A polyphasic approach to study the diversity and vertical distribution of sulfur-oxidizing Thiomicrospira species in coastal sediments of the German Wadden Sea

Recently, four Thiomicrospira strains mere isolated from a coastal mud flat of the German Wadden Sea (T, Brinkhoff and G. Muyzer, Appl, Environ. Micro biol, 63:3789-3796, 1997), Were we describe the use of a polyphasic approac h to investigate the functional role of these closely related bacteria. Mic rosensor measurements showed that there mas oxygen penetration into the sed iment to a depth of about 2.0 mm, The pH decreased from 8.15 in the overlay ing water to a minimum value of 7.3 at a depth of 1.2 mm, Further down in t he sediment the pH increased to about 7.8 and remained constant. Most-proba ble-number (MPN) counts of chemolithoautotrophic sulfur-oxidizing bacteria revealed nearly constant numbers along the vertical profile; the cell conce ntration ranged from 0.93 x 10(5) to 9.3 x 10(5) cells per g of sediment. A specific PCR was used to detect the presence of Thiomicrospira cells in th e MPN count preparations and to determine their 16S rRNA sequences, The con centration of Thiomicrospira cells dial: not decrease with depth, It was fo und that Thiomicrospira strains were not dominant sulfur-oxidizing bacteria in this habitat, Denaturing gradient gel electrophoresis (DGGE) of PCR-amp lified 16S ribosomal DNA fragments followed by hybridization analysis with a genus-specific oligonucleotide pn,be revealed the diversity of Thiomicros pira strains in the MPN cultures, Sequence analysis of the highest MPN dilu tions in which the genus Thiomicrospira was detected revealed that there me re four clusters of several closely related sequences, Only one of the 10 T hiomicrospira sequences retrieved was related to sequences of known isolate s from the same habitat, Slot blot hybridization of rRNA isolated from diff erent sediment layers showed that, in contrast to the concentration of Thio microspira cells, the concentration of Thiomicrospira-specific rRNA decreas ed rapidly in the region below the oxic layer of the sediment, This study r evealed the enormous sequence diversity of closely related microorganisms p resent in one habitat, which so far has been found only by sequencing molec ular isolates, in addition, it showed that most of the Thiomicrospira popul ations in the sediment studied mere quiescent.