Genomic Characterization of Potential Plant Growth-Promoting Features of Sphingomonas Strains Isolated from the International Space Station

Abstract

In an ongoing microbial tracking investigation of the International Space Station (ISS), several Sphingomonas strains were isolated. Based on the 16S rRNA gene sequence, phylogenetic analysis identified the ISS strains as Sphingomonas sanguinis (n = 2) and one strain isolated from the Kennedy Space Center cleanroom (used to assemble various Mars mission spacecraft components) as Sphingomonas paucimobilis. Metagenomic sequence analyses of different ISS locations identified 23 Sphingomonas species. An abundance of shotgun metagenomic reads were detected for S. sanguinis in the location from where the ISS strains were isolated. A complete metagenome-assembled genome was generated from the shotgun reads metagenome, and its comparison with the whole-genome sequences (WGS) of the ISS S. sanguinis isolates revealed that they were highly similar. In addition to the phylogeny, the WGS of these Sphingomonas strains were compared with the WGS of the type strains to elucidate genes that can potentially aid in plant growth promotion. Furthermore, the WGS comparison of these strains with the well-characterized Sphingomonas sp. LK11, an arid desert strain, identified several genes responsible for the production of phytohormones and for stress tolerance. Production of one of the phytohormones, indole-3-acetic acid, was further confirmed in the ISS strains using liquid chromatography-mass spectrometry. Pathways associated with phosphate uptake, metabolism, and solubilization in soil were conserved across all the S. sanguinis and S. paucimobilis strains tested. Furthermore, genes thought to promote plant resistance to abiotic stress, including heat/cold shock response, heavy metal resistance, and oxidative and osmotic stress resistance, appear to be present in these space-related S. sanguinis and S. paucimobilis strains. Characterizing these biotechnologically important microorganisms found on the ISS and harnessing their key features will aid in the development of self-sustainable long-term space missions in the future. IMPORTANCESphingomonas is ubiquitous in nature, including the anthropogenically contaminated extreme environments. Members of the Sphingomonas genus have been identified as potential candidates for space biomining beyond earth. This study describes the isolation and identification of Sphingomonas members from the ISS, which are capable of producing the phytohormone indole-3-acetic acid. Microbial production of phytohormones will help future in situ studies, grow plants beyond low earth orbit, and establish self-sustainable life support systems. Beyond phytohormone production, stable genomic elements of abiotic stress resistance, heavy metal resistance, and oxidative and osmotic stress resistance were identified, rendering the ISS Sphingomonas isolate a strong candidate for biotechnology-related applications.

Keywords: International Space Station; Sphingomonas; phytohormones; plant growth promotion.

FIG 1

The abundance of Sphingomonas species across different flights and locations on the ISS based on metagenome-derived genomic reads. The numbers on top of bars represent the number of Sphingomonas species identified from that particular location in the corresponding flight number (F1, F2, or F3). Different locations on the ISS from where samples were collected: location 1, port panel next to cupola (node 3); location 2, waste and hygiene compartment (node 3); location 3, advanced resistive exercise device (ARED) foot platform (node 3); location 4, dining table (node 1); location 5, zero G stowage rack (node 1); location 7, panel near portable water dispenser (LAB); and location 8, port crew quarters, bump out exterior aft wall (node 2).

FIG 2

Circular genome representations were constructed using BLAST Ring Image Generator (BRIG) version 0.95, with Sphingomonas sp. LK11 selected as the reference genome (chromosome and plasmids). The innermost ring (black) shows the GC content of the reference Sphingomonas sp. LK11 genome. Subsequent rings illustrate the percent identity of the selected Sphingomonas genomes calculated using blastn (E value: 10, lower identity cutoff: 70%, upper identity cutoff: 90%). Starting from ring two, the order of the illustrated genomes is as follows: 2, S. paucimobilis FKI-L5-BR-P1; 3, S. paucimobilis LCT-SP1; 4, S. paucimobilis NCTC 11030^T; 5, S. sanguinis IIF7SW-B3A; 6, S. sanguinis IIF7SW-B5; 7, S. sanguinis ISS-IIF7SWP (MAG); 8, S. sanguinis NBRC 13937^T. The outermost ring displays randomly selected annotations originating from the Sphingomonas sp. LK11 chromosome.

FIG 3

Pangenome analysis of S. sanguinis, S. paucimobilis genomes obtained from space-relevant surfaces with their type strains, and Sphingomonas sp. LK11, generated by the Anvi’o software. Primary functional annotation in Anvi’o was conducted using the anvi-script-FASTA-to-contigs-db and anvi-run-ncbi-cogs commands. Subsequent pangenome gene clustering was carried out using blastp via the anvi-pan-genome command (–num-threads 2, –mcl-inflation 6, –min-bit 0.5, –use-ncbi-blast). Ordering of the pangenome display was determined using a Euclidean distance clustering algorithm and the provided ward linkage method. Beginning from the innermost ring and moving outward, rings 1 to 8 correspond to gene clusters identified in each of the analyzed Sphingomonas genomes in the following order: 1, Sphingomonas sp. LK11; 2, S. paucimobilis FKI-L5-BR-P1; 3, Sphingomonas LCT-SP1; 4, S. paucimobilis NCTC 11030^T; 5, S. sanguinis IIF7SW-B3A; 6, S. sanguinis IIF7SW-B5; 7, S. sanguinis ISS-IIF7SWP (MAG); 8, S. sanguinis NBRC 13937^T. Ring 9 shows a density plot of the number of genes within the identified gene clusters, and ring 10 highlights single-copy gene clusters (black). Rings 11 and 12 correspond to the geometric homogeneity index and functional homogeneity indices of similarity, with the former accounting for gaps in alignments, while the latter scores functional similarities by residue within the aligned sequences. The outermost ring represents the location of known Clusters of Orthologous Genes (COG) functional categories. The right-most bar chart corresponds to the number of singleton gene clusters, number of gene clusters, number of genes, total length, and GC content for each genome present in the analysis.

FIG 4

(A) Zeaxanthin and nostoxanthin pathway. (B) Genes discussed relevant to trehalose metabolism in S. sanguinis and S. paucimobilis. In the first step of the otsA/B pathway, a condensation reaction between glucose 6-phosphate and UDP-glucose synthesizes trehalose 6-phosphate (otsA). Second, a dephosphorylation of trehalose 6-phosphate catalyzed by otsB yields the product trehalose. Additionally, trehalose can also be formed from the rearrangement of the α(1,4) glycosidic bonds of glucose polymers to α(1) glycosidic bonds via treY, allowing for the hydrolysis of the terminal trehalose disaccharide via treZ. (C) Proposed pathway for IAA production in S. paucimobilis and S. sanguinis using functional predictions generated by eggNOG-MapperV2. Enzyme classes are denoted in brackets, with gene, enzyme, or protein names provided where appropriate. Purple coloring denotes identified enzyme classes that were identified only in the analyzed S. paucimobilis spaceflight strains. Question marks (??) denote yet-to-be-validated reaction mechanisms in the proposed pathway. Chemical structures were constructed using the ChemDraw software.

FIG 5

(A) LC-MS profile of IAA produced by S. sanguinis and S. paucimobilis flight isolates, compared to the standard. (B) LC-MS/MS spectrum of IAA standard compared to that produced by S. sanguinis and S. paucimobilis flight isolates, all generating similar fragmentation patterns.

FIG 6

Hierarchical clustering of the relative abundances for eggNOG functional categories for each of the selected Sphingomonas strains. Counts of each functional category were normalized to the total number of annotated proteins within each genome. The y-axis displays eggNOG functional category letter codes corresponding to the following: A, RNA processing and modification; B, chromatin structure and dynamics; C, energy production and conversion; D, cell cycle control, cell division, chromosome partitioning; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; K, transcription; L, replication, recombination and repair; M, cell wall/membrane/envelope biogenesis; N, cell motility; O, posttranslational modification, protein turnover, chaperones; P, inorganic ion transport and metabolism; Q, secondary metabolites biosynthesis, transport and catabolism; R, general function prediction only; S, function unknown; T, Signal transduction mechanisms; U, intracellular trafficking, secretion, and vesicular transport; V, defense mechanisms; W, extracellular structures; Y, nuclear structure; Z, cytoskeleton.

FIG 7

(A) UpSetR plot showing the intersection of named eggNOG predicted proteins from S. sanguinis and S. paucimobilis genomes obtained from space-relevant surfaces and their respective type strains. Total named eggNOG protein counts from each strain are shown in a horizontal bar plot (left) and the number of shared named eggNOG proteins among the strains are represented in the vertical bar plot (right). (B) JVENN plot displaying the intersection of named EggNOG proteins according to their species and environment (S: space-relevant surface, T: type strain), where the named eggNOG proteins were pooled by category for simpler comparisons. Color schemes denote both the species and their environment of origin (green: S. paucimobilis space-relevant surface isolate, blue: S. paucimobilis type strain, pink: S. sanguinis space-relevant surface isolate, yellow: S. sanguinis type strain).