Determine whether aromatics originate from polyketide or shikimate pathway

Determine whether aromatics originate from polyketide or shikimate pathway

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Is there a way to determine whether an aromatic compound originates from the polyketide or shikimate pathway by looking at its structure? If so, how?

Aromatics that stem from the Shikimat Pathway result in a structure like this

Whilst aromatics originating from the Polyketide Pathway have an alternativ structure in the distribution of the OH groups.

If both pathways produce the same molecule, you can't tell: It's the same molecule.

You could knock out some enzymes on either side and see which ones affect output. Or you could spike the cells with a radiolabeled precursor of only one pathway, and then see if the output is also labeled.

However, you say "by looking at its structure", so I'm assuming no additional experiment can be done - then no, except for the trivial case where only one of the pathways produces the molecule (in which case it obviously came from that one).

Evolution of a secondary metabolic pathway from primary metabolism: shikimate and quinate biosynthesis in plants

The shikimate pathway synthesizes aromatic amino acids essential for protein biosynthesis. Shikimate dehydrogenase (SDH) is a central enzyme of this primary metabolic pathway, producing shikimate. The structurally similar quinate is a secondary metabolite synthesized by quinate dehydrogenase (QDH). SDH and QDH belong to the same gene family, which diverged into two phylogenetic clades after a defining gene duplication just prior to the angiosperm/gymnosperm split. Non-seed plants that diverged before this duplication harbour only a single gene of this family. Extant representatives from the chlorophytes (Chlamydomonas reinhardtii), bryophytes (Physcomitrella patens) and lycophytes (Selaginella moellendorfii) encoded almost exclusively SDH activity in vitro. A reconstructed ancestral sequence representing the node just prior to the gene duplication also encoded SDH activity. Quinate dehydrogenase activity was gained only in seed plants following gene duplication. Quinate dehydrogenases of gymnosperms, represented here by Pinus taeda, may be reminiscent of an evolutionary intermediate since they encode equal SDH and QDH activities. The second copy in P. taeda maintained specificity for shikimate similar to the activity found in the angiosperm SDH sister clade. The codon for a tyrosine residue within the active site displayed a signature of positive selection at the node defining the QDH clade, where it changed to a glycine. Replacing the tyrosine with a glycine in a highly shikimate-specific angiosperm SDH was sufficient to gain some QDH function. Thus, very few mutations were necessary to facilitate the evolution of QDH genes.

1 Introduction

Aromatic and aromatics-derived compounds (ADC) are valuable bulk or fine chemicals with a myriad of important applications. They serve as building blocks for many valuable compounds including polymers such as plastics, resins, and fibers and are used as lubricants, dyes, and pesticides. Moreover, they are needed for the production of indispensable nutra- and pharmaceuticals. [ 1-4 ] Their vast majority is produced from petroleum in energy-intensive and environment-polluting processes. The increasing demand, the limitation of fossil resources, and the current environmental crisis have drawn rising attention to the much-required shift toward a sustainable, bio-based production of fuels and chemicals from renewable feedstocks. [ 5 ] Next to green chemistry, microbial biocatalysis is a promising strategy to produce aromatics at ambient temperatures and pressure without the use of toxic catalysts, thereby reducing energy consumption and waste generation. [ 6 ] The progress in this field has been recently reviewed in multiple publications giving a broad overview for several host organisms. [ 1-4, 7, 8 ] The here presented review specifically highlights the application of different Pseudomonas species as microbial cell factory for the production of industrially relevant aromatics and ADC. This includes the de novo synthesis of such from renewable and abundantly available feedstocks and associated genetic engineering strategies enabling efficient bioconversion. Moreover, biotransformation approaches of natural and fabricated aromatic substrates into value-added products are elucidated, with a focus on the unique stress resistance of Pseudomonas enabling the use of organic solvents in biphasic fermentations. The valorization of lignin- and plastic-derivable aromatics and metabolic funneling of heterogeneous mixtures thereof is also discussed.


1. A method for identification of a test agent that can inhibit one or more enzymes that are part of a shikimate pathway, the method comprising introducing the test agent into modified Saccharomyces yeast, the modified Saccharomyces yeast comprising:

i) a disruption of an endogenous Saccharomyces yeast gene that encodes the shikimate pathway enzyme, and ii) a supplemental gene encoding a heterologous enzyme that is homologous to the enzyme encoded by the endogenous Saccharomyces yeast gene wherein an inhibition of growth of the modified Saccharomyces yeast relative to a control is indicative that the test agent inhibits the shikimate pathway enzyme, and wherein the control comprises growth of the modified Saccharomyces yeast into which the test agent is introduced, and wherein the growth of the modified Saccharomyces yeast is performed on a medium that is supplemented with one or more aromatic amino acids such that growth of the modified yeast is independent of the function of the supplemental gene.

2. The method of claim 1, wherein a plurality of distinct test agents are introduced into a plurality of distinct Saccharomyces yeast cultures.

3. The method of claim 2, wherein the plurality of test agents are distinct test agents.

4. The method of claim 1, wherein the endogenous Saccharomyces yeast gene that is disrupted or deleted is ARO1.

5. The method of claim 1, wherein the supplemental gene is homologous to the endogenous Saccharomyces yeast gene that is disrupted or deleted and is from a species that is infectious to mammals, insects, birds, fish or plants.

6. The method of claim 1, wherein the supplemental gene is from a prokaryotic pathogen.

7. The method of claim 1, wherein the supplemental gene is from a eukaryotic pathogen.

Regulatory architecture of phenylpropanoid biosynthesis

Because of the extensive information available on its structural and regulatory genes, the phenylpropanoid pathway serves as an excellent system for developing an understanding of how to genetically manipulate complex natural product pathways in plants. However, we still lack important information concerning the points of flux control at and within the various branch pathways depicted in Fig. 1 and the potential cross-talk between pathways. Also important is the extent to which sets of reactions are organized in metabolic channels or ‘metabolons’, resulting in the sequestration of intermediates from diffusible cytosolic pools ( Srere, 1987 ). All of these factors may strongly impact the outcome of attempts to increase or decrease the level of a particular compound by transgenic approaches. Addressing these questions will require interdisciplinary approaches involving molecular, cellular, and structural biology.

Our understanding of flux control and cross-talk in phenylpropanoid biosynthesis has come primarily from studies in which specific enzymes in the pathway have been over-expressed or down-regulated in transgenic plants. Such an approach has shown that the entry point enzyme PAL is directly rate limiting for the production of chlorogenic acid (CGA, caffeoyl quinic acid) in tobacco leaves, but that factors in addition to PAL control flux into flavonoids and lignin ( Howles et al., 1996 ). CGA has been implicated in resistance to both microbes and insects ( Yao et al., 1995 ), although PAL over-expressing plants with elevated CGA appear to show impaired resistance to insect herbivory as a result of cross-talk between the salicylate and jasmonate signal pathways ( Felton et al., 1999 ).

In potato tubers, the creation of an artificial sink for tryptophan through the transgenic expression of a tryptophan decarboxylase gene resulted in lowered phenylalanine pools and reduced levels of wound-induced CGA and lignin, with a resulting increase in susceptibility to Phytophthora infestans ( Yao et al., 1995 ). CGA levels are also reduced in tobacco by down-regulation of C4H, the second enzyme in the phenylpropanoid pathway, and this is accompanied by a feedback inhibition of PAL activity, possibly as a result of feedback inhibition of PAL expression by cinnamate or some derivative thereof ( Blount et al., 2000 ). In contrast, over-expression of C4H did not consistently result in increased levels of CGA ( Blount et al., 2000 ), confirming that PAL rather than C4H is the flux control point into the phenylpropanoid pathway in tobacco leaves.

Chalcone isomerase (CHI) catalyses a near-diffusion-limited reaction that can also occur spontaneously at cellular pH, and is not therefore generally viewed as a potential rate-limiting enzyme for flavonoid biosynthesis. However, over-expression of CHI in tomato fruit peel leads to an 80-fold increase in the levels of flavonols ( Muir et al., 2001 ), and threefold increases in flavonol levels can be obtained by the expression of alfalfa CHI in Arabidopsis (C.J. Liu and R.A. Dixon, unpublished results). CHI would therefore appear to be a component of flux control into the flavonoid branch of phenylpropanoid biosynthesis.

The phenylpropanoid pathway presents some of the best-characterized examples of metabolic channelling in plant metabolism. Metabolic channelling involves the physical organization of successive enzymes in a metabolic pathway into complexes through which pathway intermediates are channelled without diffusion into the bulk of the cytosol ( Srere, 1987 ). Such complexes are loose, however, and many of the enzymes involved may be operationally soluble. The complexes allow for efficient control of metabolic flux, and protect unstable intermediates from non-productive breakdown or access to enzymes from potentially competing pathways. Such complexes may involve direct physical interactions between the various enzymes, as recently demonstrated for enzymes of flavonoid biosynthesis in Arabidopsis ( Winkel-Shirley, 1999 ), or may be associated with the colocalization of enzymes on membranes or other surfaces ( Liu and Dixon, 2001 ). In both cases, channelling can be demonstrated by double labelling or isotope dilution experiments in which exogenously applied intermediates are less efficient precursors of downstream products than their upstream substrates. Such criteria have confirmed channelling between PAL and C4H at the entry point into the phenylpropanoid pathway ( Czichi and Kindl, 1975 Hrazdina and Jensen, 1992 Hrazdina and Wagner, 1985 Rasmussen and Dixon, 1999 ), and between isoflavone synthase (IFS) and IOMT at the entry point into the isoflavonoid phytoalexin pathway ( Liu and Dixon, 2001 ). In both cases, the involvement of a membrane-associated cytochrome P450 enzyme (C4H or IFS), that might act to ‘anchor’ the complex to the endoplasmic reticulum, should be noted.

Metabolic channelling can impact plant defence responses in two ways. First, it is possible that intermediates destined to become a particular metabolic end product, such as a phenylpropanoid-derived phytoalexin, may be channelled in such a way that they utilize different ‘pools’ of metabolic enzymes than other products that may share some of the same biosynthetic steps. This could be achieved by utilizing different isoenzymic forms of the various pathway enzymes in different complexes. Such a model would predict that the multiple genes for many of the pathway enzymes described below might have both distinct and overlapping functions, a hypothesis that remains to be tested. If this were true, measurement of changes in gene transcripts, using probes that do not distinguish between all possible forms of the encoded enzyme, might lead to results that do not correlate with defence metabolism, as observed for flavonoid/isoflavonoid defences in bacterially infected alfalfa ( Sallaud et al., 1997 ). Second, although metabolic channelling might improve the efficiency of induced defences, it also presents a potential barrier to efficient metabolic engineering, in that channelled intermediates may not be accessible to the enzyme products of transgenes introduced in order to divert a pathway into the formation of a novel bioactive compound.

Author information

These authors contributed equally: Jin-Quan Huang, Xin Fang.


State Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, University of CAS, Chinese Academy of Sciences, Shanghai, China

Jin-Quan Huang, Xiu Tian, Ping Chen, Jia-Ling Lin, Xiao-Xiang Guo, Jian-Xu Li, Zhen Fan, Wei-Meng Song, Fang-Yan Chen, Ruzha Ahati, Ling-Jian Wang & Xiao-Ya Chen

State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China

School of Life Science and Technology, ShanghaiTech University, Shanghai, China

Shanghai Key Laboratory of Plant Functional Genomics and Resources, Shanghai Chenshan Botanical Garden, Shanghai Chenshan Plant Science Research Center, Chinese Academy of Sciences, Shanghai, China

1. Introduction

The prenylation of small aromatic molecules is a pivotal step in the biosynthesis of many natural products. The linkage of isoprenoid precursors, derived from the mevalonate or the methylerythritol phosphate pathway, with aromatic precursors, mostly derived from the polyketide or shikimate pathway, has provided the basis for the evolution of an impressive diversity of secondary metabolites in bacteria, fungi and plants.

Recently, a new class of enzymes catalyzing the prenylation of aromatic compounds has been discovered in Streptomy-cetes, gram-positive soil bacteria which are prolific producers of important natural products [1]. This enzyme class comprises CloQ and NovQ, which attach a dimethylallyl side chain to C-3 of 4-hydroxyphenylpyruvate in aminocoumarin antibiotic biosynthesis [2], and Orf2 of naphterpin biosynthesis, which attaches a geranyl side chain to various phenolic compounds [3]. The genuine substrate of Orf2 is yet unknown.

X-ray crystallographic investigations proved that the enzymes of this class display a new type of antiparallel β,α barrel fold. In contrast to the TIM barrel, the prenyltranferase barrel shows 10 antiparallel (rather than parallel) β strands forming a central, solvent-filled barrel which contains the binding sites for the aromatic and isoprenoid substrates [3,4]. Modelling studies suggested that a previously hypothetical protein of Streptomyces coelicolor A3(2), termed HypSc, shares the same protein structure, and prenyltransferase activity could indeed be demonstrated for this protein [3]. The members of this enzyme class are soluble, monomeric biocatalysts which show unusual promiscuity for their aromatic substrates. It has been pointed out that the discovery of this class may have a considerable impact on the synthesis of prenylated aromatic compounds since these enzymes may be used to create libraries of prenylated aromatic compounds for drug development [5,6].

We here report a new enzyme of this class, Fnq26, which catalyzes C- and O-prenylations of different phenolic substrates. In contrast to Orf2 of naphterpin biosynthesis, the Fnq26 reaction is independent of Mg 2+ ions, and as the first member of this class, Fnq26 is capable to carry out a ‘‘reverse’’ prenylation, i.e. the attachment of C-3 rather than C-1 of geranyl diphosphate to the aromatic substrate. Such a reverse prenylation is consistent with the presumed role of Fnq26 in the biosynthesis of furanonaphthoquinone I (FNQ I) ( Fig. 1 ), a natural product of mixed isoprenoid/polyketide origin from Streptomyces cinnamonensis DSM 1042 [7].

Biosynthesis of FNQ I and naphterpin.

SW and XZ conceived and designed the experiments. SW performed experiments, analyzed the experimental data, and drafted the manuscript. CF, KL, and JC assisted in experimental work and manuscript writing. HH and WW contributed reagents & materials. XZ revised the manuscript. All authors contributed to the final paper.

This work was financed by the National Key Science Research Projects (Grant No. 2019YFA09004302) and the National Natural Science Foundation of China (Grant No. 31670033).


Various animals are associated with specific endosymbiotic microorganisms that provide the host with essential nutrients or confer protection against natural enemies. Genomic analyses of the many endosymbioses that are found in plant sap-feeding hemipteran insects have revealed independent acquisitions — and occasional replacements — of endosymbionts, such that many of these endosymbioses involve two or more microbial partners. In this Review, I discuss how partitioning of the genetic capacity for metabolic function between different endosymbionts has sustained nutritional function in multi-partner endosymbioses, and how the phenotypic traits of these endosymbionts can be shaped by co-evolutionary interactions with both co-occurring microbial taxa and the host, which often operate over long evolutionary timescales.

Results and Discussion

The anabolic shikimic acid pathway has seven steps [supporting information (SI) Scheme 1], which may be catalyzed by seven different polypeptides or by fewer multifunctional polypeptides (22). The enzymes for five of the biosynthetic steps are homologous in all organisms that possess the pathway. For two of the steps, there are two different enzymes known for each, and every organism expressing the pathway has a homologue to one of these enzymes. Furthermore, there are two additional considerations in detecting genes encoding shikimic acid pathway enzymes in N. vectensis: (i) the evolutionary origin of the genes would be uncertain so that the sequences might have diverged considerably from any comparison sequences used and (ii) the genomic sequence may contain introns.

To obtain the greatest sensitivity of interrogation, the HMMER suite of programs (23) was used to search for consensus protein sequences by using hidden Markov model profiles. This method provides greater weight to evolutionary conserved residues, and local profiles reveal the protein fragments in coding exons. The genome sequence of N. vectensis was translated in all six reading frames and searched by using nine profiles covering all seven enzymes of the shikimic acid pathway obtained from the Pfam database (24). Two alignments (“hits”) were found in large scaffolds with HMMER using the aroA and aroB profiles (SI Dataset 1). The aroA hit occurred in scaffold_33 (1.4 Mbp). When the predicted protein sequence was used for a BLAST search, it aligned with the murA gene product of a variety of bacteria with ≈40% amino acid identity. This bacterial gene encodes UDP-N-acetylglucosamine 1-carboxyvinyltransferase (SI Dataset 2), an enzyme related to aroA (3-phosphoshikimate 1-carboxyvinyltransferase), whereas the MurA enzyme is involved in the biosynthesis of the peptidoglycan cell wall. This finding initially suggested that the aligned sequence might originate from bacterial contamination. However, close examination of the HMMER results showed that the predicted protein lacked ≈20 conserved amino acids at the C terminal, and that the missing amino acid sequence was located ≈1 kb downstream in the scaffold. Visual comparison of the genomic sequence with consensus sequences for vertebrate introns revealed plausible splice sites (AGGTRA and AGG, respectively) that would produce mRNA encoding a full-length murA homologue having a close fit to the search profile. The presence of introns thus eliminates the question of bacterial contaminants or symbionts as the proximal source of this gene.

The 1.4-Mbp scaffold containing the aroA-like homolog was translated in all six reading frames and scanned by using HMMER with the entire Pfam library. This process showed the presence of a variety of typical eukaryotic domains including reverse transcriptase, EGF, calcium binding EGF domain, defensin-like peptide, actin, and the fork-head domain, again supporting the idea that the aroA-like homolog is contained in the N. vectensis genome itself. The sequence of the predicted protein was used to construct a phylogenetic tree to compare with the closest bacterial sequences found in the BLAST search and with Tenacibaculum sp. MED152 and Escherichia coli W3110 (Fig. 1). The aroA-like sequence of N. vectensis did not cluster with homologs from any group of bacteria tested, but showed a sequence divergence from bacterial sequences comparable with those of murA genes between different bacterial groups. Whether this gene in N. vectensis directs biosynthesis of peptidoglycan or shikimate pathway intermediates is yet unknown.

Phylogenetic tree showing the relationship of the predicted protein sequence of the N. vectensis aroA-like gene to the predicted murA protein sequences of the seven best hits in a BLAST analysis and to those in E. coli and Tenacibaculum. Distances were calculated from a CLUSTAL W alignment using the Jones-Taylor-Thornton matrix, and the tree was constructed by using the neighbor-joining algorithm in programs of the PHYLIP package (version 3.63). The distance is proportion of amino acid substitutions.

The second alignment, related to aroB, was present on scaffold-85 (0.8 Mbp). When the predicted protein sequence was used for a BLAST search (SI Dataset 3), the closest fit was with the dinoflagellate Oxyrrhis marina (66% amino acid sequence identity). In this dinoflagellate, the aroB enzyme (3-dehydroquinate synthase) is present in the chloroplast and is fused to an O-methyltransferase (25). When the complete fusion protein sequence from O. marina was used in a BLAST search against the translated DNA of N. vectensis, it was evident that a fusion protein gene was also present in N. vectensis (SI Dataset 4). This gene contains five introns. When the aroB segment of the gene was used to construct a phylogenetic tree with the closest BLAST hits (Fig. 2), the N. vectensis sequence emerged as being closest to those of two dinoflagellates (O. marina and Heterocapsa triquetra) that each possess the complete fusion gene. Again, this gene could be involved in the synthesis of precursors leading to shikimate pathway-derived secondary metabolites, most notably 3-dehydroquinate, the putative intermediate branchpoint to MAA biosynthesis (5).

Phylogenetic tree showing the relationship of the deduced protein sequence of the aroB part of the AroB-O-methyltransferase protein of N. vectensis to homologous dinoflagellate proteins. Sequences were aligned with CLUSTALW and the tree was constructed by using the neighbor-joining algorithm with distances derived from the Jones-Taylor-Thornton model (using PHYLIP version 3.63). The tree was rooted by using Anabaena variabilis as an out group. The distances are the proportion of amino acid substitutions, and the bootstrap values based on 100 samples are shown.

Because endosymbiotic dinoflagellates are often associated with cnidarians, the possibility had to be considered that there was an undetected dinoflagellate contaminating the N. vectensis sequence. The predicted protein sequences derived from the neighboring genes on either side of the aroB-like homolog [encoding a RuvB-like protein and a probable malate synthase (MS), respectively] were used for BLAST searches. The closest alignments were with various vertebrates and with sequences from the sea urchin Strongylocentrotus purpuratus, which makes it unlikely that these shikimate pathway genes in the host metazoan's genome are from contamination by the genome of an associated dinoflagellate (SI Dataset 5). In addition, three putative protein sequences [β-tubulin, heat shock protein 90 (HSP-90), and proliferating cell nuclear antigen (PCNA)] from O. marina were used for BLAST searches against N. vectensis. The best hits were used to construct a phylogenetic tree, and in no case were the N. vectensis and O. marina sequences closely related (Fig. 3). It must be stressed, however, that additional evidence is necessary to determine the assumed function of these genes and for proof of their acquisition by horizontal gene transfer (HGT) in N. vectensis, particularly because cnidarians reputedly have conserved genes that they inherited from nonmetazoan ancestors (26). Although the importance of HGT in eukaryotic evolution remains controversial, there is independent evidence for the occurrence of another HGT event in N. vectensis. Comparative genomic examination of glyoxylate cycle enzymes has revealed the likely transfer of a bifunctional isocitrate lyase (ICL) and a MS, encoded by a fused ICL-MS gene from a bacterial precursor, to the N. vectensis genome (27). Our findings are similar to those of others reporting evidence of gene transfer to freshwater cnidarian (Hydra) species from multiple ancestral eukaryotic partners (18, 28).

Phylogenetic tree of PCNA protein sequences. The sequence of the PCNA protein of O. marina was used for a BLAST search against the translated genomic sequences of N. vectensis. The BLAST alignments were used to assemble the protein sequence from N. vectensis. The sequences from the two species were used for BLAST searches of GenBank, and a selection of the best hits for each species was used to construct a phylogenetic tree by using the neighbor-joining algorithm in programs of the PHYLIP package (version 3.63). The distance is proportion of nucleotide substitutions.

Our genomic mining of N. vectensis revealed another surprise beyond the transfer of genes from a bacterium and a dinoflagellate to the cnidarian's genome. We found seven good sequence alignments corresponding to five potential genes of the shikimic acid pathway. Among these were four very strong alignments corresponding to the genes aroA, aroB, aroC, and aroE of E. coli (SI Dataset 6). The predicted protein sequences of these genes were used in BLAST search queries (29) against the National Center for Biotechnology Information (NCBI) GenBank database to reveal related sequences. In all four cases, the best matches were to the genes of the shikimic acid pathway in Flavobacteria, having ≈70% amino acid identity (SI Dataset 7). In most cases Tenacibaculum sp. MED152, whose genome is being sequenced (, was the best match, although a strict similarity may be influenced by database bias for this bacterium. A fifth gene in N. vectensis corresponded to the aroF-H genes of E. coli, which encode isoenzymes for 3-deoxy- d -arabinoheptulosonate-7-phosphate synthase (DAHPS). However, BLAST searches showed the best hits (90% amino acid identity) to be the kdsA genes of Flavobacteria these encode other isoenzymes of the DAHPS family that are involved in lipopolysaccharide synthesis.

The high similarity of the N. vectensis gene sequences to those of the bacterial shikimate pathway could be explained by either a recent HGT event or bacterial DNA contamination in the N. vectensis genome sequences. The codon usage was similar to Tenacibaculum rather than N. vectensis. Two sequences were identified that appeared to be significant fragments of bacterial 16S rRNA genes. One 16S rRNA sequence (985 bp SI Dataset 8a ) showed closest similarity to Pseudomonas sequences. However, as it did not belong to a scaffold containing other bacterial sequences and there were no other Pseudomonas-like genomic sequences detected, it is likely that it is derived from a sequencing contaminant. Because the original shotgun sequencing data were not available to us, we could not analyze the N. vectensis genome by using a recently released version of the Glimmer gene annotation tool ( ref. 30), which would have been a useful way to quantify the percentage of the hologenome encoded on small scaffolds and likely, therefore, to be from living bacteria.

The other 16S rRNA sequence belonged to a scaffold, which also contained 23S rRNA sequences in an arrangement typical of rRNA operons (720 bp SI Dataset 8b ), and a phylogenetic tree of the 16S rRNA portion (Fig. 4) showed that it came from a flavobacterium, but it could not be assigned to a known genus. Phylogenetic trees were also constructed for the aroA, aroB, aroC, and aroE sequences, with similar results. A further consideration was that much of the genome of N. vectensis was organized into large scaffolds, whereas these 16S rRNA fragments were present in small scaffolds from which short contigs were sequenced, so that only incomplete gene sequences were revealed. This result gave the first indication that these 16S rRNA fragments might be from bacterial contamination rather than from genomic DNA of N. vectensis in the strict sense. The Tenacibaculum genome project has identified most of its genes, and the 2,679 predicted protein sequences from the genomic annotation were used for a BLAST search against translated N. vectensis DNA. When a stringent expected value of <10 −30 was used, 509 of the Tenacibaculum sequences (19%) gave positive hits. However, a less stringent cutoff (10 −10 ) gave 1,563 (58%) hits. In many of these cases, higher expected values were associated with partial sequences, as the hits were in smaller scaffolds having small contigs with many bases in the scaffolds not being determined. In fact, the aroE and kdsA genes were at the ends of contigs so that their sequences were truncated and lacked the last 40 or 25 aa, respectively. Although an accidental contamination of the original N. vectensis template cannot be ruled out, an exciting possibility is that the sequences come from a previously unsuspected flavobacterial associate similar to Tenacibaculum.

Phylogenetic tree showing the relationship of the 16S rRNA gene sequence found in the N. vectensis genome sequence (720-bp fragment in entry c429301624.Contig1 of StellaBase, SI Dataset 8a ) to the sequences of the closest type strains in Ribosomal Data Base Project II (release 9.52 Distances were calculated from a CLUSTAL W alignment using the F84 model, and the tree was constructed as in Fig. 3.

There is independent support for our contention that the foregoing sequences in the published genome for N. vectensis may come from bacteria associated with the early developmental stages of the anemone. The authors of the reported Nematostella vectensis genome (20), in their supporting online material (Supplement S2 in, did explicitly state that they prepared genomic DNA from larvae to avoid contamination by the commensals or symbionts that have been reported for the adults, although they gave no reference for the latter statement regarding such associates. Despite this precaution, there are separate findings that DNA isolates from embryos and early planula larvae of this sea anemone contain 16S rRNA sequences obtained from PCR amplicons attributed to bacteria, including those of the same groups (Flavobacteria and Pseudomonas) that we report here (H. Marlow and M. Q. Martindale, personal communication).

Bacterial associates of cnidarians have been known for at least 30 years (e.g., refs. 31 and 32), and most recently they have been visualized microscopically as epibionts and endosymbionts in two species of freshwater Hydra (33) and as envelope-wrapped aggregates in caverns between ectodermal cells of the nominally nonsymbiotic sea anemone Metridium senile (34). Such an intimate association with metazoan cells lacking an external physical barrier lends itself to direct host–microbe interactions manifested variously as pathogenicity in corals (35), the development of the immune response in Cnidaria (33), and a close symbiotic integration culminating in HGT from bacteria to host cnidarian as demonstrated here. Virtually nothing is known of the biosynthetic or other metabolic function of bacteria symbiotic with cnidarian hosts, a topic that, like so many others in modern marine microbiology, warrants investigation.

HGT between bacteria and certain metazoans (ecdysozoans, including insects and nematodes) was recently demonstrated by Baldo et al. (36) to be more widespread than suspected. They noted that bacterial sequences have been regarded previously as contamination and systematically excluded by eukaryotic genome sequencing projects, possibly masking the importance of such transfer in diverse invertebrates. Earlier, the genome sequence of the bacterial endosymbiont Carsonella ruddii found in aphids was made public (37, 38). Comparison of this genome sequence with that of another bacterial endosymbiont of aphids, Buchnera aphidicola, showed that both genomes had undergone considerable deletion, including loss of some genes encoding essential metabolic pathways. One such missing pathway leading to the formation of the aromatic amino acid tryptophan in C. ruddii caught our attention. According to dogma (10), precursors for this essential amino acid should be synthesized via the shikimic acid pathway in the commensal bacteria. Again, we searched global sequence alignments for genes encoding enzymes of the shikimic acid pathway in these bacterial genomes. We found one gene encoding a putative 5-enolpyruvylshikimate-3-phosphate phospholyase in C. ruddii (although whether this gene would transcribe a functional product is debatable owing to the large number of stop codons in the sequence), and only three (those encoding shikimate 5-dehydrogenase, 5-enolpyruvylshikimate-3-phosphate phospholyase, and 5-enolpyruvylshikimate-3-phosphate synthase) of the seven genes for the pathway were apparent in the B. aphidicola genome (SI Dataset 9). Taken together with our findings for the putative Tenacibaculum-like symbiont and its host N. vectensis, this evidence strongly suggests that the loss of essential metabolic function in the endosymbiont is an ongoing process of gene transfer and deletion in the evolution of symbioses that could ultimately lead to extinction of the symbiont by progressive assimilation of its genetic material into the host genome (37, 38).

The elucidation of “shared metabolic adaptations,” where the production of essential metabolites involves input by the partners of a symbiosis (even if one is degenerate), will require further genomic dissection of the unique organization and molecular functioning of invertebrate-microbial symbioses. This is highlighted by our finding that two of the genes for enzymes of the shikimic pathway, classically said to be absent from “animals,” are encoded in the metazoan host's genome. The extent to which such HGT, or the involvement of unsuspected bacterial consorts, may account for the apparent metabolic anomalies in cnidarians described in the Introduction, warrants further investigation. Understanding these processes may additionally provide critical insight into the cause of metabolic dysfunction evoked by climate change and environmental stress, particularly in the fragile symbioses of tropical corals and other marine cnidarians.

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