Molecular biological detection and quantification of novel Fibrobacter populations in freshwater lakes

Abstract

PCR and quantitative PCR (qPCR) primers targeting the 16S rRNA gene were used to detect and quantify members of the genus Fibrobacter in lake water, sediment and colonized cotton taken from two freshwater lakes. Phylogenetic analysis identified two groups of sequences; those clustered with Fibrobacter succinogenes, the type species, and a defined cluster of clones loosely associated with several Fibrobacter sequences observed previously in clone libraries from freshwater environments. 16S rRNA gene sequences recovered in the same way from soil samples and ovine feces in the surrounding land were all F. succinogenes and did not include any from this group of the "freshwater" Fibrobacteres. In all cases, nested PCR was required to detect Fibrobacter 16S rRNA genes, and qPCR analysis of reverse transcribed bacterial community RNA confirmed their very low relative abundance on colonized cotton baits in the water column (at 0, 3, 7, 11, and 13 m) and on the sediment surface (<0.02% of total bacterial rRNA). However, in Esthwaite Water sediment itself, the relative abundance of fibrobacters was 2 orders of magnitude higher (ca. 1% of total bacterial rRNA). The presence of fibrobacters, including the cellulolytic rumen species F. succinogenes, on colonized cellulose samples and in lake sediment suggests that these organisms may contribute to the primary degradation of plant and algal biomass in freshwater lake ecosystems.

FIG. 1.

Data on the nested PCR amplification of community DNA extracted from colonized cotton, membrane-filtered lake water, and sediment samples using the Fibrobacter genus-specific 16S rRNA gene primer set (Fib1F and Fib2AR). The 17-m-depth sample represents the lake sediment, and 0- to 13-m samples represent the water column. Percentage oxygen saturation and temperature readings of the water column and sediment of Esthwaite Water in the English Lake District were also measured. +, nested PCR amplification product obtained; −, no PCR amplification observed; Sed, lake sediment sample.

FIG. 2.

Maximum-likelihood tree of 16S rRNA gene sequences amplified from lake water, sediment, and colonized cotton samples using the Fibrobacter genus-specific Fib1 and Fib2A PCR primer set. Contiguous sequences were aligned with their three nearest neighbor sequences from both cultured and uncultured isolates using the Greengenes NAST aligner (7, 8), in addition to a number of representatives from other bacterial phyla. The alignment was imported into the Arb software program (21) and manually optimized. A final alignment corresponding to E. coli 16S rRNA gene positions 153 to 1017 was exported from Arb, and a maximum-likelihood tree was constructed by PhyML online (12) using the HKY85 substitution model and the Shimodaira-Hasegawa (SH)-like aLRT branch support method. The tree was imported back into Arb, where non-Fibrobacteres sequences were omitted from the tree to better demonstrate the phylogeny of the Fibrobacteres phylum. Fibrobacteres subphylum 2 contains sequences drawn from previous studies of the termite gut (13, 33). Filled circles indicate those nodes at which a bootstrap value of >95% was observed, and unfilled circles denote nodes with bootstrap values between 75 and 95%. The accession number of each sequence is displayed in parentheses alongside the sequence. Numbers shown on collapsed branches indicate the number of sequences within the branch. Sequences shown in bold were amplified using the Fib1 and Fib2A primer set applied in this study. The scale bar represents 0.1 base substitution per nucleotide.