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struct including a myelin gene to drive animmune response in the direction of suppress-ing autoimmunity to myelin. Others have usedimmune constructs to target cytokines to spe-cific addresses, delivering the cytokine to thecorrect ‘mailbox’ where it will have maximaleffect7.

The use of ‘immunocytokines’ and othernovel cytokine constructs should enhance theefficacy of these natural products in fightingdisease. Other enzymatic cleavage sites on thecytokine engineered by Chernajovsky and col-leagues include substrates of thrombospondinand transglutaminase. Enzymes like transglut-aminase are more abundant at sites of neu-rodegeneration8. Thus, cytokine therapy mightbe enhanced for treatment of diseases likeAlzheimer and Huntington disease associatedwith increased transglutaminase8. A motif inthe engineered β-interferon construct encod-ing the peptide sequence arginine, glycine,aspartate (RGD) facilitates interaction withintegrins. This provides additional capabilitiesto target adhesion molecules and extracellularmatrix proteins9.

One of the possible negative aspects ofextensively engineering any natural molecule isthe risk that the newly engineered structureswill appear sufficiently different to theimmune system to elicit limiting antibodyresponses or allergies. Even small modifica-

tions to accommodate an enzymatic cleavagesite or to allow recognition by a crucial targetcould alter a self-molecule sufficiently to raisepotential problems.

Despite these caveats, optimizing enzymesprovides exciting new opportunities inbiotechnology. The use of protein engineeringto create new receptor constructs for tumornecrosis factor-α or to engineer chimeric mon-oclonal antibodies against this cytokine hasrevolutionized the treatment of autoimmunediseases, such as rheumatoid arthritis. This featwas acknowledged in this year’s Lasker Prizefor Medical Research to Feldmann andMaini10. Ever more spectacular uses of thesenatural molecules and their antagonists willcontinue to add to our arsenal against disease.

1. Adams, G., Vessillier, S., Dreja, H. & Chernajovsky, Y.Nat. Biotechnol. 21, 1314–1320 (2003).

2. Jacobs, L. et al. Arch. Neurol. 44, 589–595 (1987).3. van Meurs, J. et al. Arth. Rheum. 42, 2074–2084

(1999).4. Gijbels, K., Galardy, R. & Steinman, L. J. Clin. Invest.

94, 2177–2182 (1994).5. Croxford, J.L. et al. J. Immunol. 160, 5181–5187

(1998).6. Garren, H. et al. Immunity 15, 15–22 (2001).7. Lode, H.N., Xiang, R., Becker, J.C., Gillies, S.D.

& Risfeld, R.A. Pharmacol. Ther. 80, 277–292(1998).

8. Karpuj, M.V. et al. Nat. Med. 8, 143–149 (2002).9. von Adrian, U. & Engelhart, B. N. Engl. J. Med. 348,

68–73 (2003).10. Feldmann, M. & Maini, R.N. Nat. Med. 9,

1245–1250 (2003).

source for the nematode. After one to threegenerations, depending on insect host size,nematodes in the infective juvenile stage leavethe depleted insect carcass in search of a newhost, carrying P. luminescens in their gut.

Like Bacillus thuringiensis, P. luminescensand its carrier nematode H. bacteriophora havebeen exploited for their insect-killing proper-ties as a biocontrol agent against insect pests4.The nematode-bacterium complex is mosteffective against a number of soil-inhabitinginsects, but its high production costs limit itsapplication to high-value niche markets.Moreover, poor shelf life, lack of persistence inthe soil and the necessity of high applicationrates have constrained large-scale use in agri-cultural settings.

The DNA sequence of the P. luminescensgenome has exceeded expectations as moreputative toxin-encoding genes have beenfound than in any other bacterial genome sofar examined. A large number of these geneshave been identified as encoding putative tox-ins based on similarities to other known bacte-rial toxins. P. luminescens had previously beenshown to carry a number of toxin complexgenes encoding high-molecular-weight secre-ted proteins that are toxic to Manduca sexta,the tobacco hornworm5, and additional toxincomplex genes have been discovered in thegenome sequence of P. luminescens. AnotherPhotorhabdus protein encoded by the gene mcf(makes caterpillars floppy) triggers apoptosisin insect hemocytes and midgut epithelium6.The P. luminescens genome sequence has fur-ther identified two homologs of insect juvenilehormone esterases3. Such proteins would bepredicted to cause inappropriate developmentof the insect host and thus may represent anovel insecticidal strategy. Indeed, the prod-ucts of these genes, when expresssed inEscherichia coli and fed to mosquitoes andcaterpillars cause death within 48 hours. Thus,the presence of multiple types of proteins withdiverse modes of toxicity indicates that P. lumi-nescens has evolved or acquired a wide range ofstrategies leading to the rapid death of a rangeof insects after infection1.

The P. luminescens genome sequence hasidentified several categories of genes that helpexplain its success in leading such a specializedlifestyle. Sequences encoding polyketide andnonribosomal peptide synthases probably syn-thesize antibiotics to protect against microbialcompetitors. Genes encoding antimicrobialcolicin-like factors and immunity proteinshave also been identified. Genes encodingsecreted enzymes predicted to have a role inbioconversion and metabolism of the insectcadaver have been identified, as well as tran-scriptional regulators of their expression. The

1294 VOLUME 21 NUMBER 11 NOVEMBER 2003 NATURE BIOTECHNOLOGY

The bacterium P. luminescens is pathogenic to awide range of insects1. It is transmitted to thebody cavity of an insect host by the nematodeHeterorhabditis bacteriophora, an organismwith which it has a complex symbiotic rela-tionship2. The ability of P. luminescens toswitch from symbiont in the nematode gut tovirulent pathogen in the insect host—a processin which the microorganism unleashes a veri-table trove of toxins to fight off microbial com-

petitors and invertebrate scavengers—hasmade it a compelling candidate for genomesequencing. In this issue, Kunst et al.3 reportthe genome sequence of P. luminescens, pro-viding a first comprehensive look into how thisspecialized microbe controls its different lifestages and identifying several genes that couldpotentially be exploited in new approaches tofight insect pests.

The complex lifecycle of P. luminescensis summarized in Figure 1. Once introducedinto an insect by infectious H. bacteriophorajuveniles (Fig. 1), the bacterium produces abattery of toxins that kill the host within 48 hours. Hydrolysis of the insect’s body by P. luminescens produces a rich food source forthe bacterium, which in turn becomes a food

Sequence of a symbiontValerie M Williamson & Harry K Kaya

The complete genome sequence of the insect pathogen and nematodesymbiont Photorhabdus luminescens identifies a trove of antibiotic and toxingenes.

Valerie M. Williamson and Harry K. Kaya are in the Department of Nematology, University of California, Davis, One Shields Ave., Davis,California 95616, USA.e-mail: vmwilliamson@ucdavis.edu

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bacterium forms an intimate association withthe gut of the nematode, and a large number ofgenes with potential roles in this symbioticinteraction have been identified, includingthose encoding proteins that probably func-tion in surface adhesion. The availability ofthe complete sequence of the P. luminescensgenome thus represents an important mile-stone toward exploiting the potential of thisbacterium as a biocontrol agent.

Access to new gene sequences encodingpotential protein toxins has further impli-cations for bioengineering insect resistance in plants. Transgenic plants expressing B. thuringiensis genes are among the most suc-cessful and widely applied biotechnologicalproducts7. In some instances, transgenic B. thuringiensis crops have had a large impacton yield and have resulted in less pesticide use.But there is concern that insect resistance to B. thuringiensis toxins in transgenic plants, aris-ing from changes in insect populations, will re-duce the effectiveness of this toxin and its tran-sgenic products. In addition, B. thuringiensistoxin proteins are generally effective against anarrow range of insects, and toxins have notbeen identified or developed against someinsect pests. Several of the predicted toxin pro-teins in P. luminescens have been shown to haveoral toxicity, but toxicity upon expression intransgenic plants has not yet been reported.The best-characterized toxic proteins from P. luminescens are large, and their expression inplants may be problematic as was the case ini-tially with B. thuringiensis peptides8.

Perhaps the most fascinating story yet to betold from the analysis of the P. luminescencegenome is how this organism came to acquirethe genes that allow it to fill its specializedniche so successfully. Comparison with the

genomes of related bacteria indicates that ex-tensive horizontal gene transfer has occurred.For example, Yersinia pestis, a flea-colonizingbacterium and the causal agent of plague, is aclose relative. Other clues to the evolution ofthe P. luminescens genome are provided by themultitude of pathogenicity islands, phageremains and abundant transposable elementsfound in the P. luminescens genome.

Further genomic analyses will provideanswers to such questions as how and whyhomologs of an insect juvenile hormoneesterase gene were incorporated into thegenome of P. luminescens and how P. lumi-nescens implements and regulates all its differ-ent insecticidal capabilities. The answers willprovide the tools to exploit this organism’scapabilities to fight insect pests in new,untested ways.

1. ffrench-Constant, R. et al. FEMS Microbiol. Rev. 26,433–456 (2003).

2. Forst, S. & Clarke, D. in EntomopathogenicNematology (ed. Gaugler, R.) 35–56 (CABIPublishing, Oxon, UK, 2002).

3. Duchaud, E. et al. Nat. Biotechnol. 21, 1307–1313(2003).

4. Kaya, H.K. & Gaugler, R. Annu. Rev. Entomol.38,181–206 (1993).

5. Bowen, D. et al. Science 280, 2129–2132 (1998).6. Daborn, P.J. et al. Proc. Natl. Acad. Sci. USA 99,

10742–10747 (2002).7. Sheldon, A.M., Zhao, J.-Z. & Roush, R.T. Annu. Rev.

Entomol. 47, 845–881 (2002).8. Estruch, J.J. et al. Nat. Biotechnol. 15, 137–141

(1997).

NATURE BIOTECHNOLOGY VOLUME 21 NUMBER 11 NOVEMBER 2003 1295

Figure 1 Symbiont or pathogen? During its complex life cycle, P. luminescens rotates between being anematode symbiont, an insect pathogen and a nematode food source.

P. luminescensLIFE CYCLE

NEM

ATOD

E

NEM

AT

OD

ESYM

BIONTFOOD

SOU

RC

ENematodes reproduce

inside insect cadaver, feeding on bacteria and degraded host tissues.

Bacteria produce toxins that kill the host, and secrete enzymes that hydrolize

the cadaver.

Bacteria reproduce, feeding on richresources of cadaver.

Nematode infective juveniles with bacteria in their guts emerge from depleted cadaver.

Infective juveniles carryingbacteria search for and find

new hosts

Nematode infective juveniles penetrate into the insect host

and release bacteria.

INSECT PATHOGEN

Erin

Boy

le

A central challenge in genomic biology is todetermine how cells coordinate the expressionof thousands of genes throughout their lifecycle or in response to external stimuli, such asnutrients or pheromones. In eukaryotes, geneexpression is modulated by various transcrip-tion factors that bind to the promoter regions,and different combinations of transcriptionfactors may alternatively activate or repress

gene expression. This is analogous to an elec-tronic circuit, in which components areswitched on and off by a network of transis-tors. In this issue, Bar-Joseph and colleagues1

report a computational approach to show thatin yeast, genes are indeed regulated in networksthat are controlled by groups of transcriptionfactors. Furthermore, they show that these reg-ulatory networks also have a modular structurein which groups of genes under the control ofthe same regulators tend to behave similarly.

Genetic regulation and its mechanisms havebeen investigated since the days of Jacob andMonod and the discovery of the lac operon.Traditionally, such studies are labor-intensiveand gene-specific and often require years of

Zhaolei Zhang and Mark Gerstein are at theDepartment of Molecular Biophysics andBiochemistry, Yale University, 266 WhitneyAvenue, New Haven, Connecticut 06520-8114,USA. e-mail: Mark.Gerstein@yale.edu

Reconstructing genetic networks inyeastZhaolei Zhang & Mark Gerstein

By combining data from gene expression and DNA-binding experiments, acomputational algorithm identifies the genetic regulatory network in yeast.

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