Family-wide Structural Characterization and Genomic Comparisons Decode the Diversity-oriented Biosynthesis of Thalassospiramides by Marine Proteobacteria

Abstract

The thalassospiramide lipopeptides have great potential for therapeutic applications; however, their structural and functional diversity and biosynthesis are poorly understood. Here, by cultivating 130 Rhodospirillaceae strains sampled from oceans worldwide, we discovered 21 new thalassospiramide analogues and demonstrated their neuroprotective effects. To investigate the diversity of biosynthetic gene cluster (BGC) architectures, we sequenced the draft genomes of 28 Rhodospirillaceae strains. Our family-wide genomic analysis revealed three types of dysfunctional BGCs and four functional BGCs whose architectures correspond to four production patterns. This correlation allowed us to reassess the "diversity-oriented biosynthesis" proposed for the microbial production of thalassospiramides, which involves iteration of several key modules. Preliminary evolutionary investigation suggested that the functional BGCs could have arisen through module/domain loss, whereas the dysfunctional BGCs arose through horizontal gene transfer. Further comparative genomics indicated that thalassospiramide production is likely to be attendant on particular genes/pathways for amino acid metabolism, signaling transduction, and compound efflux. Our findings provide a systematic understanding of thalassospiramide production and new insights into the underlying mechanism.

Keywords: bacteria; biosynthesis; genomics; natural product; neuroprotection.

FIGURE 1.

Sampling locations of the Thalassospira and Tistrella strains. In total, 76 Thalassospira strains and 27 Tistrella strains were collected.

FIGURE 2.

Chemical structures and activities of thalassospiramides. A, the 21 newly discovered thalassospiramides are indicated by red text. Blue structural portions indicate N-terminal amino acids used to distinguish the A- and B-like molecules. Green portions highlight a 4-amino-3,5-dihydroxy-pentanoic acid motif, red portions indicate a common valine residue, and black portions are conserved throughout the family. Absolute configurations of these cyclopeptides are drawn on the basis of the previous report of 16 known thalassospiramides (4). Thalassospiramides A2, A3, C1, F1, H1, and I1 have saturated fatty acids at the N terminus, and A10 and A11 have fatty acids of different lengths (placement of double bond within the side chain was not determined in A11). Structures: A, r = OH, R1 = H, R2 = OH; A1, r = hydroxyPh, R1 = H, R2 = OH; A2, r = OH, R1 = H, R2 = OH, saturated; A3, r = hydroxyPh, R1 = H, R2 = OH, saturated; A4, r = iPr, R1 = H, R2 = OH; A5, r = Ph, R1 = H, R2 = OH; A6, r = hydroxyPh, R1 = CH3, R2 = OH; A7, r = Ph, R1 = CH3, R2 = OH; A8, r = OH, R1 = CH3, R2 = OH; A9, r = OH, R1 = H, R2 = OH, FA = CO(CH2)6CH3; A10, r = OH, R1 = H, R2 = OH, FA = CO(CH2)6CH3; A11, r = OH, R1 = H, R2 = OH, FA (fatty acids) = CO(CH2)6(CH = CH)CH3. C, r = hydroxyPh; C1, r = hydroxyPh, saturated; C2, r = OH. E, n = 1; E1, n = 2. H, m = 2, n = 1; H1, m = 2, n = 1, saturated; H2, m = 1, n = 1; H3, m = 1, n = 2. B, r = Ph, R1 = H; B1, r = hydroxyPh, R1 = H; B2, r = OH, R1 = CH3; B4, r = hydroxyPh, R1 = CH3; B5, r = OH, R1 = CH3. D, r = H; D1, r = OH. F, r = Ph, R1 = H; F1, r = hydroxyPh, R1 = H, saturated; F2, r = hydroxyPh, R1 = CH3; F3, r = Ph, R1 = CH3. B, in the calpain inhibitory activity assay, Z-Leu-Leu-Tyr-fluoromethylketone was used as a positive control (P-control), whereas samples without compound were defined as a negative control (N-control). All tested compounds showed significant differences from the negative control. C, the neuroprotective assay was performed using mouse primary neurons. EdU/MAP2 indicates the relative amount of DNA residing in newly replicated neuron cells and acts as a proxy for numbers of neuronal cells re-entering the cell cycle. Lower EdU/MAP2 ratios suggest neuroprotection, potentially through calpain protease inhibition. P-control, no thalassospiramide or conditioned medium added; N-control, no thalassospiramide added. All significance values are based on comparison with values shown for the negative control. Experiments were performed in triplicate. Statistics were performed using Student's t tests (*, p < 0.05; **, p < 0.01; ***, p < 0.005).

FIGURE 3.

Seven putative thalassospiramide BGCs in the Rhodospirillaceae family. Bacterial strains and molecule production patterns are specified after the cluster name. Truncated domains (A domains in modules 1a and 5; for a detailed description of these truncated domains see supplemental Fig. S1) are denoted by a dotted circle, and missing domains are denoted by a cross. Cs, starter condensation; C, condensation; A, adenylation; T, thiolation; KS, ketosynthase; AT, acyltransferase; KR, ketoreductase; MT, methyltransferase; TE, thioesterase.

FIGURE 4.

Phylogenetic analysis of Rhodospirillaceae strains. A, trees based on functional genes (2C domains, left) and 28 concatenated conserved single-copy genes (right) are compared. Protein BLAST analysis using thalassospiramide A and C domains identified the NRPS proteins in the bacteria Tolypothrix, Lonsdalea, and Ralstonia; therefore, genes encoding these proteins are serving as the out groups. Bootstrap values based on 1000 replicates are shown at the nodes. Squares with different colors distinguish bacterial strains with functional gene clusters for thalassospiramide biosynthesis, whereas triangles with different colors distinguish strains with dysfunctional gene clusters. Solid lines indicate consistency between the gene tree and the species tree, whereas dashed lines indicate inconsistency between the two trees, likely because of HGT events. B–D, protein sequences of the ribosomal protein S3 (RpsC) and the 2C domain from the strains DSM21159, 1A00383, and DSM21160 were compared by protein BLAST to sequences in other strains to confirm their relative phylogenetic distances.

FIGURE 5.

Genes differentiating the thalassospiramide-producing and -non-producing bacteria. Genes were identified based on comparison between 18 genomes of thalassospiramide-producing bacteria and 14 genomes of non-producing bacteria using Student's t test (p < 0.001). A large proportion of these KEGG-annotated genes belong to the categories of amino acid metabolism (KEGG number in red), signal transduction (KEGG number in green), and compound transport (KEGG number in blue). All of the 108 significantly changed genes are listed in supplemental Table S2.

FIGURE 6.

Representation of the proposed biosynthetic pathways of gene clusters 1, 2, 4, and 7. The white ribbons represent the linear sequences of the gene products, black solid lines identify the modules found within the same protein, the black dashed line suggests that the module has no function, red arrows show the movement of the growing peptide chain along the megasynthase, and green arrows show the movement of amino acids from supplementing the A domain to the T domain.