Evidence for the biosynthesis of bryostatins by the bacterial symbiont "Candidatus Endobugula sertula" of the bryozoan Bugula neritina

The marine bryozoan, Bugula neritina, is the source of the bryostatins, a f amily of macrocyclic lactones with anticancer activity. Bryostatins have lo ng been suspected to be bacterial products. B. neritina harbors the unculti vated gamma proteobacterial symbiont "Candidatus Endobugula sertula." In th is work several lines of evidence are presented that show that the symbiont is the most likely source of bryostatins. Bryostatins are complex polyketi des similar to bacterial secondary metabolites synthesized by modular type I polyketide synthases (PKS-I). PKS-I gene fragments were cloned from DNA e xtracted from the B. neritina-"E. sertula" association, and then primers sp ecific to one of these clones, KSa, were shown to amplify the KSa gene spec ifically and universally from total B. neritina DNA. In addition, a KSa RNA probe was shown to bind specifically to the symbiotic bacteria located in the pallial sinus of the larvae of B. neritina and not to B. neritina cells or to other bacteria. Finally, B. neritina colonies grown in the laborator y were treated with antibiotics to reduce the numbers of bacterial symbiont s. Decreased symbiont levels resulted in the reduction of the KSa signal as well as the bryostatin content. These data provide evidence that the symbi ont E. sertula has the genetic potential to make bryostatins and is necessa ry in full complement for the host bryozoan to produce normal levels of bry ostatins. This study demonstrates that it may be possible to clone bryostat in genes from B. neritina directly and use these to produce bryostatins in heterologous host bacteria.