T4 lysozyme was thought to destroy bacteria by its muramidase activity. However, we demonstrate here that amphipathic helix stretches in the C-terminus of T4 lysozyme mediate its bactericidal and fungistatic activities. In heatdenatured T4 lysozyme, the enzymatic activity is completely abolished but unexpectedly, the antimicrobial functions remain preserved. Small synthetic peptides corresponding to amphipathic C-terminal domains of T4 lysozyme show a microbicidal activity. Its membrane disturbing activity was directly demonstrated for bacterial, fungal and plant cells but not in a hemolysis assay. Comparable results were obtained with hen egg white lysozyme. This opens up many new opportunities for optimization of lysozymes as antimicrobial agents in various applications by protein engineering.z 1999 Federation of European Biochemical Societies.

The phytopathogenic bacterium Erwina carotovora spp. which infects potato plants causes severe losses in agriculture. No protective means or resistance traits usable for plant breeding are known. Introduction of a new resistance gene into potato by gene technology leads to a reduced susceptibility of the transgenic plants towards Erwinia carotovora atroseptica infection. Bacteriophage T4 lysozyme is the most active member of a class of bacteriolytic enzymes also detected in several plant species. Secretion of the foreign T4 lysozyme into the intercellular spaces of transgenic potato plants effects a resistance against the phytopathogenic bacterium already at low expression levels.

Phosphoenolpyruvate carboxylase (PEPC) genes from Corynebacterium glutamicum (cppc), Escherichia coli (eppc) or Flaveria trinervia (fppc) were transferred to Solanum tuberosum. Plant regenerants producing foreign PEPC were identified by Western blot analysis. Maximum PEPC activities measured in eppc and fppc plants grown in the greenhouse were doubled compared to control plants. For cppc a transgenic plant line could be selected which exhibited a fourfold increase in PEPC activity. In the presence of acetyl-CoA, a known activator of the procaryotic PEPC, a sixfold higher activity level was observed. In cppc plants grown in axenic culture PEPC activities were even higher. There was a 6-fold or 12-fold increase in the PEPC activities compared to the controls measured in the absence or presence of acetyl-CoA, respectively. Comparable results were obtained by transient expression in Nicotiana tabacum protoplasts. PEPC of C. glutamicum (PEPC C.g.) in S. tuberosum leaf extracts displays its characteristic K(m) (PEP) value. Plant growth was examined with plants showing high expression of PEPC and, moreover, with a plant cell line expressing an antisense S. tuberosum (anti-sppc) gene. In axenic culture the growth rate of a cppc plant cell line was appreciably diminished, whereas growth rates of an anti-sppc line were similar or slightly higher than in controls. Malate levels were increased in cppc plants and decreased in antisense plants. There were no significant differences in photosynthetic electron transport or steady state CO2 assimilation between control plants and transformants overexpressing PEPC C.g. or anti-sppc plants. However, a prolonged dark treatment resulted in a delayed induction of photosynthetic electron transport in plants with less PEPC. Rates of CO2 release in the dark determined after a 45 min illumination period at a high proton flux density were considerably enhanced in cppc plants and slightly diminished in anti-sppc plants. When CO2 assimilation rates were corrected for estimated rates of mitochondrial respiration in the light, the electron requirement for CO2 assimilation determined in low CO2 was slightly lower in transformants with higher PEPC, whereas transformants with decreased PEPC exhibited an appreciably elevated electron requirement. The CO2 compensation point remained unchanged in plants (cppc) with high PEPC activity, but might be increased in an antisense plant cell line. Stomatal opening was delayed in antisense plants, but was accelerated in plants overexpressing PEPC C.g. compared to the controls.

No abstract

The release of transgenic organisms has evoked an unusual legal process in that laws governing it are prospective on perceived risks rather than retrospective on experienced risks as is the usual case with legislating against problems. Most countries undertaking transgenic releases have adopted a regulatory structure usually comprising controlled releases to address questions of perceived risks followed by uncontrolled commercial releases. There has been an increasing number of commercial releases from approximately 11 million hectares of transgenic crops in 1997 to more than 27 million hectares in 1998. Most of these commercial releases have been in industrialized countries with only a small proportion in developing countries. The controlled releases, together with laboratory experiments, have addressed a range of perceived risks which can be put into three groups: risks to humans and domesticated animals, risks tO the environment, and commercial risks. These perceived risks have to be assessed against the baseline of current and projected farming practices with non-transgenic crops. Few, if any, of these perceived risks have been shown to be real risks which are significantly more important than the non-transgenic situation. The situation with plants transgenically protected against virus infection was discussed. In some countries, the discussions on transgenic crop releases have entered the public domain. The debate has raised various ethical issues and reflects the wish of society to be involved in the adoption of new technologies. [L]