Development and testing of improved suicide functions for biological containment of bacteria.

We have developed very efficient suicide functions for biological containment based on the lethal Escherichia coli relF gene. The suicide functions are placed in duplicate within a plasmid and arranged to prevent inactivation by deletion, recombination, and insertional inactivation. The efficiency of this concept was tested in a plasmid containment system that prevents transfer of plasmids to wild-type bacteria. Protection against plasmid transfer was assayed in test tubes and in rat intestine. Protection was efficient and refractory to inactivation by mutation and transposons. The efficiency of the suicide system was also tested in soil and seawater. We show that unprecedented suicide efficiency can be achieved in soil and seawater after suicide induction by IPTG (isopropyl-beta-D-thiogalactopyranoside). More than 7 orders of magnitude reduction in suicide bacteria was achieved.

Development of efficient suicide mechanisms for biological containment of bacteria.

To optimize plasmid containment, we have systematically investigated the factors that limit the killing efficiency of a suicide system based on the relF gene from Escherichia coli controlled by inducible lac promoters and placed on plasmids. In induction experiments with this suicide system, killing efficiency was unaffected by temperature and growth medium; there was no requirement for great promoter strength or high plasmid copy number. We could demonstrate that the factors limiting killing were the mutation rate of the suicide function and the reduced growth rate caused by a basal level of expression of the suicide gene during normal growth, which can give a selective growth advantage to cells with mutated suicide functions. The capacity of the plasmid-carried killing system to contain the plasmid was tested in transformation, transduction, and conjugational mobilization. The rate of plasmid transfer detected in these experiments seemed too high to provide adequate biological containment. As expected from the induction experiments, plasmids that escaped containment in these transfer experiments turned out to be mutated in the suicide function. With lac-induced suicide as a test, the efficiency of the system was improved by tightening the repression of the suicide gene, thereby preventing selection of cells mutated in the killing function. Reduction of the mutational inactivation rate of the suicide system by duplication of the suicide function augmented the efficiency of the suicide dramatically. These results permit the construction of extremely efficient biological containment systems.

Sequence of the relB transcription unit from Escherichia coli and identification of the relB gene.

Escherichia coli relB mutants react to amino acid starvation by several abnormal responses, including accumulation of a translational inhibitor. We have isolated a relB-complementing plasmid from the Clarke and Carbon E. coli DNA library. From this plasmid we sequenced a 2140-bp segment which included the relB gene by the following two criteria: (i) it complements chromosomal relB mutations, (ii) the corresponding DNA segment cloned from chromosomal DNA of three relB mutants was defective in relB complementation. All three mutations fell within an open reading frame of 79 amino acids. A polypeptide of 9 kd compatible with this open reading frame was synthesized in maxicells and is in all probability the product of the relB gene. By nuclease S1 mapping we have determined the transcription start and stop of an 870 base transcript of the relB gene.

Isolation and characterization of the dut gene of Escherichia coli. I. Cloning in thermoinducible plasmids.

We have constructed thermoinducible plasmids carrying the gene (dut) for the enzyme deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase, EC 3.6.1.23) from Escherichia coli. A 9.4-kb BamHI restriction enzyme fragment carrying the dut gene was inserted into the runaway-replication plasmid pKN402A (Uhlin et al., Gene 6 (1979) 91-106). Strains carrying such plasmids increased their dUTPase activity considerably. In minimal medium a 200-fold increase was demonstrated. A smaller (1.5-kb) SacI-BamHI fragment from the dut region was also cloned into pKN402A. The dUTPase production in dut mutant strains carrying this plasmid (pKK141) was only at about wild-type level after temperature shift. To test the hypothesis that the SacI cleavage used affects a control region for the dut gene, we recloned the dut fragment by transferring it from pKK141 into pHUB2 (Bernard et al., Gene 5 (1979) 59-76), a plasmid carrying the phage lambda pL promoter. A 3.6-kb EcoRI-BamHI fragment from pKK141, including the 1.5-kb SacI-BamHI segment from the dut region, was inserted downstream from the pL promoter. When this plasmid was present in a strain containing a thermosensitive lambda repressor gene, thermoinduction of dUTPase was negligible, apparently due to the presence of some termination signals between pL and dut. Therefore, we removed a 1.9-kb EcoRI-SacI fragment from the region between pL and the dut gene and replaced it with a 0.22-kb EcoRI-SacI fragment, obtained from the b2 region of lambda. Strains carrying such a shortened dut-pHUB2 derivative and a temperature-sensitive lambda repressor overproduced dUTPase very dramatically after heat induction. The final level reached was 300-400 times the wild-type level, corresponding to 10% of the total soluble protein. The information obtained, together with analysis of plasmid-directed polypeptide products described by Lundberg et al. (Gene 22 (1983) 127-131) shows that the SacI site is indeed on the promoter-proximal side of the dut gene.

Isolation and characterization of the dut gene of Escherichia coli. II. Restriction enzyme mapping and analysis of polypeptide products.

Restriction endonuclease mapping of previously constructed dut plasmids has been carried out using the enzymes PvuI, PvuII and SacI. Various dut plasmids were also tested in the "maxicell" protein-synthesizing system. They all show two protein bands in common, one of Mr 16000 in agreement with the size previously reported for the purified dUTPase subunit (Shlomai and Kornberg, 1978). With the information obtained the structural gene for dUTPase can be assigned to a 950-bp SacI-PvuII fragment of the E. coli genome. Studies, described in the preceding paper, on the overproduction of dUTPase by bacterial strains carrying different dut plasmids strongly suggest that the dut gene is transcribed in the direction from the SacI site towards the PvuII site and that the SacI site is located within the dut control region. The second protein band observed in the "maxicell" experiments has an Mr of 23500. Its identity is unknown but it may represent a precursor of dUTPase or the product of a separate gene located between dut and pyrE.

Nucleotide sequence of the structural gene for dUTPase of Escherichia coli K-12.

The nucleotide sequence of the dUTPase structural gene, dut, of Escherichia coli has been determined. The DNA sequence predicts a polypeptide chain of 150 amino acid residues (mol. wt. 16 006) corresponding in size and composition to the purified dUTPase subunit. In a tentative promoter region preceding the dut gene, the -35 and -10 regions are separated by a SacI (SstI) site. Cloning of the dut gene utilization this SacI site was previously shown to reduce dut expression dramatically. The nucleotide sequence also contains a 210-codon open reading frame 106 bp downstream of dut and co-directional with dut. Previous protein synthesis experiments using dut plasmids allocated the gene of a polypeptide of mol. wt. 23 500 to this DNA region. The open reading frame thus may correspond to a protein of unknown function co-transcribed with the dut gene.

Bacteriophage T4-induced shut-off of host-specific translation.

To study the mechanism by which bacteriophage T4 inhibits the synthesis of inducible host enzymes we measured the formation of beta-galactosidase from preformed lac mRNA. Beta-Galactosidase was induced with isopropyl-beta-D-thiogalactopyranoside in the presence of 7-azatryptophan, a tryptophan analogue that is incorporated into proteins and renders the beta-galactosidase formed inactive. The accumulated las mRNA was measured by capacity to form active beta-galactosidase after a chase of the analogue with excess tryptophan. After T4 infection the ability to form beta-galactosidase from the preformed lac mRNA was rapidly lost even when T4 infection took place in the presence of rifampin. This restriction was dependent on the multiplicity of infection. At a multiplicity of infection of 8.6, 90% of the ability to express preformed lac mRNA was lost within 30 s. The kinetics of cessation of beta-galactosidase synthesis after T4 infection indicate that infection blocks initiation of lac mRNA translation.