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

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 ΔpppA 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.