Klaus Bartsch

Molecular organization of the Escherichia coli gab cluster: nucleotide sequence of the structural genes gabD and gabP and expression of the GABA permease gene

We have determined the nucleotide sequences of two structural genes of the Escherichia coli gab cluster, which encodes the enzymes of the 4-aminobutyrate degradation pathway: gabD, coding for succinic semialdehyde dehydrogenase (SSDH, EC 1.2.1.16) and gabP, coding for the 4-aminobutyrate (GABA) transport carrier (GABA permease). We have previously reported the nucleotide sequence of the third structural gene of the cluster, gabT, coding for glutamate: succinic semialdehyde transaminase (EC 2.6.1.19). All three gab genes are transribed unidirectionally and their orientation within the cluster is 5′-gabD-gabT-gabP-3′. gabT and gabP are separated by an intergenic region of 234-bp, which contains three repetetive extragenic palindromic (REP) sequences. The gabD gene consists of 1,449 nucleotides specifying a protein of 482 amino acids with a molecular mass of 51.7 kDa. The protein shows significant homologies to the NAD+-dependent aldehyde dehydrogenase (EC 1.2.1.3) from Aspergillus nidulans and several mammals, and to the tumor associated NADP+-dependent aldehyde dehydrogenase (EC 1.2.1.4) from rat. The permease gene gabP comprises 1,401 nucleotides coding a highly hydrophobic protein of 466 amino acids with a molecular mass of 51.1 kDa. The GABA permease shows features typical for an integral membrane protein and is highly homologous to the aromatic acid carrier from E. coli, the proline, arginine and histidine permeases from Saccharomyces cerevisiae and the proline transport protein from A. nidulans. Uptake of GABA was increased ca. 5-fold in transformants of E. coli containing gabP plasmids. Strong overexpression of the gabP gene under control of the isopropyl-2-d-thiogalactoside (IPTG) inducible tac promoter, however, resulted in a severe growth inhibition of the transformed strains. The GABA carrier was characterized using moderately overexpressing transformants. The Km of GABA uptake was found to be 11.8 μM and the Vmax 0.33 nmol/min · mg cells. Uptake of GABA was stimulated by ammonium sulfate and abolished by 2,4-dinitrophenol. Aspartate competed with GABA for uptake.

Safeners recruit multiple signalling pathways for the orchestrated induction of the cellular xenobiotic detoxification machinery in Arabidopsis

Safeners enhance herbicide tolerance in crop plants but not in target weeds, thus improving herbicide selectivity. The safeners isoxadifen-ethyl and mefenpyr-diethyl protect cereal crops from sulfonyl urea herbicides in postemergence application. The two safeners were shown here to induce the cellular xenobiotic detoxification machinery in Arabidopsis thaliana when applied to leaves in a way mimicking field application. Gene expression profiling revealed the induction of 446 genes potentially involved in the detoxification process. Transgenic Arabidopsis plants expressing a reporter gene under control of a safener-responsive maize promoter were used as a model system to study the safener signalling pathway. Reporter gene analysis in the tga2/3/5/6, sid2-2 and npr1 mutants as compared with the wild-type background showed that safener inducibility required TGA transcription factors and salicylic acid (SA) in a NON-EXPRESSOR of PR-1 (NPR1)-independent pathway converging on two as-1 promoter elements. For the majority of the safener-responsive Arabidopsis genes, a similar dependence on TGA transcription factors and/or SA was shown by gene expression profiling in wild-type plants as compared with the tga2/3/5/6 and sid2-2 mutants. Thirty-eight percent of the genes, however, were induced by safeners in a TGA/SA-independent manner. These genes are likely to be controlled by WRKY transcription factors and cognate W-boxes in their promoters.