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

Abstract

-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.