Genetic manipulation of Kluyveromyces lactis linear DNA plasmids: gene targeting and plasmid shuffles

Abstract

Genetic manipulation of yeast linear DNA plasmids, particularly of k1 and k2 from the non-conventional dairy yeast Kluyveromyces lactis, has been advanced by the recent establishment of DNA transformation-mediated one-step gene disruption and allele replacement techniques. These methods provide the basis for a strategy for the functional analysis of plasmid genes and DNA elements. By use of double selection regimens, these single-gene procedures have been extended to effect disruption of individual genes on plasmid k2 and transplacement of a functional copy onto plasmid k1, resulting in the production of yeast strains with an altered plasmid composition. This cytoplasmic gene shuffle system facilitates the introduction of specifically modified alleles into k1 or k2 in order to study the function, expression (from UCS promoters) and regulation of cytoplasmic linear plasmid genes. Additionally, identification, characterization and localization of plasmid gene products of interest are made possible by shuffling GFP-, epitope-or affinity purification-tagged alleles between k2 and k1. The gene shuffle approach can also be used for vector development and heterologous protein expression in order to exploit the biotechnical potential of the K. lactis k1/k2 system in yeast cell factory research. ß

Self-synthesizing DNA transposons in eukaryotes

“…As we reported here, Polintons share their main structural characteristics with ''selfish'' linear plasmids, bacteriophages, and adenoviruses that multiply by using their protein-primed DNA polymerases. Linear plasmids can be split into two groups: (i), plasmids that exist in mitochondria of plants and fungi and (ii), plasmids that exist in the yeast cytoplasm (37). Although it is likely that mitochondrial linear plasmids evolved from bacteriophages during the evolution of mitochondria from bacteria, understanding the evolution of cytoplasmic plasmids is hampered by different equally plausible scenarios (38,39).…”

Diphtheria Disease and Genes Involved in Formation of Diphthamide, Key Effector of the Diphtheria Toxin

“…Prominent examples for phage-dependent toxin expression and disease formation include exotoxin-associated scarlet fever by Streptocoocus pyogenes (Johnson et al, 1986;Broudy et al, 2001), dysentery causing Shiga toxins Stx1 and Stx2 from Shigella dysenteriae (Newland et al, 1985;Willshaw et al, 1985;Huang, et al, 1986), phage CTXφ encoded cholera toxin from Vibrio cholera (Waldor & Mekalanos, 1996;Faruque & Nair, 2002) and diphtheria toxin encoded on a beta prophage from lysogens of Corynebacterium diphtheriae (Holmes & Barksdale, 1969;Bishai & Murphy 1988). Together 334 with tRNase ribotoxins and anticodon nucleases from prokaryal and eukaryal microbes that have been shown to be encoded by transposable elements as well as circular and nonconventional linear DNA plasmids (Tokunaga et al, 1990;Kaufmann, 2000;Schaffrath & Meinhardt, 2005;Schaffrath et al, 1999), all of these genetic constellations implicate scenarios in which killer phenotypes have been evolved and spread by way of viral DNA transduction pathways or other forms of horizontal gene transfer. In support of this notion, certain cytoplasmic killer plasmids and their associated toxin phenotypes can be transferred between distinct yeast genera by means of cytoduction (Gunge 1983;Sugisaki et al, 1985) and horizontal transfer of the diphtheria toxin encoding tox gene from phages has been assigned to in situ lysogenic conversion of non-toxigenic to virulent corynebacteria (Freeman, 1951).…”

Kluyveromyces lactis cytoplasmic plasmid pGKL2: heterologous expression of Orf3p and proof of guanylyltransferase and mRNA–triphosphatase activities

“…In contrast to plants and filamentous fungi, almost all of the yeast linear dsDNA elements are cytoplasmically localized; they have been isolated from numerous genera, such as Botryoascus, Debaryomyces, Kluyveromyces, Pichia, Saccharomyces, Trichosporon and Wingea (Cong et al, 1994;Fukuhara, 1995). The genetic organization of yeast linear elements appeared to be quite uniform (Hishinuma and Hirai, 1991;Klassen, 2001) with the killer plasmid pair pGKL1 and pGKL2 of Kluyveromyces lactis (also referred to as k1 and k2) the most thoroughly characterized Gunge, 1995;Schaffrath et al, 1999;Meinhardt and Schaffrath, 2001). K. lactis cells containing pGKL1 and pGKL2 differ from plasmid-less cells by their ability to kill sensitive yeasts belonging either to the same or a different species .…”