Analysis of actinomycete communities by specific amplification
of genes encoding 16S rRNA and gel-electrophoretic separation
in denaturing gradients

J 1997

Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients

HEUER, H, Martin KRSEK, Patrick James BAKER, K SMALLA, EMH WELLINGTON et. al.

Základní údaje

Originální název

Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients

Autoři

HEUER, H, Martin KRSEK, Patrick James BAKER, K SMALLA a EMH WELLINGTON

Vydání

Applied and Environmental Microbiology, WASHINGTON, AMER SOC MICROBIOLOGY, 1997, 0099-2240

Další údaje

Jazyk

angličtina

Typ výsledku

Článek v odborném periodiku

Utajení

není předmětem státního či obchodního tajemství

Impakt faktor

Impact factor: 3.336

UT WoS

A1997XP06700042

Změněno: 8. 1. 2016 11:14, doc. Ing. Martin Krsek, CSc., MSc

Anotace

V originále

A group-specific primer, F243 (positions 226 to 243, Escherichia coli numbering), was developed by comparison of sequences of genes encoding 16S rRNA (16S rDNA) for the detection of actinomycetes in the environment with PCR and temperature or denaturing gradient gel electrophoresis (TGGE or DGGE, respectively). The specificity of the forward primer in combination with different reverse ones was tested with genomic DNA from a variety of bacterial strains. Most actinomycetes investigated could be separated by TGGE and DGGE, with both techniques giving similar results. Two strategies were employed to study natural microbial communities. First, we used the selective amplification of actinomycete sequences (E. coli positions 226 to 528) for direct analysis of the products in denaturing gradients. Second, a nested PCR providing actinomycete-specific fragments (E. coli positions 226 to 1401) was used which served as template for a PCR when conserved primers were used. The products (E. coli positions 968 to 1401) of this indirect approach were then separated by use of gradient gels. Both approaches allowed detection of actinomycete communities in soil. The second strategy allowed the estimation of the relative abundance of actinomycetes within the bacterial community. Mixtures of PCR-derived 16S rDNA fragments were used as model communities consisting of five actinomycetes and five other bacterial species. Actinomycete products were obtained over a 100-fold dilution range of the actinomycete DNA in the model community by specific PCR; detection of the diluted actinomycete DNA was not possible when conserved primers were used. The methods tested for detection were applied to monitor actinomycete community changes in potato rhizosphere and to investigate actinomycete diversity in different soils.

Citovat

HEUER, H, Martin KRSEK, Patrick James BAKER, K SMALLA a EMH WELLINGTON. Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients. Applied and Environmental Microbiology. WASHINGTON: AMER SOC MICROBIOLOGY, 1997, roč. 63, č. 8, s. 3233-3241. ISSN 0099-2240.

@article{1323297,
   author = {Heuer, H and Krsek, Martin and Baker, Patrick James and Smalla, K and Wellington, EMH},
   article_location = {WASHINGTON},
   article_number = {8},
   language = {eng},
   issn = {0099-2240},
   journal = {Applied and Environmental Microbiology},
   title = {Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients},
   volume = {63},
   year = {1997}
}

TY  - JOUR
ID  - 1323297
AU  - Heuer, H - Krsek, Martin - Baker, Patrick James - Smalla, K - Wellington, EMH
PY  - 1997
TI  - Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients
JF  - Applied and Environmental Microbiology
VL  - 63
IS  - 8
SP  - 3233-3241
EP  - 3233-3241
PB  - AMER SOC MICROBIOLOGY
SN  - 00992240
N2  - A group-specific primer, F243 (positions 226 to 243, Escherichia coli numbering), was developed by comparison of sequences of genes encoding 16S rRNA (16S rDNA) for the detection of actinomycetes in the environment with PCR and temperature or denaturing gradient gel electrophoresis (TGGE or DGGE, respectively). The specificity of the forward primer in combination with different reverse ones was tested with genomic DNA from a variety of bacterial strains. Most actinomycetes investigated could be separated by TGGE and DGGE, with both techniques giving similar results. Two strategies were employed to study natural microbial communities. First, we used the selective amplification of actinomycete sequences (E. coli positions 226 to 528) for direct analysis of the products in denaturing gradients. Second, a nested PCR providing actinomycete-specific fragments (E. coli positions 226 to 1401) was used which served as template for a PCR when conserved primers were used. The products (E. coli positions 968 to 1401) of this indirect approach were then separated by use of gradient gels. Both approaches allowed detection of actinomycete communities in soil. The second strategy allowed the estimation of the relative abundance of actinomycetes within the bacterial community. Mixtures of PCR-derived 16S rDNA fragments were used as model communities consisting of five actinomycetes and five other bacterial species. Actinomycete products were obtained over a 100-fold dilution range of the actinomycete DNA in the model community by specific PCR; detection of the diluted actinomycete DNA was not possible when conserved primers were used. The methods tested for detection were applied to monitor actinomycete community changes in potato rhizosphere and to investigate actinomycete diversity in different soils.
ER  -

HEUER, H, Martin KRSEK, Patrick James BAKER, K SMALLA a EMH WELLINGTON. Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients. \textit{Applied and Environmental Microbiology}. WASHINGTON: AMER SOC MICROBIOLOGY, 1997, roč.~63, č.~8, s.~3233-3241. ISSN~0099-2240.

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