A Chromosome Integration System for Stable Gene Transfer into Thermus flavus

Weber, Jakob; Johnson, S P; Vonstein, V; Casadaban, Malcolm J.; Demirjian, David C.

doi:10.1038/nbt0395-271

Cited by 14 publications

(13 citation statements)

References 27 publications

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“…At the same time, Malcolm was engaged in founding and developing a biotechnology company, ThermoGen Inc., exploring the genes of the archaeon Thermus flavus for thermostable enzymes with industrial uses (40,41). This work was carried forward by David C. Demirjian, Malcolm's last Ph.D. student.…”

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confidence: 99%

Remembering Malcolm J. Casadaban

2010

J Bacteriol

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Malcolm J. Casadaban died on 13 September 2009 from an infection and was found to have a weakened strain of the bacterium Yersinia pestis in his blood. This tragic event took the life of one of the most creative and influential geneticists of our time. In the late 1970s and '80s, Malcolm invented novel approaches which changed the way many of us did science. Jon Beckwith, Tom Silhavy, and Olaf Schneewind have chronicled his scientific life from graduate school to his death and give us insight into Malcolm's genius. Philip Matsumura Editor in Chief, Journal of Bacteriology

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confidence: 99%

Remembering Malcolm J. Casadaban

2010

J Bacteriol

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“…This species belongs to the proposed family "Rhodothermaceae" of the phylum Bacteroidetes (13) and has no close relatives for which genetic methodologies have been developed. The thermoadapted kanamycin resistance determinant frequently used for selection in thermophiles (11,24) is unsuitable for R. marinus because of its natural resistance to aminoglycosides (1). Therefore, an endogenous marker was chosen, the R. marinus trpB gene.…”

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Generation of Targeted Deletions in the Genome of Rhodothermus marinus

Björnsdóttir

Friðjónsson

Hreggviðsson

et al. 2011

Appl Environ Microbiol

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The aim of this work was to develop an approach for chromosomal engineering of the thermophile Rhodothermus marinus. A selection strategy for R. marinus had previously been developed; this strategy was based on complementing a restriction-negative trpB strain with the R. marinus trpB gene. The current work identified an additional selective marker, purA, which encodes adenylosuccinate synthase and confers adenine prototrophy. In a two-step procedure, the available Trp ؉ selection was used during the deletion of purA from the R. marinus chromosome. The alternative Ade ؉ selection was in turn used while deleting the endogenous trpB gene. Since both deletions are unmarked, the purA and trpB markers may be reused. Through the double deletant SB-62 (⌬trpB ⌬purA), the difficulties that are associated with spontaneous revertants and unintended chromosomal integration of marker-containing molecules are circumvented. The selection efficiency in R. marinus strain SB-62 (⌬trpB ⌬purA) was demonstrated by targeting putative carotenoid biosynthesis genes, crtBI, using a linear molecule containing a marked deletion with 717 and 810 bp of 5 and 3 homologous sequences, respectively. The resulting Trp ؉ transformants were colorless rather than orange-red. The correct replacement of an internal crtBI fragment with the trpB marker was confirmed by Southern hybridization analysis of the transformants. Thus, it appears that target genes in the R. marinus chromosome can be readily replaced with linear molecules in a single step by double-crossover recombination.

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“…They possess a natural transformation system (17) and are competent regardless of their growth phase (12). Genetic systems based on the application of autonomously replicating plasmids, as well as integration vectors or vectors containing cassettes, for chromosomal integration have been established (13,18,22,23,35,40). So far, the only antibiotic selection markers described for Thermus bacteria are thermostabilized variants of the kanamycin nucleotidyltransferase gene derived from a thermophilic Bacillus gene (28) or from the kan gene of Staphylococcus aureus (24).…”

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Production of Recombinant α-Galactosidases in Thermus thermophilus

Friðjónsson¹,

Mattes

2001

Appl Environ Microbiol

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A Thermus thermophilus selector strain for production of thermostable and thermoactive ␣-galactosidase was constructed. For this purpose, the native ␣-galactosidase gene (agaT) of T. thermophilus TH125 was inactivated to prevent background activity. In our first attempt, insertional mutagenesis of agaT by using a cassette carrying a kanamycin resistance gene led to bacterial inability to utilize melibiose (␣-galactoside) and galactose as sole carbohydrate sources due to a polar effect of the insertional inactivation. A Gal ؉ phenotype was assumed to be essential for growth on melibiose. In a Gal ؊ background, accumulation of galactose or its metabolite derivatives produced from melibiose hydrolysis could interfere with the growth of the host strain harboring recombinant ␣-galactosidase. Moreover, the AgaT ؊ strain had to be Km s for establishment of the plasmids containing ␣-galactosidase genes and the kanamycin resistance marker. Therefore, a suitable selector strain (AgaT ؊ Gal ؉ Km s ) was generated by applying integration mutagenesis in combination with phenotypic selection. To produce heterologous ␣-galactosidase in T. thermophilus, the isogenes agaA and agaB of Bacillus stearothermophilus KVE36 were cloned into an Escherichia coli-Thermus shuttle vector. The region containing the E. coli plasmid sequence (pUC-derived vector) was deleted before transformation of T. thermophilus with the recombinant plasmids. As a result, transformation efficiency and plasmid stability were improved. However, growth on minimal agar medium containing melibiose was achieved only following random selection of the clones carrying a plasmid-based mutation that had promoted a higher copy number and greater stability of the plasmid.␣-Galactosidases catalyze the hydrolysis of ␣-1,6-linked ␣-galactose residues from oligosaccharides and polymeric galactomannans (9,25,26,42). They have considerable potential in various industrial applications, e.g., in the sugar industry for the elimination of D-raffinose from sugar beet syrup. Due to the elevated temperatures used during the sugar manufacturing process, as well as in other industrial applications, stability and activity at high temperatures are important properties of ␣-galactosidases.We have been studying ␣-galactosidases from various bacteria with regard to their application as oligosaccharidehydrolyzing enzymes (9,11,14). Our intention was to subject ␣-galactosidase to thermoadaptation by introducing genes encoding enzymes inactive at high temperatures into a thermophilic bacterium for subsequent selection of enzyme variants active at high temperatures. We chose Thermus thermophilus as a host due to its high transformation ability (17) and ability to use melibiose (␣-galactoside) as a sole carbohydrate source (10). Thermus species have been used for expression of heterologous genes and selection of thermostable enzyme variants (16,19,34,36). They possess a natural transformation system (17) and are competent regardless of their growth phase (12). Genetic systems based on the applic...

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A Chromosome Integration System for Stable Gene Transfer into Thermus flavus

Cited by 14 publications

References 27 publications

Remembering Malcolm J. Casadaban

Remembering Malcolm J. Casadaban

Generation of Targeted Deletions in the Genome of Rhodothermus marinus

Production of Recombinant α-Galactosidases in Thermus thermophilus

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