Molecular biological detection and characterization of Clostridium populations in municipal landfill sites

Abstract

Primer sets specific for 16S rRNA genes were designed for four phylogenetic groups of clostridia known to contain mesophilic cellulolytic species. Specific amplification of these groups from landfill leachate DNA extracts demonstrated the widespread occurrence of clostridia from the Clostridium thermocellum and C. leptum groups. In contrast, the C. botulinum group was never detected, and the C. coccoides-C. lentocellum group was only occasionally detected. Amplification products were analyzed by temporal thermal gel electrophoresis to generate profiles of the clostridial groups and to identify dominant bands. Sequence analysis of 17 landfill clones confirmed that the primers were specific for the clostridial subgroups and that the cloned sequences had a close relationship with known cellulose-degrading clostridia. The primers have therefore been authenticated for use in the rapid identification of clostridia in anaerobic environments.

FIG. 1.

TTGE profiles of 16S rRNA genes of Clostridium clusters III, IV, and XIVab. Amplification products using group-specific primers were reamplified using primer TTGE-1 (cluster III) or TTGE-2 (clusters IV and XIVab) and were separated by TTGE analysis. The profiles comprise amplification products from landfill sites R, C, H, W, B, P, Br, S, and So. Cloned sequences that matched bands marked with arrows on the TTGE gel were sequenced and used for phylogenetic analysis.

FIG. 2.

TTGE profiles of 16S rRNA genes of Clostridium cluster III amplified from Br and So landfill leachate DNAs. The lanes labeled “clones” contain amplification products of DNAs cloned from sites Br and So.

FIG. 3.

Phylogenetic tree showing relationships of 16S rRNA genes from Clostridium cluster III. Sequences recovered from landfill sites are in boldface. The tree was constructed using the Jukes-Cantor distance matrix and the neighbor-joining method. The scale bar represents a 2% difference in nucleotide sequence positions.

FIG. 4.

Phylogenetic tree showing relationships of 16S rRNA genes from Clostridium cluster IV. Sequences recovered from landfill sites are in boldface. The tree was constructed as described in the legend to Fig. 3.