Heterotrophic symbionts of phototrophic consortia: members of a novel diverse cluster of Betaproteobacteria characterized by a tandem rrn operon structure.

ABSTRACT

Phototrophic consortia represent the most highly developed type of interspecific association of bacteria and consist of green sulfur bacterial epibionts attached around a central colourless rod-shaped bacterium. Based on 16S rRNA gene sequencing, the central bacterium of the consortium 'Chlorochromatium aggregatum' was recently shown to represent a novel and phylogenetically isolated lineage of the Comamonadaceae within the beta-subgroup of the Proteobacteria. To date, 19 types of phototrophic consortia are distinguished based on the different 16S rRNA gene sequences of their epibionts, but the diversity and phylogenetic relationships of the heterotrophic partner bacteria are still unknown. We developed an approach based on the specific rrn (ribosomal RNA) operon structure of the central bacterium of 'C. aggregatum' to recover 16S rRNA gene sequences of other central bacteria and their close relatives from natural consortia populations. Genomic DNA of the central bacterium of 'C. aggregatum' was first enriched several hundred-fold by employing a selective method for growth of consortia in a monolayer biofilm followed by a purification of the genome of the central bacterium by cesium chloride-bisbenzimidazole equilibrium density gradient centrifugation. A combination of inverse PCR, cloning and sequencing revealed that two rrn operons of the central bacterium are arranged in a tandem fashion and are separated by an unusually short intergenic region of 195 base pairs. This rare gene order was exploited to screen various natural microbial communities by PCR. We discovered a diverse and previously unknown subgroup of Betaproteobacteria in the chemoclines of freshwater lakes. This group was absent in other freshwater and soil samples. All the 16S rRNA gene sequences recovered are related to that of the central bacterium of 'C. aggregatum'. Fluorescence in situ hybridization indicated that two of these sequences originated from central bacteria of different phototrophic consortia, which, however, were only distantly related to the central bacterium of 'C. aggregatum'. Based on a detailed phylogenetic analysis, these central bacterial symbionts of phototrophic consortia have a polyphyletic origin.