Complete characterisation of Tn 5530 from Burkholderia cepacia strain 2a (pIJB1) and studies of 2,4-dichlorophenoxyacetate uptake by the organism

Abstract

The complete genetic characterisation of Tn 5530 in Burkholderia cepacia strain 2a (pIJB1) has been accomplished, indicating that it is a Tn 3-like transposon with a complex structure bearing operons for the catabolism of 2,4-dichlorophenoxyacetate (2,4-D) and malonate. Tn 5530 is terminated at both ends by the IS 1071::IS 1471 element and the 2,4-D- and malonate-dissimilatory operons are separated by a region encoding a putA and lrp gene and a gene encoding a chloride channel protein. The chloride channel protein may have a role in the expulsion of chloride ions liberated by the dissimilation of 2,4-D. In addition, a putative transposase with a high level of sequence similarity to those of plasmid pGH1 from Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. glycinea, and a transcription factor similar to those of the TetR family with low but significant levels of sequence similarity to those identified in a number of other organisms was observed. The entire Tn 5530 sequence length, including the IS 1071::IS 1471 elements, was found to be 40,956 bp, and pIJB1 was replicon-typed and otherwise characterised as being of the IncP-1β subgroup, bearing merA and merD genes conferring resistance to mercuric chloride. The rate of uptake of 2,4-D by B. cepacia strain 2a was observed to proceed more readily at acid pH, suggesting involvement of the undissociated form of the compound. Uptake did not show saturation kinetics, was concentration-dependent, and appeared to occur in two stages; an initial accumulation followed by a linear second phase. Uptake could be inhibited by sodium azide but not by arsenate, N, N ′-dicyclohexylcarbodi-imide (DCCD) or carbonylcyanide m-chlorophenyl-hydrazone (CCCP) suggesting that it is not energy-dependent.