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.