The Majority of Active Rhodobacteraceae in Marine Sediments Belong to Uncultured Genera: A Molecular Approach to Link Their Distribution to Environmental Conditions

Abstract

General studies on benthic microbial communities focus on fundamental biogeochemical processes or the most abundant constituents. Thereby, minor fractions such as the Rhodobacteraceae are frequently neglected. Even though this family belongs to the most widely distributed bacteria in the marine environment, their proportion on benthic microbial communities is usually within or below the single digit range. Thus, knowledge on these community members is limited, even though their absolute numbers might exceed those from the pelagic zone by orders of magnitudes. To unravel the distribution and diversity of benthic, metabolically active Rhodobacteraceae, we have now analyzed an already existing library of bacterial 16S rRNA transcripts. The dataset originated from 154 individual sediment samples comprising seven oceanic regions and a broad variety of environmental conditions. Across all samples, a total of 0.7% of all 16S rRNA transcripts was annotated as Rhodobacteraceae. Among those, Sulfitobacter, Paracoccus, and Phaeomarinomonas were the most abundant cultured representatives, but the majority (78%) was affiliated to uncultured family members. To define them, the 45 most abundant Rhodobacteraceae-OTUs assigned as "uncultured" were phylogenetically assembled in new clusters. Their next relatives particularly belonged to different subgroups other than the Roseobacter group, reflecting a large part of the hidden diversity within the benthic Rhodobacteraceae with unknown functions. The general composition of active Rhodobacteraceae communities was found to be specific for the geographical location, exhibiting a decreasing richness with sediment depth. One-third of the Rhodobacteraceae-OTUs significantly responded to the prevailing redox regime, suggesting an adaption to anoxic conditions. A possible approach to predict their physiological properties is to identify the metabolic capabilities of their nearest relatives. Those need to be proven by physiological experiments, as soon an isolate is available. Because many uncultured members of these subgroups likely thrive under anoxic conditions, in future research, a molecular-guided cultivation strategy can be pursued to isolate novel Rhodobacteraceae from sediments.

Keywords: benthic; diversity; microbial communities; phylogeny; pyrosequencing.

FIGURE 1

Sampling sites worldwide (large panel) and in the Gulf of Mexico (small panel). Sediments were collected from 2007 to 2012 during several expeditions. The color scale represents water depths at the various sites. The map was created using OceanDataView (Schlitzer, 2018).

FIGURE 2

Composition of the benthic, metabolically active microbial community based on bacterial 16S rRNA transcripts in the entire dataset displayed in the different phylogenetic levels (n = sum of sequences in the respective phylogenetic level, “others” includes all phyla < 2%).

FIGURE 3

Maximum likelihood tree highlighting the position of consensus sequences of the 45 most abundant Rhodobacteraceae-OTUs assigned as “uncultured” relative to other members of the family. The tree was created using ARB (Ludwig et al., 2004) and rooted by sequences of the genus Rhizobium. To keep the tree clear and readable, type strains that were not related to the “uncultured” sequences were collapsed into single groups. Sequences in green could clearly be related to next relatives, orange ones changed positions in comparison to neighbor joining and maximum parsimony trees. Blue sequences always formed consistent clusters.

FIGURE 4

Non-metric multidimensional scaling plot (NMDS based on Bray–Curtis distances) of the active Rhodobacteraceae community compositions based on the different sampling sites. Cycles show the community compositions at the specific site and the color of the cycles indicates increasing sediment depths (light blue: surface, dark blue: down to 100 m below seafloor).

FIGURE 5

Distribution of the OTUs affiliated to cultured representatives within the Rhodobacteraceae. OTUs are sorted by their relative abundance on the total bacterial community. Sample locations are arranged by water depth and then by sediment depth. The affiliation of OTUs to the phylogenetic subgroups is displayed by red = Roseobacter, orange = Stappia, green = Amaricoccus, purple = Rhodobacter, and blue = Paracoccus.

FIGURE 6

Distribution of the OTUs affiliated to uncultured representatives within the Rhodobacteraceae. OTUs are sorted by their relative abundance on the total bacterial community. Sample locations are arranged by water depth and then by sediment depth. The affiliation of OTUs to the phylogenetic subgroups is displayed by red = Roseobacter, orange = Stappia, green = Amaricoccus, black = cluster I, gray = cluster II, and blue = unclear assignment.

FIGURE 7

Significant correlation (p ≤ 0.05) of single OTUs to the environmental parameters TOC, sulfate, sulfide, and iron (Fe²⁺). All OTUs related to cultured and uncultured Rhodobacteraceae were included in the analysis. Displayed are the Spearman’s rank correlation coefficients (r_s). While increasing blue colors show positive correlations, red colors indicate negative values.