Effect of primers hybridizing to Different evolutionarily conserved regions of the small-subunit rRNA gene in PCR-based microbial community analyses and genetic profiling

Genetic profiling techniques of microbial communities based on PCR-amplifie d signature genes, such as denaturing gradient gel electrophoresis or singl e-strand-conformation polymorphism (SSCP) analysis, are normally done with PCR products of less than 500-bp. The most common target for diversity anal ysis, the small-subunit rRNA genes, however, are larger, and thus, only par tial sequences can be analyzed. Here, we compared the results obtained by P CR targeting different variable (V) regions (V2 and V3, V4 and V5, and V6 t o V8) of the bacterial 16S rRNA gene with primers hybridizing to evolutiona rily conserved flanking regions. SSCP analysis of single-stranded PCR produ cts generated from 13 different bacterial species showed fewer bands with p roducts containing V4-V5 (average, 1.7 bands per organism) than with V2-V3 (2.2 bands) and V6-V8 (2.3 bands). We found that the additional bands (>1 p er organism) were caused by intraspecies operon heterogeneities or by more than one conformation of the same sequence. Community profiles, generated b y PCR-SSCP from bacterial-cell consortia extracted from rhizospheres of fie ld-grown maize (Zea mays), were analyzed by cloning and sequencing of the d ominant bands. A total of 48 sequences could be attributed to 34 different strains from 10 taxonomical groups. Independent of the primer pairs, we fou nd proteobacteria (alpha, beta, and gamma subgroups) and members of the gen us Paenibacillus (low G+C gram-positive) to be the dominant organisms. Othe r groups, however, were only detected with single primer pairs. This study gives an example of how much the selection of different variable regions co mbined with different specificities of the flanking "universal" primers can affect a PCR-based microbial community analysis.