The Eastern Nebraska Salt Marsh Microbiome Is Well Adapted to an Alkaline and Extreme Saline Environment

Abstract

The Eastern Nebraska Salt Marshes contain a unique, alkaline, and saline wetland area that is a remnant of prehistoric oceans that once covered this area. The microbial composition of these salt marshes, identified by metagenomic sequencing, appears to be different from well-studied coastal salt marshes as it contains bacterial genera that have only been found in cold-adapted, alkaline, saline environments. For example, Rubribacterium was only isolated before from an Eastern Siberian soda lake, but appears to be one of the most abundant bacteria present at the time of sampling of the Eastern Nebraska Salt Marshes. Further enrichment, followed by genome sequencing and metagenomic binning, revealed the presence of several halophilic, alkalophilic bacteria that play important roles in sulfur and carbon cycling, as well as in nitrogen fixation within this ecosystem. Photosynthetic sulfur bacteria, belonging to Prosthecochloris and Marichromatium, and chemotrophic sulfur bacteria of the genera Sulfurimonas, Arcobacter, and Thiomicrospira produce valuable oxidized sulfur compounds for algal and plant growth, while alkaliphilic, sulfur-reducing bacteria belonging to Sulfurospirillum help balance the sulfur cycle. This metagenome-based study provides a baseline to understand the complex, but balanced, syntrophic microbial interactions that occur in this unique inland salt marsh environment.

Keywords: Marichromatium; Prosthecochloris; Rubribacterium; Salt Creek; Sulfurimonas; Sulfurospirillum; microbiome; salt marsh; sulfur cycling.

Figure 1

(A) Map of the sampling locations for this study. Samples were taken at the valley of Salt Creek and Little Salt Creek near Lincoln, Nebraska. The sampling area within the salt marsh is marked in red. All maps were generated using Google Maps (B) Overview of the NE Salt Marsh sampling locations: SM2B, SM2C, SM2A, and SM2E.

Figure 2

A dendrogram showing the hierarchical clustering of the metagenomes from the three sampling locations, based on genus-level classifications of 16S rRNA gene amplicon samples. Only families with >3% of the sequencing reads are represented in the figure legend.

Figure 3

Whole-genome-based phylogenetic tree of all sequenced Prosthecochloris species. The phylogenetic tree was generated using the CodonTree method within PATRIC [16], which used PGFams as homology groups. The support values for the phylogenetic tree were generated using 100 rounds of the “Rapid bootstrapping” option of RaxML [19]. Chlorobaculum sp. 24CR was used as an outgroup [63]. The branch length tree scale is defined as the mean number of substitutions per site, which is the average across both nucleotide and amino acid changes. The NE Salt Marsh Prosthecochloris sp. SM2 is shown in red.

Figure 4

Whole genome-based phylogenetic tree of sequenced Chromatiaceae species. The phylogenetic tree was generated using the CodonTree method within PATRIC [16], which used PGFams as homology groups. The support values for the phylogenetic tree were generated using 100 rounds of the “Rapid bootstrapping” option of RaxML [19]. Halochromatium sp. A10 was used as an outgroup. The branch length tree scale is defined as the mean number of substitutions per site, which is the average across both nucleotide and amino acid changes. The NE Salt Marsh Marichromatium sp. SM2 is shown in red.

Figure 5

Simplified overview of the proposed sulfur cycling in microbial ecosystems. Bacterial genera identified in the NE Salt Marsh samples that are assumed to be involved are indicated for each group. AER: aerobic reactions; ANAER: anaerobic reactions. Image created using BioRender.com.