ABSTRACT
Ribosomal gene sequences were obtained from bryozoans in the genus
Bugula
and their bacterial symbionts; analyses of host and symbiont phylogenetic trees did not support a history of strict cospeciation. Symbiont-derived compounds known to defend host larvae from predation were only detected in two out of four symbiotic
Bugula
species.
ABSTRACT
The bryozoans
Bugula neritina
and
Bugula simplex
harbor bacteria in the pallial sinuses of their larvae as seen by electron microscopy. In
B. neritina
, the bacterial symbiont has been characterized as a gamma-proteobacterium, “
Candidatus
Endobugula sertula.” “
Candidatus
E. sertula” has been implicated as the source of the bryostatins, polyketides that provide chemical defense to the host and are also being tested for use in human cancer treatments. In this study, the bacterial symbiont in
B. simplex
larvae was identified by 16S rRNA-targeted PCR and sequencing as a gamma-proteobacterium closely related to and forming a monophyletic group with “
Candidatus
E. sertula.” In a fluorescence in situ hybridization, a 16S ribosomal DNA probe specific to the
B. simplex
symbiont hybridized to long rod-shaped bacteria in the pallial sinus of a
B. simplex
larva. The taxonomic status “
Candidatus
Endobugula glebosa” is proposed for the
B. simplex
larval symbiont. Degenerate polyketide synthase (PKS) primers amplified a gene fragment from
B. simplex
that closely matched a PKS gene fragment from the bryostatin PKS cluster. PCR surveys show that the symbiont and this PKS gene fragment are consistently and uniquely associated with
B. simplex
. Bryostatin activity assays and chemical analyses of
B. simplex
extracts reveal the presence of compounds similar to bryostatins. Taken together, these findings demonstrate a symbiosis in
B. simplex
that is similar and evolutionarily related to that in
B. neritina
.
ABSTRACT
The marine bryozoan,
Bugula neritina
, is the source of the bryostatins, a family of macrocyclic lactones with anticancer activity. Bryostatins have long been suspected to be bacterial products.
B. neritina
harbors the uncultivated gamma proteobacterial symbiont “
Candidatus
Endobugula sertula.” In this work several lines of evidence are presented that show that the symbiont is the most likely source of bryostatins. Bryostatins are complex polyketides similar to bacterial secondary metabolites synthesized by modular type I polyketide synthases (PKS-I). PKS-I gene fragments were cloned from DNA extracted from the
B. neritina-“E. sertula”
association, and then primers specific to one of these clones, KSa, were shown to amplify the KSa gene specifically 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 laboratory were treated with antibiotics to reduce the numbers of bacterial symbionts. Decreased symbiont levels resulted in the reduction of the KSa signal as well as the bryostatin content. These data provide evidence that the symbiont
E. sertula
has the genetic potential to make bryostatins and is necessary in full complement for the host bryozoan to produce normal levels of bryostatins. This study demonstrates that it may be possible to clone bryostatin genes from
B. neritina
directly and use these to produce bryostatins in heterologous host bacteria.