations with à la carte genetic traps: translating dehydrochlorination of lindane (gamma-hexachlorocyclohexane) into lacZ-based phenotypes.
The ability of the product of a desired reaction to activate a bacterial transcriptional regulator was exploited to develop genetic traps that render the catalytic activity born by a DNA clone into a selectable/scorable phenotype. We established this strategy with a system to expose the activity of dehydrochlorinases acting upon gamma-hexachlorocyclohexane (gamma-HCH or lindane). To this end, the effector-binding protein, XylR, was evolved by gene shuffling plus mutagenic polymerase chain reaction to be optimally responsive to the major product of gamma-HCH dehydrochlorination, 1,2,4-trichlorobenzene (TCB). We then derived Escherichia coli strains that constitutively expressed the modified XylR variant (named XylR5) and had lacZ under control of the Pu promoter, which is activated by XylR. A robotic beta-galactosidase assay indicated that when the resulting strain was transformed with a linA+ clone (expressing a gamma-HCH dehydrochlorinase from Sphingomonas paucimobilis UT26), it had levels of beta-galactosidase that were dependent on the gamma-HCH concentration. This à la carte host thus translated the conversion of gamma-HCH to TCB into upregulation of lacZ. An alternate host additionally expressing LacY grew efficiently on lactose only when LacZ was upregulated in a fashion dependent on TCB or other effectors of XylR5. These results demonstrated the power of deriving a host for the genetic scrutiny, rather than enzymatic screening, of clones expressing a given catabolic enzyme.
Department of Microbiology and Immunology, University of British Columbia, 300-6174 University Blvd., Vancouver, BC V6T 1Z3, Canada.
William W
WW
Mohn
Junkal
J
Garmendia
Teca C
TC
Galvao
Víctor
V
de Lorenzo
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001673:Biodegradation, Environmental; D002722:Chlorobenzenes / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000096:biosynthesis; D015870:Gene Expression; D001556:Hexachlorocyclohexane / Q000378:metabolism*; D007763:Lac Operon; D007785:Lactose / Q000378:metabolism; D008190:Lyases / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis; D016296:Mutagenesis; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D020578:Sphingomonas / Q000201:enzymology / Q000235:genetics; D027981:Symporters / Q000096:biosynthesis; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D001616:beta-Galactosidase / Q000032:analysis* / Q000235:genetics
D001426:Bacterial Proteins; D002722:Chlorobenzenes; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D014157:Transcription Factors; C074322:XylR protein, Pseudomonas; C009947:1,2,4-trichlorobenzene; D001556:Hexachlorocyclohexane; D001616:beta-Galactosidase; D008190:Lyases; C019994:dehydrochlorinases; D007785:Lactose