Synthetic temperature-inducible lethal gene circuits in Escherichia coli.

Temperature sensitivity is often used as a way to attenuate micro-organisms to convert them into live vaccines. In this work, we explore the use of temperature-sensitive (TS) genetic circuits that express lethal genes as a widely applicable approach to TS attenuation. We tested different combinations of TS repressors and cognate promoters controlling the expression of genes encoding restriction endonucleases inserted at four different non-essential sites in the Escherichia coli chromosome. We found that the presence of the restriction endonuclease genes did not affect the viability of the host strains at the permissive temperature, but that expression of the genes at elevated temperatures killed the strains to varying extents. The chromosomal insertion site of the lethal cassettes affected their functionality, and insertion at one site, ycgH, rendered them ineffective at inducing death at high temperature. Induction of a TS circuit in a growing culture led to a reduced cell mass and a reduction of the number of cells that could exclude a dye that indicated viability. Incubation of cells carrying a TS lethal gene circuit initially grown at low temperature and then suspended in phosphate buffered saline at high temperature led to about 100-fold loss of cell viability per day, compared to a minimal loss of viability for the parental strain. Strains carrying either one or two TS lethal circuits could generate mutants that survived at high temperature. These mutants included complete deletions of the lethal gene circuits.

Temperature-Sensitive Salmonella enterica Serovar Enteritidis PT13a Expressing Essential Proteins of Psychrophilic Bacteria.

Synthetic genes based on deduced amino acid sequences of the NAD-dependent DNA ligase (ligA) and CTP synthetase (pyrG) of psychrophilic bacteria were substituted for their native homologues in the genome of Salmonella enterica serovar Enteritidis phage type 13a (PT13a). The resulting strains were rendered temperature sensitive (TS) and did not revert to temperature resistance at a detectable level. At permissive temperatures, TS strains grew like the parental strain in broth medium and in macrophage-like cells, but their growth was slowed or stopped when they were shifted to a restrictive temperature. When injected into BALB/c mice at the base of the tail, representing a cool site of the body, the strains with restrictive temperatures of 37, 38.5, and 39°C persisted for less than 1 day, 4 to 7 days, and 20 to 28 days, respectively. The wild-type strain persisted at the site of inoculation for at least 28 days. The wild-type strain, but not the TS strains, was also found in spleen-plus-liver homogenates within 1 day of inoculation of the tail and was detectable in these organs for at least 28 days. Intramuscular vaccination of White Leghorn chickens with the PT13a strain carrying the psychrophilic pyrG gene provided some protection against colonization of the reproductive tract and induced an anti-S. enterica antibody response.