Serratia liquefaciens FG3 isolated from a metallophyte plant sheds light on the evolution and mechanisms of adaptive traits in extreme environments

Abstract

Serratia liquefaciens strain FG3 (SlFG3), isolated from the flower of Stachytarpheta glabra in the Brazilian ferruginous fields, has distinctive genomic, adaptive, and biotechnological potential. Herein, using a combination of genomics and molecular approaches, we unlocked the evolution of the adaptive traits acquired by S1FG3, which exhibits the second largest chromosome containing the largest conjugative plasmids described for Serratia. Comparative analysis revealed the presence of 18 genomic islands and 311 unique protein families involved in distinct adaptive features. S1FG3 has a diversified repertoire of genes associated with Nonribosomal peptides (NRPs/PKS), a complete and functional cluster related to cellulose synthesis, and an extensive and functional repertoire of oxidative metabolism genes. In addition, S1FG3 possesses a complete pathway related to protocatecuate and chloroaromatic degradation, and a complete repertoire of genes related to DNA repair and protection that includes mechanisms related to UV light tolerance, redox process resistance, and a laterally acquired capacity to protect DNA using phosphorothioation. These findings summarize that SlFG3 is well-adapted to different biotic and abiotic stress situations imposed by extreme conditions associated with ferruginous fields, unlocking the impact of the lateral gene transfer to adjust the genome for extreme environments, and providing insight into the evolution of prokaryotes.

Figure 1

Circular map of chromosome (a) and plasmids (b) of SlFG3. Chromosome - dark blue and light bars represent, respectively, genes annotated in the + and - DNA strand; Green blocks - anomalous regions; Dark gray circle - GC content; Salmon and wine lines - GC cumulative content. Plasmids - arrows determine the strand encoding the genes with respective functions presented in the legend; Tra - conjugation genes (light green); Fim - fimbriae genes (red bar). (c) Histogram that determines the representativeness of the plasmid genes for the genres identified as best comparison hits.

Figure 2

Comparative analysis of SlFG3 genome with other complete genomes of bacteria from the Serratia genus. (a) Flower plot that highlights the number of unique, core, and flexible genes. (b) Phylogenomic analysis determined from the core genome. Yersinia pestis strain 8081 and Escherichia coli strain K12 were used as an external group. *genomes that have clustered outside the clade of their respective species. (c) Venn diagram highlighting the unique, flexible, and core genes for the genomes present in the clade of S. liquefaciens (SlATCC27592, SlFDAARGOS, and SlHUMV21), including S. proteamaculans 568 (Sp568). In white, the number of unique single copy genes is highlighted, and the unique genes in multiple copies are in blue. For the genomes of SlFG3 and Sp568 a more detailed analysis was established, classifying the unique genes present in the chromosomal or plasmid units. (d) Bidirectional best hit analysis (BBH) between the genes that make up the SlFG3 genome in relation to the other genomes of S. liquefaciens and Sp568, using RAST. Colors range from dark blue (100%) to light green (70%), representing the degree of conservation of the sequences. The red and green bars identify putative (intact or questionable) phage regions according to analysis established by the PHAST program. (e) Comparative analysis of SlFG3 genes relative to the genomes of SlATCC27592, SlFDAARGOS, SlHUMV21, and Sp568 according to the functional classifications of RAST. Categories: 1 - Amino Acids and Derivatives (531); 2 - Carbohydrates (693); 3 - Cell Division and Cell Cycle (42); 4 - Cell Wall and Capsule (193); 5 - Cofactors, Vitamins, Prosthetic Groups, Pigments (292); 6 - DNA Protection and Metabolism (128); 7 - Dormancy and Sporulation (3); 8 - Fatty Acids, Lipids, and Isoprenoids (162); 9 - Iron Acquisition and Metabolism (110); 10 - Membrane Transport (183); 11 - Metabolism of Aromatic Compounds (95); 12 - Miscellaneous (50); 13 - Motility and Chemotaxis (86); 14 - Nitrogen Metabolism (44); 15 - Nucleosides and Nucleotides (149); 16 - Phages, Prophages, Transposable Elements, Plasmids (148); 17 - Phosphorus Metabolism (73); 18 - Photosynthesis (0); 19 - Potassium Metabolism (37); 20 - Protein Metabolism (295); 21 - Regulation and Cell Signaling (168); 22 - Respiration (171); 23 - RNA Metabolism (252); 24 - Secondary Metabolism (8); 25 - Stress Response (183); 26 - Sulfur Metabolism (68); 27 - Virulence, Disease and Defense (126). Six functional subcategories were highlighted by variation in the number of annotated genes and will be discussed throughout the text (DNA repair, DNA phosphorothioation (PT), chloroaromatic degradation, catechol β-ketoadipate, and phages and prophages).

Figure 3

Analysis of the genes and metabolic pathways associated with metabolism of phenolic compounds in the genome of SlFG3. (a) Distribution of the SlFG3 genes in the metabolic pathway subcategories associated with degradation of phenolic compounds. (b) Comparison of the genes quantitatively presented in A in relation to the genomes of the other species present in the clade of S. liquefaciens. The cat and pca genes involved, respectively, with the chloroaromatic degradation pathway, and catechol B-ketoadipate pathway are highlighted: these are present exclusively in plant-associated genomes (SlFG3 and Spro568). (c) Analysis of the syntenia and function of each of these genes in the metabolism of 4-hydroxybenzoate (4-HB) and chloroaromatic compounds. 3,4-DHB-3,4-dihydroxybenzoate (protochatecuate); 3-cycloM-3-carboxy-cis, cis-muconate; 4-CML-4-carboxymuconolactone; 3-OEL-3-oxoadipate enol lactone; 3-AO-3-oxoadipate; 3-AO-coA-3-oxoadipyl-CoA; TCA - tricarboxylic acid cycle.

Figure 4

Comparative analysis of sulfur metabolism pathways. (a) Presence and absence of genes involved in cysteine biosynthesis and metabolism involving the five genomes present in the clade of S. liquefaciens. Squares represent the presence of the respective investigated genes, which may be present in single copy, or as multiple copies according to the number within these squares. (b) Integrated metabolism of cysteine biosynthetic and metabolic pathways that culminate in three important routes: biosynthesis of glutathione that, in turn, would be associated with protection against oxidative stress; RNA modification, as an additional protection of the addition of a sulfur molecule at position 34; and DNA protection against phosphorothioation–mediated events. (c) Functional analysis of DNA protection of SlFG3 by phosphothioation events when submitted to stress conditions (For details see Supplementary Fig. S1). (d) Analysis of the possible region associated with horizontal gene tranfer where the genes associated with DNA PT (dndBCDE) in the genome of SlFG3 are inserted. DR - direct repeats; tRNALeu – Leucine tRNA gene; DptFGH - genes involved with DNA restriction and modification mediated by PT DNA.

Figure 5

Analysis of oxidative stress metabolism and DNA repair in the genome of SlFG3. (a) Comparison involving the presence and absence of genes involved in DNA repair, involving the five genomes present in the clade of S. liquefaciens. In red is highlighted the exclusive recT gene of SlFG3 and an additional copy of dam, dinI and radC genes, the latter present in the HGT region where the DNA PT genes are inserted (Fig. 3). (b) Tolerance of SlFG3 exposed for 30, 60, and 90 seconds of UV and incubated in the presence and absence of light, compared to E. coli. (c) Comparison involving the presence and absence of genes involved in oxidative stress adaptation (red), enzymes (orange) and GSH biosynthesis and recycling (brown), involving the five genomes present in the S. liquefaciens clade. (d) Integrative metabolism of pathways associated with oxidative stress in the SlFG3 genome. It is possible to observe a complete repertoire of genes involved in protection against cell damage induced by reactive oxygen species (ROS). (e) Functional analysis of the ability of SlFG3 to protect against H₂O₂-induced damage in comparison to E. coli in the absence and increasing presence of 1, 2 and 5 mM H₂O₂ (f) Evaluation of resistance to acute exposure to 1 mM H₂O₂ SlFG3 compared to E. coli.

Figure 6

Structural and functional analysis of SlFG3 gene cluster related to cellulose production. (a) Analysis of the composition of a gene cluster associated with cellulose synthesis. The clustering involves nine genes: 2 regulatory (blue), 4 associated with export and cellulose synthesis (pink), and 3 associated with quorum sensing and modulation of cellulose synthesis responses (green). In each of the genes that are part of the cluster was verified the presence of functional domains. (b) Plates demonstrating the production of cellulose by SlFG3 when compared to E. coli, revealed by the bright blue methods of Coomassie (above) and calcofluor (below).

Figure 7

Analysis of the composition of gene clusters associated with synthesis of secondary metabolites in the genome of SlFG3. Of the 35 clusters identified by AntiSmash tool, four were highlighted - among them: biosynthesis of turmebactin (a), pseudomonine (b), malleobactin (c) and a cluster associated with an unidentified NRP (d). In green background, the gene clusters that are part of the biosynthetic core (c) and accessory genes (a) involved in the synthesis of these compounds are highlighted. The legend below the figure highlights the classification of these genes and the characterization of functional domains. It is possible to observe that all four clusters were identified in other species from Serratia genus, sometimes maintaining a high degree of conservation, as is the case of the cluster of turnebactin (a), sometimes with a low degree of maintenance of the genus composition, as is the case for the malleobactin synthesis cluster (c).

Figure 8

Schematic representation of the integrated metabolism of SlFG3. The arrows determine the flow of metabolic information. The colors of these arrows are differentiated only to allow a better understanding of the processes involved. The tandem arrows identify the presence of gene clusters associated with their respective characterized functions. The numbers in parentheses determine the total number of genes associated with the respective function presented.