Microheterogeneity in 16S ribosomal DNA-defined bacterial populations from a stratified planktonic environment is related to temporal changes and to ecological adaptations - PubMed (original) (raw)

Emilio O Casamayor et al. Appl Environ Microbiol. 2002 Apr.

Abstract

Temporal changes of the bacterioplankton from a meromictic lake (Lake Vilar, Banyoles, Spain) were analyzed with four culture-independent techniques: epifluorescence microscopy, PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting, fluorescence in situ whole-cell hybridization and flow cytometry sorting. Microscopically, blooms of one cyanobacterium (Synechococcus sp.-like), one green sulfur bacterium (Chlorobium phaeobacteroides-like), and one purple sulfur bacterium (Thiocystis minor-like) were observed at different depths and times. DGGE retrieved these populations and, additionally, populations related to the Cytophaga-Flavobacterium-Bacteroides phylum as predominant community members. The analyses of partial 16S ribosomal DNA sequences from the DGGE fingerprints (550 bp analyzed) revealed higher genetic diversity than expected from microscopic observation for most of these groups. Thus, the sequences of two Synechococcus spp. (both had a similarity of 97% to Synechococcus sp. strain PCC6307 in 16S rRNA), two Thiocystis spp. (similarities to Thiocystis minor of 93 and 94%, respectively), and three Cytophaga spp. (similarities to Cytophaga fermentans of 88 and 89% and to Cytophaga sp. of 93%, respectively) were obtained. The two populations of Synechococcus exhibited different pigment compositions and temporal distributions and their 16S rRNA sequences were 97.3% similar. The two Thiocystis populations differed neither in pigment composition nor in morphology, but their 16S rRNA sequences were only 92.3% similar and they also showed different distributions over time. Finally, two of the Cytophaga spp. showed 96.2% similarity between the 16S rRNA sequences, but one of them was found to be mostly attached to particles and only in winter. Thus, the identity of the main populations changed over time, but the function of the microbial guilds was maintained. Our data showed that temporal shifts in the identity of the predominant population is a new explanation for the environmental 16S rRNA microdiversity retrieved from microbial assemblages and support the hypothesis that clusters of closely related 16S rRNA environmental sequences may actually represent numerous closely related, yet ecologically distinct, populations.

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Figures

FIG. 1.

FIG. 1.

Depth profiles of temperature, conductivity, oxygen, and sulfide from Lake Vilar for three selected dates in 1996.

FIG. 2.

FIG. 2.

Negative image of ethidium bromide-stained DGGE gel containing PCR-amplified segments of 16S rRNA genes obtained by using universal bacterial primers. Lanes E, M, and H correspond to the aerobic epilimnion, the oxic-anoxic metalimnion, and the anaerobic rich-sulfide hypolimnion, respectively. Small circles correspond to the bands excised from the gel and then sequenced, and numbers are given for these sequences.

FIG. 3.

FIG. 3.

Seasonal changes in the relative contributions of selected DGGE bands to the total intensity for each sample in Fig. 2. (A) Bands from Synechococcus spp. in the aerobic epilimnion; (B) bands from Thiocystis spp. in the oxic-anoxic interface; (C) bands of Cytophaga spp. in the anaerobic rich-sulfide hipolimnion.

FIG. 4.

FIG. 4.

FISH analysis with specific probes for both Thiocystis spp. (CHR-452 and CHR-626; upper and middle panels, respectively) and one Cytophaga sp. (CFB563, lower panels). Bars, 5 μm. Positive Thiocystis cells appear white; autofluorescent negative cells of Thiocystis appear gray. Cytophagas are shown to correspond to the DAPI-stained cells attached to particles.

FIG. 5.

FIG. 5.

Negative image of ethidium bromide-stained DGGE gel containing PCR-amplified segments of 16S rRNA genes obtained by using cyanobacterium-chloroplast primers. Lanes A and B correspond to the sorted populations from spring and summer, respectively. Identification of the bands excised from the gel is presented in Table 2.

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