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A path to biodiscovery: isolating Antarctic ascidian-associated bacteria and screening genomes for secondary metabolite-encoded biosynthetic gene clusters and signatures of cold adaptation
AdvisorMurray, Alison E
Cell and Molecular Biology
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Natural products synthesized by host-associated microbes from polar regions pose tremendous potential for addressing human health challenges. One prominent example is the ascidian Synoicum adareanum, found on the seafloor of coastal regions of Antarctica, of interest in part due to one compound, palmerolide A. Palmerolide A is a cytotoxic macrolide with specific activity towards cancer cells which occurs in high concentration in its tissues. It has since been determined that one core bacterial member of the moderately diverse Synoicum adareanum microbiome is responsible for the biosynthesis of palmerolide A. This finding reinforces the significance of investigating natural product potential in host-associated microbes from high-latitude ecosystems. One goal of the Synoicum adareanum research program is establishing a sustainable biological source of palmerolide A either through cultivation or bioengineering solutions. Secondly, exploring the microbiome for additional cold-water biosynthesized natural products is of interest given recent metagenome results that suggest substantial natural product biosynthetic potential. To advance this research requires investigating cultivation strategies for the Verrucomicrobia-affiliated palmerolide A producer, establishing a more comprehensive understanding of the biosynthetic potential of other microbes in the Synoicum adareanum microbiome, and ascertaining the cold-adaptive characteristics of the genome and encoded proteome evolution of host-associated high latitude marine microorganisms. Here, we implemented a minimal media cultivation and dereplication strategy to isolate bacteria, rapidly screen for palmerolide A-encoding biosynthetic gene clusters using PCR and determine the identity of the novel isolates with long read 16S rRNA phylogenetic analyses. This work added phylogenetic breadth to previously cultivated bacterial classes Gammaproteobacteria and Alphaproteobacteria from the Synoicum adareanum microbiome, to now include Bacilli, Acidimicrobiia, Actinobacteria and Bacteroidia. Through sequencing representative genomes of 22 isolates, 135 biosynthetic gene cluster types across eight compound categories were identified within this expanded culture collection using bioinformatic approaches. Comparative genomic analyses between orthologous proteins predicted between our high-latitude, Antarctic isolates and representative low/mid latitude marine microorganisms of the same genera revealed signatures of cold adaptation in the Antarctic genomes. We found an overall decrease in proline residues that form rigid kinks in protein sequences. Likewise, charged amino acids and arginine/(arginine+lysine) content, which form protein-stabilizing salt bridges believed to hinder protein function at low temperatures were reduced. The predicted Antarctic proteins also followed the established trend of increased glycine and serine content in cold adapted proteins, understood to increase protein dynamics in cold through their small size and reduced ionic charge. Interestingly, significant numbers of coding sequences with lower GC content were found in the Antarctic genomes even if genome-wide GC contents were similar, or even higher, for the Antarctic organism. Also surprising, more predicted proteins with higher intrinsic disorder were observed in the low/mid latitude genomes, perhaps indicative of a possible environmental temperature threshold for disorder which balances protein function and cold denaturation resistance. These cultivation, phylogenomic and bioinformatic results provide a deeper view into this Antarctic marine ascidian, the diversity and potential for secondary metabolite production within its microbial community, and trends observed in protein and genome sequences of cold adapted organisms.