Mol Gen Genet (1983) 191:10-16 © Springer-Verlag 1983 A Functional Map of the Nopaline Catabolism Genes on the Ti Plasmid of Agrobacterium tumefaciens C58 C.L. Schardl* and C.I. Kado Davis Crown Gall Group, Department of Plant Pathology, University of California, Davis, CA 956/6, USA Summary. The nopaline catabolism (noc) genes are located in a 14.4 kb region on the pTiC58 plasmid ofA. tumefaciens C58. These genes permit the bacterium to grow on nopaline NZ-(1,3-dicarboxylpropyl) arginine, a substrate produced in plant tumors initiated by strain C58. The functions of the noc genes include the use of nopaline and L-ornithine as sole carbon and nitrogen sources. Using Tn5 insertional mutants, we have identified and mapped the positions of the genes that are responsible for nopaline catabolism (NopC), ornithine catabolism (OrnC) and nopaline uptake (NopU). A polar relationship was found between these phenotypes, which extended leftward over the noc region to the T-region. The NopC mutants were also deficient in nopaline oxidase, an enzyme that liberates free arginine from nopaline. The noc region also encodes the synthesis of a periplas- mic protein, nl that was induced by nopaline. Tn5 inser- tional mutations and molecular cloning were used to map the nl production locus. The recombinant plasmids, pSa4480 and pSa4481, containing the 8.9 kb right-hand end of the noc region, conferred nl production when introduced into a pTi-free strain of A. tumefaciens.' Production of nl by the strains carrying these plasmids required nopaline induction. We have identified in toto three noc loci: nocB, nocC, and nocA, which confer nl production, nopaline oxidase production and ornithine catabolism respectively. A model is proposed whereby the noc genes of pTiC58 are contained on a leftward reading operon in the order nocB, nocC, and nocA. Introduction Crown gall tumor induction requires the presence of the tumor-inducing plasmid (pTi) in A. tumefaciens. A segment of pTi called the T-region is transferred to the plant host genome apparently as part of the crown gall tumor forma- tion process (Chilton et al. 1977). Several research groups have identified T-region genes that encode the production of opines such as nopaline and octopine in transformed plant cells. Nopaline-type Ti plasmids, such as pTiC58, con- tain genes for both the synthesis and degradation of nopa- * Present address. Plant Breeding Institute, Marls Lane, Trump- ington, Cambridge CB2 2LQ, UK Offprint requests to: C.I. Kado line (Bomhoffet al. 1976). The nopaline synthesis (nos) gene is contained on the T-region and functions in the plant cell (Holsters et al. 1980). The nopaline catabolism (noc) genes, which map adjacently to the right of the T-region of pTiC58 (Holsters et al. 1980), also confer ornithine ca- tabolism on the bacterium (Ellis et al. 1979). The noc genes allow the bacterium to use these substrates as sole nitrogen and carbon sources in vitro and, presumably, in the crown gall tumor. The analogous octopine N2-(1-carboxyethyl) arginine catabolism (occ) system has been partly characterized. Oc- topine uptake is induced by octopine in strain A. tumefa- ciens B6S3 (Klapwijk et al. 1977). Arginine and glutamate are intermediates of octopine catabolism (Petit et al. 1970. Klapwijk et al. (1978) and Bomhoff (1974) have described an octopine oxidase, which degrades octopine to arginine and pyruvate. A related activity, lysopine oxidase, has also been described (Jubier 1975). In this paper we have identified and mapped the pheno- types of the pTiC58 noc-region, which includes nopaline uptake (NopU), utilization of nopaline as a carbon source (NopC), or as a nitrogen source (NopN) and ornithine cata- bolism (OrnC). In addition, nopaline oxidase (Nox) and an inducible periplasmic protein nl were found to be en- coded by this region. We present evidence that the loci affecting these phenotypes are contained on a single operon. Materials and Methods Chemicals. [3H]-nopaline, labeled in the arginine moiety, was prepared by the method of J. Temp6 (personal commu- nication) and Bomhoff (1974). Crown gall tobacco callus tissue samples, designated CG-1CTer and originally ob- tained from Milton Gordon, were placed onto fresh MS agar without hormones (Murashige and Skoog 1962) and grown for 3 weeks in the dark at room temperature. One mCi [3H]-L-arginine (sp. act. > 300 mCi/mmol, New En- gland Nuclear) was added to each tissue sample and further incubated for 2 days. The tissues were ground in 60% etha- nol yielding an extract that was filtered and concentrated by flash evaporation at 50 ° C. Nopaline was separated from arginine by preparative electrophoresis (Liu and Kado 1979). The product was quantified by a diacetyl method (Bomhoff et al. 1976). Unlabeled nopaline was chemically synthesized by the method of Jensen et al. (1977) with slight modifications as follows: After cyanoborohydride reduction, the reaction