Affiliation:
1. Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada
Abstract
ABSTRACT
We have assembled a collection of 13 psychrophilic
ligA
alleles that can serve as genetic elements for engineering mesophiles to a temperature-sensitive (TS) phenotype. When these
ligA
alleles were substituted into
Francisella novicida
, they conferred a TS phenotype with restrictive temperatures between 33 and 39°C. When the
F. novicida
ligA
hybrid 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. Four
F. novicida ligA
hybrid strains generated no temperature-resistant variants at a detectable level. These results suggest that multiple mutations are needed to create temperature-resistant variants of these
ligA
gene products. One
ligA
allele was isolated from a
Colwellia
species that has a maximal growth temperature of 12°C, and this allele supported growth of
F. novicida
only as a hybrid between the psychrophilic and the
F. novicida
ligA
genes. However, the full psychrophilic gene alone supported the growth of
Salmonella enterica
, imparting a restrictive temperature of 27°C. We also tested two
ligA
alleles from two
Pseudoalteromonas
strains for their ability to support the viability of a
Saccharomyces cerevisiae
strain 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 of
ligA
alleles should be useful in engineering bacteria, and possibly eukaryotic microbes, to predictable TS phenotypes.
Funder
Gouvernement du Canada | Natural Sciences and Engineering Research Council of Canada
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Cited by
3 articles.
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