Analysis of Bacterial Communities in Soil by Denaturing Gradient Gel Electrophoresis and Clone Libraries as Influenced by Different Reverse Primers

Department of Microbial Ecology, Centre of Ecological and Evolutionary Studies (CEES), University of Groningen, Kerklaan 30, 9751 NN Haren, the Netherlands

^* To whom correspondence should be addressed. Email: j.d.van.elsas{at}rug.nl.

	Abstract

To assess soil bacterial diversity, PCR systems consisting of several slightly different reverse primers together with forward primer F968-GC, have been used for subsequent DGGE or clone library analyses. In this study, a set of 13 hitherto-used and novel reverse primers was tested with the canonical forward primer as to the DGGE fingerprints obtained from a 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 using reverse primers with either an A or a G at position 14, denoted 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 (under which novel) bacterial groups from soil. Most clones fell into two main phyla, the Firmicutes and the Proteobacteria. Within the Firmicutes, the majority of the clones belonged to the genus Bacillus. Within the Proteobacteria, the majority of the clones fell into the proteobacterial alpha or gamma subgroups, whereas a few were delta and beta Proteobacteria. 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 the reverse primers R1401-1b and R1401-1a both produced libraries with highest diversities, yet amplified different types. Their concomitant use is recommended.