Computational Insight into Intraspecies Distinctions in Pseudoalteromonas distincta: Carotenoid-like Synthesis Traits and Genomic Heterogeneity

Abstract

Advances in the computational annotation of genomes and the predictive potential of current metabolic models, based on more than thousands of experimental phenotypes, allow them to be applied to identify the diversity of metabolic pathways at the level of ecophysiology differentiation within taxa and to predict phenotypes, secondary metabolites, host-associated interactions, survivability, and biochemical productivity under proposed environmental conditions. The significantly distinctive phenotypes of members of the marine bacterial species Pseudoalteromonas distincta and an inability to use common molecular markers make their identification within the genus Pseudoalteromonas and prediction of their biotechnology potential impossible without genome-scale analysis and metabolic reconstruction. A new strain, KMM 6257, of a carotenoid-like phenotype, isolated from a deep-habituating starfish, emended the description of P. distincta, particularly in the temperature growth range from 4 to 37 °C. The taxonomic status of all available closely related species was elucidated by phylogenomics. P. distincta possesses putative methylerythritol phosphate pathway II and 4,4'-diapolycopenedioate biosynthesis, related to C30 carotenoids, and their functional analogues, aryl polyene biosynthetic gene clusters (BGC). However, the yellow-orange pigmentation phenotypes in some strains coincide with the presence of a hybrid BGC encoding for aryl polyene esterified with resorcinol. The alginate degradation and glycosylated immunosuppressant production, similar to brasilicardin, streptorubin, and nucleocidines, are the common predicted features. Starch, agar, carrageenan, xylose, lignin-derived compound degradation, polysaccharide, folate, and cobalamin biosynthesis are all strain-specific.

Keywords: Pseudoalteromonas; aryl polyene hybrid; biosynthetic gene clusters; marine bacteria; metabolic pathways; metabolic reconstruction; pan-genome; phenotype variability; phylogenomic relationships.

Figure 1

The strains P. distincta KMM 638^T (left, diffusible black pigmentation), P. distincta KMM 3548 (formerly P. paragorgicola KMM 3548) (middle, cell-bound slight yellow-orange pigmentation), and P. distincta KMM 701 (right, whitish) were cultivated on marine agar under the daylight for 7 (a) and 9 (b) days at 24 °C.

Figure 2

Maximum-likelihood phylogeny of the genus Pseudoalteromonas based on 400 universal markers selected by PhyloPhlAn3.0 and reconstructed by RAxML with non-parametric bootstrapping using 100 replicates, including Bar, with 0.1 substitutions per amino acid position. The corresponding GenBank accession numbers for genomes are given in parentheses. Representatives with novel genomes are in bold.

Figure 3

Similarity matrix for pairwise genome comparisons plotted by OrthoVenn2: the heatmap shows the ortholog clusters between any pair of genomes.

Figure 4

The pan-genome of 21 Pseudoalteromonas strains. Clustering of the genomes based on the presence/absence patterns of 10,045 pan-genomic clusters. The genomes are organized in radial layers as core, soft-core, shell, and cloud gene clusters (Euclidean distance; Ward linkage), which are defined by the gene tree in the center. The genome of the type strain P. distincta ATCC 700518^T is colored very dark blue (hex code #04353d), the genomes of other P. distincta strains are colored sherpa blue (hex code #065563), the genome of P. arctica A 37-1-1^T is colored cyan blue (hex code #10abc7), and the genomes of other Pseudoalteromonas species are colored grey (hex code #4f4a4f). The heatmap displays pairwise values of average nucleotide (ANI) and amino acid (AAI) identities in percentages calculated using the online server ANI/AAI-Matrix.

Figure 5

(A) Maximum-likelihood phylogeny of twenty-one Pseudoalteromonas strains based on 1369 core genes selected by Roary and reconstructed by IQ-TREE and (B) distribution of secondary metabolism biosynthetic gene clusters (BGCs) across the genomes.

Figure 6

Putative biosynthetic gene clusters for RiPP-like peptides (1 cluster: nucleocidin-like; 2 cluster: brasilicardin-like) in the strains P. distincta and P. arctica A 37-1-2^T.

Figure 7

Comparative analysis of synteny between resorcinol and/or aryl polyene biosynthetic gene clusters (ape BGC), which were found in ten Pseudoalteromonas genomes: Parc A 37-1-2^T, P. arctica A 37-1-2^T; Pdis 2-2A-13, P. distincta 2-2A-13; Ppar KMM 3548^T, P. paragorgicola KMM 3548^T; Paga DSM 14585^T, P. agarivorans DSM 14585^T; Ptel DSM 16098, “P. telluritireducens” DSM 16098; Pnig NBRC 103036^T, P. nigrifaciens NBRC 103036^T; Ptra KMM 520^T, P. translucida KMM 520^T; Pdis ANT/505, P. distincta ANT/505; Psp TAE79, Pseudoalteromonas sp. TAE79; Psp TAE80, Pseudoalteromonas sp. TAE80. Genes were colored based on their annotations, as indicated at the bottom of the figure.

Figure 8

The distribution of carbohydrate-active enzymes (CAZymes) in the genomes of P. distincta and P. arctica A 37-1-2^T. The heat map shows the number of genes assigned to individual CAZyme families. Rows are clustered using Euclidean distances. Parc A 37-1-2^T, P. arctica A 37-1-2^T; Pdis 2-2A-13, P. distincta 2-2A-13; Ppar KMM 3548^T, P. paragorgicola KMM 3548^T; Paga DSM 14585^T, P. agarivorans DSM 14585^T; Ptel DSM 16098, “P. telluritireducens” DSM 16098; Pnig NBRC 103036^T, P. nigrifaciens NBRC 103036^T; Ptra KMM 520^T, P. translucida KMM 520^T; Pdis ANT/505, P. distincta ANT/505; Psp TAE79, Pseudoalteromonas sp. TAE79; Psp TAE80, Pseudoalteromonas sp. TAE80.

Figure 9

Functional annotation of unique genes among strains of P. distincta and P. arctica A 37-1-1^T (the P. distincta TB25 orthologues were mostly not found): Genes were assigned to the following COG categories: C, energy production and conversion; D, cell cycle control and mitosis; E, amino acid metabolism and transport; F, nucleotide metabolism and transport; G, carbohydrate metabolism and transport; H, coenzyme metabolism, I, lipid metabolism; J, translation; K, transcription; L, replication and repair; M, cell wall/membrane/envelope biogenesis; N, cell motility; O, post-translational modification, protein turnover, chaperone functions; P, inorganic ion transport and metabolism; Q, secondary structure; T, signal transduction; U, intracellular trafficking and secretion; V, defense mechanisms; X, mobilome: prophages, transposons; R, general functional prediction only; S, function unknown; W, extracellular structures.

Figure 10

Strain-specific metabolic pathways in P. distincta and P. arctica A 37-1-1^T (pathway signatures are depicted in the corresponding Supplementary Table S5): 1—P. distincta 16-SW-7 (GCA_005877035.1; seawater); 2—P. distincta ANT 505 (GCA_000212655.3; seawater); 3—P. distincta ATCC 700518^T (GCA_000814675.1; marine sponge); 4—P. distincta U2A (GCA_008370225.1; brown algae surface); 5—P. elyakovii SM1926 (GCA_007786285.1; surface seawater); 6—P. paragorgicola KMM3548 (GCA_014918315.1); 7—Pseudoalteromonas sp. AC163 (GCA_000497935.1; marine sponge); 8—Pseudoalteromonas sp. TAE79 (GCA_000498015.1; water column); 9—Pseudoalteromonas sp. TAE80 (GCA_000498035.1; water column); 10—Pseudoalteromonas sp. TB25 (GCA_000497995.1; Antarctic marine sponge); 11—P. arctica A 37 1 2T (GCA_000238395.4; seawater); 12—P. distincta 2-2A-13.