Aroa

Abstract: An efficient transformation system was developed for tomato by studying several factors that affect the rate of Agrobacterium mediated transformation of explant tissue. Cotyledons of tomato were co–cultivated with Agrobacterium tumefaciens harboring a binary vector with two neomycin phosphotransferase (NPT) II genes and a mutant aroA gene. Over 100 transgenic plants were regenerated and rooted on medium containing kanamycin and eighty percent of these plants exhibited both NPT II enzyme activity and produced the mutant aroA protein. Progeny of aroA positive plants were tolerant to glyphosate at concentrations of 0.84 kg active ingredient/ha. The tolerance phenotype segregated in a manner consistent with Mendelian inheritance.

Abstract: Enhanced resistance to glyphosate, an inhibitor of the aromatic amino acid biosynthesis pathway, is imparted to a glyphosate sensitive host. A mutated aroA gene is employed which expresses 5-enolpyruvyl-3- phosphoshikimate synthase (EC: 2.5.1.19) (ES-3-P synthase). Methods are provided for obtaining the aroA mutation which provides the enzyme resistant to inhibition by glyphosate, means for introducing the structural gene into a sensitive host, as well as providing a method of producing the enzyme. The E. coli strain C600(pPMG1) has been deposited at the A.T.C.C. on Dec. 14, 1982 and given A.T.C.C. Accession No. 39256.

Abstract: Due to the dependency on aromatic precursors, the growth of Salmonella typhimurium aroA- is limited in immunocompetent mice. Here we show that H-21-A beta-/- mice (lacking MHC class II molecules and thus devoid of mature CD4+ TCR-alpha beta cells), TCR-beta-/- mice (devoid of TCR-alpha beta cells), and IFN-gamma R-/- mice (unresponsive to IFN-gamma) are highly susceptible to S. typhimurium aroA- infection compared with heterozygous controls. In contrast, beta 2m-deficient mice (lacking surface MHC class I and thus devoid of conventional CD8+ T cells) or TCR-delta-/- mice (devoid of TCR-gamma delta cells) were equally as resistant to S. typhimurium aroA- infection as their heterozygous littermates. These findings emphasize the vital role of CD4+ TCR-alpha beta cells and IFN-gamma in resistance against S. typhimurium aroA-. Sublethal inocula of S. typhimurium aroA- led to permanent infection in H-21-A beta-/- mice, suggesting that bacterial starvation is insufficient for sterile clearance in immunocompetent mice and that MHC class II-dependent immune mechanisms are required for pathogen eradication. The TCR-beta-/- mice suffered from salmonellosis more severely than the MHC class II-deficient mutants, suggesting an auxiliary function of CD8+ T cells. Recombinant S. typhimurium aroA-, secreting listeriolysin (Hly) of Listeria monocytogenes, are capable of escaping from the phagosome into the cytosol of the host cell. However, the course of infection of these recombinant S. typhimurium SL7207 Hlys and control strains did not differ in beta 2m-/- mutants. This finding argues against direct correlation of cytosolic location of S. typhimurium SL7207 Hlys with CD8+ T cell dependency of protection.

Abstract: Stable transposon-generated auxotrophic mutations in aroA, purA, and purE or aroA and purA together were introduced into Salmonella typhimurium strains which were virulent in mice. Strains harboring any of these mutations were attenuated when tested in BALB/c mice. purE strains were less attenuated than aroA or purA strains. Both aroA and purA mutants persisted for several weeks in the livers and spleens of the mice after intravenous infection, although the numbers of viable cells detected at various times after infection differed. aroA strains persisted at a higher level than purA strains and were effective live vaccines given intravenously or orally. purA strains were ineffective as oral vaccines and were poor intravenous vaccines. Strains harboring both aroA and purA mutations together were ineffective vaccines when administered orally or intravenously even though they persisted in the livers and spleens of the mice for long periods after intravenous infection.