title: Developing a genetic manipulation system for the Antarctic archaeon, Halorubrum lacusprofundi: investigating acetamidase gene function.
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.
authors.affiliation: School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, New South Wales, 2052, Australia.
authors.forename: Y
authors.initials: Y
authors.lastname: Liao
authors.affiliation: School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, New South Wales, 2052, Australia.
authors.forename: T J
authors.initials: TJ
authors.lastname: Williams
authors.affiliation: School of Physics, The University of New South Wales, Sydney, New South Wales, 2052, Australia.|The ithree institute, University of Technology Sydney, Broadway, New South Wales, 2007, Australia.
authors.forename: J C
authors.initials: JC
authors.lastname: Walsh
authors.affiliation: School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, New South Wales, 2052, Australia.
authors.forename: M
authors.initials: M
authors.lastname: Ji
authors.affiliation: Bioanalytical Mass Spectrometry Facility, The University of New South Wales, Sydney, New South Wales, Australia.
authors.forename: A
authors.initials: A
authors.lastname: Poljak
authors.affiliation: School of Physics, The University of New South Wales, Sydney, New South Wales, 2052, Australia.
authors.forename: P M G
authors.initials: PM
authors.lastname: Curmi
authors.affiliation: The ithree institute, University of Technology Sydney, Broadway, New South Wales, 2007, Australia.
authors.forename: I G
authors.initials: IG
authors.lastname: Duggin
authors.affiliation: School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, New South Wales, 2052, Australia.
authors.forename: R
authors.initials: R
authors.lastname: Cavicchioli
mesh_terms: D000581:Amidohydrolases / Q000172:deficiency / Q000235:genetics*; D000864:Antarctic Regions; D019843:Archaeal Proteins / Q000235:genetics* / Q000378:metabolism; D001673:Biodegradation, Environmental; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D017353:Gene Deletion; D019848:Gene Expression Regulation, Archaeal; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000737:chemistry* / Q000378:metabolism; D055352:Halorubrum / Q000145:classification / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D010802:Phylogeny; D010969:Plastics / Q000378:metabolism; D015183:Restriction Mapping; D014170:Transformation, Genetic; D014874:Water Pollutants, Chemical / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D003470:Culture Media; D010969:Plastics; D014874:Water Pollutants, Chemical; D000581:Amidohydrolases; C021445:acetamidase; C101846:formamidase