Isolation of Two Polyketide Synthase Gene Fragments from the Uncultured Microbial Symbiont of the Marine Bryozoan Bugula neritina

"Candidatus Endobugula sertula," the uncultured microbial symbiontof the bryozoan Bugula neritina, produces ecologically and biomedicallyimportant polyketide metabolites called bryostatins. We isolatedtwo gene fragments from B. neritina larvae that have high levelsof similarity to polyketide synthase genes. These gene fragmentsare clearly associated with the symbiont and not with the host.

INTRODUCTION

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Introduction

The marine bryozoan Bugula neritina lives in temperate habitatsthroughout the world. Its uncultured symbiont "Candidatus Endobugulasertula" produces polyketide secondary metabolites called bryostatins(4, 12). These compounds defend the host's larvae from predationby fish (12). The molecular structure of the bryostatins suggeststhat they are assembled by enzymes called polyketide synthases(PKS). These large multifunctional modular enzymes are madeof repeating domains, and the nascent polyketide grows in anassembly line fashion and is modified chemically depending onthe presence of certain domains as it "moves down" the enzymecomplex (5). Davidson and coworkers (4) found a portion of apolyketide synthase gene cluster, a ketosynthase (KS) domain,in DNA isolated from deep California (CA) populations of B.neritina/"Ca. Endobugula sertula." They demonstrated that thereis expression of this KS gene in "Ca. Endobugula sertula" inthe pallial sinus, a groove on the top anterior pole of thehost larvae where an inoculum of "Ca. Endobugula sertula" isfound. Hildebrand and coworkers subsequently isolated a largeopen reading frame (bryA) from deep and shallow CA populationsof B. neritina/"Ca. Endobugula sertula" that encodes an enzymethat is thought to catalyze the loading and initial elongationand modification steps of bryostatin biosynthesis (9). To date,efforts to culture the symbiont for gene knockout and complementationstudies to unequivocally demonstrate that the PKS gene clusterproduces bryostatins have not been successful. The CA shallowand deep populations of B. neritina/"Ca. Endobugula sertula"have different suites of bryostatins that have the same macrolactonecore but different pendant groups (3). The bryostatins in populationsfrom North Carolina (NC) are similar to those in CA shallowpopulations (3, 12, 13). Here we report the isolation of twoPKS gene fragments from NC populations of B. neritina/"Ca. Endobugulasertula," one of which is almost identical to a portion of deepCA bryA and the other of which has not been reported previously.

Identification of polyketide synthase gene fragments.

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Identification of polyketide...

Adult colonies of the bryozoan B. neritina were collected fromRadio Island Jetty near Morehead City, NC. These colonies werekept in flowing seawater tables at the University of North Carolina'sInstitute of Marine Science. Lopanik et al. (12) described larvalspawning and collection methodologies previously. Genomic DNAwas extracted from B. neritina larvae using an Isoquick DNAextraction kit (Orca Research, Inc.). Degenerate PCR primersfor a PKS gene were developed based on proteobacterial KS aminoacid sequences deposited in the GenBank database (accessionnumbers AF081920 [three KS genes], AF239749 [two KS genes],and AF319998) (Table 1). These sequences were aligned usingthe ClustalW method, and regions where there were high levelsof similarity were identified. The amino acid residues M/L/SDPQQRand LGDPIEI/L were used to design the forward and reverse primers,respectively, and the corresponding DNA sequences were alignedto determine the amount of degeneracy at each oligonucleotideposition. Primers based on these sequences (Table 1) were usedto amplify B. neritina larval DNA in a 25-µl PCR mixturewith a "hot-start" Taq polymerase enzyme (JumpStart Taq; Sigma-Aldrich).The first two cycles consisted of melting at 94°C for 2min, annealing at 37°C for 2 min, and extension at 72°Cfor 3 min, and this was followed by 39 cycles of denaturationat 94°C for 30 s, annealing at 54°C for 45 s, and extensionfor 1 min and a final extension for 5 min. The products wereseparated by electrophoresis on a 1% agarose gel, and a bandat $~$ 700 bp was excised from the gel. The PCR product was purifiedfrom the gel (Supelco GenElute agarose spin column) and cloned(Invitrogen TOPO TA cloning kit) into Escherichia coli. Theseven resulting clones were screened by colony PCR using vector-specificprimers M13F and M13R to amplify the insert. The PCR productswere analyzed by restriction enzyme digestion with AluI (NewEngland Biolabs). Five clones had unique inserts which weresequenced using an Applied Biosystems, Inc. 310 genetic analyzer.The resulting sequences were searched against the GenBank database.Internal primers based on the DNA sequences were synthesized,and the clone DNA was amplified with the corresponding M13For M13R primer (Table 1). The products were sequenced, whichresulted in the complete DNA sequence of the insert in boththe forward and reverse directions. The complete sequences werealigned with similar sequences using ClustalX (19), and a phylogenetictree was generated with Mega3.1 (11) using the minimum-evolutionmethod and bootstrapping 10,000 times.

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TABLE 1. Sequences of primers used in this study

Of the five clones that had unique DNA inserts, the sequencesof two of the inserts (designated BKS1 and BKS2) had high levelsof similarity to other bacterial PKS genes. The other threeclones contained noncoding DNA sequences. The amino acid sequencesencoded by BKS1 and BKS2 were 49.5% identical to each other(Fig. 1); the DNA sequences were 54.4% identical. BKS1 was almostidentical (98.7% amino acid identity, 97.2% DNA identity) toKS2 of bryA, the putative bryostatin biosynthetic gene recentlyisolated from deep CA populations of B. neritina/"Ca. Endobugulasertula," and very similar (80% identical amino acids, 82.1%DNA identity) to KS2 of bryA isolated from shallow CA populationsof B. neritina (Fig. 2) (9). In contrast, the BKS2-encoded aminoacid sequence was 49.1% identical to the KS2-encoded amino acidsequence of deep CA BryA and 48.7% identical to the KS2-encodedamino acid sequence of shallow BryA (the DNA sequences were52.7% and 53.7% similar, respectively). Additionally, the twoinserts were fairly similar to PKS genes from the Paederus beetlebacterial symbiont, a member of the ${gamma}$ -Proteobacteria (Fig. 2)(16, 17), and to the onnamide cluster from a symbiont isolatedfrom the marine sponge Theonella swinhoei (Fig. 2) (18). Thetwo clones were closely related to PKS gene clusters that havediscrete acyltransferase (AT) domains, such as those of thepederin PKS clusters (PedF and PedI) (16, 17), the onnamidecluster (OnnB) (18), the leinamycin cluster (LmnJ) (2), andthe mupirocin cluster (MmpD) (6) (Fig. 2); in fact, BryA doesnot have integrated ATs (9). In typical type I PKS gene systems,the AT domains, which are responsible for attaching the extenderunit to the enzyme, are embedded within each module of the PKSpolypeptide (5); however, in several systems, there is onlyone AT domain, and it is found in a separate open reading frameupstream or downstream of the open reading frame encoding thePKS polypeptides (1, 2, 6, 16-18). The B. neritina/"Ca. Endobugulasertula" KS genes were also fairly similar (>48% identityat the amino acid level) to KS genes from bacteria that arenot closely related according to 16S rRNA gene sequences, includingMicrocystis aeruginosa (cyanobacteria) and Streptomyces spp.(actinobacteria) (Fig. 2). This is not surprising as evolutionarypressures such as horizontal gene transfer and environmentalfactors may affect secondary metabolite biosynthetic gene clusterevolution differently than they affect 16S rRNA evolution (7,10, 15).

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FIG. 1. Alignment of amino acid sequences of two KS domain genes isolated from B. neritina/"Ca. Endobugula sertula" with BryA KS2 from shallow and deep California populations. S, shallow; D, deep.

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FIG. 2. Minimum-evolution tree based on amino acid sequences of PKS genes from B. neritina/"Ca. Endobugula sertula" and related organisms. The accession numbers of sequences obtained from GenBank are indicated. The sequences in boldface type are described in this paper. Bootstrap analysis (performed 10,000 times) percentages greater than 50% are indicated at the nodes. Scale bar = 0.1 amino acid substitution per site. The suffix NC indicates North Carolina, and the suffix CA indicates California.

Association of PKS gene fragments with "Ca. Endobugula sertula."

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Association of pks gene...

In spring 2002, three replicate groups of B. neritina larvaeand, after they settled onto polystyrene petri dishes, newlysettled juveniles were exposed for 10 h to the antibiotic gentamicin(75 µg ml^–1) on 10 consecutive days. Individualstreated with gentamicin and control individuals (larvae andjuveniles exposed to filtered seawater lacking antibiotic) weretransplanted into the field and grown until they were reproductiveadults. Lopanik et al. (12) provided a full description of theexperimental details previously. Aliquots of the next-generationlarvae from experimental and control adults were collected,and DNA was extracted for analysis. Purified DNA was amplifiedusing (i) primers specific for the B. neritina cytochrome oxidaseI gene, (ii) primers specific for the "Ca. Endobugula sertula"16S rRNA gene (8), and (iii) primers developed from the twoPKS gene DNA sequences isolated from B. neritina/"Ca. Endobugulasertula" (BKS1 and BKS2) (Table 1). For each reaction, the presenceof the gene was assessed by electrophoresis of the reactionproducts through an agarose gel. The B. neritina cytochromeoxidase I gene was amplified from all next-generation larvaecollected from the control and gentamicin-treated B. neritinacolonies (data not shown). Both the BKS1 and BKS2 genes appearedto be associated with "Ca. Endobugula sertula," as they werenot detected in larvae spawned from the gentamicin-treated group,in which the symbiont loads were reduced by >99% (12), butwere present in larvae with "Ca. Endobugula sertula" (Fig. 3).In the aposymbiotic larvae the bryostatin concentrations werefound to be reduced by 97%, and these larvae were not chemicallydeterrent to fish compared to control larvae (12). Our datasupport the findings of Davidson et al. (4), who performed asimilar symbiont knockout experiment, in which they observeda 95% reduction in "Ca. Endobugula sertula" levels in the treatmentcolonies and a 50% reduction in bryostatin levels in adult B.neritina colonies grown from larvae treated with antibiotics.

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FIG. 3. PCR of symbiotic and aposymbiotic B. neritina larval DNA with primers specific for 16S rRNA genes (8) (a) and the polyketide synthase gene fragments BKS1 (b) and BKS2 (c). Primers are described in Table 1. Lanes 1 and 2, control (symbiotic; n = 2); lanes 3, 4, and 5, treatment (aposymbiotic; n = 3); lane 6, no-template control; lane L, molecular weight ladder.

We isolated two KS genes from B. neritina/"Ca. Endobugula sertula"DNA that were very similar to KS genes from other bacteria,including members of the ${gamma}$ -Proteobacteria (e.g., Paederus symbiontand Pseudomonas fluorescens) (Fig. 1). BKS1 was almost identicalto KS2 of bryA isolated from deep CA populations of B. neritina/"Ca.Endobugula sertula." This observation is particularly interestingbecause the populations of B. neritina/"Ca. Endobugula sertula"used in this study (shallow NC) are genetically more similarto shallow CA populations based on the B. neritina cytochromeoxidase I gene and the "Ca. Endobugula sertula" 16S rRNA genesequences (3, 14). Furthermore, the shallow CA and NC populationshave the most similar bryostatin profiles (3, 12, 13). Whilethe differences in the KS genes between shallow NC and shallowCA populations of B. neritina/"Ca. Endobugula sertula" are unlikelyto result in differences in bryostatin composition in thesepopulations (3) as these KS genes likely produce the polyketidecore common to all of the bryostatins, the results may supportthe notion that secondary metabolism genes are often the resultof horizontal gene transfer and recombination (7, 10, 17).

Elimination of the symbiont from B. neritina adults and larvaeresulted in the elimination of these KS genes (Fig. 3). We cannotunequivocally demonstrate that the KS genes described from CA(9) and NC (Fig. 1) populations of B. neritina/"Ca. Endobugulasertula" are involved in the biosynthesis of the bryostatinsuntil the symbiont is cultivated and traditional gene knockoutand complementation studies confirm this or the PKS clusteris expressed in a heterologous host and bryostatin productionis established. However, because BKS1 is very closely relatedto bryA, the PKS gene isolated in bryostatin-producing CA populationsof B. neritina/"Ca. Endobugula sertula" (Fig. 2) (9), we hypothesizethat these genes may be involved in bryostatin biosynthesis.bryA is too short to produce the entire backbone of bryostatinbased on conventional type I PKS biosynthetic models (5); thus,other PKS genes are probably involved. It is possible that BKS2resides in one of these other genes.

Nucleotide sequence accession numbers.

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Nucleotide sequence accession...

The GenBank accession numbers for the BKS1 and BKS2 nucleotidesequences are DQ663570 and DQ663571, respectively.

ACKNOWLEDGMENTS

We are grateful to Barb Campbell, Kathy Coyne, and Lisa Waidnerfor their advice concerning the molecular portions of this project.We thank Jeremy Weisz, Channing Jones, and Glen Safrit for assistancewith collection of B. neritina.

Sigma Xi grants-in-aid of research to N.B.L. and SG projectR/F-9 (grant to N.M.T.) funded portions of this project.

FOOTNOTES

* Corresponding author. Present address: Life Sciences Institute, University of Michigan, 210 Washtenaw Avenue, Ann Arbor, MI 48109-2216. Phone: (734) 647-8988. Fax: (734) 615-3641. E-mail: nlopanik{at}umich.edu.

Published ahead of print on 22 September 2006.

REFERENCES

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