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Patin, NV, Floros DJ, Hughes CC, Dorrestein PC, Jensen PR.  2018.  The role of inter-species interactions in Salinispora specialized metabolism. Microbiology-Sgm. 164:946-955.   10.1099/mic.0.000679   AbstractWebsite

Bacterial genome sequences consistently contain many more biosynthetic gene clusters encoding specialized metabolites than predicted by the compounds discovered from the respective strains. One hypothesis invoked to explain the cryptic nature of these gene clusters is that standard laboratory conditions do not provide the environmental cues needed to trigger gene expression. A potential source of such cues is other members of the bacterial community, which are logical targets for competitive interactions. In this study, we examined the effects of such interactions on specialized metabolism in the marine actinomycete Salinispora tropica. The results show that antibiotic activities and the concentration of some small molecules increase in the presence of co-occurring bacterial strains relative to monocultures. Some increases in antibiotic activity could be linked to nutrient depletion by the competitor as opposed to the production of a chemical cue. Other increases were correlated with the production of specific compounds by S. tropica. In particular, one interaction with a Vibrio sp. consistently induced antibiotic activity and was associated with parent ions that were unique to this interaction, although the associated compound could not be identified. This study provides insight into the metabolomic complexities of bacterial interactions and baseline information for future genome mining efforts.

Bruns, H, Crusemann M, Letzel AC, Alanjary M, McInerney JO, Jensen PR, Schulz S, Moore BS, Ziemert N.  2018.  Function-related replacement of bacterial siderophore pathways. Isme Journal. 12:320-329.   10.1038/ismej.2017.137   AbstractWebsite

Bacterial genomes are rife with orphan biosynthetic gene clusters (BGCs) associated with secondary metabolism of unrealized natural product molecules. Often up to a tenth of the genome is predicted to code for the biosynthesis of diverse metabolites with mostly unknown structures and functions. This phenomenal diversity of BGCs coupled with their high rates of horizontal transfer raise questions about whether they are really active and beneficial, whether they are neutral and confer no advantage, or whether they are carried in genomes because they are parasitic or addictive. We previously reported that Salinispora bacteria broadly use the desferrioxamine family of siderophores for iron acquisition. Herein we describe a new and unrelated group of peptidic siderophores called salinichelins from a restricted number of Salinispora strains in which the desferrioxamine biosynthesis genes have been lost. We have reconstructed the evolutionary history of these two different siderophore families and show that the acquisition and retention of the new salinichelin siderophores co- occurs with the loss of the more ancient desferrioxamine pathway. This identical event occurred at least three times independently during the evolution of the genus. We surmise that certain BGCs may be extraneous because of their functional redundancy and demonstrate that the relative evolutionary pace of natural pathway replacement shows high selective pressure against retention of functionally superfluous gene clusters.

Amos, GCA, Awakawa T, Tuttle RN, Letzel AC, Kim MC, Kudo Y, Fenical W, Moore BS, Jensen PR.  2017.  Comparative transcriptomics as a guide to natural product discovery and biosynthetic gene cluster functionality. Proceedings of the National Academy of Sciences of the United States of America. 114:E11121-E11130.   10.1073/pnas.1714381115   AbstractWebsite

Bacterial natural products remain an important source of new medicines. DNA sequencing has revealed that a majority of natural product biosynthetic gene clusters (BGCs) maintained in bacterial genomes have yet to be linked to the small molecules whose biosynthesis they encode. Efforts to discover the products of these orphan BGCs are driving the development of genome mining techniques based on the premise that many are transcriptionally silent during normal laboratory cultivation. Here, we employ comparative transcriptomics to assess BGC expression among four closely related strains of marine bacteria belonging to the genus Salinispora. The results reveal that slightly more than half of the BGCs are expressed at levels that should facilitate product detection. By comparing the expression profiles of similar gene clusters in different strains, we identified regulatory genes whose inactivation appears linked to cluster silencing. The significance of these subtle differences between expressed and silent BGCs could not have been predicted a priori and was only revealed by comparative transcriptomics. Evidence for the conservation of silent clusters among a larger number of strains for which genome sequences are available suggests they may be under different regulatory control from the expressed forms or that silencing may represent an underappreciated mechanism of gene cluster evolution. Coupling gene expression and metabolomics data established a bioinformatic link between the salinipostins and their associated BGC, while genetic manipulation established the genetic basis for this series of compounds, which were previously unknown from Salinispora pacifica.

Machado, H, Tuttle RN, Jensen PR.  2017.  Omics-based natural product discovery and the lexicon of genome mining. Current Opinion in Microbiology. 39:136-142.   10.1016/j.mib.2017.10.025   AbstractWebsite

Genome sequencing and the application of omic techniques are driving many important advances in the field of microbial natural products research. Despite these gains, there remain aspects of the natural product discovery pipeline where our knowledge remains poor. These include the extent to which biosynthetic gene clusters are transcriptionally active in native microbes, the temporal dynamics of transcription, translation, and natural product assembly, as well as the relationships between small molecule production and detection. Here we touch on a number of these concepts in the context of continuing efforts to unlock the natural product potential revealed in genome sequence data and discuss nomenclatural issues that warrant consideration as the field moves forward.

Letzel, AC, Li J, Amos GCA, Millan-Aguinaga N, Ginigini J, Abdelmohsen UR, Gaudencio SP, Ziemert N, Moore BS, Jensen PR.  2017.  Genomic insights into specialized metabolism in the marine actinomycete Salinispora. Environmental Microbiology. 19:3660-3673.   10.1111/1462-2920.13867   AbstractWebsite

Comparative genomics is providing new opportunities to address the diversity and distributions of genes encoding the biosynthesis of specialized metabolites. An analysis of 119 genome sequences representing three closely related species of the marine actinomycete genus Salinispora reveals extraordinary biosynthetic diversity in the form of 176 distinct biosynthetic gene clusters (BGCs) of which only 24 have been linked to their products. Remarkably, more than half of the BGCs were observed in only one or two strains, suggesting they were acquired relatively recently in the evolutionary history of the genus. These acquired gene clusters are concentrated in specific genomic islands, which represent hot spots for BGC acquisition. While most BGCs are stable in terms of their chromosomal position, others migrated to different locations or were exchanged with unrelated gene clusters suggesting a plug and play type model of evolution that provides a mechanism to test the relative fitness effects of specialized metabolites. Transcriptome analyses were used to address the relationships between BGC abundance, chromosomal position and product discovery. The results indicate that recently acquired BGCs can be functional and that complex evolutionary processes shape the micro-diversity of specialized metabolism observed in closely related environmental bacteria.

Schorn, MA, Alanjary MM, Aguinaldo K, Korobeynikov A, Podell S, Patin N, Lincecum T, Jensen PR, Ziemert N, Moore BS.  2016.  Sequencing rare marine actinomycete genomes reveals high density of unique natural product biosynthetic gene clusters. Microbiology-Sgm. 162:2075-2086.   10.1099/mic.0.000386   AbstractWebsite

Traditional natural product discovery methods have nearly exhausted the accessible diversity of microbial chemicals, making new sources and techniques paramount in the search for new molecules. Marine actinomycete bacteria have recently come into the spotlight as fruitful producers of structurally diverse secondary metabolites, and remain relatively untapped. In this study, we sequenced 21 marine-derived actinomycete strains, rarely studied for their secondary metabolite potential and under-represented in current genomic databases. We found that genome size and phylogeny were good predictors of biosynthetic gene cluster diversity, with larger genomes rivalling the well-known marine producers in the Streptomyces and Salinispora genera. Genomes in the Micrococcineae suborder, however, had consistently the lowest number of biosynthetic gene clusters. By networking individual gene clusters into gene cluster families, we were able to computationally estimate the degree of novelty each genus contributed to the current sequence databases. Based on the similarity measures between all actinobacteria in the Joint Genome Institute's Atlas of Biosynthetic gene Clusters database, rare marine genera show a high degree of novelty and diversity, with Corynebacterium, Gordonia, Nocardiopsis, Saccharomonospora and Pseudonocardia genera representing the highest gene cluster diversity. This research validates that rare marine actinomycetes are important candidates for exploration, as they are relatively unstudied, and their relatives are historically rich in secondary metabolites.

Tang, XY, Li J, Millan-Aguinaga N, Zhang JJ, O'Neill EC, Ugalde JA, Jensen PR, Mantovani SM, Moore BS.  2015.  Identification of thiotetronic acid antibiotic biosynthetic pathways by target-directed genome mining. Acs Chemical Biology. 10:2841-2849.   10.1021/acschembio.5b00658   AbstractWebsite

Recent genome sequencing efforts have led to the rapid accumulation of uncharacterized or "orphaned" secondary metabolic biosynthesis gene clusters (BGCs) in public databases. This increase in DNA-sequenced big data has given rise to significant challenges in the applied field of natural product genome mining, including (i) how to prioritize the characterization of orphan BGCs and (ii) how to rapidly connect genes to biosynthesized small molecules. Here, we show that by correlating putative antibiotic resistance genes that encode target-modified proteins with orphan BGCs, we predict the biological function of pathway specific small molecules before they have been revealed in a process we call target-directed genome mining. By querying the pan-genome of 86 Salinispora bacterial genomes for duplicated house-keeping genes colocalized with natural product BGCs, we prioritized an orphan polyketide synthase-nonribosomal peptide synthetase hybrid BGC (tlm) with a putative fatty acid synthase resistance gene. We employed a new synthetic double-stranded DNA-mediated cloning strategy based on transformation-associated recombination to efficiently capture tlm and the related Mu BGCs directly from genomic DNA and to heterologously express them in Streptomyces hosts. We show the production of a group of unusual thiotetronic acid natural products, including the well-known fatty acid synthase inhibitor thiolactomycin that was first described over 30 years ago, yet never at the genetic level in regards to biosynthesis and autoresistance. This finding not only validates the target-directed genome mining strategy for the discovery of antibiotic producing gene clusters without a priori knowledge of the molecule synthesized but also paves the way for the investigation of novel: enzymology involved in thiotetronic, acid natural product biosynthesis.

Gallagher, KA, Jensen PR.  2015.  Genomic insights into the evolution of hybrid isoprenoid biosynthetic gene clusters in the MAR4 marine streptomycete clade. Bmc Genomics. 16   10.1186/s12864-015-2110-3   AbstractWebsite

Background: Considerable advances have been made in our understanding of the molecular genetics of secondary metabolite biosynthesis. Coupled with increased access to genome sequence data, new insight can be gained into the diversity and distributions of secondary metabolite biosynthetic gene clusters and the evolutionary processes that generate them. Here we examine the distribution of gene clusters predicted to encode the biosynthesis of a structurally diverse class of molecules called hybrid isoprenoids (HIs) in the genus Streptomyces. These compounds are derived from a mixed biosynthetic origin that is characterized by the incorporation of a terpene moiety onto a variety of chemical scaffolds and include many potent antibiotic and cytotoxic agents. Results: One hundred and twenty Streptomyces genomes were searched for HI biosynthetic gene clusters using ABBA prenyltransferases (PTases) as queries. These enzymes are responsible for a key step in HI biosynthesis. The strains included 12 that belong to the 'MAR4' clade, a largely marine-derived lineage linked to the production of diverse HI secondary metabolites. We found ABBA PTase homologs in all of the MAR4 genomes, which averaged five copies per strain, compared with 21 % of the non-MAR4 genomes, which averaged one copy per strain. Phylogenetic analyses suggest that MAR4 PTase diversity has arisen by a combination of horizontal gene transfer and gene duplication. Furthermore, there is evidence that HI gene cluster diversity is generated by the horizontal exchange of orthologous PTases among clusters. Many putative HI gene clusters have not been linked to their secondary metabolic products, suggesting that MAR4 strains will yield additional new compounds in this structure class. Finally, we confirm that the mevalonate pathway is not always present in genomes that contain HI gene clusters and thus is not a reliable query for identifying strains with the potential to produce HI secondary metabolites. Conclusions: We found that marine-derived MAR4 streptomycetes possess a relatively high genetic potential for HI biosynthesis. The combination of horizontal gene transfer, duplication, and rearrangement indicate that complex evolutionary processes account for the high level of HI gene cluster diversity in these bacteria, the products of which may provide a yet to be defined adaptation to the marine environment.