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.     

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.     

Active cell membrane mechanisms involved in the exclusion of RH 123 allow distinction between normal and tumoral cells

Human cell lines derived from three epithelial carcinomas (CaSki, HeLa, SiHa), one B lymphoma (BL60), one promyelocytic (HL60), one monocytic (U937) leukemia, one chronic myelogenous leukemia (sensitive K562S; multichemoresistant K562R) and normal human skin fibroblasts were compared for their capacity of staining with rhodamine 123 (Rh 123) and their kinetics of dye exclusion. Cells were exposed for 30 min to 10 g/ml of Rh 123 in culture medium; fluorescence intensity was measured by flow cytometry immediately or 1, 2, 3 and 4 h after staining. The highest fluorescence intensity was observed in carcinoma cell lines; there was no incorporation in multichemoresistant K562R cells. Exclusion of Rh 123 was evaluated from 0 to 4 h, both by flow cytometry and by fluorimetry. Fluorescence intensity measured by flow cytometry decreased slightly in carcinoma and leukemia cells and rapidly in fibroblasts. In all cell lines Rh 123 exclusion was inhibited by 40 mol/L verapamil and 5 mmol/L probenecid. Thus, incorporation and exclusion of Rh 123 allows distinction between normal and tumoral cells; moreover, inhibition of exclusion by verapamil and probenecid favors the involvement of active cell membrane mechanisms in the exclusion process.Abbreviations PBS phosphate-buffered saline - Rh 123 rhodamine 123     

Effect of ovarian antral follicle cauterization on the interestrus interval of the gilt

The objective of the present study was to determine if destruction of ovarian antral follicles by laser-cauterization affects CL lifespan during the estrous cycle of the gilt. Cyclic gilts were randomly assigned to either SHAM, laser (L) or laser-estradiol (L-E2) treatment groups, with the L-E2 group receiving a 5-mg intramuscular (i.m.) injection of estradiol-17beta cypionate at the time of the first surgery. Ovarian antral follicles were laser-cauterized on either Days 12 and 14 (L12) or Days 14 and 17 (L14) of the estrous cycle. In the L12-E2 group, 3 of 4 gilts had extended mean interestrus intervals of more than 22 days compared with 0 of 4, 0 of 6, 0 of 7 and 1 of 5 gilts in the SHAM, L12, L14 and L14-E2 groups, respectively. The L12-E2 gilts had a longer (P<0.05) mean interestrus interval (23.5+/-1.3 days) than the L12 (20.0+/-1.1 days), L14 (20.7+/-1.0 days) and SHAM (20.5+/-1.3 days). The mean interestrus interval of L14-E2 gilts (21.8+/-1.2 days) did not differ from those of the L12-E2 group or the L12, L14 and SHAM group gilts. Six additional gilts were injected with 5 mg estradiol cypionate-17beta to serve as nonsurgical controls for E2 treatment. Gilts (3 of 3) given an E2 injection on Day 12 had extended mean interestrus interval (26.0+/-2.6 days), while 2 of 3 gilts injected with E2 on day 14 had extended mean interestrus intervals (27.7+/-2.1 days). These results indicate that in cyclic gilts destruction of ovarian follicles by laser-cauterization did not affect CL lifespan, and that luteolysis is not dependent on the presence of antral follicles.