Identification of and spatio-temporal differences between microbial assemblages from two neighboring sulfurous lakes: comparison by microscopy and denaturing gradient gel electrophoresis

Abstract

The microbial assemblages of Lake Cisó and Lake Vilar (Banyoles, northeast Spain) were analyzed in space and time by microscopy and by performing PCR-denaturing gradient gel electrophoresis (DGGE) and sequence analysis of 16S rRNA gene fragments. Samples obtained from different water depths and at two different times of the year (in the winter during holomixis and in the early spring during a phytoplankton bloom) were analyzed. Although the lakes have the same climatic conditions and the same water source, the limnological parameters were different, as were most of the morphologically distinguishable photosynthetic bacteria enumerated by microscopy. The phylogenetic affiliations of the predominant DGGE bands were inferred by performing a comparative 16S rRNA sequence analysis. Sequences obtained from Lake Cisó samples were related to gram-positive bacteria and to members of the division Proteobacteria. Sequences obtained from Lake Vilar samples were related to members of the Cytophaga-Flavobacterium-Bacteroides phylum and to cyanobacteria. Thus, we found that like the previously reported differences between morphologically distinct inhabitants of the two lakes, there were also differences among the community members whose morphologies did not differ conspicuously. The changes in the species composition from winter to spring were also marked. The two lakes both contained sequences belonging to phototrophic green sulfur bacteria, which is consistent with microscopic observations, but these sequences were different from the sequences of cultured strains previously isolated from the lakes. Euryarchaeal sequences (i.e., methanogen- and thermoplasma-related sequences) also were present in both lakes. These euryarchaeal group sequences dominated the archaeal sequences in Lake Cisó but not in Lake Vilar. In Lake Vilar, a new planktonic population related to the crenarchaeota produced the dominant archaeal band. The phylogenetic analysis indicated that new bacterial and archaeal lineages were present and that the microbial diversity of these assemblages was greater than previously known. We evaluated the correspondence between the abundances of several morphotypes and DGGE bands by comparing microscopy and sequencing results. Our data provide evidence that the sequences obtained from the DGGE fingerprints correspond to the microorganisms that are actually present at higher concentrations in the natural system.

FIG. 1

Vertical profiles for temperature, conductivity, oxygen concentration, sulfide concentration, light penetration, and cell counts on two different days in Lake Cisó. (A) Winter mixing (19 February 1996). (B) Spring stratification (9 April 1996). The sampling depths used for the molecular survey are indicated by arrows. Cell counts are on a logarithmic scale.

FIG. 2

Vertical profiles for temperature, conductivity, oxygen concentration, sulfide concentration, light penetration, and cell counts on two different days in Lake Vilar. (A) Winter (19 February 1996). (B) Spring (9 April 1996). The sampling depths used for the molecular survey are indicated by arrows. Cell counts are on a logarithmic scale.

FIG. 3

Negative images of ethidium bromide-stained DGGE gels containing PCR-amplified segments of 16S rRNA genes obtained by using three primer sets. The band patterns were obtained with bacterial primers (A), cyanobacterium-chloroplast primers (B), and archaeal primers (C) from the oxic epilimnion (lanes E) and from the anoxic metalimnion (lanes M) and hypolimnion (lanes H). Lanes E1, M1, and H1 and lanes E2, M2, and H2 correspond to winter and spring, respectively, in Lake Vilar. Lane W and lanes E and M correspond to winter and spring, respectively, in Lake Cisó. Bands were designated as described in the text.

FIG. 4

Phylogenetic affiliations within the domain Bacteria of the excised bands obtained from the gels in Fig. 3A and 3B. The tree was constructed by adding by parsimony analysis the partial sequence data to a previously validated and optimized tree. Scale bar = 0.10 mutation per nucleotide position.

FIG. 5

Phylogenetic affiliations within the domain Archaea of excised bands obtained from the gel in Fig. 3C. The tree was constructed by adding by parsimony analysis the partial sequence data to a previously validated and optimized tree. Scale bar = 0.10 mutation per nucleotide position.