Metagenomic Signatures of Bacterial Adaptation to Life in the Phyllosphere of a Salt-Secreting Desert Tree

Abstract

The leaves of Tamarix aphylla, a globally distributed, salt-secreting desert tree, are dotted with alkaline droplets of high salinity. To successfully inhabit these organic carbon-rich droplets, bacteria need to be adapted to multiple stress factors, including high salinity, high alkalinity, high UV radiation, and periodic desiccation. To identify genes that are important for survival in this harsh habitat, microbial community DNA was extracted from the leaf surfaces of 10 Tamarix aphylla trees along a 350-km longitudinal gradient. Shotgun metagenomic sequencing, contig assembly, and binning yielded 17 genome bins, six of which were >80% complete. These genomic bins, representing three phyla (Proteobacteria,Bacteroidetes, and Firmicutes), were closely related to halophilic and alkaliphilic taxa isolated from aquatic and soil environments. Comparison of these genomic bins to the genomes of their closest relatives revealed functional traits characteristic of bacterial populations inhabiting the Tamarix phyllosphere, independent of their taxonomic affiliation. These functions, most notably light-sensing genes, are postulated to represent important adaptations toward colonization of this habitat.

Importance: Plant leaves are an extensive and diverse microbial habitat, forming the main interface between solar energy and the terrestrial biosphere. There are hundreds of thousands of plant species in the world, exhibiting a wide range of morphologies, leaf surface chemistries, and ecological ranges. In order to understand the core adaptations of microorganisms to this habitat, it is important to diversify the type of leaves that are studied. This study provides an analysis of the genomic content of the most abundant bacterial inhabitants of the globally distributed, salt-secreting desert tree Tamarix aphylla Draft genomes of these bacteria were assembled, using the culture-independent technique of assembly and binning of metagenomic data. Analysis of the genomes reveals traits that are important for survival in this habitat, most notably, light-sensing and light utilization genes.

FIG 1

Clustering of 5,122 contigs (minimum length, 5 kbp; total of 103.15 Mbp) coassembled from all phyllosphere metagenomic data (n = 20) based on tetranucleotide frequencies and coverage values. Contigs longer than 40 kbp were split into pieces of 20 kbp (see splits layer) in order to optimize the view of well-assembled genomes. Length, GC content, and taxonomy (when detected) are displayed for each contig as independent layers. Taxonomy was inferred using myRAST function svr_assign_to_dna_using_figfams. The mean coverage (left panel) and portion coverage (right panel) of each contig are displayed across biological samples (n = 10). Finally, 19 genomic selections were made for clusters larger than 0.5 Mbp (outer layer) and colored based on RAST taxonomy results.

FIG 2

Relative abundance of SSU reads. Left, cross domain relative abundance based on rRNA gene (SSU) read counts in metagenomic data sets. Right, comparison between bacterial SSU profiles from amplicon (top panels) and shotgun metagenomic (middle panels) data sets, normalized to the number of bacterial SSU copies in the metagenomic data set. The bottom panel shows the proportion of reads mapped from each metagenomic data set to genomic bins.

FIG 3

Nonmetric multidimensional scaling (NMDS) ordination of Bray-Curtis similarity matrix based on V6 data at the family taxonomic level. The spatial proximity of samples (black dots) on the graph is proportional to the similarity in community composition. Bacterial families are shown as bubbles, positioned according to their relative abundance among samples. Bubbles are colored according to class and sized by relative abundance. Classes in gray scale were not represented in genomic bins. The inset graph displays the negative correlation between the abundances at the different sampling sites of the two major classes, Bacilli and Gammaproteobacteria.

FIG 4

Comparative view of gene content tree (based on Get_homologue analysis) (left) and phylogenetic tree (based on a concatenated alignment of 9 ribosomal proteins) (right). Both trees include genomic bins and reference genomes used for comparative genomics. Branches are colored by class. Bootstrap values are shown on the nodes of the phylogenetic tree.

FIG 5

Metabolic diagram of the main inhabitants of the Tamarix phyllosphere, based on mapping of the KEGG profile onto metabolic maps. Compounds appearing in the diagram are source compounds that can be linked to the central metabolic modules. Membrane transport includes ATP binding cassette (ABC) transporters and phosphotransferase systems (PTS). For ABC transporters, it was required that at least two of the three components are present; for PTS with more than one component, both were required to be present in order to be included in the model. Brown, Gram positive; blue, Gram negative; black, core.

FIG 6

Functions unique to Tamarix. Functions that are ubiquitous in Tamarix bins and are restricted to one class or less in closely related reference genomes are shown. The functions shown include only those that are shared by at least three separate taxonomic classes among genome bins, allowing occurrence in only one taxonomic class among the reference genomes. Color is proportional to the prevalence of the gene within each class.