Nutrient supplementation experiments with saltern microbial communities implicate utilization of DNA as a source of phosphorus

Abstract

All environments including hypersaline ones harbor measurable concentrations of dissolved extracellular DNA (eDNA) that can be utilized by microbes as a nutrient. However, it remains poorly understood which eDNA components are used, and who in a community utilizes it. For this study, we incubated a saltern microbial community with combinations of carbon, nitrogen, phosphorus, and DNA, and tracked the community response in each microcosm treatment via 16S rRNA and rpoB gene sequencing. We show that microbial communities used DNA only as a phosphorus source, and provision of other sources of carbon and nitrogen was needed to exhibit a substantial growth. The taxonomic composition of eDNA in the water column changed with the availability of inorganic phosphorus or supplied DNA, hinting at preferential uptake of eDNA from specific organismal sources. Especially favored for growth was eDNA from the most abundant taxa, suggesting some haloarchaea prefer eDNA from closely related taxa. The preferential eDNA consumption and differential growth under various nutrient availability regimes were associated with substantial shifts in the taxonomic composition and diversity of microcosm communities. Therefore, we conjecture that in salterns the microbial community assembly is driven by the available resources, including eDNA.

Fig. 1. Microbial growth and associated changes in available nutrients in the experimental microcosms.

a–c Growth curves under experimental treatments. Each line represents a separate experimental treatment. For each treatment, the added nutrients are denoted as “+” followed by a nutrient symbol of carbon (C), nitrogen (N), phosphorus (P_i), Hfx. volcanii DS2 DNA (H) and E. coli dam⁻/dcm⁻ DNA (E). Starvation growth curve (S) serves as a control. Error bars represent the standard error of the mean. b Growth curves of the slow-growing microcosms from (a) plotted on a different scale. a, c Two independent sets of experiments. d, e Changes in carbon, nitrogen and phosphorus after the microbial growth has stopped. The plotted changes are mean values from two experimental replicates. The experimental treatments (X axis) are grouped into three categories based on whether carbon and/or nitrogen were provided. Nutrient abbreviations: TOC, total organic carbon; TN, total nitrogen; TP, total phosphorus; P_i, inorganic phosphorus. Organic phosphorus (P_o) was computed as by subtracting P_i from TP. For “+P_i” treatment, the calculated value of P_o is replaced with an asterisk, due to errors associated with measurements of TP and P_i for that treatment. Actual values for the measured nutrient concentrations and the associated calculations are shown in Supplementary Tables S1 and S2.

Fig. 2. OTU composition of the microbial communities (ICC) and of eDNA in the associated water columns (ICW).

a Relative abundance of 16S rRNA-based OTUs in the pre-incubation community (X), after starvation (S), and after nutrient-addition experiments. For experimental treatment abbreviations see Fig. 1 legend. Only a few selected taxonomic groups are highlighted, while other OTUs are pooled into “Other Archaea” and “Other Bacteria” categories. b Overlap of 16S rRNA-based OTUs in ICC and ICW across all samples combined. c Relative abundances of 16S rRNA-based OTUs that constitute ≥ 1% of at least one sample (designated as “abundant OTUs”) in comparison to the OTUs with < 1% abundance (denoted as “Other OTUs”). d Relative abundances of rpoB-based OTUs from class Halobacteria that constitute ≥1% of at least one sample (designated as “ahOTUs”) in comparison to the Halobacterial OTUs with <1% abundance (denoted as “Other Halobacterial OTUs”).

Fig. 3. Comparison of the rpoB-based OTU compositions of the analyzed samples.

a Principal Coordinate Analysis (PCoA) of all samples using the pairwise Bray-Curtis dissimilarity as a distance metric. The distances were calculated using all OTUs in a sample. Circles denote microbial community samples, while triangles refer to the water column samples. Within these, samples for treatments with added DNA are shown using filled symbols, while all remaining samples are shown using open symbols. For the PCoA analyses carried out using 16S rRNA-based OTUs, see Supplementary Fig. S3. b–d PCoA of the samples within each of the three clusters. Same distance measure and same symbol notations as in a. e Taxonomic composition of samples within each of the three clusters. See Supplementary Table S3 for the actual relative abundance values.

Fig. 4. Relative abundance of ahOTUs across three clusters.

a, b Relative abundance of ahOTUs in three clusters summarized in a ternary plot. Each vertex represents one of the three clusters. Each circle represents an ahOTU. The position of a circle is determined by relative abundance (RA) of the ahOTU in three clusters, and the circle size is proportional to the ahOTU’s average relative abundance across all 69 samples. In a, ahOTUs with significantly higher abundance in Cluster 1, 2, or 3 than in the other two clusters are colored in green, orange and brown, respectively, while OTUs without significant difference in abundance are shown in gray. In b, the circles are colored according to the taxonomic assignment of the ahOTUs. c–e Aggregated relative abundances (aRA) of ahOTUs with significantly higher abundance in one cluster across each of the three clusters. Each point represents an ahOTU. For each OTU its aRA in a cluster i (i = 1, 2, 3) is defined as $aR{A}_i=\frac{R{A}_i}{{\sum }_{j=1}^3\left(R{A}_j\right)}$, where RA_i is the relative abundance of the ahOTU in the cluster i. The distribution of aRAs within a cluster is summarized by a probability density function and a box-and-whisker plot, with whiskers extending to 1.5 of the interquartile range.