Chromosomal gene inactivation in the green sulfur bacterium Chlorobium tepidum by natural transformation

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Chromosomal gene inactivation in the green sulfur bacterium Chlorobium tepidum by natural transformation. / Frigaard, N-U; Bryant, D A.

In: Applied and Environmental Microbiology, Vol. 67, No. 6, 2001, p. 2538-2544.

Research output: Contribution to journal › Journal article › Research › peer-review

Harvard

Frigaard, N-U & Bryant, DA 2001, 'Chromosomal gene inactivation in the green sulfur bacterium Chlorobium tepidum by natural transformation', Applied and Environmental Microbiology, vol. 67, no. 6, pp. 2538-2544. https://doi.org/10.1128/AEM.67.6.2538-2544.2001

APA

Frigaard, N-U., & Bryant, D. A. (2001). Chromosomal gene inactivation in the green sulfur bacterium Chlorobium tepidum by natural transformation. Applied and Environmental Microbiology, 67(6), 2538-2544. https://doi.org/10.1128/AEM.67.6.2538-2544.2001

Vancouver

Frigaard N-U, Bryant DA. Chromosomal gene inactivation in the green sulfur bacterium Chlorobium tepidum by natural transformation. Applied and Environmental Microbiology. 2001;67(6):2538-2544. https://doi.org/10.1128/AEM.67.6.2538-2544.2001

Author

Frigaard, N-U ; Bryant, D A. / Chromosomal gene inactivation in the green sulfur bacterium Chlorobium tepidum by natural transformation. In: Applied and Environmental Microbiology. 2001 ; Vol. 67, No. 6. pp. 2538-2544.

Bibtex

@article{32f64ea0962611de8bc9000ea68e967b,
title = "Chromosomal gene inactivation in the green sulfur bacterium Chlorobium tepidum by natural transformation",
abstract = "Conditions for inactivating chromosomal genes of Chlorobium tepidum by natural transformation and homologous recombination were established. As a model, mutants unable to perform nitrogen fixation were constructed by interrupting nifD with various antibiotic resistance markers. Growth of wild-type C. tepidum at 40 degrees C on agar plates could be completely inhibited by 100 microg of gentamicin ml(-1), 2 microg of erythromycin ml(-1), 30 microg of chloramphenicol ml(-1), or 1 microg of tetracycline ml(-1) or a combination of 300 microg of streptomycin ml(-1) and 150 microg of spectinomycin ml(-1). Transformation was performed by spotting cells and DNA on an agar plate for 10 to 20 h. Transformation frequencies on the order of 10(-7) were observed with gentamicin and erythromycin markers, and transformation frequencies on the order of 10(-3) were observed with a streptomycin-spectinomycin marker. The frequency of spontaneous mutants resistant to gentamicin, erythromycin, or spectinomycin-streptomycin was undetectable or significantly lower than the transformation frequency. Transformation with the gentamicin marker was observed when the transforming DNA contained 1 or 3 kb of total homologous flanking sequence but not when the transforming DNA contained only 0.3 kb of homologous sequence. Linearized plasmids transformed at least an order of magnitude better than circular plasmids. This work forms a foundation for the systematic targeted inactivation of genes in C. tepidum, whose 2.15-Mb genome has recently been completely sequenced.",
author = "N-U Frigaard and Bryant, {D A}",
note = "Keywords: Chlorobi; Chromosomes, Bacterial; Drug Resistance, Microbial; Gene Silencing; Genes, Bacterial; Genetic Markers; Nitrogen Fixation; Recombination, Genetic; Selection (Genetics); Transformation, Bacterial",
year = "2001",
doi = "10.1128/AEM.67.6.2538-2544.2001",
language = "English",
volume = "67",
pages = "2538--2544",
journal = "Applied and Environmental Microbiology",
issn = "0099-2240",
publisher = "American Society for Microbiology",
number = "6",

}

RIS

TY - JOUR

T1 - Chromosomal gene inactivation in the green sulfur bacterium Chlorobium tepidum by natural transformation

AU - Frigaard, N-U

AU - Bryant, D A

N1 - Keywords: Chlorobi; Chromosomes, Bacterial; Drug Resistance, Microbial; Gene Silencing; Genes, Bacterial; Genetic Markers; Nitrogen Fixation; Recombination, Genetic; Selection (Genetics); Transformation, Bacterial

PY - 2001

Y1 - 2001

N2 - Conditions for inactivating chromosomal genes of Chlorobium tepidum by natural transformation and homologous recombination were established. As a model, mutants unable to perform nitrogen fixation were constructed by interrupting nifD with various antibiotic resistance markers. Growth of wild-type C. tepidum at 40 degrees C on agar plates could be completely inhibited by 100 microg of gentamicin ml(-1), 2 microg of erythromycin ml(-1), 30 microg of chloramphenicol ml(-1), or 1 microg of tetracycline ml(-1) or a combination of 300 microg of streptomycin ml(-1) and 150 microg of spectinomycin ml(-1). Transformation was performed by spotting cells and DNA on an agar plate for 10 to 20 h. Transformation frequencies on the order of 10(-7) were observed with gentamicin and erythromycin markers, and transformation frequencies on the order of 10(-3) were observed with a streptomycin-spectinomycin marker. The frequency of spontaneous mutants resistant to gentamicin, erythromycin, or spectinomycin-streptomycin was undetectable or significantly lower than the transformation frequency. Transformation with the gentamicin marker was observed when the transforming DNA contained 1 or 3 kb of total homologous flanking sequence but not when the transforming DNA contained only 0.3 kb of homologous sequence. Linearized plasmids transformed at least an order of magnitude better than circular plasmids. This work forms a foundation for the systematic targeted inactivation of genes in C. tepidum, whose 2.15-Mb genome has recently been completely sequenced.

AB - Conditions for inactivating chromosomal genes of Chlorobium tepidum by natural transformation and homologous recombination were established. As a model, mutants unable to perform nitrogen fixation were constructed by interrupting nifD with various antibiotic resistance markers. Growth of wild-type C. tepidum at 40 degrees C on agar plates could be completely inhibited by 100 microg of gentamicin ml(-1), 2 microg of erythromycin ml(-1), 30 microg of chloramphenicol ml(-1), or 1 microg of tetracycline ml(-1) or a combination of 300 microg of streptomycin ml(-1) and 150 microg of spectinomycin ml(-1). Transformation was performed by spotting cells and DNA on an agar plate for 10 to 20 h. Transformation frequencies on the order of 10(-7) were observed with gentamicin and erythromycin markers, and transformation frequencies on the order of 10(-3) were observed with a streptomycin-spectinomycin marker. The frequency of spontaneous mutants resistant to gentamicin, erythromycin, or spectinomycin-streptomycin was undetectable or significantly lower than the transformation frequency. Transformation with the gentamicin marker was observed when the transforming DNA contained 1 or 3 kb of total homologous flanking sequence but not when the transforming DNA contained only 0.3 kb of homologous sequence. Linearized plasmids transformed at least an order of magnitude better than circular plasmids. This work forms a foundation for the systematic targeted inactivation of genes in C. tepidum, whose 2.15-Mb genome has recently been completely sequenced.

U2 - 10.1128/AEM.67.6.2538-2544.2001

DO - 10.1128/AEM.67.6.2538-2544.2001

M3 - Journal article

C2 - 11375161

VL - 67

SP - 2538

EP - 2544

JO - Applied and Environmental Microbiology

JF - Applied and Environmental Microbiology

SN - 0099-2240

IS - 6

ER -

ID: 14095658