RESUMEN
Using microplates as pressure and cultivation vessels, a high-throughput method was developed for analyzing the high-pressure inactivation kinetics of microorganisms. The loss of viability from a high-pressure treatment, measured based on the growth delay during microplate cultivation, showed reproducibility with the conventional agar plate method and was applicable for the kinetics analysis.
Asunto(s)
Desinfección/métodos , Viabilidad Microbiana , Presión , Cinética , Reproducibilidad de los ResultadosRESUMEN
Rhodococcus jostii RHA1 accumulates chlorobenzoates (CBA) during the degradation of polychlorinated biphenyls (PCBs). CBA degradation is considered one of the rate-limiting steps in the complete degradation of PCBs. To reduce the accumulation of CBAs, the upper pathway enzyme genes for PCB degradation of RHA1 were introduced into a CBA-degrading bacterium, Burkholderia sp. NK8. The resulting recombinant strain exhibited no biphenyl 2,3-dioxygenase (BphA) activity encoded by bphAaAbAcAd genes, which encode the large and small subunits of the terminal oxygenase component and the ferredoxin and reductase subunits responsible for electron transfer from NADH to the large subunit. The remaining enzyme genes involved in the transformation of biphenyl to benzoate, bphB2C1D1, which encode dehydrogenase, ring-cleavage dioxygenase and hydrolase, conferred activities to NK8. To obtain the BphA activity of RHA1 in NK8, sets of BphA genes were constructed by combining the bphAaAbAcAd genes of RHA1 and bphA3A4 of Pseudomonas pseudoalcaligenes KF707, encoding the ferredoxin and reductase subunits. Hybrid derivatives of BphA containing the KF707 bphA3 conferred BphA activity to NK8, and a derivative containing the RHA1 bphAaAb and KF707 bphA3A4 genes exhibited the highest BphA activity. A plasmid containing the RHA1 bphAaAb and KF707 bphA3A4 genes plus the RHA1 bphB2C1D1 genes was constructed and introduced into NK8. The resulting recombinant strain efficiently degraded 2-, 3- and 4-chlorobiphenyls with an apparent reduction in CBA accumulation in comparison to the recombinant mutant strain, which had an insertion in the cbeA gene to inactivate CBA dioxygenase.
Asunto(s)
Burkholderia/metabolismo , Contaminantes Ambientales/metabolismo , Bifenilos Policlorados/metabolismo , Biodegradación Ambiental , Burkholderia/genética , Clorobenzoatos/metabolismo , Dioxigenasas/genética , Dioxigenasas/metabolismo , Ferredoxinas/genética , Ferredoxinas/metabolismo , Genes Bacterianos , Hidrolasas/genética , Hidrolasas/metabolismo , Redes y Vías Metabólicas/genética , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Oxigenasas/genética , Oxigenasas/metabolismo , Rhodococcus/enzimología , Rhodococcus/genéticaRESUMEN
Rhodococcus jostii RHA1 is a polychlorinated biphenyl degrader. Multi-component biphenyl 2,3-dioxygenase (BphA) genes of RHA1 encode large and small subunits of oxygenase component and ferredoxin and reductase components. They did not express enzyme activity in Escherichia coli. To obtain BphA activity in E. coli, hybrid BphA gene derivatives were constructed by replacing ferredoxin and/or reductase component genes of RHA1 with those of Pseudomonas pseudoalcaligenes KF707. The results obtained indicate a lack of catalytic activity of the RHA1 ferredoxin component gene, bphAc in E. coli. To determine the cause of inability of RHA1 bphAc to express in E. coli, the bphAc gene was introduced into Rosetta (DE3) pLacI, which has extra tRNA genes for rare codons in E. coli. The resulting strain abundantly produced the bphAc product, and showed activity. These results suggest that codon usage bias is involved in inability of RHA1 bphAc to express its catalytic activity in E. coli.