Lack of Overt Genome Reduction in the Bryostatin-ProducingBryozoan Symbiont “Candidatus Endobugula sertula”

Ian J. Miller,a Niti Vanee,b Stephen S. Fong,b Grace E. Lim-Fong,c Jason C. Kwana

Pharmaceutical Sciences Division, University of Wisconsin—Madison, Madison, Wisconsin, USAa; Department of Chemical and Life Science Engineering, VirginiaCommonwealth University, Richmond, Virginia, USAb; Department of Biology, Randolph-Macon College, Ashland, Virginia, USAc

ABSTRACT

The uncultured bacterial symbiont “Candidatus Endobugula sertula” is known to produce cytotoxic compounds called bryo-statins, which protect the larvae of its host, Bugula neritina. The symbiont has never been successfully cultured, and it wasthought that its genome might be significantly reduced. Here, we took a shotgun metagenomics and metatranscriptomics ap-proach to assemble and characterize the genome of “Ca. Endobugula sertula.” We found that it had specific metabolic deficien-cies in the biosynthesis of certain amino acids but few other signs of genome degradation, such as small size, abundant pseudo-genes, and low coding density. We also identified homologs to genes associated with insect pathogenesis in othergammaproteobacteria, and these genes may be involved in host-symbiont interactions and vertical transmission. Metatranscrip-tomics revealed that these genes were highly expressed in a reproductive host, along with bry genes for the biosynthesis of bryo-statins. We identified two new putative bry genes fragmented from the main bry operon, accounting for previously missing enzy-matic functions in the pathway. We also determined that a gene previously assigned to the pathway, bryS, is not expressed inreproductive tissue, suggesting that it is not involved in the production of bryostatins. Our findings suggest that “Ca. Endobu-gula sertula” may be able to live outside the host if its metabolic deficiencies are alleviated by medium components, which is con-sistent with recent findings that it may be possible for “Ca. Endobugula sertula” to be transmitted horizontally.

IMPORTANCE

The bryostatins are potent protein kinase C activators that have been evaluated in clinical trials for a number of indications, in-cluding cancer and Alzheimer’s disease. There is, therefore, considerable interest in securing a renewable supply of these com-pounds, which is currently only possible through aquaculture of Bugula neritina and total chemical synthesis. However, theseapproaches are labor-intensive and low-yielding and thus preclude the use of bryostatins as a viable therapeutic agent. Our ge-nome assembly and transcriptome analysis for “Ca. Endobugula sertula” shed light on the metabolism of this symbiont, poten-tially aiding isolation and culturing efforts. Our identification of additional bry genes may also facilitate efforts to express thecomplete pathway heterologously.

Marine invertebrates, such as tunicates and sponges, areknown to harbor symbiotic communities of bacteria. Be-

cause these animals are sessile and have limited physical de-fenses, their microbiomes are thought to serve defensive func-tions, and bacterial symbionts in these invertebrates have beenimplicated in the production of many bioactive small mole-cules (1, 2). Such small molecules can possess therapeuticallyrelevant activities, so there is great interest in studying the bio-synthetic potential and symbiotic functions of these defensivemicroorganisms (1, 2). However, most symbionts (and mostenvironmental bacteria in general [1, 3, 4]) are difficult to cul-ture, which renders their bioactive metabolites challenging toaccess on an industrial or clinically useful scale. Due to fastid-ious or cryptic growth requirements, the study of these systemsis possible only through culture-independent methods, such asdirect sequencing of environment-derived DNA (termed shot-gun metagenomics).

In the present work, we used shotgun metagenomics to gain agreater understanding of the uncultured bacterial symbiont “Can-didatus Endobugula sertula,” which resides within the marinebryozoan Bugula neritina (Fig. 1A), where it is known to producedefensive compounds called bryostatins (5–8) (Fig. 1B). Althoughthe adult bryozoan is covered in a protective layer of chitin, thelarvae require chemical defense after release (9–11), and high con-

centrations of “Ca. Endobugula sertula” have been observedwithin free-swimming larvae (10).

Despite many unsuccessful attempts to culture the producingorganism in laboratory settings, the bryostatins remain a target oftherapeutic interest (7), as they are cytotoxic highly potent proteinkinase C activators that have been investigated in clinical trials forthe treatment of cancer, Alzheimer’s disease, and HIV infection(Fig. 1B) (7). The bry pathway for bryostatin production is what isknown as a trans-acetyltransferase (AT)-type polyketide synthase(PKS) pathway (8, 12). PKSs are related to fatty acid synthases,and they construct a molecule in a similar fashion from two-car-

Received 15 June 2016 Accepted 25 August 2016

Accepted manuscript posted online 2 September 2016

Citation Miller IJ, Vanee N, Fong SS, Lim-Fong GE, Kwan JC. 2016. Lack of overtgenome reduction in the bryostatin-producing bryozoan symbiont“Candidatus Endobugula sertula.” Appl Environ Microbiol 82:6573– 6583.doi:10.1128/AEM.01800-16.

Editor: H. L. Drake, University of Bayreuth

Address correspondence to Jason C. Kwan, jason.kwan@wisc.edu.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.01800-16.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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November 2016 Volume 82 Number 22 aem.asm.org 6573Applied and Environmental Microbiology

bon units derived from malonate (13), using multiple enzymaticdomains in a typical PKS protein. Most PKS pathways include ATdomains within PKS proteins that are used to load the activatedS-coenzyme A (CoA) thioester form of malonate, but in trans-ATsystems, the AT exists on a separate protein. Portions of the brypathway for the bryostatins were recovered previously using clonelibrary methods (6), but the complete genome sequence, includ-ing missing pieces of the bry pathway, has remained elusive due torecalcitrance of the symbiont to culture. A number of factors arethought to contribute to this recalcitrance, including the possibil-ity of substantial genome reduction (7), which has been reportedin other invertebrate symbioses (14–16). Indeed, the divergence ofsymbionts in genetically isolated hosts (17), as well as restrictionto B. neritina and allied species, the vertical mode of symbionttransmission, and the ongoing inability to isolate and culture “Ca.Endobugula sertula” suggested a lifestyle of extreme host depen-dence that can lead to gene loss and genome reduction (18). How-

ever, we recently found that under some circumstances, “Ca. En-dobugula sertula” may be horizontally transferred between B.neritina individuals (19), which is not consistent with strict hostdependency that is associated with extreme genome reduction(18).

Our objective was to assemble the genome of “Ca. Endobugulasertula” in order to determine whether the symbiosis has evolvedto a state of codependency, evidenced by bacterial genome degra-dation, as in some other defensive symbioses (14–16). ShotgunDNA and RNA sequencing revealed a genome with few signs ofreduction, largely intact mainstream metabolic pathways, and aputative mechanism of vertical transmission of “Ca. Endobugulasertula” to host larvae.

MATERIALS AND METHODSCollection of biological material. Collection of samples AB1_ovicells andMHD_larvae has been described elsewhere (76). Briefly, an adult individ-

a. b. c.Bryostatin R1 R2

4 a b5 a b6 c b7 b b8 c c9 b c10 a H11 b H13 c H14 a OH16 a H17 a H18 a H19 a c20 a H

Bryostatins 19 and 20

A

B

O O

R1

OO

O

R2

O

O

OH

HO

OH OH

O

O

O O

R1

OO

O

O

OHR2

O

O

OH

HO

OH OH

Bryostatins 4-11,13-14, and 16-18

O

O

O

O

O

O

Ovicells

LophophoresZooids

FIG 1 Morphology and chemotype of the Bugula neritina shallow genotype (type S). (A) Micrograph showing the morphology of zooids and ovicells in a B.neritina colony. Colonies consist of individual animals (zooids) that are specialized for feeding, substrate attachment, or reproduction. Food particles arecaptured by feeding zooids using pulsating projections called lophophores. Reproductive zooids brood developing embryos in chambers called ovicells beforethey are released as free-swimming larvae. (B) Bryostatin structures found in the shallow genotype (type S) Bugula neritina (73, 74). Bryostatins 4 to 11, 13, 14,and 16 to 18 are associated with this genotype, and these structures vary at positions R1 and R2, with some possessing the ester side chains shown. Bryostatins 19and 20 possesses an extra cyclization of one �-branch, and they are also found in type S individuals.

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ual (AB1) was collected by hand from the sides of a floating dock inNovember 2013 near Morehead City, NC, at site Atlantic Beach (AB;34°42=24.527�N 76°44=18.286�W). Mature larval brooding chambers(ovicells) were dissected from AB1 and combined to form sampleAB1_ovicells. Additional B. neritina individuals were collected at More-head City Docks (MHD; 34°43=8.879�N 76°42=49.838�W), also in No-vember 2013. Approximately 20 of these B. neritina colonies were used forcombined larval collection. The collected larvae were combined to formsample MHD_larvae. Both samples were genotyped using Linneman etal.’s protocol (19) and found to be the “shallow” (S) genotype. Both sam-ples were preserved in RNAlater and stored at �80°C.

Nucleic acid extraction, sequencing, and assembly. DNA was ex-tracted from RNAlater-preserved tissue samples using a procedure previ-ously optimized for tunicate shotgun metagenomics (20). A portion ofAB1_ovicells was ground with a mortar and pestle under liquid nitrogenbefore being resuspended in 5 ml of 2 mg/ml lysozyme in Tris-EDTA (TE)buffer. A portion of MHD_larvae was added directly to 5 ml of 2 mg/mllysozyme in TE. In both cases, extractions were incubated at 30°C, withshaking, for 1 h. After this time, 1.2 ml of 0.5 M EDTA was added to eachtube along with proteinase K (final concentration, 0.2 mg/ml; Qiagen),and mixtures were incubated at 30°C for 5 min. After the addition of 650�l of 10% SDS, the mixtures were incubated at 37°C with shaking over-night. NaCl (1.2 ml of 5 M) was then added to each tube, along with 1.0 mlof cetyltrimethylammonium bromide (CTAB)-NaCl solution (10%CTAB in 0.7 M NaCl), and the tubes were incubated at 65°C for 20 min.Mixtures were extracted twice with 1:1 phenol-chloroform, and 1 volumeof isopropanol was added to the aqueous fraction, which was then storedat 4°C overnight. Tubes were spun down at 3,220 � g for 30 min at 0°C.Supernatants were carefully removed, and 2 ml of 70% ethanol in waterwas added to each tube before they were spun down again. The superna-tants were removed, and tubes were inverted for 20 min before 500 �l ofTE was added. The tubes were left overnight at 4°C to allow DNA todissolve, before extractions were subjected to repurification by Genomic-tip 100/G (Qiagen), according to the manufacturer’s instructions. Thisprocedure yielded 9.4 �g of DNA from AB1_ovicells and 882 ng of DNAfrom MHD_larvae (both in 500 �l, with the concentration measured bythe PicoGreen assay). For metagenomic analysis, TruSeq libraries wereprepared with �300-bp inserts and then subjected to sequencing in 2 �100-bp runs on a HiSeq 2000 (Illumina). RNA was extracted from ap-proximately 40 mg of AB1_ovicells. After grinding with a mortar andpestle under liquid nitrogen, the material was resuspended in 600 �l ofbuffer RLT (Qiagen) containing 6 �l of �-mercaptoethanol. The mixturewas homogenized by drawing a sterile 20-G needle up and down 15 timesbefore being spun down at 16,800 � g for 3 min. Total RNA was thenpurified from the crude lysate using the RNeasy minikit (Qiagen), utiliz-ing the optional DNase step. This procedure yielded 1.6 �g of RNA (in 100�l). The resulting RNA was flash-frozen in liquid nitrogen and stored at�80°C. Prokaryotic and eukaryotic rRNA was depleted with the Ribo-Zero rRNA removal (epidemiology) kit (Epicentre), and eukaryotic poly-adenylated transcripts were depleted with poly(T) beads. RNA in the re-sulting eluate was recovered and purified with Agencourt RNAClean XPbeads. Stranded RNA sequencing (RNA-seq) Illumina libraries were pre-pared with �300-bp inserts and subjected to two Illumina HiSeq 2000sequencing runs, one at 2 � 101 bp and one at 2 � 151 bp. Sequence yieldsare shown in Table S1 in the supplemental material. Reads were qualityfiltered and then assembled with SPAdes (21), and contigs were taxonom-ically classified by using MEGAN (22) to process the results of BLASTP(protein-protein Basic Local Alignment Search Tool) searches of all pre-dicted open reading frames (ORFs) against the NCBI nonredundant pro-tein database (NR), as previously described (76). These classificationsallowed bacterial contigs to be separated from eukaryotic and “unclassi-fied kingdom” contigs likely originating from the host genome.AB1_ovicells and MHD_larvae metagenomes were compared by aligningraw MHD_larvae sequence reads to bacterial AB1_ovicells contigs of �3kbp in length. Contigs with �1� read coverage in the resulting alignment

were visualized in R (23) as points on a graph of GC% versus coverage(see Fig. S1 in the supplemental material). Outlier contigs to the singleapparent cluster were removed using mclust (24) in R (23). This clusterwas determined to be the “Ca. Endobugula sertula” genome, based onphylogeny and inclusion of known “Ca. Endobugula sertula” genes (seeResults).

PCR amplification and screening to confirm connectivity betweencontigs. Primers (see Table S3 in the supplemental material) were de-signed to have an annealing temperature of �55°C manually and usingthe Primer3 algorithm (25) to test various aspects of genomic assemblies.For a 10-�l reaction matrix, the following volumes and concentrations ofeach component were used: 5 �l of 2 KOD buffer, 2 �l of 2 mM dinucleo-side triphosphates (dNTPs), 0.2 �l of KOD Xtreme Hot Start DNA poly-merase (Merck Group, Darmstadt, Germany), 1 �l of 3 �M forwardprimer, 1 �l of 3 �M reverse primer, and 0.8 �l of template (1:10 dilutionof the AB1_ovicells DNA extraction). Reactions were carried out on aBio-Rad (Hercules, CA) C1000 Touch thermal cycler in 200-�l 8-striptubes using a thermocycling program consisting of 94°C for 2 min; 35cycles of 98°C for 10 s, 55°C for 30 s, and custom extension time (1 min perkbp expected product) at 68°C; and then 68°C for 10 min with an indefi-nite hold at 12°C upon thermocycling completion.

“Candidatus Endobugula sertula” genome annotation, RNA-seqalignment, and functional category assignment. RNA-seq data were fil-tered with SeqyClean (https://github.com/ibest/seqyclean), using the pa-rameter “-polyat.” The resulting filtered reads were aligned with Bowtie 2(26) to contigs in the “Ca. Endobugula sertula” assembly using the end-to-end alignment options “-very-sensitive -no-discordant -no-unal.”Contigs were annotated with the Prokka pipeline (27) and combined withRNA-seq alignment files in Geneious (Biomatters Ltd., San Francisco,CA) to visualize transcript abundance. Reads aligned to each ORF werecounted in Geneious, and for each gene, normalized reads per kilobasepair of gene per million (RPKM) reads aligning to annotated ORFs in the“Ca. Endobugula sertula” genome (28) values were calculated.

MEGAN (22) was used to assign functional Kyoto Encyclopedia ofGenes and Genomes (KEGG) categories to predicted protein sequences.KEGG trees were uncollapsed two levels in MEGAN, and all assignmentsexcept for “organismal systems” and “human diseases” were exported toa .csv file (with the columns “read name” and “KEGG name”). CalculatedRPKM values and the MEGAN .csv table were used to calculate propor-tions of the “Ca. Endobugula sertula” transcriptome that corresponded toeach KEGG category. Where multiple KEGG categories were assigned toone predicted gene, that gene’s RPKM value was split equally among theassigned categories.

Homolog analysis in “Ca. Endobugula sertula.” Predicted genes inthe draft “Ca. Endobugula sertula” assembly with homologs in other gam-maproteobacteria (Fig. 2; see also Fig. S4 and Table S2 in the supplementalmaterial) were identified with a procedure previously used to determinehomologs conserved between an intracellular symbiont and its closestfree-living relative (47). Predicted protein sequences from the “Ca. En-dobugula sertula” assembly were used as queries in BLASTP searchesagainst the NR database, and the ranks of proteins from target bacteriawere tabulated with a custom script. Genes were counted as having ho-mologs in the target sequence set if a target sequence had a rank of 100in the BLAST results. The best BLAST hit from the target sequence set wascounted as the homolog of the protein query from the “Ca. Endobugulasertula” assembly.

Bioinformatic analysis for metabolic modeling. Genes identified byannotation and homology were referenced to UniProt to associate andconfirm Enzyme Commission (EC) numbers with each gene. The identi-fied EC numbers were mapped to biochemical pathways and comparedusing BioCyc and KEGG pathways. The functional continuity of bio-chemical pathways was tested using a draft constraint-based model of“Ca. Endobugula sertula” containing 1,774 biochemical reactions using abiomass objective function and flux balance analysis (FBA).

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Construction of “Ca. Endobugula sertula” phylogenomic tree.AMPHORA2 (30, 31) was used to scan the “Ca. Endobugula sertula”assembly for 104 phylogenetic marker genes, which were extracted andmanually examined to resolve instances of multiple hits. The markergenes were individually aligned to the internal reference database suppliedwith AMPHORA2. Genes from gammaproteobacterium IMCC1989(32) were manually added, as this bacterium was not present in theAMPHORA2 database. The marker genes were individually aligned to theinternal reference database supplied with AMPHORA2. The set of indi-vidual marker alignments was filtered such that only reference genomeswith �75% of the marker genes were retained, and then marker genesrepresented in 75% of these genomes were removed. The resulting 29marker alignments were concatenated, and residues not aligning toAMPHORA2’s hidden Markov models (HMMs), signified by lowercaseresidues in the resulting alignment file, were removed from the alignment.Trees were then constructed with FastTree 2 (33) using the parameters“-gamma -slow -spr 10 -mlacc 3 -bionj.” After FastTree 2 runs were com-plete, accession numbers were substituted for strain designations accord-ing to entries in the RefSeq database. The resulting tree was rooted arbi-trarily at the divergence of the phylum Deinococcus-Thermus and other

bacteria, as others have done previously (30). The tree was manipulatedusing the Interactive Tree of Life server (34).

Accession number(s). Raw shotgun metagenome reads for AB1_ovicellsand MHD_larvae were deposited in the Sequence Read Archive (SRA).The accession numbers for AB1_ovicells are SRR4020077, SRR4020078,SRR4020081, SRR4020082, SRR4020083, and SRR4020084; the accessionnumbers for MHD_larvae are SRR4020079, SRR4020080, SRR4020086,SRR4020087, and SRR4020088. Metatranscriptomic RNA-seq reads forAB1 were deposited in the SRA under accession numbers SRR4020085and SRR4125604. The annotated draft genome for AB1_endobugula as-sembly is accessible through the NCBI under the accession numberMDLC00000000. All these data sets are accessible under BioProjectPRJNA322176 and BioSample numbers SAMN05039512 (AB1_ovicells)and SAMN05513359 (MHD_larvae).

RESULTSAssembly of “Ca. Endobugula sertula” genome. DNA from twoseparate samples was sequenced: free-swimming larvae from apooled sample of B. neritina colonies (MHD_larvae), and ovicells

4115 47

937168 529

172

n = 3,049

1,140

Teredinibacter turnerae T7901 γ-proteobacterium IMCC1989

Photorhabdus/ Xenorhabdus

Other protein-coding genes

A B0.1

Pseudomonas aeruginosa PA7

Pseudomonas aeruginosa PAO1Pseudomonas aeruginosa UCBPP-PA14

Pseudomonas aeruginosa LESB58

Azotobacter vinelandii DJPseudomonas stutzeri ATCC 17588Pseudomonas stutzeri A1501

Pseudomonas putida S16Pseudomonas entomophila L48

Pseudomonas putida W619Pseudomonas putida GB-1Pseudomonas putida KT2440Pseudomonas putida F1

Pseudomonas protegens Pf-5Pseudomonas brassicacearum subsp. brassicacearum

Pseudomonas fluorescens SBW25Pseudomonas syringae pv. tomato str. DC3000Pseudomonas syringae pv. syringae B728aPseudomonas syringae pv. phaseolicola 1448A

Pseudomonas fluorescens Pf0-1

Pseudomonas fulva 12-XPseudomonas mendocina ympPseudomonas mendocina NK

Halomonas elongata DSM 2581Chromohalobacter salexigens DSM 3043

Hahella chejuensis KCTC 2396Marinobacter hydrocarbonoclasticus VT8

Marinomonas mediterranea MMB-1Marinomonas sp. MWYL1

Marinomonas posidonica IVIA-Po-181γ-proteobacterium IMCC1989“Candidatus Endobugula sertula” AB1Cellvibrio japonicus Ueda107

Saccharophagus degradans 2-40Teredinibacter turnerae T7901

100

100

10091

100

99

100

100

100

100

9999

100919598100

90

100

100

86100

100

96

100100

100100

85100

Protein homologs in “Ca. E. sertula”

5 kb

NODE28

Yen-Tcchi1 yenA1 yenA2 chi2 yenB yenC1 yenC2

CAB835_02580 AB835_02575 AB835_02570 AB835_02565 AB835_02460 AB835_02555 AB835_02550 AB835_02545

60%

30%

FIG 2 Phylogenomic analysis of “Ca. Endobugula sertula.” (A) An approximately maximum-likelihood tree generated by FastTree 2 from 29 concatenatedsingle-copy-marker gene protein sequences from the “Ca. Endobugula sertula” genome and 1,336 other reference genomes. Bootstrap proportions greater than70% are expressed to the left of each node as a percentage of 1,000 replicates. (B) Venn diagram of protein-coding genes in the “Ca. Endobugula sertula” genome,showing homologs of predicted proteins in T. turnerae T7901, gammaproteobacterium IMCC1989, and Photorhabdus/Xenorhabdus. (C) Comparison of Tc genecluster on NODE28 with a chitinase-containing Tc locus in the insect pathogen Y. entomophaga, showing corresponding regions of protein sequence homologybetween the two clusters (65). The levels of shading represent amino acid identity.

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dissected from a single adult colony (AB1_ovicells). Reference-based assembly of 16S sequences using EMIRGE (35, 36) recon-structed a 16S rRNA sequence with 99% identity to that of “Ca.Endobugula sertula” (type S strain, also termed BnSP; accessionnumber AF006606 [37]) in both AB1_ovicells and MHD_larvae,but it suggested the presence of multiple bacteria in theAB1_ovicells sample that were not found in MHD_larvae.Through 16S rRNA amplicon sequencing, it was found that this“Ca. Endobugula sertula” sequence accounted for 2.4% and 5.6%of reads in AB1_ovicells and MHD_larvae, respectively, and apartfrom this symbiont, there was virtually no overlap in the widermicrobiome between these two samples (76). Reads from bothAB1_ovicells and MHD_larvae were assembled separately usingSPAdes (21). Contigs from the AB1_ovicells assembly, which hadgenerally higher-quality sequence assembly characteristics (largeroverall assembly size, higher N50, etc.) than the pooled MHD_lar-vae sample (see Table S1 in the supplemental material), were clas-sified taxonomically based on the homology of their open readingframes (ORFs) to the NCBI NR database. To simplify the overallmetagenomic assembly of AB1_ovicells, contigs smaller than3,000 bp and contigs that were classified as eukaryotic (�135 Mbpin total) or unclassified at the kingdom level based on these taxo-nomic assignments were removed.

Because the EMIRGE-reconstructed 16S sequences suggestedthat “Ca. Endobugula” was the only shared bacterium between theAB1_ovicells and MHD_larvae assembly, contigs with read cov-erage in both samples were identified, which should include se-quences belonging to “Ca. Endobugula sertula” and any host ge-nome contigs misclassified as bacteria. AB1_ovicells contigs thathad MHD_larvae coverage appeared to form two discrete clustersbased on GC% and coverage (see Fig. S1 in the supplementalmaterial). These two clusters were automatically separated using atechnique where data are fitted to a certain number of groupsbased on discrete distribution patterns, known as normal-mixturemodeling (24). The accuracy of this clustering approach was eval-uated using the assigned ORF (see Fig. S2 in the supplementalmaterial) and contig-based (see Fig. S3 in the supplemental mate-rial) taxonomy of these two clusters, as well as single-copy-markeranalysis (Table 1). The contigs belonging to one of these groups(clusters 1 and 2) contained bry pathway components and hadORF taxonomic classifications consistent with previous 16S-basedtaxonomies of “Ca. Endobugula sertula,” such as known relativesgammaproteobacterium IMCC1989 (32) and Teredinibacter turn-erae (38).

Phylogenomic and symbiotic characteristics of the “Candi-datus Endobugula sertula” genome. The draft genome of “Ca.Endobugula sertula” is 3.4 Mbp in size (112 contigs), has 41.2%GC content, and is estimated to be 100% complete by single-copy-marker gene analysis (39) (Table 1). The “Ca. Endobugulasertula” 16S rRNA gene sequence reconstructed by EMIRGE wasjoined to one of the putative “Ca. Endobugula sertula” contigsthrough PCR and Sanger sequencing. In addition, a whole-ge-nome phylogenetic tree constructed from concatenated bacterialmarker genes placed the genome in a clade with known “Ca. En-dobugula sertula” relatives gammaproteobacterium IMCC1989,T. turnerae, Saccharophagus degradans, and Cellvibrio japonicus(Fig. 2A) (17, 38, 40). All of these relatives are free living except forT. turnerae, which is an intracellular symbiont of wood-feedingshipworms (38), although it should be noted that T. turnerae can

be grown in the laboratory, in contrast to “Ca. Endobugulasertula.”

“Candidatus Endobugula sertula” possesses many proteinswith homologs in related gammaproteobacteria (Fig. 2B), but alarge portion, including the bry pathway, are also not shared withthe closest free-living relatives. Teredinibacter turnerae (38), S. de-gradans (41), and C. japonicus (42) are all specialized in the deg-radation of complex polysaccharides, but “Ca. Endobugulasertula” does not appear to possess homologs of the �100 glycosylhydrolase (GH) domains found in T. turnerae, which are used todigest wood for its host. “Candidatus Endobugula sertula” alsodoes not appear to possess homologs of the nitrogen fixation genesfound in T. turnerae (38), which compensate for a nitrogen-poorwood diet. However, a group of genes were identified that showedhomology to proteins found in Photorhabdus and/or Xenorhabdusspecies (Fig. 2B), which are predominantly insect pathogens thatassociate with nematode vectors (43). These genes included sev-eral toxin complex (Tc) gene clusters (Fig. 2C), which have beenassociated with both host specificity and pathogenesis (44). One ofthese loci, on contig NODE28, was found to contain two chitinasegenes.

Reductive evolution and metabolic and metatranscriptomicanalysis. “Candidatus Endobugula sertula” has, to our knowl-edge, never been cultured in the laboratory, and it has been pre-viously suggested that its genome might be reduced (7), similar tosome long-term symbionts of insects (15, 18) and marine tuni-cates (14, 16). Although the “Ca. Endobugula sertula” genome isthe smallest out of several close relatives (Table 2), there is littleother evidence to indicate genome degradation or reduction. It is3.4 Mbp in size, larger than the previously predicted 2 Mbp basedon flow cytometry (7), and despite the high AT content (�70%)of certain regions in the bryostatin pathway (6, 7), the genome asa whole is only 58.4% AT. Furthermore, the coding density ofthe “Ca. Endobugula sertula” genome is 85.1%, which falls intothe average bacterial coding density range (85 to 90% [45]). Wecompared the lengths of genes in T. turnerae and gammaproteo-bacterium IMCC1989 to homologs in “Ca. Endobugula sertula”in an attempt to identify potential pseudogenes that were 80%

TABLE 1 “Candidatus Endobugula sertula” genome assemblycharacteristics

Genome featurea Value

No. of contigs 112Assembly size (bp) 3,350,348GC content (%) 41.63N50 (bp) 49,663Longest contig (bp) 153,360Coverage 6.2No. of tRNA genes 49No. of rRNA genes 1No. of CDSs 2,850Coding density (%) 84.2No. of conserved single-copy genes

(expected/observed)139/139

No. of repeated conserved single-copy genes 1No. of ORFs with functional annotation 3,049No. of ORFs without functional prediction 1,576Avg CDS length (bp) 972a The coverage quoted here is k-mer coverage reported by the SPAdes assembler, wherek � 77. CDSs, protein-coding genes.

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of the length of their homologs (46, 47). A low number of genes,30, were identified as potential pseudogenes, and most of thesewere annotated with functions associated with motility and regu-latory sensors (see Fig. S4 and Table S2 in the supplemental ma-terial). Bioinformatic analysis and metabolic modeling, however,indicated potential metabolic insufficiencies in amino acid metab-olism, as we could not find complete pathways to synthesize me-thionine and threonine (Fig. 3).

To gain a functional snapshot of “Ca. Endobugula sertula,” weextracted and sequenced RNA from the AB1_ovicells sample (Fig.4). In “Ca. Endobugula sertula,” 12.1% of the functionally as-signed transcriptome is dedicated collectively to putative symbi-otic functions: bryostatin synthesis (3.6%) and the expression ofTc genes (8.5%). Despite apparent insufficiencies in amino acidbiosynthesis, the single largest fraction (14.7%), by KEGG cate-gory, of the symbiont’s transcriptome was dedicated to processesin amino acid metabolism (Fig. 4). A number of enzymes in theamino acid metabolism category are involved in arginine biosyn-thesis. For instance, argininosuccinate synthase (AB835_04910)and acetylornithine aminotransferase (AB835_00040) are the twomost highly expressed enzymes in this amino acid metabolismcategory. Another highly expressed enzyme involved in amino

acid biosynthesis was aspartate transaminase (AB835_06295),which is a multifunctional enzyme that is involved in aspartate,tyrosine, phenylalanine, and tryptophan metabolism. While thepathways for histidine and lysine appeared to be complete, pres-ent, and transcribed, lower expression levels of certain enzymes inthese pathways (Fig. 3; see also Data Set S1 in the supplementalmaterial) make the actual level of amino acid biosynthesis moreuncertain, given that transcript levels often do not accurately pre-dict downstream protein abundance (48).

De novo assembly and repeat resolution of the bryostatinbiosynthetic pathway. The bry pathway was previously se-quenced as a contiguous locus in the type S genotype of “Ca.Endobugula sertula,” containing five genes encoding PKS pro-teins (BryBCXDA) and various accessory genes, including thetrans-AT bryP and genes involved in �-branching (bryQR, Fig.5C) (6). The known sequence included several exact repeats,which complicated de novo assembly of this region in theAB1_ovicells metagenome. A procedure devised by Albertsen etal. (49) was used to resolve the repeats by identifying paired readsaligning at the ends of two (or more) contigs (Fig. 5A). Relativeread coverage of repeats versus unique regions, as well as contigorientation, was used to construct a connection map and restrictthe sequence to two possible structures (Fig. 5B). The correct brylocus structure, as determined by PCR (see Tables S3 and S4 in thesupplemental material), was in agreement with the published se-quence (accession no. EF032014). The published pathway, how-ever, is missing some key components needed for �-branching toproduce the methylated esters seen in the final bryostatin struc-tures. In polyketides made by trans-AT PKS pathways, �-alkylchains are commonly introduced by a set of proteins minimallyincluding a standalone acyl-carrier protein (ACP), a decarboxyla-tive ketosynthase (KS), a 3-hydroxy-3-methylglutaryl-CoA syn-thase (HMGS) and 1-2 enoyl-CoA-hydratases (ECH) (50) (Fig.5C). A previously unknown ECH and an adjacent ACP were

TABLE 2 Comparison of “Ca. Endobugula sertula” genome with thoseof close relatives

Genome (reference)Size(Mbp)

GCcontent(%)

No. ofgenes Habitat Lifestyle

“Ca. Endobugula sertula” 3.34 41.6 2,850 Marine SymbiontGammaproteobacterium

IMCC1989 (32)3.94 42.1 2,844 Marine Free-living

T. turnerae T7901 (38) 5.19 50.8 4,690 Marine SymbiontS. degradans 2-40 (41) 5.06 45.8 4,017 Marine Free-livingC. japonicus Ueda107 (42) 4.58 52.0 3,790 Terrestrial Free-livingH. chejuensis KCTC 2396 (75) 7.22 54.8 6,783 Marine Free-living

erythrose-4-Pshikimate chorismate

tryptophan

phenylalanine

tyrosine

5-phosphoribosyl1-pyrophosphate ornithine

histidine

pyruvate2-ketovaline

valine

leucine

2-oxobutanoate isoleucine

aspartate lysine

threonine

homoserine

homocysteine

cysteine

cystathioninemethionine

Absent

RPKM < 100

100 < RPKM < 1000

RPKM >1000

glutamateornithine

arginine

FIG 3 Amino acid biosynthesis pathways found in the “Ca. Endobugula sertula” genome. Enzyme commission (EC) numbers are colored based on presence andRPKM values.

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found in a separate locus (bryTU), and these are predicted to act inthe dehydration of the new side chain and as a carrier for themalonate �-branch, respectively. These genes are homologous tocorresponding genes in other trans-AT PKS pathways, with theclosest homologs to bryT and bryU being batD (batumin/kaliman-tacin pathway [51], 53% amino acid identity) and calX (calyculinpathway [52], 45% amino acid identity), respectively.

Interestingly, all bry mRNA transcripts were detected throughmetatranscriptomics, except bryS, a methyltransferase previouslythought to carry out the O-methylation of �-branches (6). Unlikeother bry genes, bryS has a homolog in gammaproteobacteriumIMCC1989 that is annotated as a tRNA-methyltransferase (acces-sion no. WP_040805166) and therefore may not be part of the brypathway, as previously predicted.

DISCUSSION

Reductive evolution is known to occur in a number of settings,including intracellular symbiosis (18, 53), intracellular pathogen-esis (54), and free-living pelagic microbes (55). In strictly intracel-lular organisms, reduction is thought to occur due to genetic driftas a consequence of genetic isolation and strict vertical transmis-

sion, low effective population sizes, and frequent bottlenecks (18).In free-living examples, it is thought that reduction is driven byselection rather than drift (56), potentially due to a selective ad-vantage in not investing in the production of a metabolite that hasbecome a “public good” in the community (57). In the “BlackQueen” model (57), when a metabolic pathway becomes rareenough in the community, further loss is selected against to pre-vent loss of community fitness as a whole.

Features in the “Ca. Endobugula sertula” genome were incon-sistent with extreme reduction due to sequence drift. The earlystages of such a process are characterized by a proliferation ofpseudogenes and an accompanying low coding density (18). Incontrast to the hundreds of pseudogenes identified in Sodalisglossinidius, a tsetse fly symbiont in the early stages of genomedegradation (58), only 30 potential pseudogenes were found in the“Ca. Endobugula sertula” genome. Furthermore, the “Ca. En-dobugula sertula” genome did not have a particularly low GCcontent but did have a coding density within the typical bacterialrange (45) and a genome size substantially larger than the previ-ously predicted 2 Mbp (7). However, careful examination of themetabolic capabilities implied by the “Ca. Endobugula sertula”genome suggested that it lacked a number of enzymes required forthe synthesis of certain amino acids (Fig. 3), potentially explainingits dependence on its host environment and, perhaps, other mi-crobial constituents of the host microbiome. Metatranscriptomicanalysis suggested that the symbiont was actively involved in met-abolic processes related to amino acid metabolism, such as argi-nine biosynthesis. High expression levels of the arginine biosyn-thesis pathway may help explain an interesting observation madeby a previous study that found that B. neritina larvae do not be-come depleted of nitrogen during the swimming stage prior tosettlement, compared to the larvae of other Bugula species that areaposymbiotic (59). Perhaps then, the host’s larval nutrition maybe augmented through nitrogen recycling by “Ca. Endobugulasertula” in the form of ammonia assimilation as carbamoyl phos-phate (60) before storage as arginine (61).

Previously, it was found that different populations and siblingspecies of Bugula harbor different strains of “Ca. Endobugulasertula” (7), which is a pattern of distribution consistent with avertical mode of symbiont transmission, where respective popu-lations have been genetically isolated since host divergence. How-ever, we previously found evidence that horizontal transmission islikely also possible (19). We found that two sibling species of B.neritina, northern (N) and shallow (S), previously thought to beallopatrically distributed, actually coexist along the Western At-lantic. Type N populations are divergent from type S animals andare aposymbiotic in their typical northern range. However, inWestern Atlantic populations, both type N and type S individualscan be found harboring 100% identical “Ca. Endobugula sertula”strains (as measured by 16S and internal transcribed spacer [ITS]sequences). The most parsimonious explanation for this observa-tion is the horizontal transfer of symbionts from type S to type Nindividuals.

The noncongruence of host and symbiont phylogenies (17, 38,40) also argues against a long evolutionary history of strict verticaltransmission. Although “Ca. Endobugula sertula” has proven dif-ficult to isolate and culture, the lack of extensive genome reduc-tion suggests that a transient host-free existence may be possible, aplausible explanation for the symbiont acquisition by type N hostsfound alongside symbiotic type S hosts at low latitudes (19). A

CategoryCarbohydrate Metabolism

Energy Metabolism

Lipid Metabolism

Nucleotide Metabolism

Amino Acid Metabolism

Metabolism of Other Amino Acids

Glycan Biosynthesis and Metabolism

Metabolism of Cofactors and Vitamins

Metabolism of Terpenoids and Polyketides

Biosynthesis of Other Secondary Metabolites

Xenobiotics Biodegradation and Metabolism

Transcription

Translation

Folding, Sorting and Degradation

Replication and Repair

Membrane Transport

Signal Transduction

Transport and Catabolism

Cell Motility

Cell Growth and Death

Cell Communication

Bry

Chitinase and Tc

FIG 4 Metatranscriptomic analysis of “Ca. Endobugula sertula.” Proportionof normalized reads, expressed as reads per kilobase of gene per million readsaligning to annotated ORFs in the “Ca. Endobugula sertula” draft genome(RPKM; 28), aligned to genes with assigned function.

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ACP

KSKS

HMGS

ECH

PKS

AT1

ACP

S

O

ACP

BryU

BryQ

BryP

S

O

R

O

BryR

PKS

S

O

R

O

O-

PKS

S

O

O-O

R OH

BryT

O

R1

O

O

OH

O O

OHO

OO

O

HO

R2

HO

S

O

O-

O

SH

**

C

s

e1

e

sA

e

s2

se 3s

e

B

se 4

s

e5

s

e 6

Cs

e

e

s7

es

7 Ce s

4e s

Be s

6s e

Ce s

5e s

Be s

3e s s e

Ae s

2s e

A 1e s

es

7 Ce s

4e s

Be s

6s e

Ce s

5e s

Be s

3e s s e

Ae s

2s e

A 1e s

A

B

166_R 12197_F 12197_R 6199_F 6199_R 139658_F

166_R 139658_F 139658_R 6199_F 6199_R 12197_F

PCR amplicons observed

PCR amplicons not observed

FIG 5 De novo assembly and repeat resolution of the bryostatin pathway, and discovery of new bry genes. Contigs aligning to the published bry gene clusterfrom type S “Ca. Endobugula sertula” (accession no. EF032014) were identified in the AB1 ovicells metagenome. Paired reads were realigned to thosecontigs, and connections were identified as suggested by paired alignments to multiple contig ends, using a script published by Albertsen et al. (49), andvisualized in Cytoscape (72). (A) Connection map of bry contigs 1 to 7 and repeats A to C (see Table S4 in the supplemental material for details on thesecontigs). Each contig is represented by two dots connected by a red line, arbitrarily marked “s” (start) and “e” (end). Dot size is scaled to contig length,and the color is scaled according to coverage (white � �12�, black � �6� k-mer coverage reported by SPAdes, where k � 77). Green lines representconnections between assembled contigs suggested by alignment of paired reads across the indicated ends. (B) Diagram showing the two possibleresolutions to the connection map in panel A, given the assumption that the correct resolution is a single contiguous sequence. The two resolutions differin the placement of contigs 4 and 5. Primers were designed for the locations shown, and PCR amplicons were only observed for the primer pairingsconsistent with the lower solution, agreeing with EF032014. All amplicons were confirmed by Sanger end sequencing. (C) Scheme showing predictedfunctions of newly discovered bry genes bryT and bryU, which may be involved in forming the �-branches seen in bryostatin structures (positionshighlighted in the bottom left structure). BryU is proposed as a standalone acyl-carrier protein (ACP), which carries a malonate unit as it is decarboxylatedby BryQ and before the resulting acetate is condensated onto the PKS-bound substrate by BryR. BryT is an enoyl-CoA dehydratase, which is proposed togenerate the �-�-unsaturated acid from the intermediate shown. AT, acyltransferase; ECH, enoyl-CoA-reductase; HMGS, 3-hydroxy-3-methylglutaryl-CoA synthase; KS, ketosynthase; PKS, polyketide synthase.

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similar situation is found in the tunicate Lissoclinum patella, whichharbors a photosynthetic symbiont, Prochloron didemni (62). Aswith “Ca. Endobugula sertula,” P. didemni has never been culti-vated in the laboratory, but its large genome size and the lack ofevidence for genetic drift and accelerated evolution suggest that itmight not be genetically isolated inside individual hosts (63).“Candidatus Endobugula sertula” appears to have specific meta-bolic deficiencies, and these deficiencies might be the reason that“Ca. Endobugula sertula” is dependent on the host. If the symbi-ont is indeed able to withstand short periods of time outside thehost (i.e., it is not genetically isolated within individual hosts),these metabolic deficiencies may have come about due to selectionfor not investing in “public goods” that are supplied by the host(57). However, over evolutionary time scales, further metabolicdeficiencies may develop if the symbiont becomes more strictlyrestricted to only living within the host and undergoes genomedegradation and reduction, which would likely prevent horizontaltransmission of the symbiont.

Despite few signs of genome degradation, the “Ca. Endobugulasertula” genome shows some potential adaptations to symbioticlife. Several pseudogenes were annotated with functions in flagel-lar assembly and motility, and similar functions are lost in intra-cellular Rickettsiales (64). The chitinase-containing Tc locus thatwas found on NODE28 is related to the Yen-Tc locus from theinsect pathogen Yersinia entomophaga (65) and might also be in-volved in symbiosis. The chitinases in Yen-Tc are thought to as-sociate with the secreted Tc assembly, which exhibits chitinaseactivity in vitro, to contribute to Y. entomophaga pathogenicity(65). The chitinase-containing Tc locus in “Ca. Endobugulasertula” is highly expressed in the ovicells, consistent with its use inallowing “Ca. Endobugula sertula” to move from the funicularcords within the adult host (5) through the potentially chitina-ceous ectocyst (66, 67), thus ensuring vertical transmission to thelarvae. The high expression of Tc loci in ovicells is suggestive of theimportance of these proteins to the symbiont during the repro-ductive phase of its host. Other symbiotic systems have beenshown to coopt mechanisms used in virulence and immunogenic-ity of pathogens, such as during the acquisition of the light-pro-ducing symbiont Vibrio fischeri by the Hawaiian bobtail squid,where the immunogenic bacterial products lipopolysaccharideand peptidoglycan are central to symbiont-host interactions (68,69). In a similar fashion, the other Tc loci in the “Ca. Endobugulasertula” genome might also be involved in aspects of recognitionand communication with the bryozoan host, and their presencemight signify that ancestors of “Ca. Endobugula sertula” werepathogenic.

In the present work, a draft assembly of the “Ca. Endobugulasertula” genome was recovered and estimated to be 100% com-plete by single-copy-marker analysis (39) using comparativeshotgun metagenomic data sets. Adapting a method developed byAlbertsen et al. (49), the sequence of the repeat-heavy bry biosyn-thetic pathway was confirmed to agree with the sequence previ-ously constructed using a clone library method and Sanger se-quencing (6). Although additional pathway components wereidentified, enzymes in the “Ca. Endobugula sertula” genome thatcould be responsible for the addition of the ester side chains couldnot be found (Fig. 1B), suggesting that the symbiont employseither a novel mechanism that could not be identified bioinfor-matically or enzymes annotated with roles in primary metabo-lism, as others have observed (16, 70). Alternatively, the side

chains may be installed by the host, rather than the symbiont.Additionally, because bryS showed no RNA-seq coverage, thisgene may not be involved in the biosynthesis of bryostatins. IfBryS is not responsible for O-methylation of �-branches, this con-version is most likely to be carried out by methyltransferase do-mains in BryA and BryB polyketide synthases.

Interestingly, although the majority of bacterial secondary me-tabolite pathways consist of genes clustered into one chromosomeregion (71), several symbiotic bacteria have been found to containfragmented pathways (14–16, 29), which include the previouslyknown portions of the bry pathway found to be split into twoseparate loci in the type D genotype of “Ca. Endobugula sertula”(7). The AB1 draft genome of “Ca. Endobugula sertula” does notcontain any other PKS pathway, apart from bry, that would ex-plain the presence of newly identified bryTU genes. Identifyingthese genes likely implicated in bryostatin biosynthesis highlightsthe importance of recovering complete or near-complete ge-nomes and, thus, the use of unbiased shotgun sequencing in cap-turing and understanding the fragmented biosynthetic pathwaysof uncultured microorganisms. Our identification of these twoadditional bry genes, along with the metabolic analysis of “Ca.Endobugula sertula,” may facilitate further studies into the renew-able supply of bryostatins, either through heterologous expressionor targeted symbiont culture.

ACKNOWLEDGMENTS

This research was performed in part using the computer resources andassistance of the UW-Madison Center For High Throughput Computing(CHTC) in the Department of Computer Sciences. We thank NielsLindquist (UNC) for assistance with field collections, Ben Oyserman(UW-Madison) for assistance with RNA-seq analysis, and Ahron Flowers(RMC) for assistance with B. neritina genotyping. We thank the Univer-sity of Wisconsin Biotechnology Center DNA Sequencing Facility for pro-viding sequencing facilities and library preparation services.

The CHTC is supported by UW-Madison, the Advanced ComputingInitiative, the Wisconsin Alumni Research Foundation, the WisconsinInstitutes for Discovery, and the National Science Foundation, and is anactive member of the Open Science Grid, which is supported by the Na-tional Science Foundation and the U.S. Department of Energy’s Office ofScience. Additionally, this work utilized computer resources at FutureGrid, which is supported by National Science Foundation grant 0910812.This work was supported by grant R21AI121704-01 from NIAID, as wellas funding from The Thomas F. and Kate Miller Jeffress Memorial Trust(Bank of America, Trustee) and the American Foundation for Pharma-ceutical Education (to I.J.M.), as well as the School of Pharmacy, theGraduate School, and the Institute for Clinical & Translational Researchat the University of Wisconsin-Madison.

G.E.L.-F. and J.C.K. collected and prepared samples for analysis; I.J.M.and J.C.K. designed and performed the research; I.J.M., N.V., G.E.L.-F.,S.S.F., and J.C.K. analyzed data; and I.J.M. and J.C.K. wrote the paper.

FUNDING INFORMATIONThis work, including the efforts of Jason Christopher Kwan, was fundedby Division of Intramural Research, National Institute of Allergy andInfectious Diseases (DIR, NIAID) (R21AI121704-01). This work, includ-ing the efforts of Ian J. Miller, was funded by American Foundation forPharmaceutical Education (AFPE). This work, including the efforts ofStephen Fong, Grace E. Lim-Fong, and Jason Christopher Kwan, wasfunded by Thomas F. and Kate Miller Jeffress Memorial Trust (JeffressTrust).

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Genomic Analysis of “Candidatus Endobugula sertula”

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