Journal of Bacteriology, March 2004, p . 1280-1286, Vol . 186, No . 5

Evidence for a Symbiosis Island Involved in Horizontal Acquisition of Pederin Biosynthetic Capabilities by the Bacterial Symbiont of Paederus fuscipes Beetles

Jörn Piel,^* Ivonne Höfer, and Dequan Hui

Department of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology, 07745 Jena, Germany

Received 27 August 2003/ Accepted 24 November 2003

 
Pederin belongs to a group of antitumor compounds found in terrestrial beetles and marine sponges . It is used by apparently all members of the rove beetle genera Paederus and Paederidus as a chemicaldefense against predators . However, a recent analysis of the putative pederin biosynthesis [ped] gene cluster strongly suggests that pederin is produced by bacterial symbionts . We have sequenced an extended region of the symbiont genome to gain further insight into the biology of this as-yet-unculturable bacterium and the evolution of pederin symbiosis . Our data indicate that the symbiont is a very close relative of Pseudomonas aeruginosa that has acquired several foreign genetic elements by horizontal gene transfer . Besides one functional tellurite resistance operon,the region contains a genomic island spanning 71.6 kb that harborsthe putative pederin biosynthetic genes . Several decayed insertion sequence elements and the mosaic-like appearance of the island suggest that the acquisition of the ped symbiosis genes was followed by further insertions and rearrangements . A horizontal transfer of genes for the biosynthesis of protective substancescould explain the widespread occurrence of pederin-type compoundsin unrelated animals from diverse habitats.

 
The pederin group of natural products consists of numerous members with potent antitumor and antiviral activities [Fig . 1] [15]. Remarkably, these structurally highly similar substances have been isolated from two entirely unrelated animal groups . Pederin itself occurs in species of the beetle genera Paederus and Paederidus, while almost all other members are known only from marine sponges. In the beetles, pederin serves as a chemical defense against predators such as spiders [11] . It is also the cause of Paederusdermatitis, a blistering inflammation that is contracted whenthe beetles are accidentally crushed on the human skin [20]. In regions with a warm climate the beetles can appear in enormous numbers, resulting in notorious epidemics that have forced the evacuation of entire settlements [18, 25] . Interestingly, theyhave also been proposed as a cause for 3 of the 10 biblicalplagues [16].

 
Several lines of evidence clearly indicate that pederin is not produced by the beetles themselves but by as-yet-unculturedbacterial symbionts . Kellner and Dettner have demonstrated thatwithin each examined beetle species pederin is synthesized inonly about 90% of the females, who alone can transfer this traitto their offspring [10] . According to 16S ribosomal DNA data,only these pederin-synthesizing females harbor a bacterium withthe closest relationship to Pseudomonas aeruginosa [8] . If eggsof such beetles are fed to nonproducing females, pederin isagain synthesized in their offspring [7] . Our search for thepederin biosynthesis genes, motivated by the prospect of generatingan alternative biotechnological source of these rare antitumor compounds, has resulted in the isolation of a polyketide synthase cluster spanning 54 kb [19] . This operon, designated the pedcluster, is present only in pederin-synthesizing females and their eggs and, architecturally, strikingly mirrors the chemical structure of pederin . Although the cluster had been isolated from beetle total DNA, its gene homology data and architectureimply that it is located on a bacterial genome . Notably, spotsequencing of regions adjacent to the ped cluster indicatedthe presence of open reading frames [ORFs] with remarkable homologyto genes from P . aeruginosa, which is in perfect agreement withthe 16S ribosomal DNA data provided by Kellner and Dettner [10].

Our preliminary sequence analysis had suggested that the ped cluster is part of a genomic island acquired by an P . aeruginosa-like bacterium . Genomic islands are large, horizontally transferred regions of DNA that can significantly enhance metabolic and colonizing capabilities and allow bacterial evolution in quantum leaps . Many of them play a crucial role in the acquisition of pathogenic traits in bacteria [5] . However, in contrast to the large number of islands involved in pathogenicity, our knowledge about the biologically related symbiosis islands is restricted to a single example reported for rhizobia [23] . In light of the absence of ped-type genes in P . aeruginosa and of the advantageof pederin biosynthesis for the survival of the beetle host,we suspected that the ped genes could be located on such anisland . In the work presented here, we fully sequenced this locus to better understand its function in symbiosis . Additionally, we present a sequence analysis of regions outside the putative island to obtain further information on the biology of the as-yet-unculturable Paederus symbiont . These data corroborate that the ped clusteris a foreign element in the symbiont genome and provide insightinto the evolution of pederin symbiosis.

 
Isolation of bacteria from Paederus fuscipes. 100 P . fuscipes beetles collected at Aydin, Turkey, were groundin liquid nitrogen and resuspended in 5 ml of Luria-Bertanimedium . The mixture was kept for 10 min on ice to let beetleresidues settle . The supernatant was centrifuged at 100 x g for 10 min to sediment eukaryotic cells . The supernatant was then passed through a 40-µm-pore-size nylon filter [Millipore],and the bacteria were pelleted by centrifugation at 5,000 xg for 10 min . The purity of the bacterial fraction was checkedby DAPI [4',6'-diamidino-2-phenylindole] staining and microscopicinspection.

Preparation and screening of a P . fuscipes symbiont genomic library. Genomic DNA from P . fuscipes bacteria was prepared by standardsodium dodecyl sulfate lysis and used for the construction ofa cosmid library in the pWEB vector [Epicentre] according to the manufacturer's instructions . Two thousand clones were deposited in 96-well plate format and transferred to Hybond N⁺ membranes. Membranes were screened by using a fluorescein-labeled probe prepared from a 1.5-kb fragment of the ped cluster and the ECL labeling and detection system [Amersham] . Adjacent genome regions were obtained by primer walking.

Cloning and sequencing of symbiont DNA adjacent to the ped locus. Cosmids were sonicated, end repaired with BAL 31 and Klenow fragment, and size fractionated by gel electrophoresis to yield fragments of 1 to 2 kb . These were ligated into the EcoRV site of pBluescript II SK[-] [Stratagene] and end sequenced by using BigDye Terminator Ready Mix [Applied Biosystems] and an ABI3700 sequencer [Applied Biosystems] . The order and direction of contigs were determined by restriction mapping and targeted subcloningof HindIII and EcoRI fragments . Sequence analysis was performed with the BLAST, PROSITE, Pfam, FRAMEPLOT, NNPP prokaryotic,and tRNASCAN-SE algorithms and the Lasergene DNASTAR package.Only regions covered at least eightfold by sequences are describedin this work . All entries classified as pseudogenes were fullysequenced.

Expression of symbiont tellurite resistance genes in Escherichia coli. The cosmid pPD5C10, containing the entire ter cluster, was transformedinto E . coli XL1-Blue [Stratagene] by electroporation . Clonescontaining the cosmid were streaked out on Luria-Bertani platescontaining various amounts of K₂TeO₃ [Aldrich] and carbenicillinat 100 µg/ml [Roth] . E . coli containing the empty pWEBvector was used as a control.

Nucleotide sequence accession numbers. The nucleotide sequences have been deposited at GenBank underaccession numbers AY328003 to AY328024.

 
Construction and screening of a cosmid library prepared from bacteria of P . fuscipes beetles. Previously identified genes had been isolated from a large metagenomiccosmid library of beetle total DNA [18] . To further simplifyscreening and to obtain additional independent evidence thatthe ped region belongs to a bacterial genome, we constructeda new library from bacteria isolated from P . fuscipes, consistingof 2,000 clones . Screening of this library with a gene probederived from the ped cluster yielded on average 1 positive in130 clones, compared to 1 positive in 6,000 cosmids of the previousmetagenomic library . This significant enrichment of ped genesclearly shows their bacterial origin.

Sequence analysis of a 106-kb region containing the ped cluster. In order to determine the structure of the putative symbiosis island and neighboring regions, we sequenced the island in its entirety and an additional 34 kb to a coverage of 72% by atleast eight overlapping sequences, thus providing sequence datafrom a region spanning approximately 106 kb . Database searchesrevealed homologies to 64 different genes [Table 1] . Of these, however, 25 are represented only as pseudogenes that are truncated or contain frameshifts and stop codons . The large number of incomplete ORFs within and outside the putative island may indicate that the symbiont genome is undergoing degradation, as has been reported for other insect endosymbionts such as Buchnera aphidicola [2].

 
The identified homologies can be classified into two similarity groups [Fig . 2] arranged into clustered regions . The first groupcomprises 26 genes and exhibits a striking homology to genes from P . aeruginosa, which is usually 80 to 90% at the protein level and around 80% at the nucleotide level . This group encompasses almost all housekeeping genes . In previous 16S rRNA studies reported by Kellner, an uncultured bacterium with the closest relationship to P . aeruginosa, which is present only in beetles with high pederin content, was detected [8] . The location ofthe pederin biosynthesis cluster beside genes with such extraordinarysimilarity to the P . aeruginosa genome strongly suggests thatthe pederin producer is identical to this beetle bacterium.Spot sequencing of two random cosmids of the library consistentlyrevealed the presence of P . aeruginosa gene orthologs [datanot shown] . This persistence of P . aeruginosa-type genes throughoutthe genome, as opposed to an accidental local clustering closeto the ped region, allows the conclusion that the symbiont isindeed a member of the genus Pseudomonas.

 
The second homology group is only around 50 to 70% similar toknown proteins and shows very weak or no homology to databaseentries at the nucleotide level . This group consists almostentirely of genes associated with secondary metabolism [pedgenes], resistance, or gene transfer, i.e., genes that are commonlyfound to be horizontally acquired . These genes are mostly clusteredinto two regions: one centering around the ped genes [Fig . 3] and another with similarity to a tellurite resistance operon.

 
P . aeruginosa orthologs. The sequenced P . aeruginosa-like genes show an average G+C contentof 55.7%, which is much lower than the 66.6% for P . aeruginosa[22] . According to homology data, most of the identified P.aeruginosa-type genes outside the ped island are involved inthe biosynthesis of amino acids and enzyme cofactors and appearto be complete and functional . These encode enzymes putativelyinvolved in the biosynthesis of phenylalanine, tyrosine, andtryptophan [3-deoxy-D-arabionoheptulosonic acid 7-phosphate[DAHP] synthase]; sulfur metabolism and cysteine biosynthesis[CysB and CysH]; and the biosyntheses of vitamin B₆ [erythrose-4-phosphatedehydrogenase], vitamin B₁₂ [CobQ, CobP, and CobU], and coenzymeA [ketopantoate reductase] . Other presumably intact genes arehighly homologous to a regulator of the LuxR family [orthologin P . aeruginosa, PA1759], a transport protein [PA1361], anisocitrate dehydrogenase kinase/phosphatase [PA1376] presumablycontrolling the glyoxylate shunt pathway, two oxidoreductases[PA5309 and PA5312], and two homologs of PA1360 and PA5314 withunknown function . In addition, six degraded genes which haveintact orthologs in P . aeruginosa could be identified . Exceptfor the putative regulator PA1760 and the homoserine kinase PA1757 nothing is known about the function of these genes.

The P . aeruginosa-like regions exhibit only very local synteny to the P . aeruginosa genome . Upstream of the tellurite operon, the three vitamin B₁₂ biosynthesis genes are present in the same arrangement in P . aeruginosa [PA1277 to PA1279] . Located between the tellurite operon and the putative ped island is a region with extensive homology to the P . aeruginosa genome between PA1750 and PA1760 . Nine of these 11 genes were at least partially identified on the sequenced DNA . However, in additionto severe gene degradation and the deletion of two genes presentin P . aeruginosa, the upstream and downstream halves of thisregion occupy switched positions compared to the orthologoussections in P . aeruginosa . The region further downstream issyntenous to the section encoding PA1360 to PA1376 but lacks13 genes with mostly unknown functions . Two further P . aeruginosaorthologs are missing in a region otherwise highly similar toPA5309 to PA5314 downstream of the ped island.

The putative ped island. Horizontally acquired genomic regions involved in pathogenicityor symbiosis are often found integrated at tRNA genes [5] . However,no such gene could be detected at either side of the ped region. We have assigned the upstream end of the putative island toa transposase gene fragment located adjacent to the PA1376 homolog.The last gene at the downstream end with low similarity to P. aeruginosa genes is another fragmented insertion sequence [IS]-like gene adjacent to a PA5312 homolog . The low-homology region therefore spans 71.6 kb . Its average G+C content is 55.7% identical to that of the P . aeruginosa-like regions.

In addition to the ped genes described previously, a number of other homologies were detected on this section . However,of these, only five belong to intact ORFs . Notable is the largenumber of homologs to functions associated with horizontal genetransfer, a common feature of genomic islands . Three IS-likeelements were identified at the upstream end and five were identifiedat the downstream end of the putative island, showing similarityto transposases, resolvases, and a phage integrase involvedin horizontal acquisition of a Shiga toxin operon in Shigella dysenteriae [14] . All of the IS-type genes are truncated atone or both ends . These homologies strongly suggest that the ped symbiosis genes are of foreign origin . Also interspersed within the region are heavily truncated orthologs of P . aeruginosa genes . Sequences resembling fragments of the putative ABC transporter component PA4500 and of PA1689, PA5264, and PA4500 with unknown function were found . In addition, apparently intact orthologsof the small hypothetical protein PA5126 and the rRNA methylasePA5127 are present . The mosaical architecture of the region,featuring numerous insertion sequences and orphan P . aeruginosa-likegenes and pseudogenes, indicates that various integration and/orrearrangement processes have followed the acquisition of theped genes . This characteristic is known for many other genomicislands.

Located immediately downstream of the ped genes is a pseudogene with two internal stop codons which exhibits highest similarity to cytochromes P450 catalyzing the oxygenation of polyketides. Examples are ORF29, which is involved in the biosynthesis ofthe enediyne antibiotic C-1027 [13], and the erythromycin hydroxylaseEryF [1] . Since pederin is a polyketide, this pseudogene couldhave once been a part of the ped cluster and participated inthe biosynthesis of an ancestral pederin-type compound . Interestingly,the numerous members of the pederin group isolated from spongesalmost always contain an additional P450-type oxygenation [15],which may be generated by an intact version of this gene.

A small degraded operon related to the biosynthesis of unsaturated fatty acids lies adjacent to the P450 pseudogene . It contains pseudogenes of an acyl coenzyme A ligase, a 12-desaturase andtwo 9-desaturases, and an intact acyl carrier protein gene.The only other complete non-P . aeruginosa-type ORFs in the pedregion, apart from the ped genes themselves, encode a putativedioxygenase similar to asparagine ß-hydroxylase anda protein with good homology to a DNA helicase from a Pseudomonasresinovorans plasmid.

The tellurite resistance operon. The tellurite resistance operon region, with an unusually lowG+C content of 51.4%, is located upstream of the ped islandand contains seven genes with good homology at the amino acidlevel to the ter genes from Yersinia pestis, E . coli O157:H7,and other pathogens . Such genes are known to confer resistanceagainst tellurite salts, which exhibit strong antibiotic properties[24] . Upstream of the ter genes, four additional ORFs that haveno counterparts in P . aeruginosa were identified . In most bacteria with ter operons, homologs to these ORFs can be found at the identical location, which suggests that they belong to the same operon . The exact function of ter clusters and their individual proteins is not well understood . Since tellurite salts are rare in nature, it is suspected that resistance against these compoundsis only a secondary property [24].

Expression of the tellurite resistance genes in E . coli. Our available sequence data indicated only one decayed genein the ter region . To test whether the ter region is functional, we studied its properties in E . coli XL1-Blue . After transformation with pPD5C10, a cosmid carrying the ter operon, clones were streaked out on plates containing potassium tellurite . The ter cosmid resulted in resistance against this compound at a MICof 500 µg/ml . The bacteria are black due to intracellularcrystals of elementary tellurium [24] . Control clones containing only the empty pWEB vector were unable to grow on tellurite-containing media . The ter genes are therefore functional in this strain.

 
The intention of our work was to gain insight into the biologyof the uncultured Paederus symbiont and into the evolution ofpederin symbiosis . A remarkable characteristic of the symbiontis the extremely high nucleotide homology of its genome to thatof P . aeruginosa, a pathogenic bacterium that can be culturedeasily . A dominant bacterium with the closest relationship toP . aeruginosa had previously also been detected by Kellner exclusivelyin pederin-positive beetles [8] . Thus, according to these two lines of evidence, the pederin producer should be a member of the genus Pseudomonas.

Closer inspection of the analyzed 106-kb region revealed a number of characteristic differences from the P . aeruginosa genome. Notable is the degradation of several P . aeruginosa orthologs, which is in strong contrast to P . aeruginosa, with its low number of pseudogenes [12, 22] . Further sequencing will be necessaryto determine whether gene decay is indeed found genome wideor occurs only locally around the ped region . Degraded genomeshave been observed in a number of other symbionts and pathogens[17] . At the extreme end of such reductive processes are obligateintracellular symbionts such as B . aphidicola [26] . These organismshave lost almost all dispensable coding and noncoding regionsand are among the bacteria with the smallest known genomes.Although the Paederus symbiont seems to have lost a number ofproteins, it apparently contains a large amount of noncodingDNA in the form of pseudogenes and large intergenic spacers.Such bacteria are speculated to occupy an intermediate positionin the evolution towards a minimal genome [3, 21] . Indeed, theoccurrence of pederin only in the two closely related beetle genera Paederus and Paederidus suggests that pederin symbiosisis considerably less ancient than the family-wide associationbetween B . aphidicola and aphids established about 200 millionyears ago [2] . The more recent origin of pederin symbiosis ispossibly also reflected by the fact that the association isunstable and not obligatory, since about 10% of the Paederusfemales found in nature are free of symbionts [9, 10].

Interestingly, most of the intact P . aeruginosa-type genes found in the region encode proteins putatively involved in amino acid and vitamin biosynthesis . For a number of symbiotic systemsit has been shown that the supply of such compounds to the hostis an important factor governing mutualism . An example is theprovision of essential amino acids by B . aphidicola to aphids,which feed on a nutrient-poor diet [4] . Although the vicinity of nutrient-providing genes to the ped cluster could argue for a similar role in Paederus beetles, the available data do not support a host dependence of symbiont nutrients . Symbiont-free beetles found in the field are virtually indistinguishable from producing specimens, which is probably a consequence of thefact that Paederus beetles, unlike sap-sucking insects, arecarnivores with a balanced diet . However, symbiont metabolitescould possibly augment the beetle diet and enhance fitness intimes of limited nutrient supply.

Two regions with missing counterparts in the P . aeruginosa genome were identified . One is a functional tellurite resistance operon, whose lower G+C content indicates that it is of foreign origin. So far almost nothing is known about the actual role that such resistance genes play in bacterial fitness . It has been shownthat ter clusters also confer resistance against bacteriophagesand bacteriocins, i.e., antibacterial proteins [27] . Determinants of tellurite resistance are known for a large number of pathogens and are commonly exploited to selectively isolate pathogenic bacteria [24] . It is therefore speculated that these clusters protect the cell against host defense reactions via an unspecific mechanism . The identified ter region may enhance the survival of the symbiont in a similar way . Interestingly, the terE gene is not intact in the symbiont and is therefore apparently not needed for resistance . A similar finding has been documentedby Whelan et al., studying a ter cluster from the plasmid R478[27].

The second low-homology region containing the ped genes bears typical hallmarks of a symbiosis island . The ped cluster should represent one of the major factors governing pederin symbiosis, since female beetles use pederin to chemically defend themselvesand their offspring against predators [11] . The lack in P . aeruginosaof homologs in this region and the presence of a large numberof degraded IS elements indicate that a P . aeruginosa-like bacteriumhas acquired the ped genes by horizontal transfer . Hence, thisacquisition may have been a crucial event in the evolution ofa symbiotically competent microorganism . According to the mosaicalappearance of the ped island, the integration likely took placeat a recombinatorial hotspot in the genome and was followedby further insertions and rearrangements.

The production of pederin-type compounds in sponges by a symbiont has not yet been demonstrated but is very likely, as complex polyketides are typically produced by bacteria . The existenceof a highly mobile genomic element carrying genes importantfor symbiosis could explain why these metabolites are foundwidespread among unrelated animals living in entirely differentenvironments . Such genes could be transferred between differentbacteria by phage infection or other mechanisms . However, atleast in the case of the Paederus symbiont, this island apparentlyhas lost its mobility functions due to gene decay and seemsto be a stable component of the genome . Similarly stabilizedislands have been observed in several pathogens . It is believedthat they are the result of an adaptive process, during whichthe island disposes of inessential genes to eventually becomepart of the core genome [5] . In the symbiont, the degradationof almost all island genes except the ped cluster suggests asimilar mechanism.

Secondary metabolites isolated from invertebrate animals but structurally resembling natural products from bacteria are remarkably widespread in nature . For many years, bacterial symbionts havebeen proposed to be the true source of these metabolites [6], but so far not a single producing bacterium has been isolated from any of these organisms . Since many of these compounds are promising drug candidates, there is a need for a better understanding of these symbiotic systems at the genetic level . These studiescould ultimately result in solving the supply problem that existswith almost all invertebrate drug candidates, e.g., by developinga bacterial cultivation system or by heterologous expressionof biosynthesis genes from symbionts in culturable bacteria.The pederin symbiont is the first mutualist producing a pharmacologicallyactive natural product, whose putative biosynthesis gene clusterwas cloned [19] and for which extensive genetic data now exist. Through comparative genomics we should gain a better understanding of symbiont genetics and improve our ability to generate environmentally sound supplies of symbiont drug candidates . In addition, comparative studies will also help to identify the factors governing pathogenesis and symbiosis in the Paederus symbiont and the closely related opportunistic pathogen P . aeruginosa.

 
We thank H . Bapinar and E. avk for helping with the collections of Turkish beetle specimens,D . Schnabelrauch for technical assistance, and R . L . L . Kellner,K . Dettner, and S.-J . Suh for valuable discussions . We are gratefulto W . Boland for support and to C . Dale for suggestions on the manuscript.

This work was supported by research grant PI 430/1-1 from the Deutsche Forschungsgemeinschaft and by the Max Planck Society.

 
* Corresponding author . Mailing address: Max Planck Institute for Chemical Ecology, Department of Bioorganic Chemistry, Hans-Knöll-Str . 8, Beutenberg Campus, 07745 Jena, Germany . Phone: 49-3641-571213 . Fax: 49-3641-571256 . E-mail: piel@ice.mpg.de.

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