Evaluation of vectors for gene expression in Pseudovibrio bacteria and their application in Aplysina marine sponge studies
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The filter feeding capacity of marine sponges contributes to biogeochemical cycling and they are also involved in habitat formation, properties that are critical to marine ecology. Sponge-associated microbes are crucial to the functional roles provided by sponges. α-Proteobacteria belonging to the Pseudovibrio genus have been isolated from many different marine sponge genera and have been proposed to contribute to sponge health. 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 reverse genetics methods to delete genes via homologous recombination have been established, no self-replicative vectors have been reported for Pseudovibrio . We show that plasmid vectors containing three different broad-host-range replicons, RSF1010, RK2, and pBBR1, can be used in P. brasiliensis for fluorescent protein expression and consequent labeling. We then applied GFP and mCherry expressing strains to answer the question of whether pseudovibriamides affect the uptake of P. brasiliensis by Aplysina aerophoba sponges. P. brasiliensis cell counts decreased in the sponge aquaria at an equivalent rate for wild-type and pseudovibriamide-defective Δ pppA mutant strains, indicating that the sponge filters each strain indiscriminately under the conditions tested. Yet, the filtering capacity varied for each sponge individual tested, stressing the importance of performing experiments with wild-type and mutant bacterial strains in the same aquarium to allow for rigorous conclusions, which is now enabled with the methods established here.
Importance
Marine sponges are ecosystem engineers. They transform nutrients into a bioavailable form for other marine organisms. Microbes are critical to the functional roles provided by sponges because they expand the metabolic capabilities of the sponge host. Yet, most of our knowledge on sponge microbes comes from genomic studies, since cultivability and the ability to perform genetics with sponge bacterial isolates is limited. The genus Pseudovibrio of α-Proteobacteria has consistently been isolated from marine sponges and it has been hypothesized to contribute to marine sponge health. Moreover, Pseudovibrio bacteria are a source of antibiotics and other secondary metabolites with the potential to be developed into pharmaceuticals. Here we established vectors for the expression of fluorescent proteins in Pseudovibrio bacteria and demonstrated their utility in in vivo studies with marine sponges. The availability of genetic tools is important to enable us to explore the emerging ecological and biotechnological potential of Pseudovibrio bacteria.