James A. Romesser

Identification of constitutive and herbicide inducible cytochromes P-450 in Streptomyces griseolus

The elevated soluble cytochrome P-450 content of Streptomyces griseolus cells found after growth in the presence of sulfonylurea herbicides has been shown to be the result of the appearance of one predominant cytochrome P-450 form. This cytochrome P-450 is the major soluble protein found to increase in amount following herbicide treatment, and functions as part of a sulfometuron methyl hydroxylase system. A second minor inducible cytochrome P-450 has been observed only in cells grown in the presence of chlorimuron ethyl, and a third cytochrome P-450 has been found to be present in all cells independent of the presence of sulfonylurea inducers. The three cytochrome P-450 isozymes are distinguishable primarily by their anion exchange properties; however, spectral properties, substrate inducibility, and enzymatic activity provide several further distinguishing features. The recognition of these inducible, xenobiotic metabolizing cytochromes P-450 in S. griseolus provides the only known description of monooxygenase proteins related to herbicide metabolism in bacteria.

Induction of cytochrome P-450-dependent sulfonylurea metabolism in Streptomyces griseolus.

Inducible cometabolism of several sulfonylurea herbicides by Streptomyces griseolus has been shown to occur by hydroxylation, O-dealkylation, or deesterification reactions. Only after growth of the bacterium in the presence of sulfonylurea did cell-free extracts exhibit NAD(P)H-dependent sulfonylurea metabolism. These extracts were shown to contain elevated levels of soluble cytochrome P-450 and exhibit sulfonylurea induced difference spectra consistent with binding of substrate to cytochrome(s) P-450. These results establish the presence of an inducible cytochrome P-450-dependent sulfonylurea metabolizing system in S. griseolus.

Plant and Bacterial Cytochromes P-450: Involvement in Herbicide Metabolism

Although herbicides have been used in weed control for many years, our understanding of the biological chemistry involved in their metabolism and degradation is still in its infancy. These reactions take place both in plants which have been treated with herbicides, and in soil microorganisms.1–3 Metabolism in a soil microorganism can result in increased water solubility of the herbicide, with corresponding increased bioavailability and biodegradability ultimately leading to the complete degradation of the herbicide. Herbicide metabolism in plants can result in altered phytotoxicity providing a mechanism of crop selectivity based on the differential metabolism of a herbicide in crop plants as compared with weeds. This very common mechanism of selectivity, which relies largely on uncharacterized enzymatic pathways, is in stark contrast to the alternative and less common mechanism of crop selectivity in which the characteristics of the target site enzyme for the herbicide 4–7 are modified to a herbicide resistant form.8–12

Isolation and characterization of Streptomyces griseolus deletion mutants affected in cytochrome P-450-mediated herbicide metabolism

SummaryMetabolism of sulfonylurea herbicides by Streptomyces griseolus ATCC 11796 is carried out via two cytochromes P-450, P-450SU1 and P-450SU2. Mutants of S. griseolus, selected by their reduced ability to metabolize a fluorescent sulfonylurea, do not synthesize cytochrome P-450SU1 when grown in the presence of sulfonylureas. Genetic evidence indicated that this phenotype was the result of a deletion of > 15 kb of DNA, including the structural genes for cytochrome P-450SU1 and an associated ferredoxin Fd-1 (suaC and suaB, respectively). In the absence of this monooxygenase system, the mutants described here respond to the presence of sulfonylureas or phenobarbital in the growth medium with the expression of only the suhC,B gene products (cytochrome P-450SU2 and Fd-2), previously observed only as minor components in wild-type cells treated with sulfonylurea. These strains have enabled an analysis of sulfonylurea metabolism mediated by cytochrome P-450SU2 in the absence of P-450SU1, yielding an in vivo delineation of the roles of the two different cytochrome P-450 systems in herbicide metabolism by S. griseolus.