The mechanisms that ensure that Ti plasmid T-DNA genes encoding proteins involved in the biosynthesis of opines in crown gall tumors are always matched by Ti plasmid genes conferring the ability to catabolize that set of opines on the inducing Agrobacterium strains are unknown. The pathway for the biosynthesis of the opine agropine is thought to require an enzyme, mannopine cyclase, coded for by the ags gene located in the T(R) region of octopine-type Ti plasmids. Extracts prepared from agropine-type tumors contained an activity that cyclized mannopine to agropine. Tumor cells containing a T region in which ags was mutated lacked this activity and did not contain agropine. Expression of ags from the lac promoter conferred mannopine-lactonizing activity on Escherichia coli. Agrobacterium tumefaciens strains harboring an octopine-type Ti plasmid exhibit a similar activity which is not coded for by ags. Analysis of the DNA sequence of the gene encoding this activity, called agcA, showed it to be about 60% identical to T-DNA ags genes. Relatedness decreased abruptly in the 5' and 3' untranslated regions of the genes. ags is preceded by a promoter that functions only in the plant. Expression analysis showed that agcA also is preceded by its own promoter, which is active in the bacterium. Translation of agcA yielded a protein of about 45 kDa, consistent with the size predicted from the DNA sequence. Antibodies raised against the agcA product cross-reacted with the anabolic enzyme. These results indicate that the agropine system arose by a duplication of a progenitor gene, one copy of which became associated with the T-DNA and the other copy of which remained associated with the bacterium.

To investigate the effect of plant transformation on opine catabolic (and pathogenic) Agrobacterium, we designed an experimental model in which the changes of a bacterial population associated with the root system of plants could be easily monitored. The bacterial population was composed of opine catabolic and noncatabolic strains of Agrobacterium. Bacteria were cocultivated with either transformed or normal Lotus plants. The composition of the bacterial population was estimated by dilution and plating of the growth medium on various media. We demonstrated that growth of bacteria was stimulated when they were associated with transformed plants. Furthermore, growth of opine-utilizing bacteria was specifically favored when bacteria were associated with transformed plants, but not when bacteria were associated with normal plants. This work indicates that transformation of plant cells by agrobacteria indeed can favor growth of the catabolic strain. This finding may lead to creation of engineered plant-bacteria interactions in which both partners will benefit from their association.

Octopine-type strains of Agrobacterium tumefaciens degrade the opine mannopinic acid through a specific pathway which involves cleavage of the molecule at the C--N bond between the amino acid and the sugar moieties. Mannose was identified as a product of the reaction. This pathway was inducible by mannopinic and agropinic acids, but not by mannopine or agropine, the two other mannityl opines. The transport system for this pathway appeared to be specific for mannopinic acid. A second, nonspecific pathway for mannopinic acid degradation was also identified. This involved some of the catabolic functions associated with the metabolism of mannopine and agropine. This second pathway was inducible by mannopine and agropine but not by mannopinic or agropinic acids. The transport system for this pathway appeared to have a broad specificity. Transposon Tn5 insertion mutants affected in the specific catabolic pathway were isolated and analyzed. These mutants continued to catabolize mannopine and agropine. Both mapped to a region of the Ti plasmid previously shown to be associated with the catabolism of mannopinic acid. Restriction enzyme analysis of the Ti plasmid from strain 89.10, an octopine strain that is naturally unable to utilize mannopinic acid, showed a deletion in this same region encoding the specific mannopinic acid degradation pathway. Analysis of recombinant clones showed that the second, nonspecific pathway was encoded in a region of the Ti plasmid associated with mannopine and agropine catabolism. This region shared no structural overlap with the segment of the plasmid encoding the specific mannopinic acid degradative pathway.

A novel enzymic activity, responsible for the conversion of mannopine to agropine by lactonization, has been identified in Agrobacterium strains. This activity is encoded by octopine-type and agropine-type Ti or Ri plasmids, and is inducible by mannopine and agropine. In crude extracts it is stable for long periods and can be used for preparative synthesis of agropine from mannopine. The physiological role of this activity is not understood. However, it is probably involved in degradation of opines of the agropine family since it is always associated with agropine utilization in wild-type strains.

The subunits present as monomers in unreduced zein and isolated as fraction M by gel filtration, were chromatographed on sulfoethyl-cellulose. Three major subfractions were detected and characterized. Each of them, submitted to electrophoresis at pH 3.5, migrated as a single band corresponding to each of the three major electrophoretic forms seen in fraction M at the same pH. The presence of lysine in some polypeptides, suggested by amino acid composition data, was confirmed by electrophoretic analysis of carbamylated subfractions at pH 3.5. At pH 8.9 each subfractions was further resolved into three cationic bands in starch gel and three (or more) anionic bands in polyacrylamide gel. The same fractionation was also obtained by submitting the major electroforms of fraction M, as isolated at pH 3.5, to isoelectric focusing. Based on these observations, the most probable distributions of basic amino acids in subunits detected by electrophoresis at pH 8.9 were specified and compared to those recently published for several zein clones. The presence per polypeptide chain of three carboxyl groups and occasionally of one lysine would be a feature of zein originating from maize hybrid Inra 260.

Unreduced zein chromatographed on Sephadex G 200 in 8 M urea, on G 100 in 1.5 or 2.5% sodium dodecyl sulfate (SDS) and on hydroxypropylated G 100 in 70% ethanol was resolved into two minor fractions A and B and two major ones D and M irrespective of the medium. The quantitative importance of the fraction M was dependent on the isolation conditions of zein. It decreased from 53% of the proteins contained in ethanolic extract and chromatographed as they were extracted, to 40% of the purified zein. The molecular weight values obtained from SDS-polyacrylamide gel electrophoresis and amino acid compositional data indicated that fractions D and M, as isolated from purified zein in the presence of ethanol, represented respectively dimeric and monomeric forms of a mixture of Mr 22 000 and 24 000 polypeptides with threonine or phenylalanine as NH2-terminal residue. Electrophoretic analysis of selectively carbamylated fraction M on starch gel at pH 3.5 revealed that zein subunits comprised several polypeptides differing in the number and the nature of basic amino acids. At least one of these polypeptides contained one lysyl residue.

Agrobacterium tumefaciens strains harboring octopine- or agropine-type Ti-plasmids induce crown gall tumors on Kalanchoë tubiflora, sunflower or carrot, that contain agropine, mannopine, mannopinic acid, and agropinic acid. Since oxidation of these compounds on electrophoretograms by an alkaline silver nitrate reagent yields darkly stained spots due to reduction of the silver ions, they are called ‘silver nitrate-positive’. These silver nitrate-positive compounds are not present in normal plant tissues nor in tumors induced by A. tumefaciens strains that contain a nopaline-type Ti-plasmid. All four compounds are opines since octopine- and agropine-type Ti-plasmids, but not nopaline-type Ti-plasmids, confer on the bacterial host the ability to catabolize them for growth. Silver nitrate-positive opines were always present in primary octopine- or agropine-type crown gall tumors. However, cultured crown gall tumors frequently did not contain detectable silver nitrate-positive opines even though other types of opines, such as octopine, were usually present.

Reduction of the Schiff bases formed between glucose, mannose or galactose with glutamic acid yields products related both structurally and biologically to agropine and to a derivative of agropine present in crown gall tumours. The catabolism of these compounds is coded for by octopine and agropine Ti-plasmids of Agrobacterium tumefaciens.

Les spectres de rotation de cis-2-3-H2-d6-but-2-ène et cis-d8-but-2-ène ont été observés dans le domaine de fréquence 18–40 GHz. La contribution de la rotation interne des toupies est négligeable. La validité de la barrière haute est confirmée en observant que tous les transitions de rotation sont des triplets symétriques. Les constantes de rotation rigides ont été determinées précisément et la géométrie de la molécule cis-but-2-ène est discutée.