Antimicrobial potential of the marine actinomycete salinispora tropica cnb-440 in co-culture : a metabolomic, proteomic and genome engineering approach

he alarming rise of antimicrobial resistance in pathogenic strains has fuelled tremendous research efforts towards the discovery of novel bioactive molecules from untried ecological niches. In this regard, the world’s oceans have revealed to be a remarkable resource of new bacterial taxa with promising biosynthetic potential. Study of the secondary metabolism of the marine actinobacterium Salinispora, for instance, has shown the genus to be an exceptional trove of unique and bioactive natural products, rivalling the significance of its terrestrial counterpart. The isolation of new secondary metabolites from members of the genus Salinispora and other microorganism is, however, currently limited because most of the biosynthetic gene clusters (BGCs) encoded in their genomes are not expressed under standard laboratory conditions. This is well exemplified in Salinispora, as a staggering 80% of its BGCs are still orphan (i.e. not linked to their products). This observation warrants the development of new approaches to unlock the biosynthetic potential of the genus. This thesis is devoted to investigating novel ways to activate, interrogate, and manipulate the biosynthetic potential of Salinispora tropica CNB-440. First, we demonstrate how co-cultivation of S. tropica with phytoplankton could be used to elicit the production of novel cryptic compounds in the actinobacterium. Our data also reveal that S. tropica exhibits antimicrobial activity against a range of eukaryotic and prokaryotic marine phototrophs via an uncharacterized mechanism. Second, we report the first proteome dataset available in the genus Salinispora, that we explored in order to identify candidate secondary pathways responsible for the biosynthesis of the detected cryptic molecules and/or the antimicrobial activity observed in co-culture. Using high-throughput proteomics, we provide evidence that the orphan nrps1 BGC is active and upregulated upon exposure to phytoplankton. We also suggest a potential mechanistic understanding of the antimicrobial effect seen in co-culture. Finally, we implemented for the first time the CRISPR/Cas9 system in a member of the genus Salinispora, as a promising tool to link specific BGCs to the detected molecules and/or observed bioactivity described in our study. As a proof of concept, we successfully engineered S. tropica by deleting an entire BGC and a PPTase from its genome

Phytoplankton trigger the production of cryptic metabolites in the marine actinobacterium Salinispora tropica

Filamentous members of the phylum Actinobacteria are a remarkable source of natural products with pharmaceutical potential. The discovery of novel molecules from these organisms is, however, hindered because most of the biosynthetic gene clusters (BGCs) encoding these secondary metabolites are cryptic or silent and are referred to as orphan BGCs. While co‐culture has proven to be a promising approach to unlock the biosynthetic potential of many microorganisms by activating the expression of these orphan BGCs, it still remains an underexplored technique. The marine actinobacterium Salinispora tropica, for instance, produces valuable compounds such as the anti‐cancer molecule salinosporamide but half of its putative BGCs are still orphan. Although previous studies have used marine heterotrophs to induce orphan BGCs in Salinispora, its co‐culture with marine phototrophs has yet to be investigated. Following the observation of an antimicrobial activity against a range of phytoplankton by S. tropica, we here report that the photosynthate released by photosynthetic primary producers influences its biosynthetic capacities with production of cryptic molecules and the activation of orphan BGCs. Our work, using an approach combining metabolomics and proteomics, pioneers the use of phototrophs as a promising strategy to accelerate the discovery of novel natural products from marine actinobacteria

Pili allow dominant marine cyanobacteria to avoid sinking and evade predation

How oligotrophic marine cyanobacteria position themselves in the water column is currently unknown. The current paradigm is that these organisms avoid sinking due to their reduced size and passive drift within currents. Here, we show that one in four picocyanobacteria encode a type IV pilus which allows these organisms to increase drag and remain suspended at optimal positions in the water column, as well as evade predation by grazers. The evolution of this sophisticated floatation mechanism in these purely planktonic streamlined microorganisms has important implications for our current understanding of microbial distribution in the oceans and predator–prey interactions which ultimately will need incorporating into future models of marine carbon flux dynamics

CRISPR/Cas9-based methods for inactivating actinobacterial biosynthetic genes and elucidating function

The CRISPR/Cas9 technology allows fast and marker-less genome engineering that can be employed to study secondary metabolism in actinobacteria. Here, we report a standard experimental protocol for the deletion of a biosynthetic gene in a Streptomyces species, using the vector pCRISPomyces-2 developed by Huimin Zhao and collaborators. We also describe how carrying out metabolite analysis can reveal the putative biosynthetic function of the inactivated gene

A Targetron-Recombinase System for Large-Scale Genome Engineering of Clostridia

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Cell gating for flow cytometry analysis.

Graphs show pseudocolor plots used for gating of S. alvi cells. Quadrant limits were determined based on the measured fluorescence of reference cells bearing (a) the empty backbone pAC07 (no fluorescence), (b) pAC08 (GFP alone), or (c) pBTK570 (E2-crimson alone). (PDF)</p

Characterization of functional broad-host range replicons in the honey bee gut symbiont <i>B</i>. <i>apis</i>.

(a) Broad-host range plasmids have different copy numbers in B. apis. Box plots show median values of plasmid copy numbers obtained by qPCR from 3 independent experiments with 5 biological replicate each (total n = 15). Median copy number are indicated with the corresponding box plots. (b) The difference in plasmid copy number results in different protein expression levels in B. apis. Graph shows mean of E2-crimson fluorescence ± standard deviations of 5 biological replicates. Each replicate represents the average fluorescence of at least 9,000 cells measured by flow cytometry. Plasmids used for panels a and b in B. apis were pBTK570, pAC06, pAC11, and pAC04, carrying the RSF1010, RK2, pTF-FC2, and pBBR1 origins of replication, respectively. (c) Some replicons are compatible and can be cotransformed in B. apis. Matrix table indicates compatible (green boxes with check mark) and incompatible (red boxes with cross mark) replicons. Vectors were found compatible upon their successful cotransformation by conjugation in B. apis cells. The data underlying this Figure can be found in the S1 Data file, sheets “Supplementary Fig 4A” and “Supplementary Fig 4B.” (PDF)</p

Main features of the broad-host range replicons tested in <i>S</i>.<i>alvi</i> and <i>B</i>. <i>apis</i>.

Main features of the broad-host range replicons tested in S.alvi and B. apis.</p

DAPI measurements from gut tissues.

Graph shows box plots representing median value of DAPI fluorescence of S. alvi biofilms imaged from the gut of bees fed sugar water supplemented with either 0, 0.1, or 1 mM IPTG. Five bees were analyzed for each condition, and fluorescence values were averaged from 3 distinct sections of each gut. One-way ANOVA test, not significant (ns) with q-value > 0.5. The data underlying this Figure can be found in the S1 Data file, sheet “Supplementary Fig 8.” (PDF)</p

Bacterial load from feces is a proxy for levels of gut colonization.

Scatterplot shows linear regression of bacterial concentration values of engineered S. alvi found in matching samples of feces and gut homogenates (i.e., feces and gut were sourced from the same bee). Pearson correlation coefficient R and p-value are provided, for n = 22. The data underlying this Figure can be found in the S1 Data file, sheet “Supplementary Fig 1”. (PDF)</p