Analysis of bacterial communities in soil by use of denaturing gradient gel electrophoresis and clone libraries, as influenced by different reverse primers.

Applied and environmental microbiology ·Vol. 74 ·No. 9 ·2008-05-00 ·Pages 2717-27

Brons JK, van Elsas JD

Abstract

To assess soil bacterial diversity, PCR systems consisting of several slightly different reverse primers together with forward primer F968-GC were used along with subsequent denaturing gradient gel electrophoresis (DGGE) or clone library analyses. In this study, a set of 13 previously used and novel reverse primers was tested with the canonical forward primer as to the DGGE fingerprints obtained from grassland soil. Analysis of these DGGE profiles by GelCompar showed that they all fell into two main clusters separated by a G/A alteration at position 14 in the reverse primer used. To assess differences between the dominant bacteria amplified, we then produced four (100-membered) 16S rRNA gene clone libraries by using reverse primers with either an A or a G at position 14, designated R1401-1a, R1401-1b, R1401-2a, and R1401-2b. Subsequent sequence analysis revealed that, on the basis of the about 410-bp sequence information, all four primers amplified similar, as well as different (including novel), bacterial groups from soil. Most of the clones fell into two main phyla, Firmicutes and Proteobacteria. Within Firmicutes, the majority of the clones belonged to the genus Bacillus. Within Proteobacteria, the majority of the clones fell into the alpha or gamma subgroup whereas a few were delta and beta proteobacteria. The other phyla found were Actinobacteria, Acidobacteria, Verrucomicrobia, Chloroflexi, Gemmatimonadetes, Chlorobi, Bacteroidetes, Chlamydiae, candidate division TM7, Ferribacter, Cyanobacteria, and Deinococcus. Statistical analysis of the data revealed that reverse primers R1401-1b and R1401-1a both produced libraries with the highest diversities yet amplified different types. Their concomitant use is recommended.

MeSH Terms

Bacteria/classification,genetics,isolation & purification Biodiversity Cluster Analysis DNA Fingerprinting/methods DNA Primers/genetics DNA, Bacterial/chemistry,genetics DNA, Ribosomal/chemistry,genetics Electrophoresis, Polyacrylamide Gel Molecular Sequence Data Nucleic Acid Denaturation Phylogeny Polymerase Chain Reaction/methods RNA, Ribosomal, 16S/genetics Sequence Analysis, DNA Soil Microbiology

Chemicals

DNA Primers DNA, Bacterial DNA, Ribosomal RNA, Ribosomal, 16S

Authors & Affiliations

2 authors, click to expand affiliations / ORCID

Brons Jolanda K

Department of Microbial Ecology, Centre of Ecological and Evolutionary Studies, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.

van Elsas Jan Dirk

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Article Info

Journal

Applied and environmental microbiology

Abbr.

Appl Environ Microbiol

ISSN

1098-5336

DOI

10.1128/AEM.02195-07

Published

2008-05-00

Epub

2008-00-29

Pages

2717-27

Language

English

Region

United States

NLM ID

7605801

PMCID

PMC2394888

Subset

Databases

GENBANK

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PubMed source DOI: 10.1128/AEM.02195-07 PMC Full Text View journal details

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Analysis of bacterial communities in soil by use of denaturing gradient gel electrophoresis and clone libraries, as influenced by different reverse primers.

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