Phylogenetic relationships of Thiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments

Abstract

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments was used to explore the genetic diversity of hydrothermal vent microbial communities, specifically to determine the importance of sulfur-oxidizing bacteria therein. DGGE analysis of two different hydrothermal vent samples revealed one PCR band for one sample and three PCR bands for the other sample, which probably correspond to the dominant bacterial populations in these communities. Three of the four 16S rDNA fragments were sequenced. By comparison with 16S rRNA sequences of the Ribosomal Database Project, two of the DGGE-separated fragments were assigned to the genus Thiomicrospira. To identify these 'phylotypes' in more detail, a phylogenetic framework was created by determining the nearly complete 16S rRNA gene sequence (approx. 1500 nucleotides) from three described Thiomicrospira species, viz., Tms. crunogena, Tms. pelophila, Tms. denitrificans, and from a new isolate, Thiomicrospira sp. strain MA2-6. All Thiomicrospira species except Tms. denitrificans formed a monophyletic group within the gamma subdivision of the Proteobacteria. Tms. denitrificans was assigned as a member of the epsilon subdivision and was distantly affiliated with Thiovulum, another sulfur-oxidizing bacterium. Sequences of two dominant 16S rDNA fragments obtained by DGGE analysis fell into the gamma subdivision Thiomicrospira. The sequence of one fragment was in all comparable positions identical to the 16S rRNA sequence of Tms. crunogena. Identifying a dominant molecular isolate as Tms. crunogena indicates that this species is a dominant community member of hydrothermal vent sites. Another 'phylotype' represented a new Thiomicrospira species, phylogenetically in an intermediate position between Tms. crunogena and Tms. pelophila. The third 'phylotype' was identified as a Desulfovibrio, indicating that sulfate-reducing bacteria, as sources of sulfide, may complement sulfur- and sulfide-oxidizing bacteria ecologically in these sulfide-producing hydrothermal vents.