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

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.

FIG. 1.

Comparison of bacterial DGGE patterns obtained from soil sample RG by PCR with canonical primer F968 (joined to a GC clamp) and reverse primers R1401-1a, -2a, -2*a, -3a, -1378*, -1b, -2b, -2*b, and -3b (A) and the corresponding dendrogram (clustering by the unweighted-pair group method with arithmetic mean) (B). M, molecular size markers; 1a, 2a, 2*a, 3a, 1378*, 1b, 2b, 2*b, and 3b, reverse primers used.

FIG. 2.

Rarefaction curves of bacterial clone libraries of soil sample RG generated with primer sets consisting of canonical primer U968f combined with four different reverse primers (R1401-1a, -2a, -1b, and -2b). Sequences were compared to GenBank rRNA gene-based entries. The number of different OTUs (defined at a 97% similarity cutoff) in each sample is plotted versus the number of sequences sampled.

FIG. 3.

Phylogenetic trees constructed on the basis of partial 16S rRNA gene sequences (about 400 bp) generated from soil with canonical forward primer F968-GC and reverse primers R1401-1a (A, 94 sequences), R1401-2a (B, 103 sequences), R1401-1b (C, 96 sequences), and R1401-2b (D, 103 sequences). The trees were calculated with the neighbor-joining algorithm. To simplify the phylogenetic trees, clones are marked as follows: no symbol, sequences with ≥97% similarity; ▴, 95 to 96% similarity; •, 90 to 94% similarity; ○, <90% similarity. Grouping is according to phylum or class (Proteobacteria) or PN candidate divisions. Abbreviations: Acido, Acidobacteria; Actino, Actinobacteria; Bacter, Bacteroidetes; Chlam, Chlamydiae; Chloro, Chloroflexi; Chlorob, Chlorobi; Cyano, Cyanobacteria; Deino, Deinococcus; Ferri, Ferribacter; Firm, Firmicutes; Gemma, Gemmatimonadetes; α-, β-, γ-, and δ-Prot, Alpha-, Beta-, Gamma-, and Deltaproteobacteria, respectively; TM7, candidate division TM7; Verruco, Verrucomicrobia.

FIG. 3.

Phylogenetic trees constructed on the basis of partial 16S rRNA gene sequences (about 400 bp) generated from soil with canonical forward primer F968-GC and reverse primers R1401-1a (A, 94 sequences), R1401-2a (B, 103 sequences), R1401-1b (C, 96 sequences), and R1401-2b (D, 103 sequences). The trees were calculated with the neighbor-joining algorithm. To simplify the phylogenetic trees, clones are marked as follows: no symbol, sequences with ≥97% similarity; ▴, 95 to 96% similarity; •, 90 to 94% similarity; ○, <90% similarity. Grouping is according to phylum or class (Proteobacteria) or PN candidate divisions. Abbreviations: Acido, Acidobacteria; Actino, Actinobacteria; Bacter, Bacteroidetes; Chlam, Chlamydiae; Chloro, Chloroflexi; Chlorob, Chlorobi; Cyano, Cyanobacteria; Deino, Deinococcus; Ferri, Ferribacter; Firm, Firmicutes; Gemma, Gemmatimonadetes; α-, β-, γ-, and δ-Prot, Alpha-, Beta-, Gamma-, and Deltaproteobacteria, respectively; TM7, candidate division TM7; Verruco, Verrucomicrobia.

FIG. 4.

Frequency histogram of the clones found in this study on the basis of ≥97%, 95% to 96%, 90% to 94%, and <90% matching OTU. The x axis shows the major existing or possibly novel phylogenetic groups (phylum or class) found in the clone libraries with each of the reverse primers used (R1401-1a, -2a, R1401-1b, and -2b), whereas the y axis shows the number of clones found in each phylogenetic group. *, these groups matched unculturable bacteria in the NCBI database.