Nonsterile soil transforms the rye metabolite 2(3H)-benzoxazolone (BOA) into 2-amino-3H-phenoxazin-3-one, which is an order of magnitude more toxic to barnyard grass than benzoxazolone. Benzoxazolone was recovered unchanged from sterile soil. However,o-aminophenol is converted to aminophenoxazinone by both sterile and nonsterile soil in the presence of air. Aminophenoxazinone is probably produced by microbial hydrolysis of benzoxazolone intoo-aminophenol, which is oxidized to aminophenoxazinone in both sterile and nonsterile soil. No 2,2'-oxo-1,1'-azobenzene was found in any incubations of soil with benzoxazolone,o-aminophenol, oro-azophenol.

We have isolated and characterized Tn3HoHol-and TnS-induced mutants of a cosmid clone, pYDH208, which encodes the mannopine (MOP) cyclase-associated catabolism of MOP and agropine (AGR). Characterization of the transposon-induced lacZ fusion mutants by ,B-galactosidase activity and mannityl opine utilization patterns identified at least 6 genetic units associated with the catabolism of these opines. Functions for the catabolism of MOP and mannopinic acid are encoded by a 16.4-kb region, whereas those for AGR are encoded by a 9.4-kb region located within the MOP catabolic locus. The induction pattern of catabolism shown by transposon insertion derivatives suggests that the catabolism of MOP, AGR, and mannopinic acid encoded by pYDH208 is regulated by at least two independent control elements. Kinetic uptake assays indicate that the clone encodes two transport systems for MOP and AGR, one constitutive and slow and the other inducible and rapid. Analysis of 13-galactosidase activities from lacZ reporter gene fusions indicated that expression of mannityl opine catabolic genes is not strongly repressed by sugars but is repressed by succinate when ammonium is the nitrogen source. The repression exerted by succinate was relieved when MOP was supplied as the sole source of nitrogen. This suggests that genes for opine catabolism encoded by pYDH208 are regulated, in part, by nitrogen availability.Production of opines, which are unusual low-molecularweight carbon compounds, is an almost unique property of crown gall tumor and hairy root cells. The specific opines produced by the transformed plant tissues are determined by the inducing Agrobacterium strain and are correlated with opines which can be catabolized by that bacterial strain (2, 28). Although opines are not required for the formation and maintenance of plant tumors or hairy roots, at least three functions for these compounds are known. First, most of the opines can be utilized as sources of carbon, nitrogen, and energy by tumorigenic agrobacteria (2, 28). Second, certain, but not all, opines induce conjugal transfer of Ti plasmids to other agrobacteria (12, 33). Third, several opines can enhance the acetosyringone-dependent induction of virulence genes which are involved in the processing, transfer, and integration of T-DNA into the plant genome (41).Crown gall tumors induced by Agrobacterium tumefaciens strains harboring classical broad-host-range, octopine-type Ti plasmids produce mannityl opines as well as octopine. There are four members of this opine family. Mannopine (MOP) and mannopinic acid (MOA) are imine conjugates of mannose and glutamine or glutamic acid, respectively. Agropine (AGR) is a lactonized derivative of MOP, while agropinic acid (AGA) is a spontaneous lactam of MOP (32,40). These compounds are abundant in crown gall tumors induced by octopine-or agropine-type A. tumefaciens strains (14, 16) and in hairy roots induced by MOP-

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Homogenates of corn seedlings inhibit both growth of Agrobacterium tumefaciens and induction of its Ti plasmid virulence (vir) genes by acetosyringone (AS). The heat-labile inhibitor has been identified as 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), present in 2-week-old seedlings (B73) at a concentration of 1.5 mM or greater. A concentration of 0.3 mM DIMBOA is sufficient to block growth of A. tumefaciens completely for 220 hr. DIMBOA at 0.1 mM concentration completely inhibited vir gene induction by 100 microM AS and reduced growth rate by 50%. Thus, DIMBOA can be expected to have a significant effect on attempts to transform corn by using A. tumefaciens as a vector.

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