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Photorhabdus and a Host of HostsNick R. Watereld, 1 Todd Ciche, 2 and David Clarke1Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY,United Kingdom; email: [email protected] 2Department of Microbiology and Molecular Genetics, Michigan State University,East Lansing, Michigan 48824; email: [email protected] of Microbiology, University College Cork, Ireland; email: david.clarke@uc

Annu. Rev. Microbiol. 2009. 63:55774

First published online as a Review in Advance on June 23, 2009

The Annual Review of Microbiology is online at micro.annualreviews.org

This articles doi:10.1146/annurev.micro.091208.073507

Copyright c 2009 by Annual Reviews. All rights reserved

0066-4227/09/1013-0557$20.00

Key Words virulence, symbiosis, insect, immunity, toxins

Abstract Photorhabdus is a member of the family Enterobacteriaceae that lives inmutualistic association with a Heterorhabditis nematode worm. The nematode worm burrows into insect prey and regurgitates Photorhabdu which goes on to kill the insect. The nematode feeds off the growibacteria until the insect tissues are exhausted, whereupon they reassciate and leave the cadaver in search of new prey. This highly efcipartnership has been used for many years as a biological crop protetion agent. The dual nature of Photorhabdus as a pathogen and mutualismakes it a superb model for understanding these apparently exclusactivities. Furthermore, recently identied clinical isolates of Photorhabdus arehelping us tounderstandhow humanpathogenscanemerge fromthe enormous reservoir of invertebrate pathogens in the environmen As Photorhabdus has never been found outside a host animal, its nicrepresents an entirely biotic landscape. In this review we discuss wmolecular adaptations allow this bacterium to complete this fascinatiand complex life cycle.

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EPN:entomopathogenicbacteria/nematodecomplex

Pathogen: amicroorganism that causes disease

IJ: the infective juvenile developmentalform of an EPN

Hemocoel: an insectsopen blood system

Hemolymph: insect

blood Pa: Photorhabdus asymbiotica

Pl : Photorhabdus luminescens

Pt : Photorhabdus temperata

ContentsINTRODUCTION .. . . . . . . . . . . . . . . . . 558 PHOTORHABDUS . . . . . . . . . . . . . . . . . . . 558NEMATODE SYMBIONT. . . . . . . . . . . 560

Metabolic Interactions . . . . . . . . . . . . . 560 Transmission of Photorhabdus

by H. bacteriophora Nematodes. . . 560Interkingdom Signal Production. . . . 562

INSECT PREY . . . . . . . . . . . . . . . . . . . . . . 563Immune Challenge. . . . . . . . . . . . . . . . . 563 Virulence Factors and Strategies . . . . 563 Avoiding the Cellular Response . . . . . 564 Toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 564 Avoiding the Humoral Responses . . . 566

OTHER MICROBES ASCOMPETITION: RESOURCEPROTECTION.. . . . . . . . . . . . . . . . . . 567

THE HUMAN HOST . . . . . . . . . . . . . . . 567 THE POSTGENOMIC ERA. . . . . . . . . 569CONCLUSIONS . . . . . . . . . . . . . . . . . . . . 569

INTRODUCTION Photorhabdus is a gram-negative member of thefamily Enterobacteriaceae that lives in a symbi-oticassociationwithan entomopathogenic Het-

erorhabditis nematode worm (EPN) in a highly effective symbiosis of pathogens (29). Pho-torhabdus is the only known bioluminescent ter-restrial bacterium; the name means glowing-rod. In the soil Photorhabdus resides in the gut of an infective juvenile (IJ) form of the nema-tode. The IJs actively seek out insect prey in thesoil, whereupon they penetrate the body, eitherdirectly or through natural openings. Once in-side the insects open blood system, the hemo-coel, the nematodes regurgitate 50200 bacte-rial cells directly into the blood, or hemolymph(12). These few bacteria are sufcient to over-come the insects immune system and set uplethal septicemia. Once the insect host is deadthe bacteria bioconvert the tissues into morebacteria. The nematode partner feeds off thebacteria and nematode reproduction continuesthrough several generations. Although not yet

established, it is likely that environmental cditions (such as availability of food or anknown signal) stimulate the developmenIJs containing their symbiotic bacteria. Up400,000 IJs may leave a single insect cad1014 days postinfection in search of newsect prey (29). The niche in which Photorhab

has evolved represents an entirely biotic lscape in which it is challenged by the immsystems of nematode host and insect prey. Fthermore, Photorhabdus must also successfcompete with saprophytic scavenging orgisms, such as protists, fungi, nematodes, obacteria, and even other insects, that inevittry to utilize the tissues of the dead insect h

In addition to its well-described rolean insect pathogen one species of the ge Photorhabdus asymbiotica( Pa), causes infectio

otherwisehealthy humans(25, 33,36). Pa isain a symbiotic relationship witha heterorhabtid nematode, Heterorhabditis gerrardi, and other EPNs can also complete its life cycinsects (34, 55). A detailed understandinhow Pa can infect both insect and human hshouldprovide major insights into theevolutof bacterial pathogenicity and also the emgence of human pathogens (76).

PHOTORHABDUS EPNs can be found in soils worldwide, anis a simple matter to recover them in the oratory by baiting soil with insects. In 1Fischer-Le Saux et al. (28) classied Photorhdus into three species on the basis of phetypic characterization and DNA relatedn P. luminescens ( Pl ), P. temperata( Pt ), and P. asybiotica ( Pa). Limited microarray analysis (multilocus sequence typing (33), and recentnomic studies (20) have conrmed this clcation. The Pa species is further subdivinto at least two highly clonal subspecies,found in the United States and the other Australia. In addition, two European straHITandJUN, isolated from nematodespotetially form a third subspecies of Pa (Figure Recently, Pa strains associated with nematohave also been reported from Japan, and

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becoming clear that they are not restricted tothe continents of North America and Australia(50). Nevertheless, it remains to be seen if allmembers of this species are capable of causingclinical infections.

Photorhabdus species are highly motile, bio-luminescent, facultatively anaerobic rods (54).

All species grow well at 28

C on blood agar, where they uniquely produce a thin line of an-nular hemolysis. Critically, the clinical isolatesalso grow at temperatures ranging from 37 to42 C. Recent work has shown that althoughmotility is not required for eitherpathogenicity or symbiosis, it does confer a competitive ad- vantage during colonization of the insect (21).

A unidirectional phenotypic variation hasbeen described for Pl and Pt (4). Continuedin vitro cultivation in nutrient-limited condi-tions leads to the generation of secondary vari-ant (P2) forms that are still pathogenic to in-sects but no longer support symbiosis with thenematode. They also show a change in the pat-tern of protein, light and metabolite produc-tion. The primary variant (P1) is the symbi-otically competent form that is isolated fromEPNs (45). With this in mind Joyce & Clarke(45) used P1 and P2 variants to provide cluesregarding the molecular basis behind symbio-sis and pathogenicity. They identied a gene with homology to hexA from Erwinia that re-presses symbiosis factors in P2, suggesting it is a key player in the regulatory network con-trolling pathogenicity and mutualism (45, 46). More recently, two studies aimed at elucidat-ing the mechanisms behind this P1/P2 switchused microarray and proteomic approaches, al-though the precise mechanism still remainselusive (31, 64).

Bioluminescence occurs inall strains onagarplates and in liquid medium, where it peaks at the end of the exponential phase. In vivo, light emission is seen only after the insect is dead andtissue degradation is at an advanced stage (15). This bioluminescence has been hypothesized to(a) represent a signal from bacteria to bacteriaor to the nematode, synchronizing symbiosis;(b) provide a visual aposematic warning to noc-turnal scavenging animals; or even (c ) act a lure

Prey

W14

TT01

Hm/Hb

HIT/JUN

ATCC43949(USA)

Kingscliff (Australia)

K122

P. luminescens(insect)

P. asymbiotica(human + insect)

P. temperata(insect)

Figure 1 A schematic representation of the multilocus sequence typing phylogeny ofgenus Photorhabdus . Model type strains of three different species are indicatin their different clades. P. asymbiotica contains at least two subclades,

representing U.S. and Australian isolates. A third clade comprising the HITand JUN isolates from mainland Europe conrms these genotypic forms arnot restricted to the two continents.

Mutualism: abenecial relationshbetween different species

to attract further insect prey to the infected in-sect cadaver. The luciferase reaction is highly efcient at utilizing molecular oxygen, and it has also been suggested that the primary rolefor bioluminescence is as a sink for molecu-lar oxygen. This would potentially starve thehost immune cells of a resource required for theproduction of free radicals that could otherwisebe used in their phagocytic destruction. Sev-eral observations argue against this role, how-ever. First, strong light emission occurs only after insect death (although it is possible that light production can occur at a local level dur-ing the early stages of infection). Second, most Photorhabdus species resist phagocytic uptake

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anyway. Furthermore, if this represented a gen-eral mechanism to evade phagocytic destruc-tion, then it is unlikely to have been restrictedto Photorhabdus only and we would expect to see many more bioluminescent terrestrialpathogens. Finally, most bioluminescent bac-teria known to date are associated with animal

symbiosis, suggesting a cryptic underlying se-lective force.

NEMATODE SYMBIONT

Metabolic Interactions

Most bacteria-host interactions involve somelevel of metabolic interaction. In the environ-ment, the Heterorhabditis - Photorhabdus interac-tion appears to be obligate, although the bac-teria can be readily cultured in vitro using

standard peptone-based media. Moreover, thenematodes can be cultured in vitro by inoculat-ing Photorhabdus biomass grown on agar plates with either IJs or eggs. The nematodes will not grow or develop on Photorhabdus cultured onLuria-Bertani agar, yet they grow readily on thesame bacteria cultured on lipid agar (nutrient agar supplemented with cod liver oil and cornsyrup). Therefore, Photorhabdus is required, but not sufcient, for nematode growth, and development is also dependent on factors present

in the insect. Exogenous sterols are important for thedevelopment of thebacteriophorousne-matode Caenorhabditis elegans , and it is likely that Heterorhabditis will have the same require-ment (8). In this regard, Photorhabdus producesa signicant amount of iso-branched fatty acids(BCFAs), with isoC15:0 being the dominant fatty acid found in Photorhabdus phospholipidsproduced by the bkdABC operon (44). Theproduction of BCFA is not widespread in the Enterobacteriaceae and the presence of BCFA in

Photorhabdus may represent an adaptation tosymbiosis. BCFAs play an important role in thedevelopment of C. elegans, in which the nema-tode itself encodes the genes required for itsproduction (48, 49). One interesting possibil-ity is that Photorhabdus may have taken on theresponsibility of producing BCFAs required by their nematode partner.

Photorhabdus also produces large quantiof crystalline inclusion proteins (CipA CipB) that have a potential role in nematnutrition (3). Mutants in cipA and cipB unable to support nematode growth development, although they are pleiotromaking it difcult to denitively dene an ex

role in symbiosis for these proteins (3)interesting aspect of the metabolic interactbetween Photorhabdus and the nematode is t Heterorhabditis will not grow on just any stof Photorhabdus , rather it usually requiresspecic cognate bacterial strain. The molecubasis of this nutritional specicity remto be elucidated but some recent work implicated a role for iron (77).

Transmission of Photorhabdus by H. bacteriophora Nematodes Photorhabdus bacteria are transmitted by specialized IJ stage of thenematode, which hbors them in the intestinal lumen as it dispefrom an insect cadaver in search of a newsect host (9, 12). The IJ is an alternative tlarval stage of the nematode that is devementally arrested and resistant to environmtal stresses. IJs are formed inside the matenematode body cavities, causing matricid

process termed endotokia matricida (9, 12), indicating that Photorhabdus is maternally tramitted by an elaborate sequence of selecevents (13).

The transmission cycle starts when an IJters an insect and regurgitates the Photorhabinto the insect hemocoel (13). Every bactecell is released into the hemocoel, perhapsecting the challenge they face from the inimmune system (12). After regurgitation,nematodes switch to feeding behavior andgest symbiont bacteria, a few of which adto the posterior nematode intestine (Figure Pulse-chase experiments revealed that theherent cells formed a persistent infection biolm while other symbiont cells were tsiently present in the intestine. After persisfor up to 38 h, the adherent bacteria invthree rectal gland cells, where they mult


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O ff s

p r i n

g ( F 1 )

M a t e r n

a l ( P

0 )

10 m

10 m

10 m

10 m

10 m

5 m

5 m

5 m

11

t = 0 hInfective

juvenile (IJ)t = 4 h

Regurgitation of intestinal symbionts

t = 6 hComplete symbionts release

t = 8 h

Symbiont adherence and worm feeding

t = 36 hBio lm formation and adherence

t = 42 hInvasion of rectal gland cells

t = 48 hIntracellular symiont growth

t = 72 hSymbiont and vacuole have multiplied

t = 112 hSymbionts released into pseudocoelom

t = 120 hSymibont adherence to the anterior IJ,IJs develop inside the maternal body

t = 136 hPharyngeal-intestinal valve cell invasion

1

2

3

4

567

8

9

Photorhabdustransmission

cycle

t = 136240 hExit and symbiont growth

in the intestinal lumen

10 Nematode Bacteria

Figure 2 Transmission cycle of Photorhabdus luminescens subsp. laumondii in a nematode host. t = hours post-infective juvenile (IJ) addition tosymbiont lawns. Nematodes in steps 38 were chased with unlabeled symbionts so that only persistent bacteria are visible. Step 1:

Symbionts are regurgitated from the intestine until (Step 2) no symbionts remain. (In Step 2 only, green color is autouorescence onematode intestine, visible because the exposure of the epiuorescent micrographs is 10 times greater than the other micrographs.)Step 3: Nematodes ingest bacteria and a few adhere to the posterior intestine. Step 4: Symbionts grow as a biolm while additionasymbionts adhere. Step 5: Invasion of the rectal gland cells by adherent symbionts. Steps 6 and 7: Intracellular growth of symbiontbacteria and vacuole replication. Step 8: Lysis of the apical side of the rectal gland cells releasing symbionts to pre-IJs developing maternal body cavity. Step 9: Symbiont adherence to the pharyngeal-intestinal valve cell. Step 10: Invasion of the pharyngeal-intes valve cells. Step 11: Exit and growth of symbiont cells in the IJ intestinal lumen.


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potential for novel and/or unusual PKS-relatedbiochemistry in Photorhabdus . The 5.5 Mbgenome of Pl TT01 has been sequenced(http://genolist.pasteur.fr/PhotoList/ ), andanalysis reveals that nearly 6% of the genomeis given over to genes predicted to be involved in the production of secondary metabo-

lites (20). This proportion is greater than the3.8% observed in Streptomyces , the model or-ganism for secondary metabolite production(and the source of > 90% of clinically impor-tant antibiotics). Therefore, there is signicant potential in Photorhabdus for the production of novel bioactive molecules. A novel virulencemapping technique termed RVA, discussed be-low, has provided the rst step in isolatingandcharacterizing a numberof these secondary metabolites (75).

INSECT PREY

Immune Challenge

The EPN complex shows little specicity inits choice of insect prey species. Furthermore,the release of at most only 50200 bacteriain a normal infection means that the bacte-ria are adapted to be highly virulent and canresist immune systems from diverse genetic

backgrounds. These requirements appear to bereected in the genomeof Pl TT01,which con-tained a greater number and diversity of toxingenes than any other sequenced genome at thetime (20).

Upon release into the insect hemocoel, Pho-torhabdus bacteria face thefast-acting innate im-mune system (41). The innate immune systemsof insects and mammals share many commonfeatures (47), the implications of which becomeobvious when considering the evolution of Pa, which is both an insect and human pathogen.Innate immune factors include cellular re-sponses, such as phagocytic destruction usingfree radicals and killer proteases, and humoralresponses, including recognition of pathogen-associated molecular patterns (PAMPs) by re-ceptors and the production of antimicrobialpeptides (AMPs) and lysozyme. A mainstay of

PAMP: pathogen-associated moleculapattern

PO: phenol oxidase

Hemocyte: an inse

immune cell

the insect immune system is the melanizationand encapsulation response mediated by phe-noloxidase (PO) (37). This system represents acascade of serine protease signaling moleculesinitiated by PAMP recognition and terminat-ing in the cleavage of pro-PO to active PO. This enzyme deposits melanin onto invading

organisms, encapsulating them and inictingfree radical damage in the process. In Mand-uca sexta, melanization typically occurs aroundgroups of hyperphagocytichemocytes, forminga nodule on the surface of tissues (17). In many ways the PO system is reminiscent of the com-plement cascade of vertebrates,andit is possiblethat they share a common evolutionary origin(30, 38). Photorhabdus has evolved multiple vir-ulence factors and strategies to deal with imme-diate immune threatsand to kill the host,which

we discuss below.

Virulence Factors and Strategies Two model insects most commonly used tostudy Photorhabdus are the large lepidopteranhosts Galleria mellonella (greater wax moth)and Manduca sexta (tobacco hornworm). Morerecent studies have used the dipteran host Drosophila melanogaster , which has the advan-tage of a fully sequenced genome and well-

developed genetic tools (39, 47, 65, 67).Prior infection of M. sexta larvae withnonpathogenic Escherichia coli elicits effectiveimmunity against subsequent infection withthe otherwise lethal Pl TT01. This protec-tion correlates with the upregulation of PAMPmolecules such as hemolin, immulection-2, andpeptidoglycan recognition protein, and also AMPs including attacin and cecropin. Prevent-ing the upregulation of these PAMP molecules with RNAi abolished this protection, whereassuppression of specic AMP production hadlittle effect (23). This suggests that M. sextacan in fact ght off a Photorhabdus infectionif given a head start. Other studies have con-rmed that M. sexta recognizes a Photorhabdus infection, as seen by an increase in mRNA forPAMPs, but that Photorhabdus can neverthelessovercome this response (24).


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TTSS: Type threesecretion system

Macrophage: amammalianprofessional

phagocytic immunecell

PVC: Photorhabdus virulence cassette

Avoiding the Cellular Response A detailed study by Silva et al. (61) followedthe fate of Pl W14 after injection into M. sextalarvae. Following injection the bacteria multi-plied rapidly both on the midgut and in thehemolymph (61). Colonization of the bloodside of the midgut is initiated at the ante-

rior and then proceeds posteriorly along itslength as infection time continued. Electronmicroscopy revealed that the bacteria occupy aspecic niche between the extracellular matrixand basal membrane of the midgut epithelium,in tight grooves made by infolding of the ep-ithelium. Although this site may be difcult forthe hemocytesto enterbecause of theirsize, not all the bacteria reside here, and all are open tohemocyte attack, at least during transit to thegut. Several toxin systems that disable orkill the

hemocytes have been characterized.

ToxinsImmunohistochemistry revealed that withinthe gut niche the bacteria express at least two virulence factors, the highly secreted gut-activetoxin complex A (Tca) and a metalloprotease,PrtA (15, 61). Close association of bacteria withtheepithelium,andsecretionwithinsuchanen-closed niche, may facilitate the rapid destruc-

tion of the gut, which is a prominent featureof Photorhabdus infection. All strains of Pho-torhabdus encode multiple homologuesof theTcgenes, suggesting they play an important role inthe biology of the organism, although the func-tions of these homologues remains obscure inmost cases (27, 68, 69, 72).

Another gut-active toxin produced by allstrains of Photorhabdus is the multidomain Mcf1toxin. This potent toxin is taken up into target cells by endocytosis, where it induces apoptosisby a novel BH3-domain-mediated effect (16, 19). This protein toxin has little cellspecicity and can also induce apoptosis inhemocytes and a range of cultured human cells,suggesting multiple roles in infection.The mcf1gene is present in all strains so far examined but appears to be encoded at different genomic lociin each case, suggesting independent acquisi-

tion (71). At least two of the Pl strains, Wand TT01, also have a homologue called M This protein differs from Mcf1 in the toactive site domain but is also believed to inapoptosis because it possesses homologyproapoptotic Pseudomonas toxin, HrmA (73

Like manypathogenicgram-negative bac

ria, Photorhabdus encodes a type three secresystem (TTSS). In Pl TT01, the TTSS is i volved in attachment to hemocytes and in deery of at least one effector molecule,LopT, ithese cells, preventing their phagocytic uptLopT is a close homolog of YopT from Yers pestis , in which it also serves to inhibit phcytosis by macrophages. This shows a sing conservation of immune avoidance meanisms in a mammalian or insect pathogenOther TTSS effector molecules are yet to

identied in Pl TT01. The TTSS operon the recently sequenced clinical strain Pa ATC43949 does not encode the lopT gene, insteaencodes a close homologue of ExoU, a poful TTSS-delivered Pseudomonas cytotoxin (2Furthermore, a subtractive DNA hybridizatstudy between Pa and Pl TT01 identied sopB gene homologue in the clinical strain (SopBinSalmonellais a TTSS-deliveredeffecinvolved in intracellular persistence in humacrophages (80).

A range of TTSS effector-like moleccan be seen in the genomes of Photorhabstrains; however, these are associated witalternative secretion system called Photorhab virulence cassettes (PVCs). PVC genomilands supercially resemble prophage and csist of around 15 genes, conserved betweePVC elements, homologous to either phor other macromolecular assembly genesthe 3 end of each conserved region is a load region that encodes one or more gethat typically show homology to the active of known toxins, reminiscent of TTSS eftors (74, 79) (Figure 3 a). There are muple, diverse PVC elements in all Photorhdus genomes so far examined, suggesting are mobile and acquired by horizontal tran(Figure 3 b). When heterologously expresfrom cosmidclones in E. coli,thePVCeleme


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Putative toxin geneOpen reading frames

Conserved region Toxin gene

Pa PVC pnf

5 kb

pnf sepC- likePhage tail

FlagellaM-ring

VgrG

Baseplate

Transcriptregulation

Fimbrial usher

Adenovirusber

AAA-ATPase

Conserved region Payload region

afp1 afp18afp17 Se afp

afp16

a

hvnA

10 kb

P. asymbiotica ATCC 43949

P. luminescens TT01

Type 4 pil

mukB

phxA

PaPVC phx

PaPVC lopT

tccC-like

PaPVC cif

lopT

toxA-like

dnt- like cif-like

PtPVC lopT PtPVC cif

sepC-like

phxA'

hvnA

phxAtcd- like

mcf- like

Type 4 pil PtPVC unit1 PtPVC unit2 PtPVC unit3 PtPVC unit4

pnf sepC-like

PaPVC pnf

lumt

PaPVC lumt

cif-like

dnt- like lopT

toxA-likesepC-like

mukB

b

Figure 3

(a) A schematic representation of a typical Photorhabdus virulence cassettes (PVC) element (PVC pnf ). Open boxes represent openreading frames (ORFs). Note the conserved region common to all PVC elements and the variable payload region with putative toxgenes shown in red. The homologous Serratia entomophila (Se) afp element is shown, and similar ORFs have been color coded. Thethick red arrows below the afp diagram indicate where insertional mutagenesis abolished toxic activity, while the thin blue arrows amutants that retained function. (b) A diagram showing the range of PVC elements in Photorhabdus luminescens ( Pl ) TT01 and P. asymbiotica ( Pa) ATCC 43949. Note several elements are common, whereas others are unique to each species. Note the tandemarrangement of four PVC elements in TT01 between a type four DNA conjugation pilus operon ( yellow box) and the mukB gene(blue box). In Pa ATCC 43949 there is only one, presumably ancestral, element in this locus. The conserved regions are shown as grboxes and the toxin genes in red. The names or closest homologues of payload genes are indicated.


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Entomopathogen: aninsect pathogen

produce a large 250-nm-long structure similartoR-typepyocins,whicharebacteriophage tailsadapted to kill related strains of bacteria. LikeR-type pyocins, the PVC elements consist of aninner stylet and an outersheath that can be seenin a relaxed or contracted injection form. Noantibacterial activity has been associated with

PVC elements, but they do have potent toxic-ity when injected into Galleria larvae (79). Lar- vae injected with the puried PVC pnf element melanize and die within minutes, with a reduc-tion in circulating hemocytes showing exten-sive actin rearrangement (79). Recent work inour lab has conrmed that PVC element tox-ins are expressed during insect infection andthat the PVC structures can attach to the sur-face of hemocytesusing SEM(N.R.Watereld,unpublished data). Our current hypothesis is

that the PVC elements resemble a freely se-creted molecular syringe that directly injectsthe payload toxin into the host cell, reminis-cent of a released TTSS. Despite the signif-icant numbers of pathogen genomes now se-quenced, the only other bacteria so far seen toencode this system is another insect pathogen,Serratia entomophila. In Serratia this is knownas the antifeeding prophage, which causes larvae of Costelytra zealandica (New Zealand grassgrub) to cease feeding (Figure 3 a). In this case

the AFP element is presumably active against gut cells rather than hemocytes (43). In Pho-torhabdus the PVC elements are tightly regu-lated at the translational level andnotexpressedat 37 C, suggesting they may be inappropriatein vertebrate infections.

Strains of Pl and Pa encode binary toxinsystems, which comprise two proteins requiredfor toxicity. These include the PirA and PirBproteins (20, 60) and proteins homologous to XaxA and XaxB, rst identied in the closely re-lated entomopathogen Xenorhabdus nematophila(66, 75). The PirAB toxins exhibit a rangeof activity including potent injectable activity against Galleria larvae; oral activity against thecaterpillar pest Plutella xylostella; and oral toxi-city against three different species of mosquito, Aedes aegypti , Culex pipiens, and Anopheles gam-biae (20, 70). XaxAB has cytolytic, hemolytic,

and proapoptotic effects on insect and mmalian cells. Finally, a range of homologuetwo partner hemolysin genes, such as Phlcan also be seen in the two completed Ptorhabdus genomes that have cytotoxic eff(6, 75).

Avoiding the Humoral Responses The secondary metabolite ST is also a tent inhibitor of the PO enzyme, allowing Ptorhabdus to avoid the melanization respo(22). Conversely, Pl TT01 also secretes a ptease, PrtS, during infection that stronglyduces melanization (40). The signicancthis is not yet clear, although premature nonlocalized activation of the PO system coalso represent a virulence strategy.

Other humoral factors that Photorhabmust resist include circulating lysozyme AMPs. Although Photorhabdus induces the trscription of AMP mRNA, it is not kn whether the bacterium is resistant to AMspecically or whether it blocks translatiosecretion of the peptides. Pl TT01 cells crying a mutation in the pbgPE operon (pdicted to be responsible for the modicaof LPS with l -aminoarabinose in response AMP) are avirulent, suggesting that Photorh

dus doesinfactadapttoAMPproductionduriinfection (2).Finally, all animals attempt to limit bact

replication through the tight binding and copartmentalization of iron. Like many anipathogens, Photorhabdus has several iron acsition systems including the production ofsiderophore photobactin (11). Furthermoremutation in the exbD gene of Pt K122 resulin a strain that was severely limited in its ity to acquire iron. This mutant was attenuain virulence and this attenuation was correl with the growth rate of the bacteria in thesect (77). The attenuation could be rescuedpreloading theinsectwith iron. IndeedtheLT(time taken to kill 50% of the insects) of wtype Pt K122 was reduced in insects prelo with iron, suggesting that iron is indeed liing in the insect (77).


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not seen in the other two members of thegenus. The Australian isolates of Pa also invadenonphagocytic cell lines and induce apoptosisin cells faster than the U.S. isolates (14). Thisability to invade immune cells suggests a routeof bacteremia spread in the clinical cases and would provide the bacteria with a niche inac-

cessible to many of the systemic humoral im-mune responses of the mammalian host. Thisparallels the related pathogen, Y. pestis , whichis also a facultative intracellular pathogen that induces apoptosis in macrophages, silences in-ammatory reactions, and allows dissemina-tionthrough the lymphatic system (56). Finally, we have recently demonstrated that both Paand Pl TT01 are inherently resistant to killingby commercially available human serum, sug-gesting that a defensive mechanism adopted

by the insect-only pathogens may be equally effective against human-complement-mediatedkilling (N.R. Watereld, unpublished data).

THE POSTGENOMIC ERA The availability of completed genome se-quences for Pl TT01 (20) and Pa ATCC 43949(United States) (58) has ushered in a new era inthe study of Photorhabdus . Furthermore, a Het-erorhabditis genome is also currently being se-

quenced, which will greatly assist the study of the symbiotic interaction. We have now completed a full in silico ge-

nomic comparison between Pa ATCC 43949(United States) and Pl TT01 that awaits pub-lication (N.R. Watereld, P. Wilkinson, L.Crossman, C. Corton, M. Sanchez Contreras,et al., unpublished data). Briey, the humanpathogenic strain has a smaller genome withreduced toxin diversity. In addition both strainspossess a range of different genomic islands,suggestive of signicant levels of horizontalgene transfer and differential host ranges (20).In an attempt to map genes induced upon ex-posure to insect homogenates, M unch et al.(53) identied 24 Pl TT01 promoters that were induced in G. mellonella infection. These

included promoters that drive expressionof thetc toxin components tccC1 and tcdA1; PVC pay-load genes plu2400, plu1672, and plu1645 ; themcf1 toxin; secondary metabolite genes; andpromoters for a range of metabolic operons(53).

Finally, we recently used Pa as a model of

an emerging human pathogen to develop anassumption-free approach for mapping viru-lence factors across whole pathogen genomes. This approach, termed rapid virulence anno-tation (RVA), screens large insertion genomiclibraries for toxicity against four different taxa:theprotist A. polyphaga,thenematode C.elegans ,the moth M. sexta, and a mouse macrophagecell line ( J774-2). RVA successfully identieda large number of virulence loci through theirgain of function in E. coli (75). This approach

is particularly useful in Photorhabdus because of the high level of toxin redundancy, unmasking virulence factors that would otherwise be hardto detect. The identication of a range of po-tent secondarymetabolite biosyntheticoperonsin these toxicity screens has also provided anexcellent way to understand the role of thesesmall molecules in infection, and presented anobvious route for novel drug discovery. We saw no obvious mammalian-only toxins, support-ingthe hypothesisthatvirulencefactors evolved

against insect hosts may be readily redeployedagainst humans.

CONCLUSIONS Photorhabdus provides an excellent model forthe study of both symbiosis and pathogenicity.In addition, with Pa now recognized as bothan insect and human pathogen, it also provides a superb opportunity to understand therole of invertebrates in the emergence of hu-man diseases. In the future, the availability of genomesequences and the ability to genetically manipulate the bacteria, the nematode partner,and the host insects will ultimately enable usto understand the molecular interplay betweenpathogenicity and symbiosis.


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SUMMARY POINTS

1. All strains of Photorhabdus live in a mutualistic relationship with insect pathogenic nematodes of the genus Heterorhabditis . This symbiosis is highly evolved and is highly straspecic.

2. Pl and Pt have not been found outside either the nematode or the insect hosts and so aradapted only for survival in these biotic environments.

3. Genome sequences have conrmed a large repertoire of genes involved in host interations, especially genes encoding putative toxins.

4. P. asymbiotica is capable of causing infections in humans, providing an excellent modelan emerging human pathogen.

5. Photorhabdus must also compete with a wide range of saprophytic microorganisms frothe soil, providing further selection for virulence.

6. The large repertoire of secondary metabolite gene clusters suggests these bacteria represent an excellent source of novel drug candidates.

7. Postgenomic techniques and the ability to genetically manipulate the bacterium, thsymbiont nematode, and the insect hosts provide an unparalleled model system for understanding animal-bacteria interactions.

DISCLOSURE STATEMENT The authors are not aware of any afliations, memberships, funding, or nancial holdingsmight be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

The authors would like to thank all past and present members of their research groups. DJCNRW thanks the Leverhulme Trust, BBSRC, and SFI for support. TAC thanks the Center f Microbial Pathogenesis at Michigan State University and the USDA for support.

LITERATURE CITED1. Aumann J, Ehlers R-U. 2001. Physico-chemical properties and mode of action of a signal from

symbiotic bacterium Photorhabdus luminescens inducing dauer juvenile recovery in the entomopathognematode Heterorhabditis bacteriophora. Nematology 3:84953

2. Bennett HP,ClarkeDJ. 2005. ThepbgPEoperon in Photorhabdus luminescens is requiredfor pathogenicand symbiosis. J. Bacteriol. 187(1):7784

3. Bintrim SB, Ensign JC. 1998. Insertional inactivation of genes encoding the crystalline inclusion pr

of Photorhabdus luminescens results in mutants with pleiotropic phenotypes. J. Bacteriol. 180(5):126164. Boemare RJ, Akhurst NE. 1988. Biochemical and physiological characterization of colony form va

in Xenorhabdus spp. (Enterobacteriaceae). J. Gen. Microbiol. 134:751615. Brachmann AO, Joyce SA, Jenke-Kodama H, Schw ar G, Clarke DJ, Bode HB. 2007. A type II polyk

synthase is responsible for anthraquinonebiosynthesis in Photorhabdus luminescens . Chembiochem8:17216. Brillard J, Duchaud E, Boemare N, Kunst F, Givaudan A. 2002. The PhlA hemolysin from the

tomopathogenic bacterium Photorhabdus luminescens belongs to the two-partner secretion familyhemolysins. J. Bacteriol. 184(14):387178


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7. Brugirard-Ricaud K,Duchaud E,Givaudan A,GirardPA,Kunst F,et al.2005.Site-specic antiphagocyticfunction of the Photorhabdus luminescens typeIII secretionsystem during insect colonization. Cell Microbiol.7(3):36371

8. Chitwood DJ. 1999. Biochemistry and function of nematode steroids. Crit. Rev. Biochem. Mol. Biol.34(4):27384

9. Discusses thesimilarities between thegenomics of the well

known model C. elegaand Heterorhabditis .

9. Ciche T. 2007. The biology and genome of Heterorhabditis bacteriophora (February 20, 2007),WormBook , ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook. 1. 135.1, http://www.wormbook.org/

10. Ciche TA, Bintrim SB, Horswill AR, Ensign JC. 2001. A phosphopantetheinyl transferase homolog isessential for Photorhabdus luminescens to support growth and reproduction of the entomopathogenic ne-matode Heterorhabditis bacteriophora. J. Bacteriol. 183(10):311726

11. Ciche TA, Blackburn M, Carney JR, Ensign JC. 2003. Photobactin: a catechol siderophore produced by Photorhabdus luminescens , an entomopathogen mutually associated with Heterorhabditis bacteriophora NC1nematodes. Appl. Environ. Microbiol. 69(8):470613

12. Ciche TA, Ensign JC. 2003. For the insect pathogen Photorhabdus luminescens , which end of a nematodeis out? Appl . Environ. Microbiol. 69(4):189097

13. Describes thecomplex process of symbiotic transmission of Photorhabdus by thnematode host andreveals the similarities with pathogenicinteractions.

13. Ciche TA, Kim KS, Kaufmann-Daszczuk B, Nguyen KC, Hall DH. 2008. Cell invasion and ma-tricide during Photorhabdus luminescens transmission by Heterorhabditis bacteriophora nematodes. Appl. Environ. Microbiol. 74(8):227587

14. Costa SC,Girard PA, Breh elin M, Zumbihl R. 2009. Theemerging human pathogen Photorhabdus asymbi-

otica is a facultative intracellular bacterium and induces apoptosis of macrophage-like cells. Infect. Immun.77:10223015. Daborn PJ, Watereld N, Blight MA, ffrench-Constant RH. 2001. Measuring virulence factor expression

by the pathogenic bacterium Photorhabdus luminescens in culture and during insect infection. J. Bacteriol.183(20):583439

16. Daborn DJ, Watereld N, Silva CP, Au CP, Sharma S, et al. 2002. A single Photorhabdus gene makescaterpillars oppy (mcf) allows Escherichia coli to persist within and kill insects. Proc. Natl. Acad. Sci. USA99:1074247

17. Dean P, PotterU, Richards EH,Edwards JP,CharnleyAK,Reynolds SE.2004. Hyperphagocytichaemo-cytes in Manduca sexta. J. Insect. Physiol. 50(11):102736

18. Dolan KM, Jones JT, Burnell AM. 2002. Detection of changes occurring during recovery from the dauerstage in Heterorhabditis bacteriophora. Parasitology 125(Pt. 1):7181

19. Dowling AJ, Daborn PJ, Watereld NR, Wang P, Streuli CH, et al. 2004. The insecticidal toxin Makescaterpillars oppy (Mcf) promotes apoptosis in mammalian cells. Cell Microbiol. 6(4):34553

20. Describes thegenome sequence of TT01, revealing thehuge number of genesinvolved in host interactions.

20. Duchaud E, Rusniok C, Frangeul L, Buchrieser C, Givaudan A, et al. 2003. The genome sequenceof the entomopathogenic bacterium Photorhabdus luminescens . Nat. Biotechnol. 21(11):130713

21. Easom EA, Clarke DJ. 2008. Motility is required for the competitive tness of entomopathogenic Pho-torhabdus luminescens during insect infection. BMC Microbiol. 8:168

22. Eleftherianos I, Boundy S, Joyce S, Aslam S, Marshall J, et al. 2007. An antibiotic produced by an insect-pathogenic bacterium suppresses host defenses through phenoloxidase inhibition. Proc. Natl. Acad. Sci.USA 104(7):241924

23. Eleftherianos I, Marokhazi J, Millichap PJ, Hodgkinson AJ, Sriboonlert A, et al. 2006. Prior infection of Manduca sexta with non-pathogenic Escherichia coli elicits immunity to pathogenic Photorhabdus luminescens :roles of immune-related proteins shown by RNA interference. Insect. Biochem. Mol. Biol. 36(6):51725

24. Eleftherianos I, Millichap PJ, ffrench-Constant RH, Reynolds SE. 2006. RNAi suppression of recog-nition protein mediated immune responses in the tobacco hornworm Manduca sexta causes increasedsusceptibility to the insect pathogen Photorhabdus . Dev. Comp. Immunol. 30(12):1099107

25. Farmer JJ 3rd, Jorgensen JH, Grimont PA, Akhurst RJ, Poinar GO Jr, et al. 1989. Xenorhabdus luminescens (DNA hybridization group 5) from human clinical specimens. J. Clin. Microbiol. 27(7):1594600

26. ffrench-Constant R, Watereld N, Daborn P, Joyce S, Bennett H, et al. 2003. Photorhabdus : towards afunctional genomic analysis of a symbiont and pathogen. FEMS Microbiol. Rev. 26:43356

27. ffrench-Constant RH, Watereld NR. 2006. An ABC guide to the bacterial toxin complexes. Adv. Appl. Microbiol. 58:16983


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28. Fischer-Le Saux M, Viallard V, Brunel B, Normand P, Boemare NE. 1999. Polyphasic classicatithegenus Photorhabdus andproposal of newtaxa: P. luminescens subsp. luminescens subsp. nov., P. luminesubsp. akhurstii subsp. nov., P. luminescens subsp. laumondii subsp. nov., P. temperata sp. nov., P. tempesubsp. temperata subsp. nov. and P. asymbiotica sp. nov. Int. J. Syst. Bacteriol. 49:164556

29. Forst S, Dowds B, Boemare N, Stackebrandt E. 1997. Xenorhabdus and Photorhabdus spp. : Bugs thatbugs. Annu. Rev. Microbiol. 51:4772

30. Fujita T, Matsushita M, Endo Y. 2004. The lectin-complement pathwayits role in innate immunityevolution. Immunol. Rev. 198:185202

31. Gaudriault S, Pages S, LanoisA, LarouiC, Teyssier C, et al. 2008. Plastic architectureof bacterial genorevealed by comparative genomics of Photorhabdus variants. Genome Biol. 9(7):R117

32. Gaudriault S, Thaler JO, Duchaud E, Kunst F, Boemare N, Givaudan A. 2004. Identication of arelated prophage remnant locus of Photorhabdus luminescens encoding an R-type phage tail-like part FEMS Microbiol. Lett. 233(2):22331

33. Gerrard J, Watereld N, Vohra R, ffrench-Constant R. 2004. Human infection with Photorhabdus abiotica: an emerging bacterial pathogen. Microbes Infect. 6(2):22937

34. Describe thediscovery andcharacterization of theheterorhabditidsymbiont for P. asymbiotica andconrms its status as aduel human/insect pathogen (also seeReference 55).

34. Gerrard JG, Joyce SA, Clarke DJ, ffrench-Constant RH, Nimmo GR, et al. 2006. Nematodesymbiont for Photorhabdus asymbiotica . Emerg. Infect. Dis. 12(10):156264

35. Gerrard JG, McNevin S, Alfredson D, Forgan-Smith R, Fraser N. 2003. Photorhabdus species: biolunescent bacteria as emerging human pathogens? Emerg . Infect. Dis. 9(2):25154

36. Gerrard JG, Vohra R, Nimmo GR. 2003. Identication of Photorhabdus asymbiotica in cases of hu

infection. Commun. Dis. Intell. 27(4):5404137. GillespieJP,KanostMR, Trenczek T. 1997. Biological mediatorsof insect immunity. Annu. Rev. Ento42:61143

38. Hall M, Scott T, Sugumaran M, Soderhall K, Law JH. 1995. Proenzyme of Manduca sexta phenol oxidpurication, activation, substrate specicity of the active enzyme, and molecular cloning. Proc. Natl. ASci. USA 92(17):776468

39. Discusses how the well-characterizedinsect model Drosophilamay be used to dissect the Photorhabdus infection process.

39. Hallem EA, Rengarajan M, Ciche TA, Sternberg PW. 2007. Nematodes, bacteria, and ies: atripartite model for nematode parasitism. Curr. Biol. 17(10):898904

40. Held KG, LaRock CN, DArgenio DA, Berg CA, Collins CM. 2007. A metalloprotease secreted binsect pathogen Photorhabdus luminescens induces melanization. Appl. Environ. Microbiol. 73(23):7622

41. Hoffmann D, Hoffmann JA. 1990. Cellular and molecular aspects of insect immunity. Res. Imm141:89596

42. Hu K, Li J, Webster JM. 1999. Nematicidal metabolites produced by Photorhabdus luminescens (Enobacteriaceae), bacterial symbiont of entomopathogenic nematodes. Nematology 1(5):45769

43. Hurst MR, Glare TR, Jackson TA. 2004. Cloning Serratia entomophila antifeeding genesa putadefective prophage active against the grass grub Costelytra zealandica. J. Bacteriol. 186(15):511628

44. Describes the rst nonplant biochemicalpathway for theproduction of STs andidenties ST as an interkingdom signaling molecule that controlsnematode growth anddevelopment.

44. Joyce SA, Brachmann AO, Glazer I, Lango L, Schw ar G, et al. 2008. Bacterial biosynthesis of multipotent stilbene. Angew. Chem. 47:194245

45. Joyce SA, Clarke DJ. 2003. A hexA homologue from Photorhabdus regulates pathogenicity, symbiosisphenotypic variation. Mol. Microbiol. 47(5):144557

46. Joyce SA, Watson RJ, Clarke DJ. 2006. The regulation of pathogenicity and mutualism in PhotorhabCurr. Opin. Microbiol. 9(2):12732

47. Khush RS, Lemaitre B. 2000. Genes that ght infection: What the Drosophila genome says about animmunity. Trends Genet. 16(10):44249

48. Kniazeva M, Crawford QT, Seiber M, Wang CY, Han M. 2004. Monomethyl branched-chain fatty aplay an essential role in Caenorhabditis elegans development. PLoS Biol. 2(9):E257

49. Kniazeva M, Euler T, Han M. 2008. A branched-chain fatty acid is involved in post-embryonic grcontrol in parallel to the insulin receptor pathway and its biosynthesis is feedback-regulated in C. elegGenes Dev. 22(15):210210

50. Kuwata R, Yoshiga T, Yoshida M, Kondo E. 2008. Mutualistic association of Photorhabdus asymbiotic w Japanese heterorhabditid entomopathogenic nematodes. Microbes Infect. 10(7):73441

51. Li J, Chen G, Webster JM, Czyzewska E. 1995. Antimicrobial metabolites from a bacterial symb J. Nat. Prod. 58(7):108186


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52. Marokhazi J,WatereldN,LeGoffG, Feil F,Stabler R,etal.2003.Usinga DNAmicroarray to investigatethe distribution of insect virulence factors in strains of Photorhabdus bacteria. J. Bacteriol. 185(15):464856

53. M unch A, Stingl L, Jung K, Heermann R. 2008. Photorhabdus luminescens genes induced upon insect infection. BMC Genomics 9:22

54. Peel MM, Alfredson DA, Gerrard JG, Davis JM, Robson JM, et al. 1999. Isolation, identication, andmolecularcharacterization of strains of Photorhabdus luminescens frominfectedhumans in Australia. J. Clin. Microbiol. 37(11):364753

55. Plichta KL, Joyce SA, Clarke D, Watereld N, Stock SP. 2009. Heterorhabditis gerrardi n. sp. (Ne-

matoda: Heterorhabditidae): the hidden host of Photorhabdus asymbiotica (Enterobacteriaceae: gamma-Proteobacteria). J. Helminthol. 16:11256. Pujol C, Bliska JB. 2005. Turning Yersinia pathogenesis outside in: subversion of macrophage function by

intracellular yersiniae. Clin. Immunol. 114(3):2162657. Richardson WH, Schmidt TM, Nealson KH. 1988. Identication of an anthraquinone pigment and a

hydroxystilbene antibiotic from Xenorhabdus luminescens . Appl. Environ. Microbiol. 54(6):1602558. Sanger Institute. P. asymbiotica genome sequencing project . http://www.sanger.ac.uk/Projects/

P asymbiotica/ 59. Sharma S, Watereld N, Bowen D, Rocheleau T, Holland L, ffrench-Constant R. 2002. The lumicins:

novel bacteriocins from Photorhabdus luminescens with similarity to the uropathogenic-specic protein(USP) from uropathogenic Escherichia coli . FEMS Microbiol. Lett. 214(2):24149

60. SicardM, HeringS, Schulte R, Gaudriault S, Schulenburg H. 2007. Theeffectof Photorhabdus luminescens (Enterobacteriaceae) on the survival, development, reproduction and behaviour of Caenorhabditis elegans (Nematoda: Rhabditidae). Environ. Microbiol. 9(1):1225

61. Silva CP, Watereld NR, Daborn PJ, Dean P, Chilver T, et al. 2002. Bacterial infection of a model insect: Photorhabdus luminescens and Manduca sexta. Cell. Microbiol. 6(4):32939

62. Strauch O, Ehlers R-U. 1998. Food signalproduction of Photorhabdus luminescens inducing therecovery of entomopathogenic nematodes Heterorhabditis spp. in liquid culture. Appl. Microbiol. Biotechnol. 50:36974

63. Tounsi S, Blight M, Jaoua S, de Lima Pimenta A. 2006. From insects to human hosts: identication of major genomic differences between entomopathogenic strains of Photorhabdus and the emerging humanpathogen Photorhabdus asymbiotica. Int. J. Med. Microbiol. 296(8):52130

64. Turlin E, Pascal G, Rousselle JC, Lenormand P, Ngo S, et al. 2006. Proteome analysis of the phenotypic variation process in Photorhabdus luminescens . Proteomics 6(9):270525

65. Tzou P, De Gregorio E, Lemaitre B. 2002. How Drosophila combats microbial infection: a model tostudy innate immunity and host-pathogen interactions. Curr. Opin. Microbiol. 5(1):10210

66. VigneuxR, Zumbihl R, Jubelin G, Ribeiro C, PoncetJ, et al.2007. The xaxABgenesencoding a newapop-totic toxin from the insect pathogen Xenorhabdus nematophila are present in plant and human pathogens. J. Biol. Chem. 282(13):957180

67. Vodovar N, Acosta C, Lemaitre B, Boccard F. 2004. Drosophila: a polyvalent model to decipher host-pathogen interactions. Trends Microbiol. 12(5):23542

68. Watereld N, Dowling A, Sharma S, Daborn PJ, Potter U, et al. 2001. Oral toxicity of Photorhabdus luminescens W14 toxin complexes in Escherichia coli . Appl. Environ. Microbiol. 67(11):501724

69. Watereld N, Hares M, Yang G, Dowling A, ffrench-Constant R. 2005. Potentiation and cellular pheno-types of the insecticidal toxin complexes of Photorhabdus bacteria. Cell Microbiol. 7(3):37382

70. Watereld N, Kamita SG, Hammock BD, ffrench-Constant R. 2005. The Photorhabdus Pir toxins aresimilar to a developmentally regulated insect protein but show no juvenile hormone esterase activity. FEMS Microbiol. Lett. 245(1):4752

71. WatereldNR.2004. Insect pathogenicity islands in the insect pathogenic bacterium Photorhabdus . Physiol. Entomol. 29:111

72. Watereld NR, Bowen DJ, Fetherston JD, Perry RD, ffrench-Constant RH. 2001. The tc genes of Photorhabdus : a growing family. Trends Microbiol. 9(4):18591

73. Watereld NR, Daborn PJ, Dowling AJ, Yang G, Hares M, ffrench-Constant RH. 2003. The insecticidaltoxin Makes caterpillars oppy 2 (Mcf2) shows similarity to HrmA, an avirulence protein from a plant pathogen. FEMS Microbiol. Lett. 229(2):26570

74. WatereldNR,DabornPJ, ffrench-Constant RH.2002.Genomic islands in Photorhabdus . Trends Microbiol.10(12):54145


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75. Describes apowerful postgenomictechnique for mapping virulence genes in bacterial pathogens.

75. Watereld NR, Sanchez-Contreras M, Eleftherianos I, DowlingA, Wilkinson P, et al. 2008. Rapid virulence annotation (RVA): identication of virulence factors using a bacterial genome library and multiple invertebrate hosts. Proc. Natl. Acad. Sci. USA 105(41):1596772

76. Highlights the idea

that insect pathogensprovide a reservoir of potential human pathogens, including the agents of plague,anthrax and Photorhabdus .

76. Watereld NR, Wren BW, ffrench-Constant RH. 2004. Invertebrates as a source of emerging human pathogens. Nat. Rev. Microbiol. 2(10):83341

77. Watson RL, Joyce SA, Spencer GV, ClarkeDJ. 2005.The exbDgeneof Photorhabdus temperatais requifor full virulence in insects and symbiosis with the nematode Heterorhabditis . Mol. Microbiol. 56(3):763

78. Williams JS, Thomas M, Clarke DJ. 2005. The gene stlA encodes a phenylalanine ammonia-lyase th

involved in the production of a stilbene antibiotic in Photorhabdus luminescens TT01. Microbiology 1518):254350

79. Yang G, Dowling AJ, Gerike U, ffrench-Constant RH, Watereld NR. 2006. Photorhabdus virulecassettes confer injectable insecticidal activity against the wax moth. J. Bacteriol. 188(6):225461

80. Zhou A, Chen LM, Hernandez L, Shears SB, Galan JE. 2001. A Salmonella inositol polyphosphatase in conjunction with other bacterial effectors to promote host cell actin cytoskeleton rearrangements bacterial internalization. Mol. Microbiol. 39(2):24859


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Annual Review Microbiology

Volume 63, 200ContentsFrontispiece

Lars G. Ljungdahl xii

A Life with Acetogens, Thermophiles, and Cellulolytic Anaerobes Lars G. Ljungdahl 1

Regulation of Translation Initiation by RNA Binding ProteinsPaul Babitzke, Carol S. Baker, and Tony Romeo 27

Chemotaxis-Like Regulatory Systems: Unique Rolesin Diverse Bacteria John R. Kirby 45

Aminoacyl-tRNA Synthesis and Translational Quality Control Jiqiang Ling, Noah Reynolds, and Michael Ibba 61

Resurrected Pandemic Inuenza VirusesTerrence M. Tumpey and Jessica A. Belser 79

Interspecies Chemical Communication in Bacterial Development Paul D. Straight and Roberto Kolter 99

Lipid Signaling in Pathogenic Fungi Ryan Rhome and Maurizio Del Poeta 119

Biological Insights from Structures of Two-Component Proteins Rong Gao and Ann M. Stock 133

Role of GTPases in Bacterial Ribosome Assembly Robert A. Britton 155

Gene Transfer and Diversication of Microbial Eukaryotes

Jan O. Andersson 177 Malaria Parasite Development in the Mosquito and Infection

of the Mammalian Host Ahmed S.I. Aly, Ashley M. Vaughan, and Stefan H.I. Kappe 195

How Sweet it is! Cell Wall Biogenesis and Polysaccharide CapsuleFormation in Cryptococcus neoformans Tamara Lea Doering 223

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Mitochondrial Evolution and Functions in Malaria Parasites Akhil B. Vaidya and Michael W. Mather

Probiotic and Gut Lactobacilli and Bidobacteria: Molecular Approaches to Study Diversity and Activity Michiel Kleerebezem and Elaine E. Vaughan

Global Emergence of Batrachochytrium dendrobatidis and AmphibianChytridiomycosis in Space, Time, and Host Matthew C. Fisher, Trenton W.J. Garner, and Susan F. Walker

Anaerobic Oxidation of Methane: Progress with an Unknown Process Katrin Knittel and Antje Boetius

The Trypanosoma brucei Flagellum: Moving Parasitesin New Directions Katherine S. Ralston, Zakayi P. Kabututu, Jason H. Melehani, Michael Oberholzer, and Kent L. Hill

Plants, Mycorrhizal Fungi, and Bacteria: A Network of InteractionsPaola Bonfante and Iulia-Andra Anca

Evolutionary Roles of Upstream Open Reading Frames in MediatingGene Regulation in Fungi Heather M. Hood, Daniel E. Neafsey, James Galagan, and Matthew S. Sachs

Single-Cell Ecophysiology of Microbes as Revealed by Raman Microspectroscopy or Secondary Ion Mass Spectrometry Imaging Michael Wagner

Microbiology of the Atmosphere-Rock Interface: How BiologicalInteractions and Physical Stresses Modulate a Sophisticated Microbial Ecosystem Anna A. Gorbushina and William J. Broughton

What Sets Bacillus anthracis Apart from Other Bacillus Species? Anne-Brit Kolst, Nicolas J. Tourasse, and Ole Andreas kstad

The Expanding World of Methylotrophic Metabolism Ludmila Chistoserdova, Marina G. Kalyuzhnaya, and Mary E. Lidstrom

Genomics, Genetics, and Cell Biology of Magnetosome FormationChristian Jogler and Dirk Schler

Predatory Lifestyle of Bdellovibrio bacteriovorus Renee Elizabeth Sockett

Plant-Growth-Promoting RhizobacteriaBen Lugtenberg and Faina Kamilova

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Photorhabdus and a Host of Hosts Nick R. Watereld, Todd Ciche, and David Clarke 557

Management of Oxidative Stress in Bacillus Peter Zuber 575

Sociobiology of the MyxobacteriaGregory J. Velicer and Michiel Vos 599

Index

Cumulative Index of Contributing Authors, Volumes 5963 625

Errata

An online log of corrections to Annual Review of Microbiology articles may be found at http://micro.annualreviews.org/

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