Expression of the 2,4-D degradative pathway of pJP4 in an alkaliphilic, moderately halophilic soda lake isolate, Halomonas sp. EF43

Abstract.

The broad host range plasmid pJP4, which carries genes for the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D), 2-methyl-4-chlorophenoxyacetic acid, and 3-chlorobenzoic acid, was used in conjugation experiments with mixed cultures enriched from water and sediment samples from an alkaline pond in the area of Szegedi Fehértó, a soda lake in south Hungary. pJP4-encoded mercury resistance was used as a selection marker. One of the transconjugants, the alkaliphilic, moderately halophilic strain EF43, stably maintained the plasmid and was able to degrade 2,4-D and 3-chlorobenzoate under alkaline conditions in the presence of an additional carbon source such as pyruvate, benzoate, or α-ketoglutarate, indicating that the degradative genes of pJP4 were expressed in this strain. However, it was unable to grow on these chloroaromatic substrates when the substrate was the sole source of carbon and energy. Chemostat cultivation experiments revealed that the 2,4-D degradation rate during growth on benzoate or pyruvate was limited by the low activity of chlorocatechol-degrading enzymes, particularly chloromuconate cycloisomerase. Strain EF43 was identified as Halomonas sp. on the basis of 16S rRNA sequencing and additional taxonomic studies. 16S rRNA sequence analysis revealed that strain EF43 is closely related to typical soda lake isolates belonging to the genus Halomonas.