Limited Metabolomic Overlap between Commensal Bacteria and Marine Sponge Holobionts Revealed by Large Scale Culturing and Mass Spectrometry-Based Metabolomics: An Undergraduate Laboratory Pedagogical Effort at Georgia Tech

Abstract

Sponges are the richest source of bioactive organic small molecules, referred to as natural products, in the marine environment. It is well established that laboratory culturing-resistant symbiotic bacteria residing within the eukaryotic sponge host matrix often synthesize the natural products that are detected in the sponge tissue extracts. However, the contributions of the culturing-amenable commensal bacteria that are also associated with the sponge host to the overall metabolome of the sponge holobiont are not well defined. In this study, we cultured a large library of bacteria from three marine sponges commonly found in the Florida Keys. Metabolomes of isolated bacterial strains and that of the sponge holobiont were compared using mass spectrometry to reveal minimal metabolomic overlap between commensal bacteria and the sponge hosts. We also find that the phylogenetic overlap between cultured commensal bacteria and that of the sponge microbiome is minimal. Despite these observations, the commensal bacteria were found to be a rich resource for novel natural product discovery. Mass spectrometry-based metabolomics provided structural insights into these cryptic natural products. Pedagogic innovation in the form of laboratory curricula development is described which provided undergraduate students with hands-on instruction in microbiology and natural product discovery using metabolomic data mining strategies.

Keywords: bacteria; mass spectrometry; metabolomics; natural products; sponge.

Figure 1

Microbiomes and metabolomes of sponges and bacterial isolates. (A) Morphology of the Floridian sponges A. fulva, S. aurea, and A. crassa. (B) Phylum-level microbiome architectures of the three sponge holobionts queried in technical duplicates (data reported in Ref. [16]). (C) Molecular network illustrating the structural similarity between metabolites detected in sponge tissue (blue nodes), bacterial isolates (green nodes), and metabolites that were shared between sponge tissue and bacterial isolates (brown nodes). Metabolites detected in media used for bacterial cultivation, and those between media and sponge tissue and media and bacterial isolates are represented as red nodes. (D) After subtraction of metabolites detected in media, the overlaps between metabolites detected in bacterial isolates (gray circles) and sponge tissues (white circles) are illustrated as Venn diagrams.

Figure 2

Dendogram representing 16S rRNA gene relatedness among bacteria isolated in this study. Bacteria genera are labeled.

Figure 3

Effect of (A) sponge sources and (B) cultivation media on diversity of commensal bacterial metabolomes as visualized using PCA plots. The variance explained by each component is labeled on the respective axes. The 95% confidence ellipses are included on the plots. Two distinct clusters of bacterial nodes are labeled in both plots, comprising of Pseudovibrio strains isolated from different sponges and Microbulbifer strains isolated mainly from S. aurea. The Microbulbifer strains are listed for clarity.

Figure 4

Discovery of cryptic metabolites shared between marine sponges and commensal bacteria. (A) A cluster of four nodes in which three nodes (in green) are detected in commensal bacteria only, and one node (in brown) is shared between commensal bacteria and A. fulva and S. aurea sponges. The parent masses for metabolites corresponding to the four nodes are labeled. For the m/z 567.497 node, (universal spectrum identifier (USI) 42 spectra were collated from commensal bacteria, and 16 spectra were collated from marine sponges. USI links for other nodes are as follows: m/z 541.483 node USI; m/z 581.513 node USI; m/z 593.514 node. (B) MS² spectra for the m/z 567.497 metabolite, as detected in highest abundance in the Microbulbifer strain 22AN-SA-001 (top), and the A. fulva and S. aurea sponges (middle and bottom, respectively). The molecular formulae of the parent ions, and the two most abundant daughter ions, m/z 311.258 and m/z 313.273 are labeled. At low m/z, a number of daughter ions separated by 14.016 Da are discernable which corresponds to differences in a methylene (-CH₂-) unit.

Figure 5

Cryptic peptidic natural products mined from commensal bacteria. (A) A cluster of nodes corresponding to metabolites detected only in extracts of commensal bacteria is shown with two nodes with the maximum number of MS² spectra associated with them in this cluster labeled with their respective parent ion masses. (B) MS² fragmentation spectra for metabolites with parent masses m/z 671.413 (top; USI and m/z 572.345 (bottom; USI). The proline, valine, and phenylalanine immonium ions (m/z 70.065, m/z 72.028, and m/z 120.081, respectively) are highlighted in red. The Pro-Val and Pro-Phe dipeptide oxonium ions (m/z 197.128 and m/z 245.128, respectively) are highlighted in purple. Progressing from the parent ions, mass differences between MS² fragment ions corresponding to proteinogenic amino acids are labeled.