Quantitative distributions of Epsilonproteobacteria and a Sulfurimonas subgroup in pelagic redoxclines of the central Baltic Sea

Abstract

Members of the class Epsilonproteobacteria are known to be of major importance in biogeochemical processes at oxic-anoxic interfaces. In pelagic redoxclines of the central Baltic Sea, an uncultured epsilonproteobacterium related to Sulfurimonas denitrificans was proposed to play a key role in chemolithotrophic denitrification (I. Brettar, M. Labrenz, S. Flavier, J. Bötel, H. Kuosa, R. Christen, and M. G. Höfle, Appl. Environ. Microbiol. 72:1364-1372, 2006). In order to determine the abundance, activity, and vertical distribution of this bacterium in high-resolution profiles, 16S rRNA cloning and catalyzed reporter deposition and fluorescence in situ hybridization (CARD-FISH) and quantitative PCR measurements were carried out. The results showed that 21% of the derived clone sequences, which in the present study were grouped together under the name GD17, had >99% similarity to the uncultured epsilonproteobacterium. A specific gene probe against GD17 (S-*-Sul-0090-a-A-18) was developed and used for enumeration by CARD-FISH. In different pelagic redoxclines sampled during August 2003, May 2005, and February 2006, GD17 cells were always detected from the lower oxic area to the sulfidic area. Maximal abundance was detected around the chemocline, where sulfide and nitrate concentrations were close to the detection limit. The highest GD17 numbers (2 x 10(5) cells ml(-1)), representing up to 15% of the total bacteria, were comparable to those reported for Epsilonproteobacteria in pelagic redoxclines of the Black Sea and the Cariaco Trench (X. Lin, S. G. Wakeham, I. F. Putnam, Y. M. Astor, M. I. Scranton, A. Y. Chistoserdov, and G. T. Taylor, Appl. Environ. Microbiol. 72:2679-2690, 2006). However, in the Baltic Sea redoxclines, Epsilonproteobacteria consisted nearly entirely of cells belonging to the distinct GD17 group. This suggested that GD17 was the best-adapted epsilonproteobacterium within this ecological niche.

FIG. 1.

Chemical parameters and the distribution and abundance of bacteria detected by CARD-FISH along the Farö Deep redoxcline in August 2003. (A) Concentrations of H₂S, O₂, and NO₃⁻. (B) Vertical distributions of total cell numbers (DAPI), eubacteria (EUB), GD17, and epsilonproteobacterial (EPS) cell numbers. The broken horizontal line in panel B indicates the chemocline.

FIG. 2.

Chemical parameters and the distribution and abundance of bacteria detected by CARD-FISH along the Gotland Deep redoxcline in May 2005. (A) Concentrations of H₂S, O₂, NO₃⁻, and CO₂ and dark CO₂ fixation rates. (B) Vertical distributions of total cell numbers (DAPI), eubacteria (EUB), GD17, and epsilonproteobacterial (EPS) cell numbers. GD17* cell numbers (bars) were determined by qPCR (as described in Materials and Methods). The broken horizontal line in panel B indicates the chemocline.

FIG. 3.

Chemical parameters and the distribution and abundance of bacteria detected by CARD-FISH along the Gotland Deep redoxcline in February 2006. (A) Concentrations of H₂S, O₂, NO₃⁻, and CO₂ dark fixation rates. (B) Vertical distributions of total cell numbers (DAPI), eubacteria (EUB), GD17, and epsilonproteobacterial (EPS) cell numbers. The broken horizontal line in panel B indicates the chemocline.

FIG. 4.

Unrooted tree showing relationships of subgroup GD17 and its closest phylogenetic relatives within the Epsilonproteobacteria. The tree was constructed using the neighbor-joining method based on a comparison of approximately 1,400 nucleotides. Escherichia coli K-12 was used as an outgroup. Branching points supported by neighbor-joining, maximum likelihood, and maximum parsimony algorithms are marked by a black circle. GenBank database accession numbers are given in parentheses. Bar, 10 substitutions per 100 nucleotides. The boxed area denotes subgroup GD17 as the target sequence of gene probe SUL90.

FIG. 5.

Relationship between GD17 and Epsilonproteobacteria (EPS) cell numbers in central Baltic Sea redoxclines. Data points originated from the Farö Deep in 2003 and the Gotland Deep in 2005 and 2006. The straight line is the regression line.

FIG. 6.

Box plot of grouped GD17 cell abundances from the Farö Deep in 2003, the Gotland Deep in 2005 and 2006 versus the occurrence of nitrate and sulfide in the water column. The number of data points included in each box is indicated.