Evaluation of vectors for gene expression inPseudovibriobacteria and their application inAplysinamarine sponge studies

Abstract

AbstractThe 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 thePseudovibriogenus 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 fromPseudovibrio brasiliensisAb134. The pseudovibriamide encodingpppgene cluster is found in two thirds ofPseudovibriogenomes. 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 forPseudovibrio. We show that plasmid vectors containing three different broad-host-range replicons, RSF1010, RK2, and pBBR1, can be used inP. brasiliensisfor fluorescent protein expression and consequent labeling. We then applied GFP and mCherry expressing strains to answer the question of whether pseudovibriamides affect the uptake ofP. brasiliensisbyAplysina aerophobasponges.P. brasiliensiscell counts decreased in the sponge aquaria at an equivalent rate for wild-type and pseudovibriamide-defective ΔpppAmutant 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.ImportanceMarine 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 genusPseudovibrioof α-Proteobacteria has consistently been isolated from marine sponges and it has been hypothesized to contribute to marine sponge health. Moreover,Pseudovibriobacteria 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 inPseudovibriobacteria and demonstrated their utility inin vivostudies with marine sponges. The availability of genetic tools is important to enable us to explore the emerging ecological and biotechnological potential ofPseudovibriobacteria.