Genomic Insights into the Radiation-Resistant Capability of Sphingomonas qomolangmaensis S5-59T and Sphingomonas glaciei S8-45T, Two Novel Bacteria from the North Slope of Mount Everest

Abstract

Mount Everest provides natural advantages to finding radiation-resistant extremophiles that are functionally mechanistic and possess commercial significance. (1) Background: Two bacterial strains, designated S5-59T and S8-45T, were isolated from moraine samples collected from the north slope of Mount Everest at altitudes of 5700m and 5100m above sea level. (2) Methods: The present study investigated the polyphasic features and genomic characteristics of S5-59^T and S8-45^T. (3) Results: The major fatty acids and the predominant respiratory menaquinone of S5-59^T and S8-45^T were summed as feature 3 (comprising C16:1 ω6c and/or C16:1 ω7c) and ubiquinone-10 (Q-10). Phylogenetic analyses based on 16S rRNA sequences and average nucleotide identity values among these two strains and their reference type strains were below the species demarcation thresholds of 98.65% and 95%. Strains S5-59^T and S8-45^T harbored great radiation resistance. The genomic analyses showed that DNA damage repair genes, such as mutL, mutS, radA, radC, recF, recN, etc., were present in the S5-59^T and S8-45^T strains. Additionally, strain S5-59^T possessed more genes related to DNA protection proteins. The pan-genome analysis and horizontal gene transfers revealed that strains of Sphingomonas had a consistently homologous genetic evolutionary radiation resistance. Moreover, enzymatic antioxidative proteins also served critical roles in converting ROS into harmless molecules that resulted in resistance to radiation. Further, pigments and carotenoids such as zeaxanthin and alkylresorcinols of the non-enzymatic antioxidative system were also predicted to protect them from radiation. (4) Conclusions: Type strains S5-59^T (=JCM 35564T =GDMCC 1.3193T) and S8-45^T (=JCM 34749T =GDMCC 1.2715T) represent two novel species of the genus Sphingomonas with the proposed name Sphingomonas qomolangmaensis sp. nov. and Sphingomonas glaciei sp. nov. The type strains, S5-59^T and S8-45^T, were assessed in a deeply genomic study of their radiation-resistant mechanisms and this thus resulted in a further understanding of their greater potential application for the development of anti-radiation protective drugs.

Keywords: DNA repair; Mount Everest; extremophiles; moraine; radiation resistance; whole-genome sequencing.

Figure 1

Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences of the strain S5-59^T, S8-45^T, and the type strains of other closely related species in the genus Sphingomonas and Parasphingorhabdus. Parasphingorhabdus marina FR1087^T (DQ781320) was used as an outgroup. The numbers on the tree indicate the percentages of bootstrap sampling derived from 1000 replications and the bootstrap values higher than 70% are shown. Bar, 0.01 substitutions per nucleotide position.

Figure 2

UBCG phylogenetic tree based on the up-to-date core gene set and pipeline of strains S5-59^T, S8-45^T, and strains of other closely related species in the genus Sphingomonas and Parasphingorhabdus. Parasphingorhabdus marina FR1087^T (DSM 22363^T) was used as an outgroup. Phylogenetic tree generated with UBCG using the amino acids sequences. The number at the nodes indicates the gene support index (maximum value, 92). The numbers at the nodes indicate the gene support index. Bar, 0.10 substitutions per nucleotide position.

Figure 3

Scanning electron microscope photos showing the morphological structure of the cells of strain S5-59^T (A), S8-45^T (C), and plate colonies’ morphology of S5-59^T (B), and S8-45^T (D).

Figure 4

The survival rates of strains S5-59^T and S8-45^T after irradiation with different doses of UVC and γ rays’ radiation. D₁₀–values are displayed by the red dash line; the blue numbers on the right y-axis of each image represent the normal growth time of the strains without irradiation. (A) Survival rates of S5-59^T after irradiation by different doses of UV-C; (B) survival rates of S5-59^T after irradiation by different doses of γ rays; (C) survival rates of S8-45^T after irradiation by different doses of UV-C; and (D) survival rates of S8-45^T after irradiation by different doses of γ rays.

Figure 5

Circular chromosome map and COG functional categories of the strains S5-59^T (A) and S8-45^T (B) genomes. The circles show the different descriptions of the content in metabasins, from the outside to inward: the outer circle represents the genome size, the second circle and the third circle represents the predicted protein-coding sequences and CDS regions on the plus and minus strands, respectively. The colors represent COG functional classification. The fourth circle represents the repeated sequence. The fifth circle represents tRNA and rRNA. The sixth circle shows GC content and the seventh circle exhibits the percent of GC-skew. (The detailed clusters of orthologous group information are shown in Table S3).

Figure 6

Genome comparisons of strains S5-59^T, S8-45^T, and their related reference strains including the OrthoANI value (A), AAI value (B), and dDDH value (C). Further, a–h represent S5-59^T, S8-45^T, Sphingomonas panacisoli HKS19^T, Sphingomonas asaccharolytica NBRC 15499^T, Sphingomonas panacis DCY99^T, Sphingomonas kaistensis PB56^T, Sphingomonas astaxanthinifaciens DSM 22298^T, and Sphingomonas ginsengisoli KCTC 12630^T, respectively. Additionally, i was the type species of Sphingomonas, i.e., Sphingomonas paucimobilis DSM 1098^T.

Figure 7

Comparisons of orthologous protein groups in S5-59^T, S8-45^T, and six related Sphingomonas genomes. (A) Percentage of core, dispensable, and unique genes in each of all eight genomes. (B) Venn diagram displaying the number of core and unique genes for each of the S5-59^T, S8-45^T, and related type strains.

Figure 8

The number and functional gene classification of pan genomes between different Sphingomonas strains. The upset plot shows the number and functional classification of the core and unique genes in different Sphingomonas strains. The bar chart above represents the number of core and unique genes contained in each type of group. The strip at the bottom left represents the total number of genes in different Sphingomonas strains. The dot and line at the bottom right represent the types of different combinations (where only values above 10 and annotated genes are shown; further, unknown genes were not shown).