Stereospecific production of the herbicide phosphinothricin (glufosinate) by transamination: cloning, characterization, and overexpression of the gene encoding a phosphinothricin-specific transaminase from Escherichia coli

We have cloned the gene encoding a 43-kilodalton transaminase from Escherichia coli K-12 with a specificity for L-phosphinothricin [L-homoalanine-4-yl-(methyl)phosphinic acid], the active ingredient of the herbicide Basta (Hoechst AG). The structural gene was isolated, together with its own promoter, and shown to be localized on a 1.6-kilobase DraI-BamHI fragment. The gene is subject to catabolite repression by glucose; however, repression could be relieved completely when 4-aminobutyrate (GABA) served as the sole nitrogen source. The regulation pattern obtained and a comparison of the restriction map of the initially cloned 15-kilobase SalI fragment with the physical map of the E. coli K-12 genome suggest that the cloned gene is identical with gabT, a locus on the gab gene cluster of E. coli K-12 which codes for the GABA:2-ketoglutartate transaminase (EC 2.6.1.19). A number of expression plasmids carrying the isolated transaminase gene were constructed. With these constructs, the transaminase expression in transformants of E. coli could be increased up to 80-fold compared with that in a wild-type control, and the transaminase constituted up to 20% of the total soluble protein of the bacteria. Thus, the protein crude extracts of the transformants could be used, after a simple heat precipitation step, for the biotechnological production of L-phosphinothricin in an enzyme reactor.     

Evolution of the Larval Cuticle Proteins Coded by the Secondary Sex Chromosome Pair: X2 and Neo-Y of Drosophila miranda: I. Comparison at the DNA Sequence Level

The larval cuticle protein genes (Lcps) represent a multigene family located at the right arm of the metacentric autosome 2 (2R) in Drosophila melanogaster. Due to a chromosome fusion the Lcp locus of Drosophila miranda is situated on a pair of secondary sex chromosomes, the X2 and neo-Y chromosome. Comparing the DNA sequences from D. miranda and D. melanogaster organization and the gene arrangement of Lcp1–Lcp4 are similar, although the intergene distances vary considerably. The greatest difference between Lcp1 and Lcp2 is due to the occurrence of a pseudogene in D. melanogaster which is not present in D. miranda. Thus the cluster of the four Lcp genes existed already before the separation of the melanogaster and obscura group. Intraspecific homogenizations of different cluster units must have occurred repeatedly between the Lcp1/Lcp2 and Lcp3/Lcp4 sequence types. The most obvious example is exon 2 of the Lcp3 gene in D. miranda, which has been substituted by the corresponding section of the Lcp4 gene rather recently. The homogenization must have occurred before the translocation which generated the neo-Y chromosome. Lcp3 of D. melanogaster has therefore no orthologous partner in D. miranda. Rearrangements in the promoter regions of the D. miranda Lcp genes have generated new, potentially functional CAAT-box motifs. Since three of the Lcp alleles on the neo-Y are not expressed and Lcp3 is expressed only at a reduced level, it is suggestive to speculate that the rearrangements might be involved as cis-regulatory elements in the up-regulation of the X2-chromosomal Lcp alleles, in Drosophila an essential process for dosage compensation. The Lcp genes on the neo-Y chromosome have accumulated more base substitutions than the corresponding alleles on the X2. Received: 27 December 1995 / Accepted: 30 April 1996     

Proton motive force generation by citrolactic fermentation in Leuconostoc mesenteroides.

In Leuconostoc mesenteroides subsp. mesenteroides 19D, citrate is transported by a secondary citrate carrier (CitP). Previous studies of the kinetics and mechanism of CitP performed in membrane vesicles of L. mesenteroides showed that CitP catalyzes divalent citrate HCit2-/H+ symport, indicative of metabolic energy generation by citrate metabolism via a secondary mechanism (C. Marty-Teysset, J. S. Lolkema, P. Schmitt, C. Divies, and W. N. Konings, J. Biol. Chem. 270:25370-25376, 1995). This study also revealed an efficient exchange of citrate and D-lactate, a product of citrate/carbohydrate cometabolism, suggesting that under physiological conditions, CitP may function as a precursor/product exchanger rather than a symporter. In this paper, the energetic consequences of citrate metabolism were investigated in resting cells of L. mesenteroides. The generation of metabolic energy in the form of a pH gradient (delta pH) and a membrane potential (delta psi) by citrate metabolism was found to be largely dependent on cometabolism with glucose. Furthermore, in the presence of glucose, the rates of citrate utilization and of pyruvate and lactate production were strongly increased, indicating an enhancement of citrate metabolism by glucose metabolism. The rate of citrate metabolism under these conditions was slowed down by the presence of a membrane potential across the cytoplasmic membrane. The production of D-lactate inside the cell during cometabolism was shown to be responsible for the enhancement of the electrogenic uptake of citrate. Cells loaded with D-lactate generated a delta psi upon dilution in buffer containing citrate, and cells incubated with citrate built up a pH gradient upon addition of D-lactate. The results are consistent with an electrogenic citrate/D-lactate exchange generating in vivo metabolic energy in the form of a proton electrochemical gradient across the membrane. The generation of metabolic energy from citrate metabolism in L. mesenteroides may contribute significantly to the growth advantage observed during cometabolism of citrate and glucose.     

Use of Solid-Phase Extraction To Determine Ergosterol Concentrations in Plant Tissue Colonized by Fungi

At present, the ergosterol and acetate-to-ergosterol techniques are generally considered to be the methods of choice to quantify fungal biomass, growth rate, and productivity under natural conditions. Both methods rely on the accurate isolation and quantification of ergosterol, a major membrane component of eumycotic fungi. Taking advantage of the solid-phase extraction (SPE) technique, we present a novel method to determine the ergosterol concentration in lipid extracts derived from plant tissues and dead organic matter colonized by fungi. In this method, a primary alkaline extract is acidified and passed through a reversed-phase (C(inf18)) SPE column. The column is then washed with an alkaline methanol-water solution to eliminate interfering substances and increase pH and is thoroughly dried in air. Ergosterol is eluted with alkaline isopropanol. This eluting solvent was chosen to produce a strongly basic pH of the final extract and thus confer stability on the ergosterol molecule before high-performance liquid chromatography analysis. The recovery of ergosterol from plant tissues and the O(infhf) horizon of a woodland soil ranged from 85 to 98%, and the overall extraction efficiency was similar to that obtained by a conventional procedure involving liquid-liquid extraction. Potential pitfalls of ergosterol analysis by SPE include (i) insufficient acidification before sample loading on the extraction column, resulting in a poor affinity of ergosterol for the sorbent; (ii) incomplete drying of the sorbent bed before the elution step; and (iii) chemical breakdown of ergosterol after elution, which was found to be related to a low pH of the final extract and a high concentration of matrix compounds. When these pitfalls are avoided, SPE is an attractive alternative to existing methods of ergosterol analysis of environmental samples.