Degradation of 1,3-dichloropropene by pseudomonas cichorii 170

Abstract

The gram-negative bacterium Pseudomonas cichorii 170, isolated from soil that was repeatedly treated with the nematocide 1, 3-dichloropropene, could utilize low concentrations of 1, 3-dichloropropene as a sole carbon and energy source. Strain 170 was also able to grow on 3-chloroallyl alcohol, 3-chloroacrylic acid, and several 1-halo-n-alkanes. This organism produced at least three different dehalogenases: a hydrolytic haloalkane dehalogenase specific for haloalkanes and two 3-chloroacrylic acid dehalogenases, one specific for cis-3-chloroacrylic acid and the other specific for trans-3-chloroacrylic acid. The haloalkane dehalogenase and the trans-3-chloroacrylic acid dehalogenase were expressed constitutively, whereas the cis-3-chloroacrylic acid dehalogenase was inducible. The presence of these enzymes indicates that 1, 3-dichloropropene is hydrolyzed to 3-chloroallyl alcohol, which is oxidized in two steps to 3-chloroacrylic acid. The latter compound is then dehalogenated, probably forming malonic acid semialdehyde. The haloalkane dehalogenase gene, which is involved in the conversion of 1,3-dichloropropene to 3-chloroallyl alcohol, was cloned and sequenced, and this gene turned out to be identical to the previously studied dhaA gene of the gram-positive bacterium Rhodococcus rhodochrous NCIMB13064. Mutants resistant to the suicide substrate 1,2-dibromoethane lacked haloalkane dehalogenase activity and therefore could not utilize haloalkanes for growth. PCR analysis showed that these mutants had lost at least part of the dhaA gene.

FIG. 1

Growth of strain 170 on a mixture of cis- and trans-1,3-dichloropropene. A 0.15-mmol portion of 1,3-dichloropropene (ratio of cis-1,3-dichloropropene to trans-1,3-dichloropropene, 1/1 [mol/mol]) was added to 100 ml of MMY in a 3-liter flask, which resulted in a low initial liquid phase concentration. Symbols: ▴, cis-1,3-dichloropropene concentration; ▾, trans-1,3-dichloropropene concentration; ▵, cis-3-chloroallyl alcohol concentration; ▿, trans-3-chloroallyl alcohol concentration; ○, optical density at 450 nm (OD₄₅₀); ⧫, chloride concentration. Abbreviations: CAA, 3-chloroallyl alcohol; DCPe, 1,3-dichloropropene.

FIG. 2

SDS-PAGE of crude extract of E. coli BL21(DE3)/pGEF(dhaA) (lane 2) and purified haloalkane dehalogenase from P. cichorii (lane 3). Lane 1 contained protein markers with molecular masses of 94, 67, 43, 30, and 20 kDa.

FIG. 3

Proposed pathway for the degradation of trans-1,3-dichloropropene in P. cichorii 170. 1, haloalkane dehalogenase (DhaA); 2, alcohol dehydrogenase; 3, aldehyde dehydrogenase; 4, 3-chloroacrylic acid dehalogenase; 5, decarboxylase. A similar pathway is envisaged for the cis isomer.