Temperature Sensitivity Conferred byligAAlleles from Psychrophilic Bacteria upon Substitution in Mesophilic Bacteria and a Yeast Species

We have assembled a collection of 13 psychrophilicligAalleles that can serve as genetic elements for engineering mesophiles to a temperature-sensitive (TS) phenotype. When theseligAalleles were substituted intoFrancisella novicida, they conferred a TS phenotype with restrictive temperatures between 33 and 39°C. When theF. novicidaligAhybrid strains were plated above their restrictive temperatures, eight of them generated temperature-resistant variants. For two alleles, the mutations that led to temperature resistance clustered near the 5′ end of the gene, and the mutations increased the predicted strength of the ribosome binding site at least 3-fold. FourF. novicida ligAhybrid strains generated no temperature-resistant variants at a detectable level. These results suggest that multiple mutations are needed to create temperature-resistant variants of theseligAgene products. OneligAallele was isolated from aColwelliaspecies that has a maximal growth temperature of 12°C, and this allele supported growth ofF. novicidaonly as a hybrid between the psychrophilic and theF. novicidaligAgenes. However, the full psychrophilic gene alone supported the growth ofSalmonella enterica, imparting a restrictive temperature of 27°C. We also tested twoligAalleles from twoPseudoalteromonasstrains for their ability to support the viability of aSaccharomyces cerevisiaestrain that lacked its essential gene,CDC9, encoding an ATP-dependent DNA ligase. In both cases, the psychrophilic bacterial alleles supported yeast viability and their expression generated TS phenotypes. This collection ofligAalleles should be useful in engineering bacteria, and possibly eukaryotic microbes, to predictable TS phenotypes.