Disruption of a sugar transporter gene cluster in a hyperthermophilic archaeon using a host-marker system based on antibiotic resistance

Rie Matsumi, Kenji Manabe, Toshiaki Fukui, Haruyuki Atomi, Tadayuki Imanaka

Research output: Contribution to journalArticle

75 Citations (Scopus)

Abstract

We have developed a gene disruption system in the hyperthermophilic archaeon Thermococcus kodakaraensis using the antibiotic simvastatin and a fusion gene designed to overexpress the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene (hmgTk) with the glutamate dehydrogenase promoter. With this system, we disrupted the T. kodakaraensis amylopullulanase gene (apuTk) or a gene cluster which includes apuTk and genes encoding components of a putative sugar transporter. Disruption plasmids were introduced into wild-type T. kodakaraensis KOD1 cells, and transformants exhibiting resistance to 4 μM simvastatin were isolated. The transformants exhibited growth in the presence of 20 μM simvastatin, and we observed a 30-fold increase in intracellular HMG-CoA reductase activity. The expected gene disruption via double-crossover recombination occurred at the target locus, but we also observed recombination events at the hmgTk locus when the endogenous hmgTk gene was used. This could be avoided by using the corresponding gene from Pyrococcus furiosus (hmgpf) or by linearizing the plasmid prior to transformation. While both gene disruption strains displayed normal growth on amino acids or pyruvate, cells without the sugar transporter genes could not grow on maltooligosaccharides or polysaccharides, indicating that the gene cluster encodes the only sugar transporter involved in the uptake of these compounds. The ΔapuTk strain could not grow on pullulan and displayed only low levels of growth on amylose, suggesting that ApuTk is a major polysaccharide-degrading enzyme in T. kodakaraensis.

Original languageEnglish
Pages (from-to)2683-2691
Number of pages9
JournalJournal of bacteriology
Volume189
Issue number7
DOIs
Publication statusPublished - Apr 1 2007

Fingerprint

Archaea
Multigene Family
Microbial Drug Resistance
Genes
Simvastatin
Genetic Recombination
Polysaccharides
Oxidoreductases
Plasmids
Growth
Thermococcus
Pyrococcus furiosus
Gene Components
Glutamate Dehydrogenase
Amylose
Gene Fusion
Coenzyme A
Pyruvic Acid
Anti-Bacterial Agents
Amino Acids

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology

Cite this

Disruption of a sugar transporter gene cluster in a hyperthermophilic archaeon using a host-marker system based on antibiotic resistance. / Matsumi, Rie; Manabe, Kenji; Fukui, Toshiaki; Atomi, Haruyuki; Imanaka, Tadayuki.

In: Journal of bacteriology, Vol. 189, No. 7, 01.04.2007, p. 2683-2691.

Research output: Contribution to journalArticle

Matsumi, Rie ; Manabe, Kenji ; Fukui, Toshiaki ; Atomi, Haruyuki ; Imanaka, Tadayuki. / Disruption of a sugar transporter gene cluster in a hyperthermophilic archaeon using a host-marker system based on antibiotic resistance. In: Journal of bacteriology. 2007 ; Vol. 189, No. 7. pp. 2683-2691.
@article{5a4af4590d454d00a6af9479bad11783,
title = "Disruption of a sugar transporter gene cluster in a hyperthermophilic archaeon using a host-marker system based on antibiotic resistance",
abstract = "We have developed a gene disruption system in the hyperthermophilic archaeon Thermococcus kodakaraensis using the antibiotic simvastatin and a fusion gene designed to overexpress the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene (hmgTk) with the glutamate dehydrogenase promoter. With this system, we disrupted the T. kodakaraensis amylopullulanase gene (apuTk) or a gene cluster which includes apuTk and genes encoding components of a putative sugar transporter. Disruption plasmids were introduced into wild-type T. kodakaraensis KOD1 cells, and transformants exhibiting resistance to 4 μM simvastatin were isolated. The transformants exhibited growth in the presence of 20 μM simvastatin, and we observed a 30-fold increase in intracellular HMG-CoA reductase activity. The expected gene disruption via double-crossover recombination occurred at the target locus, but we also observed recombination events at the hmgTk locus when the endogenous hmgTk gene was used. This could be avoided by using the corresponding gene from Pyrococcus furiosus (hmgpf) or by linearizing the plasmid prior to transformation. While both gene disruption strains displayed normal growth on amino acids or pyruvate, cells without the sugar transporter genes could not grow on maltooligosaccharides or polysaccharides, indicating that the gene cluster encodes the only sugar transporter involved in the uptake of these compounds. The ΔapuTk strain could not grow on pullulan and displayed only low levels of growth on amylose, suggesting that ApuTk is a major polysaccharide-degrading enzyme in T. kodakaraensis.",
author = "Rie Matsumi and Kenji Manabe and Toshiaki Fukui and Haruyuki Atomi and Tadayuki Imanaka",
year = "2007",
month = "4",
day = "1",
doi = "10.1128/JB.01692-06",
language = "English",
volume = "189",
pages = "2683--2691",
journal = "Journal of Bacteriology",
issn = "0021-9193",
publisher = "American Society for Microbiology",
number = "7",

}

TY - JOUR

T1 - Disruption of a sugar transporter gene cluster in a hyperthermophilic archaeon using a host-marker system based on antibiotic resistance

AU - Matsumi, Rie

AU - Manabe, Kenji

AU - Fukui, Toshiaki

AU - Atomi, Haruyuki

AU - Imanaka, Tadayuki

PY - 2007/4/1

Y1 - 2007/4/1

N2 - We have developed a gene disruption system in the hyperthermophilic archaeon Thermococcus kodakaraensis using the antibiotic simvastatin and a fusion gene designed to overexpress the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene (hmgTk) with the glutamate dehydrogenase promoter. With this system, we disrupted the T. kodakaraensis amylopullulanase gene (apuTk) or a gene cluster which includes apuTk and genes encoding components of a putative sugar transporter. Disruption plasmids were introduced into wild-type T. kodakaraensis KOD1 cells, and transformants exhibiting resistance to 4 μM simvastatin were isolated. The transformants exhibited growth in the presence of 20 μM simvastatin, and we observed a 30-fold increase in intracellular HMG-CoA reductase activity. The expected gene disruption via double-crossover recombination occurred at the target locus, but we also observed recombination events at the hmgTk locus when the endogenous hmgTk gene was used. This could be avoided by using the corresponding gene from Pyrococcus furiosus (hmgpf) or by linearizing the plasmid prior to transformation. While both gene disruption strains displayed normal growth on amino acids or pyruvate, cells without the sugar transporter genes could not grow on maltooligosaccharides or polysaccharides, indicating that the gene cluster encodes the only sugar transporter involved in the uptake of these compounds. The ΔapuTk strain could not grow on pullulan and displayed only low levels of growth on amylose, suggesting that ApuTk is a major polysaccharide-degrading enzyme in T. kodakaraensis.

AB - We have developed a gene disruption system in the hyperthermophilic archaeon Thermococcus kodakaraensis using the antibiotic simvastatin and a fusion gene designed to overexpress the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene (hmgTk) with the glutamate dehydrogenase promoter. With this system, we disrupted the T. kodakaraensis amylopullulanase gene (apuTk) or a gene cluster which includes apuTk and genes encoding components of a putative sugar transporter. Disruption plasmids were introduced into wild-type T. kodakaraensis KOD1 cells, and transformants exhibiting resistance to 4 μM simvastatin were isolated. The transformants exhibited growth in the presence of 20 μM simvastatin, and we observed a 30-fold increase in intracellular HMG-CoA reductase activity. The expected gene disruption via double-crossover recombination occurred at the target locus, but we also observed recombination events at the hmgTk locus when the endogenous hmgTk gene was used. This could be avoided by using the corresponding gene from Pyrococcus furiosus (hmgpf) or by linearizing the plasmid prior to transformation. While both gene disruption strains displayed normal growth on amino acids or pyruvate, cells without the sugar transporter genes could not grow on maltooligosaccharides or polysaccharides, indicating that the gene cluster encodes the only sugar transporter involved in the uptake of these compounds. The ΔapuTk strain could not grow on pullulan and displayed only low levels of growth on amylose, suggesting that ApuTk is a major polysaccharide-degrading enzyme in T. kodakaraensis.

UR - http://www.scopus.com/inward/record.url?scp=34147158720&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=34147158720&partnerID=8YFLogxK

U2 - 10.1128/JB.01692-06

DO - 10.1128/JB.01692-06

M3 - Article

C2 - 17259314

AN - SCOPUS:34147158720

VL - 189

SP - 2683

EP - 2691

JO - Journal of Bacteriology

JF - Journal of Bacteriology

SN - 0021-9193

IS - 7

ER -