Increased species diversity and extended habitat range of sulfur-oxidizing Thiomicrospira spp

Abstract

We combined traditional cultivation methods and new molecular techniques to study the diversity and habitat range of bacteria of the genus Thiomicrospira. Specific primers were designed and used in the PCR to amplify the 16S ribosomal DNA (rDNA) of Thiomicrospira spp. and thus detect the presence of these bacteria in environmental samples and enrichment cultures. By using this genus-specific PCR, we were able to amplify 722-bp-long 16S rDNA fragments from different saltwater habitats as well as from a freshwater ecosystem. Furthermore, we were able to isolate most of these bacteria in pure culture by using enrichment cultures for chemolithoautotrophic sulfur-oxidizing bacteria. With denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments followed by hybridization analysis with one of the primers as a genus-specific probe, it was possible to monitor the success of isolation. The combined approach resulted in the isolation of several chemolithoautotrophic bacteria from different habitats: e.g., a coastal sediment along the coast of Chile, a microbial mat of the hypersaline pond Solar Lake (Sinai, Egypt), and the saline spring Artern (Thuringia, Germany). In addition, four different isolates were obtained from sediment and water samples taken at Jadebusen, which is part of the German Waddensea. Comparative analysis of the nearly complete 16S rRNA sequences of these isolates indicated several new species, all grouping with the Thiomicrospira species of the gamma subdivision of the class Proteobacteria. A freshwater Thiomicrospira species could not be isolated, but sequence analysis of the PCR product obtained after amplification of the environmental DNA with the Thiomicrospira-specific primers revealed its phylogenetic affiliation. The study indicates an increased species diversity of Thiomicrospira and the ubiquity of this sulfur-oxidizing bacterium in habitats with reduced sulfur compounds.