Abstract
No systems have been reported for genetic manipulation of cold-adapted Archaea. Halorubrum lacusprofundi is an important member of Deep Lake, Antarctica (~10% of the population), and is amendable to laboratory cultivation. Here we report the development of a shuttle-vector and targeted gene-knockout system for this species. To investigate the function of acetamidase/formamidase genes, a class of genes not experimentally studied in Archaea, the acetamidase gene, amd3, was disrupted. The wild-type grew on acetamide as a sole source of carbon and nitrogen, but the mutant did not. Acetamidase/formamidase genes were found to form three distinct clades within a broad distribution of Archaea and Bacteria. Genes were present within lineages characterized by aerobic growth in low nutrient environments (e.g. haloarchaea, Starkeya) but absent from lineages containing anaerobes or facultative anaerobes (e.g. methanogens, Epsilonproteobacteria) or parasites of animals and plants (e.g. Chlamydiae). While acetamide is not a well characterized natural substrate, the build-up of plastic pollutants in the environment provides a potential source of introduced acetamide. In view of the extent and pattern of distribution of acetamidase/formamidase sequences within Archaea and Bacteria, we speculate that acetamide from plastics may promote the selection of amd/fmd genes in an increasing number of environmental microorganisms.
Highlights
No systems have been reported for genetic manipulation of cold-adapted Archaea
Mevinolin, which is derived from the fungus Aspergillus, inhibits 3-hydroxy-3-methylglutaryl coenzyme A reductase (HmgA), which is an essential enzyme in the synthesis of isoprenoid lipids in Archaea
We report the first procedure for performing gene transfer and gene knockouts for a psychrophilic member of the Archaea
Summary
No systems have been reported for genetic manipulation of cold-adapted Archaea. Halorubrum lacusprofundi is an important member of Deep Lake, Antarctica (~10% of the population), and is amendable to laboratory cultivation. Transformation systems for haloarchaea were developed in the late 1980s25, largely built around the well-studied species, Haloferax volcanii and Halobacterium salinarum[26,27,28,29], leading to the development of molecular genetic tools including selectable markers[27,30,31,32,33], shuttle vectors[30,34,35,36,37,38,39,40], reporter constructs[41,42,43,44], overexpression systems[40], and gene knockout systems[28,29,31,32]. Overexpression of the gene that encodes HmgA (hmgA) can provide resistance to the statins fluvastatin, simvastatin and pravastatin[33]
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