Protease-dependent streptomycin sensitivity in E. coli--a system for protease inhibitor selection.

We have developed a bacterial cell system in which the activity of an expressed heterologous protease confers a dominant streptomycin-sensitive (strs) phenotype on the cell. This phenotype owes its high selectivity to the fact that streptomycin (strep) resistance, which is conferred on E. coli by mutants of ribosomal protein S12, is highly recessive to strep sensitivity. Thus, when strep-resistant (strr) strains of E. coli are transformed to co-express the wild-type allele of S12 in addition to the mutant allele, their sensitivity to strep increases by a factor of 100-1000. Similarly, we found that when the same strr strains were transformed to co-express a heterologous protease and an inactive fusion of S12 with a substrate of the protease, the strep sensitivity of the cells increased approximately 100-fold. This effect was strictly dependent on correct cleavage of the S12 precursor, required only modest levels of expression of protease and substrate, and could be competitively inhibited by co-expression of an alternative substrate gene. This system thus appears to be well-suited to the identification of protease inhibitors, either by selection from libraries of endogenously expressed random peptide-encoding genes, or by screening synthetic or natural products libraries. Protease-dependent dominant phenotypes may be more sensitive and appropriate than the more commonly used recessive phenotypes for proteases which are activating enzymes.

Directed tagging of the Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene with the maize transposon activator.

The FATTY ACID ELONGATION1 (FAE1) gene of Arabidopsis is required for the synthesis of very long chain fatty acids in the seed. The product of the FAE1 gene is presumed to be a condensing enzyme that extends the chain length of fatty acids from C18 to C20 and C22. We report here the cloning of FAE1 by directed transposon tagging with the maize element Activator (Ac). An unstable fae1 mutant was isolated in a line carrying Ac linked to the FAE1 locus on chromosome 4. Cosegregation and reversion analyses established that the new mutant was tagged by Ac. A DNA fragment flanking Ac was cloned by inverse polymerase chain reaction and used to isolate FAE1 genomic clones and a cDNA clone from a library made from immature siliques. The predicted amino acid sequence of the FAE1 protein shares homology with those of other condensing enzymes (chalcone synthase, stilbene synthases, and beta-ketoacyl-acyl carrier protein synthase III), supporting the notion that FAE1 is the structural gene for a synthase or condensing enzyme. FAE1 is expressed in developing seed, but not in leaves, as expected from the effect of the fae1 mutation on the fatty acid compositions of those tissues.