Isolation and characterization of Tn5-induced mutants of Pseudomonas paucimobilis UT26 defective in gamma-hexachlorocyclohexane dehydrochlorinase (LinA).
Pseudomonas paucimobilis UT26 grows on gamma-hexachlorocyclohexane (gamma-HCH) as a sole source of carbon and energy. Tn5 mutation was introduced into UT26, and two kinds of mutants defective in gamma-HCH degradation were phenotypically isolated; one (UT64) completely lacked the activity to degrade gamma-HCH, while the other (UT61) retained a very low level of activity. Tagging and sequencing analysis showed that both mutants had a Tn5 insertion at the same site of the linA (gamma-HCH dehydrochlorinase encoding) gene. However, UT61 had an additional rearrangement, which could be the cause of its retaining a low level of activity. An in vitro complementation test with a crude extract from UT64 plus partially purified LinA protein showed that LinA was essential not only for the first-step reaction (gamma-HCH to gamma-pentachloro-cyclohexene; gamma-PCCH), but also for the second-step reaction (gamma-PCCH to compound B) of gamma-HCH degradation in UT26.
Nagata
Y
Y
Department of Agricultural Chemistry, University of Tokyo, Japan.
Imai
R
R
Sakai
A
A
Fukuda
M
M
Yano
K
K
Takagi
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D003470:Culture Media; D004251:DNA Transposable Elements; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D001556:Hexachlorocyclohexane / Q000378:metabolism*; D008190:Lyases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics*; D015183:Restriction Mapping
D003470:Culture Media; D004251:DNA Transposable Elements; D001556:Hexachlorocyclohexane; D008190:Lyases; C019990:DDT-dehydrochlorinase