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The asparagine synthetase A (EC 6.3.1.1) of E. coli (AS-A) mainly uses ammonia to produce asparagine, a key nitrogen transporter in plants. The AS-A encoding gene (asnA) was expressed constitutively in lettuce cultivar ‘Cortina’ under the control of pMAC, a chimerical promoter, to induce phenotypical alterations of plant growth and quality as a consequence of nitrogen status changes. Nine fertile\ud transgenic lines harbouring independent T-DNA insertions were recovered. Primary transformants shared new visible traits such as a higher leaf number and wider leaf surface than the wild-type. The progeny of three primary transformants stably maintained these phenotypes, to which the synthesis of both asnA transcript and protein were associated. In pMAC:asnA plants, seed germination, formation and development of leaves, bolting and flowering occurred earlier than non-transformed plants.\ud Twenty-eight days after sowing (das), transgenic \ud plants showed a ca. 1.3 increase of leaf area and dry\ud weight as compared to the wild-type. Moreover, the \ud contents of asparagine, aspartic acid and glutamine,\ud but not that of glutamic acid, of pMAC:asnA young\ud plants (21 das) were greater than the wild-type. The level of total soluble protein was higher in transgenic than in non-transformed leaves borne on plants at 35, 50 and 75 das. A decrease of nitrate was also measured in pMAC:asnA leaves with respect to non-transformed ‘Cortina’, in transgenic populations at 60 das. In pMAC:asnA genotypes, the altered content of nitrogen transport amino acids, the tolerance\ud to increasing doses of ammonium and phosphinothricin indirectly proved the AS-A enzymatic activity in lettuce

Bacterial peptide synthetases have two common features that appear to be strictly conserved. 1) The enzyme subunits are co-regulated at both transcriptional and translational level. 2) The organization of the different enzymatic domains constituting the enzyme fulfills the "colinearity rule" according to which the order of the domains along the chromosome parallels their functional hierarchy. Considering the high degree of conservation of these features, one would expect that mutations such as transcription uncoupling and domain dissociations, deletions, duplications, and reshuffling would result in profound effects on the quality and quantity of synthesized peptides. To start testing this hypothesis, we designed two mutants. In one mutant, the operon structure of surfactin synthetase was destroyed, thus altering the concerted expression of the enzyme subunits. In the other mutant, the thioesterase domain naturally fused to the last amino acid binding domain of surfactin was physically dissociated and independently expressed. When the lipopeptides secreted by the mutant Bacillus subtilis strains were purified and characterized, they appeared to be expressed approximately at the same level of the wild type surfactin and to be identical to it, indicating that specific domaindomain interactions rather than coordinated transcription and translation play the major role in determining the correct assembly and activity of peptide synthetases.

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