Phylogenetic analysis of culturable dimethyl sulfide-producing bacteria from a spartina-dominated salt marsh and estuarine water

Abstract

Dimethylsulfoniopropionate (DMSP), an abundant osmoprotectant found in marine algae and salt marsh cordgrass, can be metabolized to dimethyl sulfide (DMS) and acrylate by microbes having the enzyme DMSP lyase. A suite of DMS-producing bacteria isolated from a salt marsh and adjacent estuarine water on DMSP agar plates differed markedly from the pelagic strains currently in culture. While many of the salt marsh and estuarine isolates produced DMS and methanethiol from methionine and dimethyl sulfoxide, none appeared to be capable of producing both methanethiol and DMS from DMSP. DMSP, and its degradation products acrylate and beta-hydroxypropionate but not methyl-3-mecaptopropionate or 3-mercaptopropionate, served as a carbon source for the growth of all the alpha- and beta- but only some of the gamma-proteobacterium isolates. Phylogenetic analysis of 16S rRNA gene sequences showed that all of the isolates were in the group Proteobacteria, with most of them belonging to the alpha and gamma subclasses. Only one isolate was identified as a beta-proteobacterium, and it had >98% 16S rRNA sequence homology with a terrestrial species of Alcaligenes faecalis. Although bacterial population analysis based on culturability has its limitations, bacteria from the alpha and gamma subclasses of the Proteobacteria were the dominant DMS producers isolated from salt marsh sediments and estuaries, with the gamma subclass representing 80% of the isolates. The alpha-proteobacterium isolates were all in the Roseobacter subgroup, while many of the gamma-proteobacteria were closely related to the pseudomonads; others were phylogenetically related to Marinomonas, Psychrobacter, or Vibrio species. These data suggest that DMSP cleavage to DMS and acrylate is a characteristic widely distributed among different phylotypes in the salt marsh-estuarine ecosystem.

FIG. 1

Kinetics of DMS production from DMSP by cell suspensions of marine bacteria. Isolates were cultured in marine or tryptic soy broth; 1 ml was harvested by centrifugation and resuspended in half-strength seawater containing DMSP. All cultures tested were fully developed in 24 h on this rich medium and contained approximately the same amount of protein (0.3 mg · ml⁻¹). The headspace analyzed for DMS by gas chromatography. The experiment was performed twice (two replicas), and the average values were used for constructing the graph.

FIG. 2

Phylogenetic tree based on the partial 16S rRNA gene sequence showing the relationship among the DMS-producing isolates belonging to the α subdivision of the Proteobacteria. The tree was constructed using Jukes-Cantor distance, and the method was neighbor-joining (39). B. subtilis was used as the outgroup, and bootstrap values greater than 50% (indicated near the branch points) based on 500 pseudoreplicas were generated to establish confidence in the clustering (16). The distance scale is the corrected proportion of the nucleotide changes. All the DMS-producing strains are in bold print; those designated “JA” are from this study.

FIG. 3

Phylogenetic tree showing the relationship of the 16S rRNA gene sequence of A. faecalis strain M3A to other, closely related members of the β subdivision of the Proteobacteria. Methods were as described in the Fig. 2 legend.

FIG. 4

Phylogenetic tree showing relationships of the DMS-producing isolates to representative cultured species of the γ subdivision of the Proteobacteria in the database. The sequence analysis was identical to that described in the legend to Fig. 2.