CONSTRUCTION AND DETECTION OF THE RECOMBINANT RHODOCOCCUS SP. STRAIN RHA1 FOR MINERALIZATION OF PCBs IN SOIL

AUTOR(ES)
DATA DE PUBLICAÇÃO

2000

RESUMO

PCBs are still one of the most important environmental pollutants. We developed a strategy of combining the biphenyl and 4-chlorobenzoate (4-CBA) degradation pathways into the same microorganism for PCB degradation. The genes from Arthrobacter globiformis strain KZT1 responsible for transforming 4-CBA into 4- hydroxybenzoate were sequenced and their products expressed in E. coli. Three open reading frames, organized in an operon, were required for this activity: 4-CBA CoAligase (fcbA), 4-CBA dehalogenase (fcbB), and 4-CBA thioesterase (fcbC). These genes are cotranscribed into one polycistronic mRNA. The presence of the 3.3 kb fragment downstream of the operon repressed the expression of the fcb operon when 4-CBA was absent. Gene expression was restored to its normal level in response to 4-CBA addition, indicating that the fcb operon is regulated by a repressor. The Gram-positive bacterium Rhodococcus species, strain RHA1, which contains the biphenyl oxidation pathway, was electroporated with a plasmid containing the 4-CBA operon. The recombinant strain grew on 4-CBA and 4-chlorobiphenyl (4-CB) as the only source of carbon, with stoichiometric release of chloride and a molar growth yield on 4-CB that suggested utilization of both biphenyl rings. Similar conversion rates were observed for wild type and recombinant strains for the eight most common congeners from the anaerobic dechlorination of Arochlor 1242 (pattern M), but the recombinant strain accumulated lower amounts of chlorinated meta-cleavage products and no 4-CBA. The recombinant cell population, when added to non-sterile 4-CB contaminated soil, increased to a density consistent with the 4-CB consumed and the fcb operon remained stable. A real time PCR assay using fluorescently labeled oligonucleotides (TaqMan probes) was developed to quantify Rhodococcus sp. RHA1(fcb) in soil. The TaqMan-16S rDNA probe detected RHA1(fcb) and phylogenetically related species while TaqMan-fcb probe was specific for the recombinant strain. The method had a 6-log dynamic range of detection (102 to 107) for both probes. In other microcosms, two recombinant strains: Rhodococcus sp. RHA1(fcb) and newly engineered Burkholderia cepacia LB400 containing the 2-chlorobenzoate (ohb) degradation operon, were added to Aroclor 1242-contaminated sediment that had undergone anaerobic dechlorination. Both recombinant strains increased their populations in the PCB contaminated sediment. The recombinant RHA1 cell number and fcb gene copies were quantified over the experimental period by real time PCR and results agreed well with plate counts of this strain. Inoculation at cell densities of 104 and 106 cells g-1 sediment resulted in equivalent PCB removals, 57% and 54%, respectively. The residual PCB congener profile after 30 days was the same for both high and low cell density inoculation.

ASSUNTO(S)

microbiologia aplicada pcb biodegradation microbiologia do solo biodegradaçao mineralização pcb

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