The holin function Ejh of the pneumococcal bacteriophage EJ-1 has been characterized. It shows structural features similar to, and functionally complemented, the prototype member of the holin family. In Escherichia coli and Pseudomonas putida the Ejh product caused cellular death, and changes in cell morphology could be accounted for by lesions in the cytoplasmic membrane. Expression of ejh resulted in the inhibition of growth in a variety of phylogenetically distant bacterial genera, suggesting a broad spectrum of action. Concomitant expression of the ejh and ejl (encodes a lysin) genes led to lysis of E. coli and P. putida cells. Remarkably, the Ejl lysin was able to attack murein from bacteria lacking choline in their sacculi, which suggests that pneumococcal lysins have a broader substrate specificity than previously assumed. Furthermore, the Ejh holin was able to trigger activity of the major pneumococcal autolysin cloned and expressed in E. coli, and this raised new questions about the regulation of this model autolysin. A new function for holins in systems where the phage lysin is supposed to be associated with the membrane is proposed.

A genetic circuit to suppress the lateral spread of cloned genes from recombinant to indigenous microorganisms in the environment has been developed. It is based on the endonucleolytic activity of the bacterial toxin colicin E3, which has a distinct target at the 3' end of the 16S ribosomal RNA; this sequence is conserved in virtually all prokaryotic and many eukaryotic genera. Cleavage at this sequence separates the mRNA binding sites from the remainder of the 16S rRNA, thereby inhibiting protein synthesis. While host bacteria carrying the genes for both colicin production and colicin immunity are perfectly viable, lateral transfer of the E3 gene to non-immune recipients results in killing of such recipients. This genetic circuit decreases operational transfer frequencies of cloned genes linked to the E3 gene among a variety of bacterial genera by four to five orders of magnitude. In combination with transposon cloning vectors, the circuit is predicted to reduce the rate of lateral spread of specific genes to ecologically insignificant levels. This system therefore represents a useful tool both to explore the evolutionary and ecological consequences of experimentally reducing lateral gene spread among microorganisms, and to increase the ecological predictability of novel recombinant microorganisms.

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To restrict horizontal gene spread and, thus, create organisms whose behavior in the field is more predictable, we have combined a mini-Tn5 cloning system that allows stable insertion of foreign genes into the chromosomes of a variety of Gram-negative bacteria with a gene-containment circuit based on the universal lethal-function colicin E3. Use of the system is exemplified by the construction of a micro-organism designed to degrade polychlorinated biphenyls, important environmental pollutants. The relevant genotype of the microorganism is subject to a stringent gene-containment mechanism that provides neither an advantage nor a disadvantage to the host cell for survival, but decreases frequencies of productive chromosomal transfer frequencies by at least four orders of magnitude.

Plantlets were regenerated by adventitious shoot budding in tissue culture from leaf explants of a single genotype of sugar beet. DNA was extracted from the parental plant and from 120 regenerants. RAPD analysis was carried out using five decanucleotide primers; 4,557 RAPD marker bands were examined and two polymorphisms were observed. Thirty secondary regenerants were then derived, using the same tissue culture technique, from thirty of the primary regenerants. Again RAPD analysis was employed and a single band polymorphism was observed out of 1,050 bands examined. The overall frequency of detection of somaclonal polymorphisms using RAPD (3 in 5,607 = 0.05%) is similar to frequencies previously reported using isozyme and RFLP technologies.