In Situ Assessment on the Physiological State of Purple and Green Sulfur Bacteria through the Analyses of Pigment and 5S rRNA Content.

Time-depth distribution of the microbial anaerobic assemblage of Lake Cisó was analyzed by microscopy, pigment composition, and electrophoretic analysis of 5S rRNAs. Purple (Amoebobacter-like and Thiocystis minor-like cells) and green (Chlorobium-like) sulfur bacteria were very abundant. Both groups coexisted in depth and in time despite the fact that they compete for the same natural resources (e.g., light and sulfide). Cell abundance, group-specific pigment content, and group-specific 5S rRNA content did not change in parallel with depth. This was due to variations in the specific content of both RNA and pigments. Specific content of RNA was systematically higher in purple than in green sulfur bacteria. The latter, in turn, displayed a much higher pigment content. Specific content of both RNA and pigments changed with depth and time. Analysis of tRNA band patterns indicated no changes in the populations forming the assemblage. Changes in specific contents, therefore, were the result of physiological adaptations of the populations already present in the system. We concluded that each group of bacteria showed differential adaptations in both RNA and pigment content, and that the specific contents measured were good indicators of the physiological status of these bacteria in situ. The higher content of RNA in purple sulfur bacteria indicates that these organisms are the main contributors to anaerobic carbon fixation and sulfide oxidation processes in Lake Cisó.

5S rRNA fingerprints of marine bacteria, halophilic archaea and natural prokaryotic assemblages along a salinity gradient.

Natural prokaryotic assemblages from two multi-pond solar salterns and pure cultures of both marine bacteria and halophilic archaea were analyzed and compared by electrophoretic analysis of 5S rRNAs. A salinity gradient from seawater (3.7%) to NaCl precipitation (37%) was studied. The culture-independent, PCR-free, fingerprinting analysis covered two objectives: (i) to compare natural assemblages among them and with results previously obtained through a PCR-dependent approach and (ii) to estimate the in situ relevance of those prokaryotic groups obtained with classical culture methodologies. Natural assemblages were analyzed through cluster analysis of quantitative 5S rRNA band patterns. The resulting groups were in accordance with environmental parameters (i.e., NaCl concentration) and with the clustering obtained after a PCR-dependent approach, showing the formation of three salinity-based groups of samples (<10%, 10-25% and >25% salinity). Similarities between the laboratory strains tested and dominant community members were studied by comparing 5S rRNA band patterns. The lack of match obtained after cluster analysis indicated that the prokaryotic populations relevant in the ponds below 25% salinity were neither Flavobacteria nor haloarchaeal strains belonging to the genera Halococcus, Haloarcula and Halobacterium. Members of Proteobacteria and Gram-positive bacteria were found to match bands in these samples. The 5S rRNA fingerprint from the dominant community members in the ponds above 30% salinity did not fit any of the cultured halophilic archaea studied, in agreement with earlier PCR results. This is consistent with a greater bias introduced by culture-dependent methods than by those based on PCR, especially for archaeal populations.

Comparison of pure cultures and natural assemblages of planktonic photosynthetic sulfur bacteria by low molecular mass RNA fingerprinting.

Pure cultures of phototrophic sulfur bacteria were compared to natural populations that bloom in karstic lakes by electrophoretic analysis of low molecular mass RNA molecules (lmwRNA) and microscopy. Similarities between dominant community members, field isolates and reference strains were established by comparing the lmwRNA band patterns through dendrograms produced with Euclidean distances and the average linkage clustering method, by a single, quick, one-step method. The fingerprinting analysis had three objectives: (i) to compare microbial assemblages from different geographical locations, (ii) to compare those organisms which grow in pure culture to those forming planktonic blooms and (iii) to give a preliminary view of the identity of the predominant community members. The lmwRNA analysis yielded a number of clusters consistent with the microscopic observations and allowed rapid comparison of the microbial communities without the need to isolate their individual components. The fingerprints of natural communities were different from most of the laboratory strains tested. Purple sulfur bacteria responsible for the blooms analyzed in karstic lakes were more closely related to the Thiocystis group than to the classical strains extensively studied in the laboratory.

Identification of phototrophic sulfur bacteria through the analysis of lmwRNA band patterns

Several phototrophic sulfur bacteria were identified preliminarily through the analysis of the low-molecular-weight RNA fraction (lmwRNA) of bacterial cells. This fraction includes the ribosomal 5S RNA and several transfer RNAs. These molecules were separated by high-resolution electrophoresis in polyacrylamide gels, and the resulting band patterns were used as fingerprints for the identification of the organisms. We examined a large number of well-characterized reference strains together with a broad range of purple sulfur bacterial isolates from freshwater and marine environments. A cluster analysis was run using the similarity matrix calculated from the band patterns. Despite the shortcomings of the method, close relatives were clustered together yielding a number of groups consistent with the phylogenetic arrangement established through the analyses of a few available 16S rRNA gene sequences. Thus, the classification obtained gives further support to rearrangement of the group as the analyses of 16S rRNA gene sequences had previously suggested. We conclude that the analysis of lmwRNA band patterns is a rapid and simple tool for grouping and preliminarily identifying new isolates of phototrophic sulfur bacteria.