Biology subjects

Eustaquio, A. S.

Publications and source records attributed to Eustaquio, A. S..

5 recordsLinked to original sources

bioRxiv2025

MALDI-FISH for co-localization of brominated metabolites and Pseudovibrio spp. in Aplysina tissue

Bromotyrosine-containing natural products have long been isolated from marine sponges of the order Verongiida. Recent studies have questioned the source of these natural products whether production occurs through the sponge, its diverse microbiota, or perhaps a combination of both. While studying the uptake of engineered bacterial strains by sponges, we have observed production of fistularin-3 in sponge. To explore these results further, we employed multimodal imaging techniques to co-localize microbial metabolites with fluorescent in situ hybridization (FISH) probes. Here, we built on previously developed MALDI-FISH methods to add to the conversation on brominated metabolite production in marine sponges. Using MALDI-MSI, we measured fistularin-3, a poly-brominated natural product previously isolated from Aplysina aerophoba, present in our A. aerophoba samples that were inoculated with Pseudovibrio brasiliensis. A recent study reported that P. brasiliensis produced fistularin-3 in pure culture, but this data has not yet been reproduced. We employed MALDI-MSI and FISH on the same sponge cryosections to colocalize the spatial distribution of fistularin-3 with P. brasiliensis in sponge tissue. Despite the caveats of this study, our data suggests that perhaps both sponge and microbe may be required for production of fistularin-3.

microbiology↗

bioRxiv2025

IDBac: an open-access web platform and compendium for the identification of bacteria by MALDI-TOF mass spectrometry.

The identification and analysis of bacteria is central to the microbiological sciences. While gene sequencing methods have been the standard to achieve this, use of MALDI-TOF mass spectrometry (MS), particularly in clinical microbiology, provides high-throughput identification to the subspecies level. However, biotyping has yet to be adopted outside of clinical settings due to the lack of a centralized public database of MS protein signatures that would facilitate isolate identification via spectral comparison. Further, current platforms lack meaningful ways to compare multiple properties from large numbers of bacterial isolates. Herein we present the IDBac web platform, a crowd-sourced central knowledgebase of protein MS signatures of >1400 strains spanning 6 bacterial phyla. Accompanying the knowledgebase is analysis infrastructure to identify unknown isolates, probe relationships within culture collections using metadata integration, and visualize specialized metabolite differences within groups of closely related bacteria. To highlight this utility and encourage wide community contribution, examples of each are presented.

microbiology↗

bioRxiv2024

Evaluation of vectors for gene expression in Pseudovibrio bacteria and their application in Aplysina marine sponge studies

-Proteobacteria belonging to the Pseudovibrio genus have been isolated from different marine organisms including marine sponges, corals and algae. This genus was first described in 2004 and has since garnered attention due to the potential ecological relevance and biotechnological application of its metabolites. For instance, we recently reported specialized metabolites we named pseudovibriamides from Pseudovibrio brasiliensis Ab134. The pseudovibriamide encoding ppp gene cluster is found in two thirds of Pseudovibrio genomes. Pseudovibriamides coordinate motility and biofilm formation, behaviors that are known to be important for host colonization. Although we previously established reverse genetics methods to delete genes via homologous recombination, no self-replicative vectors have been reported for Pseudovibrio. We show that plasmid vectors containing two different broad-host-range replicons, RSF1010 and pBBR1, can be used in P. brasiliensis. The efficiency of vector transfer by electroporation averaged [~] 3 x 103 CFU/{micro}g plasmid DNA whereas the conjugation frequency from E. coli ranged from 10-3 to 10-6. We then tested the vectors for fluorescent protein expression and consequent labeling, which allowed us to observe their effects on swarming motility and to compare plasmid stability. This study expands the genetic toolbox available for Pseudovibrio which is expected to enable future ecological and biotechnological studies. ImportanceThe genus Pseudovibrio of -Proteobacteria has consistently been isolated from marine sponges and other marine organisms such as corals and algae. Pseudovibrio bacteria are a source of antibiotics and other secondary metabolites with the potential to be developed into pharmaceuticals. Moreover, the secondary metabolites they produce are important for their physiology and for interactions with other organisms. Here we expand the genetic tool box available for Pseudovibrio bacteria by establishing self-replicative vectors that can be used for the expression of e.g., fluorescent proteins. The availability of genetic tools is important to enable us to explore the emerging ecological and biotechnological potential of Pseudovibrio bacteria.

genetics↗

bioRxiv2024

Pseudovibriamides from Pseudovibrio marine sponge bacteria promote swarming motility via transcriptional modulation

Pseudovibrio -Proteobacteria have been repeatedly isolated from marine sponges and proposed to be beneficial to the host. Bacterial motility is known to contribute to host colonization. We have previously identified pseudovibriamides A and B, produced in culture by Pseudovibrio brasiliensis Ab134, and shown that pseudovibriamide A promotes flagellar motility. Pseudovibriamides are encoded in a hybrid nonribosomal peptide synthetase-polyketide synthase gene cluster that also includes several accessory genes. Pseudovibriamide A is a linear heptapeptide and pseudovibriamide B is a nonadepsipeptide derived from pseudovibriamide A. Here we define the borders of the pseudovibriamides gene cluster, assign function to biosynthetic genes using reverse genetics and test the hypothesis that pseudovibriamides impact motility by modulating gene transcription. RNA-seq transcriptomic analyses of strains having different compositions of pseudovibriamides suggested that both pseudovibriamides A and B affect genes potentially involved in motility, and that a compensatory mechanism is at play in mutants that produce only pseudovibriamide A, resulting in comparable swarming motility as the wild type. The data gathered suggest that pseudovibriamides A and B have opposite roles in modulating a subset of genes, with pseudovibriamide B having a primary effect in gene activation, and pseudovibriamide A on inhibition. Finally, we observed many differentially expressed genes (up to 29% of the total gene number) indicating that pseudovibriamides have a global effect on transcription that goes beyond motility. ImportanceMarine sponges are found throughout the oceans from tropical coral reefs to polar sea floors, playing crucial roles in marine ecosystems. Pseudovibrio bacteria have been proposed to contribute to sponge health. We have previously shown that pseudovibriamides produced by Pseudovibrio brasiliensis promote bacterial motility, a behavior that is beneficial to bacterial survival and to host colonization. The gene cluster that encodes pseudovibriamide biosynthesis is found in two thirds of Pseudovibrio genomes. This gene cluster is also present in Pseudomonas bacteria that interact with terrestrial plants and animals. Here we first assign function to pseudovibriamide biosynthetic genes using reverse genetics. We then show that pseudovibriamides play a major role in transcriptional regulation, affecting up to 29% of P. brasiliensis genes, including motility genes. Thus, this work gives insights into pseudovibriamide biosynthesis and provides evidence that they are signaling molecules relevant to bacterial motility and to other yet to be identified phenotypes.

microbiology↗

bioRxiv2020

A novel family of nonribosomal peptides modulate collective behavior in Pseudovibrio bacteria isolated from marine sponges

Collective behavior is a common feature of life. Although swarming motility and biofilms are opposed collective behaviors, both contribute to bacterial survival and host colonization. We have identified a link between motility/biofilms and a nonribosomal peptide synthetase-polyketide synthase gene cluster family (ppp) conserved in Pseudovibrio and Pseudomonas Proteobacteria known to interact with diverse eukaryotes. After developing reverse genetics for Pseudovibrio, we discovered two pseudovibriamide families, heptapeptides with a reversal in chain polarity via an ureido linkage 1-6 and related nonadepsipeptides 7-12. Imaging mass spectrometry showed that 1 was excreted whereas 7 was colony-associated. Deletion of pppA abolished production of 1-12 leading to reduced motility and increased biofilm production. pppD mutants that produced only 1-6 showed motility comparable to the wild-type and reduced biofilm formation, indicating that the excreted heptapeptides play a role in promoting motility. In contrast to lipopeptides widely known to affect swarming and biofilms, pseudovibriamides are not surfactants. Our results expand current knowledge on metabolites mediating bacterial collective behavior. Moreover, the establishment of reverse genetics will enable future exploration of the ecological and biotechnological potential of Pseudovibrio bacteria which have been proposed to contribute to marine sponge health. SignificanceBacteria contribute to health and disease of plants and animals. Specialized metabolites produced by bacteria are important in mediating their behavior and the colonization of their hosts. We have identified a conserved gene cluster family in Pseudovibrio and Pseudomonas bacteria known to colonize marine animals and terrestrial plants, respectively. Using Pseudovibrio as a model, we show the encoded metabolites, which we termed pseudovibriamides, promote motility and decrease biofilms. In contrast to lipopeptides widely known to affect motility/biofilms, pseudovibriamides are not surfactants, but instead are linear peptides with a reversal in chain polarity. The discovery of pseudovibriamides expands current knowledge of bacteria collective behavior. The establishment of reverse genetics will enable exploration of the ecological and biotechnological potential of Pseudovibrio bacteria. ClassificationBiological Sciences, Microbiology

microbiology↗