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Applied and Environmental Microbiology, May 2008, p. 2717-2727, Vol. 74, No. 9
0099-2240/08/$08.00+0     doi:10.1128/AEM.02195-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Analysis of Bacterial Communities in Soil by Use of Denaturing Gradient Gel Electrophoresis and Clone Libraries, as Influenced by Different Reverse Primers{triangledown}

Jolanda K. Brons and Jan Dirk van Elsas*

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

Received 26 September 2007/ Accepted 20 February 2008

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.

* Corresponding author. Mailing address: Department of Microbial Ecology, Centre of Ecological and Evolutionary Studies, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands. Phone: 31.50.3632151. Fax: 31.50.3632154. E-mail: j.d.van.elsas{at}rug.nl

{triangledown} Published ahead of print on 29 February 2008.

Applied and Environmental Microbiology, May 2008, p. 2717-2727, Vol. 74, No. 9
0099-2240/08/$08.00+0     doi:10.1128/AEM.02195-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Hardoim, C. C. P., Costa, R., Araujo, F. V., Hajdu, E., Peixoto, R., Lins, U., Rosado, A. S., van Elsas, J. D. (2009). Diversity of Bacteria in the Marine Sponge Aplysina fulva in Brazilian Coastal Waters. Appl. Environ. Microbiol. 75: 3331-3343 [Abstract] [Full Text]  


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