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Photorhabdus and a Host of Hosts

Nick R. Watereld,1 Todd Ciche,2 and David Clarke3
1

Annu. Rev. Microbiol. 2009.63:557-574. Downloaded from www.annualreviews.org by Pontificia Universidad Javeriana on 09/17/13. For personal use only.

Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, United Kingdom; email: bssnw@bath.ac.uk Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan 48824; email: ciche@msu.edu Department of Microbiology, University College Cork, Ireland; email: david.clarke@ucc.ie

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 in a mutualistic association with a Heterorhabditis nematode worm. The nematode worm burrows into insect prey and regurgitates Photorhabdus, which goes on to kill the insect. The nematode feeds off the growing bacteria until the insect tissues are exhausted, whereupon they reassociate and leave the cadaver in search of new prey. This highly efcient partnership has been used for many years as a biological crop protection agent. The dual nature of Photorhabdus as a pathogen and mutualist makes it a superb model for understanding these apparently exclusive activities. Furthermore, recently identied clinical isolates of Photorhabdus are helping us to understand how human pathogens can emerge from the enormous reservoir of invertebrate pathogens in the environment. As Photorhabdus has never been found outside a host animal, its niche represents an entirely biotic landscape. In this review we discuss what molecular adaptations allow this bacterium to complete this fascinating and complex life cycle.

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Contents
INTRODUCTION . . . . . . . . . . . . . . . . . . PHOTORHABDUS . . . . . . . . . . . . . . . . . . . NEMATODE SYMBIONT. . . . . . . . . . . Metabolic Interactions . . . . . . . . . . . . . Transmission of Photorhabdus by H. bacteriophora Nematodes . . . Interkingdom Signal Production . . . . INSECT PREY . . . . . . . . . . . . . . . . . . . . . . Immune Challenge . . . . . . . . . . . . . . . . . Virulence Factors and Strategies . . . . Avoiding the Cellular Response . . . . . Toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Avoiding the Humoral Responses . . . OTHER MICROBES AS COMPETITION: RESOURCE PROTECTION . . . . . . . . . . . . . . . . . . . THE HUMAN HOST . . . . . . . . . . . . . . . THE POSTGENOMIC ERA . . . . . . . . . CONCLUSIONS . . . . . . . . . . . . . . . . . . . . 558 558 560 560 560 562 563 563 563 564 564 566

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567 567 569 569

INTRODUCTION
Photorhabdus is a gram-negative member of the family Enterobacteriaceae that lives in a symbiotic association with an entomopathogenic Heterorhabditis nematode worm (EPN) in a highly effective symbiosis of pathogens (29). Photorhabdus is the only known bioluminescent terrestrial bacterium; the name means glowingrod. In the soil Photorhabdus resides in the gut of an infective juvenile (IJ) form of the nematode. The IJs actively seek out insect prey in the soil, whereupon they penetrate the body, either directly or through natural openings. Once inside the insects open blood system, the hemocoel, the nematodes regurgitate 50200 bacterial cells directly into the blood, or hemolymph (12). These few bacteria are sufcient to overcome the insects immune system and set up lethal septicemia. Once the insect host is dead the bacteria bioconvert the tissues into more bacteria. The nematode partner feeds off the bacteria and nematode reproduction continues through several generations. Although not yet
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established, it is likely that environmental conditions (such as availability of food or an unknown signal) stimulate the development of IJs containing their symbiotic bacteria. Up to 400,000 IJs may leave a single insect cadaver 1014 days postinfection in search of new insect prey (29). The niche in which Photorhabdus has evolved represents an entirely biotic landscape in which it is challenged by the immune systems of nematode host and insect prey. Furthermore, Photorhabdus must also successfully compete with saprophytic scavenging organisms, such as protists, fungi, nematodes, other bacteria, and even other insects, that inevitably try to utilize the tissues of the dead insect host. In addition to its well-described role as an insect pathogen one species of the genus, Photorhabdus asymbiotica (Pa), causes infection in otherwise healthy humans (25, 33, 36). Pa is also in a symbiotic relationship with a heterorhabditid nematode, Heterorhabditis gerrardi, and like other EPNs can also complete its life cycle in insects (34, 55). A detailed understanding of how Pa can infect both insect and human hosts should provide major insights into the evolution of bacterial pathogenicity and also the emergence of human pathogens (76).

EPN: entomopathogenic bacteria/nematode complex Pathogen: a microorganism that causes disease IJ: the infective juvenile developmental form of an EPN Hemocoel: an insects open blood system Hemolymph: insect blood Pa: Photorhabdus asymbiotica Pl: Photorhabdus luminescens Pt: Photorhabdus temperata

PHOTORHABDUS
EPNs can be found in soils worldwide, and it is a simple matter to recover them in the laboratory by baiting soil with insects. In 1999, Fischer-Le Saux et al. (28) classied Photorhabdus into three species on the basis of phenotypic characterization and DNA relatedness: P. luminescens (Pl ), P. temperata (Pt), and P. asymbiotica (Pa). Limited microarray analysis (52), multilocus sequence typing (33), and recent genomic studies (20) have conrmed this classication. The Pa species is further subdivided into at least two highly clonal subspecies, one found in the United States and the other in Australia. In addition, two European strains, HIT and JUN, isolated from nematodes potentially form a third subspecies of Pa (Figure 1). Recently, Pa strains associated with nematodes have also been reported from Japan, and it is

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becoming clear that they are not restricted to the continents of North America and Australia (50). Nevertheless, it remains to be seen if all members of this species are capable of causing clinical infections. Photorhabdus species are highly motile, bioluminescent, facultatively anaerobic rods (54). All species grow well at 28 C on blood agar, where they uniquely produce a thin line of annular hemolysis. Critically, the clinical isolates also grow at temperatures ranging from 37 to 42 C. Recent work has shown that although motility is not required for either pathogenicity or symbiosis, it does confer a competitive advantage during colonization of the insect (21). A unidirectional phenotypic variation has been described for Pl and Pt (4). Continued in vitro cultivation in nutrient-limited conditions leads to the generation of secondary variant (P2) forms that are still pathogenic to insects but no longer support symbiosis with the nematode. They also show a change in the pattern of protein, light and metabolite production. The primary variant (P1) is the symbiotically competent form that is isolated from EPNs (45). With this in mind Joyce & Clarke (45) used P1 and P2 variants to provide clues regarding the molecular basis behind symbiosis and pathogenicity. They identied a gene with homology to hexA from Erwinia that represses symbiosis factors in P2, suggesting it is a key player in the regulatory network controlling pathogenicity and mutualism (45, 46). More recently, two studies aimed at elucidating the mechanisms behind this P1/P2 switch used microarray and proteomic approaches, although the precise mechanism still remains elusive (31, 64). Bioluminescence occurs in all strains on agar plates 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 and tissue degradation is at an advanced stage (15). This bioluminescence has been hypothesized to (a) represent a signal from bacteria to bacteria or to the nematode, synchronizing symbiosis; (b) provide a visual aposematic warning to nocturnal scavenging animals; or even (c) act a lure

Prey
W14 TT01

P. luminescens (insect)

Hm/Hb

HIT/JUN ATCC43949 (USA)

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P. asymbiotica (human + insect)

Kingscliff (Australia)

K122

P. temperata (insect)

Figure 1 A schematic representation of the multilocus sequence typing phylogeny of the genus Photorhabdus. Model type strains of three different species are indicated in their different clades. P. asymbiotica contains at least two subclades, representing U.S. and Australian isolates. A third clade comprising the HIT and JUN isolates from mainland Europe conrms these genotypic forms are not restricted to the two continents.

to attract further insect prey to the infected insect cadaver. The luciferase reaction is highly efcient at utilizing molecular oxygen, and it has also been suggested that the primary role for bioluminescence is as a sink for molecular oxygen. This would potentially starve the host immune cells of a resource required for the production of free radicals that could otherwise be used in their phagocytic destruction. Several observations argue against this role, however. First, strong light emission occurs only after insect death (although it is possible that light production can occur at a local level during the early stages of infection). Second, most Photorhabdus species resist phagocytic uptake
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Mutualism: a benecial relationship between different species

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anyway. Furthermore, if this represented a general mechanism to evade phagocytic destruction, then it is unlikely to have been restricted to Photorhabdus only and we would expect to see many more bioluminescent terrestrial pathogens. Finally, most bioluminescent bacteria known to date are associated with animal symbiosis, suggesting a cryptic underlying selective force.

NEMATODE SYMBIONT Metabolic Interactions

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Most bacteria-host interactions involve some level of metabolic interaction. In the environment, the Heterorhabditis-Photorhabdus interaction appears to be obligate, although the bacteria can be readily cultured in vitro using standard peptone-based media. Moreover, the nematodes can be cultured in vitro by inoculating Photorhabdus biomass grown on agar plates with either IJs or eggs. The nematodes will not grow or develop on Photorhabdus cultured on Luria-Bertani agar, yet they grow readily on the same bacteria cultured on lipid agar (nutrient agar supplemented with cod liver oil and corn syrup). 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 the development of the bacteriophorous nematode Caenorhabditis elegans, and it is likely that Heterorhabditis will have the same requirement (8). In this regard, Photorhabdus produces a signicant amount of iso-branched fatty acids (BCFAs), with isoC15:0 being the dominant fatty acid found in Photorhabdus phospholipids produced by the bkdABC operon (44). The production of BCFA is not widespread in the Enterobacteriaceae and the presence of BCFA in Photorhabdus may represent an adaptation to symbiosis. BCFAs play an important role in the development of C. elegans, in which the nematode itself encodes the genes required for its production (48, 49). One interesting possibility is that Photorhabdus may have taken on the responsibility of producing BCFAs required by their nematode partner.
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Photorhabdus also produces large quantities of crystalline inclusion proteins (CipA and CipB) that have a potential role in nematode nutrition (3). Mutants in cipA and cipB are unable to support nematode growth and development, although they are pleiotropic, making it difcult to denitively dene an exact role in symbiosis for these proteins (3). An interesting aspect of the metabolic interaction between Photorhabdus and the nematode is that Heterorhabditis will not grow on just any strain of Photorhabdus, rather it usually requires its specic cognate bacterial strain. The molecular basis of this nutritional specicity remains to be elucidated but some recent work has implicated a role for iron (77).

Transmission of Photorhabdus by H. bacteriophora Nematodes

Photorhabdus bacteria are transmitted by the specialized IJ stage of the nematode, which harbors them in the intestinal lumen as it disperses from an insect cadaver in search of a new insect host (9, 12). The IJ is an alternative third larval stage of the nematode that is developmentally arrested and resistant to environmental stresses. IJs are formed inside the maternal nematode body cavities, causing matricide, a process termed endotokia matricida (9, 12), and indicating that Photorhabdus is maternally transmitted by an elaborate sequence of selective events (13). The transmission cycle starts when an IJ enters an insect and regurgitates the Photorhabdus into the insect hemocoel (13). Every bacterial cell is released into the hemocoel, perhaps reecting the challenge they face from the insect immune system (12). After regurgitation, the nematodes switch to feeding behavior and ingest symbiont bacteria, a few of which adhere to the posterior nematode intestine (Figure 2). Pulse-chase experiments revealed that the adherent cells formed a persistent infection as a biolm while other symbiont cells were transiently present in the intestine. After persisting for up to 38 h, the adherent bacteria invade three rectal gland cells, where they multiply

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11
10 m

10
t = 136 h Pharyngeal-intestinal valve cell invasion

t = 136240 h Exit and symbiont growth in the intestinal lumen

t=0h Infective juvenile (IJ)

Nematode

Bacteria
10 m

t=4h Regurgitation of intestinal symbionts

9
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F 1) g( in

2
t=6h Complete symbionts release

Off sp r

5 m

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

Photorhabdus transmission cycle

0)

10 m

t=8h Symbiont adherence and worm feeding

4
10 m

al er n t a M
10 m

t = 112 h Symbionts released into pseudocoelom

(P

t = 36 h Biofilm formation and adherence

6
5 m

5 m

t = 72 h Symbiont and vacuole have multiplied t = 48 h Intracellular symiont growth

t = 42 h Invasion of rectal gland cells

Figure 2 Transmission cycle of Photorhabdus luminescens subsp. laumondii in a nematode host. t = hours post-infective juvenile (IJ) addition to symbiont 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 of the nematode 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 additional symbionts adhere. Step 5: Invasion of the rectal gland cells by adherent symbionts. Steps 6 and 7: Intracellular growth of symbiont bacteria and vacuole replication. Step 8: Lysis of the apical side of the rectal gland cells releasing symbionts to pre-IJs developing in the maternal body cavity. Step 9: Symbiont adherence to the pharyngeal-intestinal valve cell. Step 10: Invasion of the pharyngeal-intestinal valve cells. Step 11: Exit and growth of symbiont cells in the IJ intestinal lumen.

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Virulence factor: a molecule produced by a pathogen that damages the host ST: stilbene molecule

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inside membrane-enclosed vacuoles for 48 h as the nematode progeny hatch and develop in the maternal body cavity. Intracellular symbionts are released to the IJs by apical lysis of the rectal gland cells, while the intestine remains intact and is relatively devoid of other intestinal symbionts (13). Less than 24 h later, usually a single symbiont cell adheres to the pharyngeal intestinal valve cells of the IJ offspring. The adherent cell(s) divides and appears to invade the valve cells before exiting and colonizing the intestinal lumen, where they persist in a presumably semidormant state before a new insect host is found. Thus, transmission of Photorhabdus by Heterorhabditis is an elaborate process involving cell-specic infection of maternal and offspring nematodes. Photorhabdus transmission by H. bacteriophora nematodes involves many events shared with pathogens that cause disease (e.g., cell-specic adhesion/invasion). It is likely that Photorhabdus employs effectors related to virulence factors in pathogens for adhesion, invasion, and intracellular growth in animal cells. A mbrial operon essential for Photorhabdus to colonize the maternal intestine and the stringent starvation protein A regulator, sspA, required for postadhesion survival in the IJ have recently been identied (T. Ciche, unpublished data). Determination of these and other factors for transmission and pathogenesis is likely to illuminate common and distinct processes involved in mutualism and infectious disease.

Interkingdom Signal Production

Recovery is the rst step in nematode development, and it refers to the postinfection recovery of the IJ into a self-fertile adult hermaphrodite. In the insect, IJ recovery is normally stimulated by uncharacterized molecule(s) present in the insect hemolymph (12, 18). There is some evidence that the neurons and signaling pathways involved in IJ recovery may be similar to those required for dauer recovery in C. elegans (39). Laser ablation of the ASJ chemosensory neuron in Heterorhabditis IJs and treatment with known
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inhibitors of muscarinic acetylcholine receptors and cGMP signaling pathways prevented IJ recovery in vitro (39). During in vitro culturing on Photorhabdus, IJ recovery is independent of insect signals but requires a food signal produced by the bacteria (1, 62). This presumably indicates to the nematode that there are enough bacteria present to support its growth and development. Recently, a component of this food signal was identied as a stilbene (ST) called 3,5-dihydroxy-4-isopropylstilbene, a major secondary metabolite produced by Photorhabdus (44). The recovery of IJs inoculated onto a lawn of Pl TT01 cells mutated in ST production is only 515% of that observed with wild-type Pl TT01 bacteria (44). However, although ST is required for IJ recovery, it is not sufcient by itself and viable Photorhabdus must also be present (44). In Pt NC19, the phosphopantethienyl (PPANT) transferase gene ngrA is required to support nematode growth reproduction (10). PPANT transferases are important for the synthesis of many secondary metabolites and the ngrA mutant is also defective in producing at least a siderophore and ST. The siderophore was determined not to be required for nematode growth (11), suggesting again the importance of the ST molecule. Nevertheless, recovered IJs that reached adulthood also failed to reproduce efciently on the ngrA mutant, suggesting that additional secondary metabolites are required for normal nematode growth and reproduction (1, 10). STs are polyketide molecules often produced by plants in response to stress and infection, and Photorhabdus is the only nonplant organism known to produce a stilbene. The biochemical pathway responsible for ST production in Photorhabdus has been characterized and is signicantly different from that observed in plants (44, 78). In addition we have recently described the characterization of a locus ( plu4185-plu4195) involved in the production of a polyketide molecule, anthraquinone, responsible for the yellow/orange pigment produced by Pl TT01 (5). Importantly, this is only the second report of Type II PKS activity in gram-negative bacteria, highlighting the

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potential for novel and/or unusual PKS-related biochemistry in Photorhabdus. The 5.5 Mb genome of Pl TT01 has been sequenced (http://genolist.pasteur.fr/PhotoList/), and analysis reveals that nearly 6% of the genome is given over to genes predicted to be involved in the production of secondary metabolites (20). This proportion is greater than the 3.8% observed in Streptomyces, the model organism for secondary metabolite production (and the source of >90% of clinically important antibiotics). Therefore, there is signicant potential in Photorhabdus for the production of novel bioactive molecules. A novel virulence mapping technique termed RVA, discussed below, has provided the rst step in isolating and characterizing a number of these secondary metabolites (75).

the insect immune system is the melanization and encapsulation response mediated by phenoloxidase (PO) (37). This system represents a cascade of serine protease signaling molecules initiated by PAMP recognition and terminating in the cleavage of pro-PO to active PO. This enzyme deposits melanin onto invading organisms, encapsulating them and inicting free radical damage in the process. In Manduca sexta, melanization typically occurs around groups of hyperphagocytic hemocytes, forming a nodule on the surface of tissues (17). In many ways the PO system is reminiscent of the complement cascade of vertebrates, and it is possible that they share a common evolutionary origin (30, 38). Photorhabdus has evolved multiple virulence factors and strategies to deal with immediate immune threats and to kill the host, which we discuss below.

PAMP: pathogenassociated molecular pattern PO: phenol oxidase Hemocyte: an insect immune cell

INSECT PREY Immune Challenge

The EPN complex shows little specicity in its choice of insect prey species. Furthermore, the release of at most only 50200 bacteria in a normal infection means that the bacteria are adapted to be highly virulent and can resist immune systems from diverse genetic backgrounds. These requirements appear to be reected in the genome of Pl TT01, which contained a greater number and diversity of toxin genes than any other sequenced genome at the time (20). Upon release into the insect hemocoel, Photorhabdus bacteria face the fast-acting innate immune system (41). The innate immune systems of insects and mammals share many common features (47), the implications of which become obvious when considering the evolution of Pa, which is both an insect and human pathogen. Innate immune factors include cellular responses, such as phagocytic destruction using free radicals and killer proteases, and humoral responses, including recognition of pathogenassociated molecular patterns (PAMPs) by receptors and the production of antimicrobial peptides (AMPs) and lysozyme. A mainstay of

Virulence Factors and Strategies

Two model insects most commonly used to study Photorhabdus are the large lepidopteran hosts Galleria mellonella (greater wax moth) and Manduca sexta (tobacco hornworm). More recent studies have used the dipteran host Drosophila melanogaster, which has the advantage of a fully sequenced genome and welldeveloped genetic tools (39, 47, 65, 67). Prior infection of M. sexta larvae with nonpathogenic Escherichia coli elicits effective immunity against subsequent infection with the otherwise lethal Pl TT01. This protection correlates with the upregulation of PAMP molecules such as hemolin, immulection-2, and peptidoglycan recognition protein, and also AMPs including attacin and cecropin. Preventing the upregulation of these PAMP molecules with RNAi abolished this protection, whereas suppression of specic AMP production had little effect (23). This suggests that M. sexta can in fact ght off a Photorhabdus infection if given a head start. Other studies have conrmed that M. sexta recognizes a Photorhabdus infection, as seen by an increase in mRNA for PAMPs, but that Photorhabdus can nevertheless overcome this response (24).
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Avoiding the Cellular Response

TTSS: Type three secretion system Macrophage: a mammalian professional phagocytic immune cell PVC: Photorhabdus virulence cassette

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A detailed study by Silva et al. (61) followed the fate of Pl W14 after injection into M. sexta larvae. Following injection the bacteria multiplied rapidly both on the midgut and in the hemolymph (61). Colonization of the blood side of the midgut is initiated at the anterior and then proceeds posteriorly along its length as infection time continued. Electron microscopy revealed that the bacteria occupy a specic niche between the extracellular matrix and basal membrane of the midgut epithelium, in tight grooves made by infolding of the epithelium. Although this site may be difcult for the hemocytes to enter because of their size, not all the bacteria reside here, and all are open to hemocyte attack, at least during transit to the gut. Several toxin systems that disable or kill the hemocytes have been characterized.

Toxins
Immunohistochemistry revealed that within the gut niche the bacteria express at least two virulence factors, the highly secreted gut-active toxin complex A (Tca) and a metalloprotease, PrtA (15, 61). Close association of bacteria with the epithelium, and secretion within such an enclosed niche, may facilitate the rapid destruction of the gut, which is a prominent feature of Photorhabdus infection. All strains of Photorhabdus encode multiple homologues of the Tc genes, suggesting they play an important role in the biology of the organism, although the functions of these homologues remains obscure in most cases (27, 68, 69, 72). Another gut-active toxin produced by all strains of Photorhabdus is the multidomain Mcf1 toxin. This potent toxin is taken up into target cells by endocytosis, where it induces apoptosis by a novel BH3-domain-mediated effect (16, 19). This protein toxin has little cell specicity and can also induce apoptosis in hemocytes and a range of cultured human cells, suggesting multiple roles in infection. The mcf1 gene is present in all strains so far examined but appears to be encoded at different genomic loci in each case, suggesting independent acquisi564 Watereld

tion (71). At least two of the Pl strains, W14 and TT01, also have a homologue called Mcf2. This protein differs from Mcf1 in the toxin active site domain but is also believed to induce apoptosis because it possesses homology to a proapoptotic Pseudomonas toxin, HrmA (73). Like many pathogenic gram-negative bacteria, Photorhabdus encodes a type three secretion system (TTSS). In Pl TT01, the TTSS is involved in attachment to hemocytes and in delivery of at least one effector molecule, LopT, into these cells, preventing their phagocytic uptake. LopT is a close homolog of YopT from Yersinia pestis, in which it also serves to inhibit phagocytosis by macrophages. This shows a striking conservation of immune avoidance mechanisms in a mammalian or insect pathogen (7). Other TTSS effector molecules are yet to be identied in Pl TT01. The TTSS operon of the recently sequenced clinical strain Pa ATCC 43949 does not encode the lopT gene, instead it encodes a close homologue of ExoU, a powerful TTSS-delivered Pseudomonas cytotoxin (26). Furthermore, a subtractive DNA hybridization study between Pa and Pl TT01 identied a sopB gene homologue in the clinical strain (63). SopB in Salmonella is a TTSS-delivered effector involved in intracellular persistence in human macrophages (80). A range of TTSS effector-like molecules can be seen in the genomes of Photorhabdus strains; however, these are associated with an alternative secretion system called Photorhabdus virulence cassettes (PVCs). PVC genomic islands supercially resemble prophage and consist of around 15 genes, conserved between all PVC elements, homologous to either phage or other macromolecular assembly genes. At the 3 end of each conserved region is a payload region that encodes one or more genes that typically show homology to the active sites of known toxins, reminiscent of TTSS effectors (74, 79) (Figure 3a). There are multiple, diverse PVC elements in all Photorhabdus genomes so far examined, suggesting they are mobile and acquired by horizontal transfer (Figure 3b). When heterologously expressed from cosmid clones in E. coli, the PVC elements

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a
Phage tail VgrG Flagella M-ring

Conserved region
Transcript regulation Adenovirus fiber AAA-ATPase

Payload region

sepC-like

pnf

Pa PVC pnf
Baseplate Fimbrial usher

afp1

afp16 afp17

afp18

Se afp

Open reading frames

Putative toxin gene

5 kb

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b
P. luminescensTT01
tcd-like sepC-like Type 4 pil PtPVC unit1 PtPVC unit2 mcf-like PtPVC unit3 PtPVC unit4 phxA' mukB toxA-like PtPVC lopT dnt-like lopT cif-like PtPVC cif hvnA hvnA phxA

P. asymbioticaATCC 43949
sepC-like toxA-like PaPVC lopT dnt-like lopT tccC-like phxA cif-like PaPVC cif mukB Type 4 pil PaPVC phx

PaPVC pnf pnf sepC-like

Conserved region Toxin gene

PaPVC lumt lumt

10 kb

Figure 3 (a) A schematic representation of a typical Photorhabdus virulence cassettes (PVC) element (PVCpnf ). Open boxes represent open reading frames (ORFs). Note the conserved region common to all PVC elements and the variable payload region with putative toxin genes shown in red. The homologous Serratia entomophila (Se) afp element is shown, and similar ORFs have been color coded. The thick red arrows below the afp diagram indicate where insertional mutagenesis abolished toxic activity, while the thin blue arrows are mutants 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 tandem arrangement 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 green boxes and the toxin genes in red. The names or closest homologues of payload genes are indicated.
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Entomopathogen: an insect pathogen

produce a large 250-nm-long structure similar to R-type pyocins, which are bacteriophage tails adapted to kill related strains of bacteria. Like R-type pyocins, the PVC elements consist of an inner stylet and an outer sheath that can be seen in a relaxed or contracted injection form. No antibacterial activity has been associated with PVC elements, but they do have potent toxicity when injected into Galleria larvae (79). Larvae injected with the puried PVCpnf element melanize and die within minutes, with a reduction in circulating hemocytes showing extensive actin rearrangement (79). Recent work in our lab has conrmed that PVC element toxins are expressed during insect infection and that the PVC structures can attach to the surface of hemocytes using SEM (N.R. Watereld, unpublished data). Our current hypothesis is that the PVC elements resemble a freely secreted molecular syringe that directly injects the payload toxin into the host cell, reminiscent of a released TTSS. Despite the significant numbers of pathogen genomes now sequenced, the only other bacteria so far seen to encode this system is another insect pathogen, Serratia entomophila. In Serratia this is known as the antifeeding prophage, which causes larvae of Costelytra zealandica (New Zealand grass grub) to cease feeding (Figure 3a). In this case the AFP element is presumably active against gut cells rather than hemocytes (43). In Photorhabdus the PVC elements are tightly regulated at the translational level and not expressed at 37 C, suggesting they may be inappropriate in vertebrate infections. Strains of Pl and Pa encode binary toxin systems, which comprise two proteins required for toxicity. These include the PirA and PirB proteins (20, 60) and proteins homologous to XaxA and XaxB, rst identied in the closely related entomopathogen Xenorhabdus nematophila (66, 75). The PirAB toxins exhibit a range of activity including potent injectable activity against Galleria larvae; oral activity against the caterpillar pest Plutella xylostella; and oral toxicity against three different species of mosquito, Aedes aegypti, Culex pipiens, and Anopheles gambiae (20, 70). XaxAB has cytolytic, hemolytic,
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and proapoptotic effects on insect and mammalian cells. Finally, a range of homologues of two partner hemolysin genes, such as PhlAB, can also be seen in the two completed Photorhabdus genomes that have cytotoxic effects (6, 75).

Avoiding the Humoral Responses

The secondary metabolite ST is also a potent inhibitor of the PO enzyme, allowing Photorhabdus to avoid the melanization response (22). Conversely, Pl TT01 also secretes a protease, PrtS, during infection that strongly induces melanization (40). The signicance of this is not yet clear, although premature and nonlocalized activation of the PO system could also represent a virulence strategy. Other humoral factors that Photorhabdus must resist include circulating lysozyme and AMPs. Although Photorhabdus induces the transcription of AMP mRNA, it is not known whether the bacterium is resistant to AMPs specically or whether it blocks translation or secretion of the peptides. Pl TT01 cells carrying a mutation in the pbgPE operon (predicted to be responsible for the modication of LPS with l-aminoarabinose in response to AMP) are avirulent, suggesting that Photorhabdus does in fact adapt to AMP production during infection (2). Finally, all animals attempt to limit bacterial replication through the tight binding and compartmentalization of iron. Like many animal pathogens, Photorhabdus has several iron acquisition systems including the production of the siderophore photobactin (11). Furthermore, a mutation in the exbD gene of Pt K122 resulted in a strain that was severely limited in its ability to acquire iron. This mutant was attenuated in virulence and this attenuation was correlated with the growth rate of the bacteria in the insect (77). The attenuation could be rescued by preloading the insect with iron. Indeed the LT50 (time taken to kill 50% of the insects) of wildtype Pt K122 was reduced in insects preloaded with iron, suggesting that iron is indeed limiting in the insect (77).

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OTHER MICROBES AS COMPETITION: RESOURCE PROTECTION

One of the major challenges for EPN symbiosis is the maintenance of a monoxenic infection in an insect cadaver in the soil. For this reason, Photorhabdus expresses a range of antimicrobial factors. The ST molecule also constitutes a broad-spectrum antibiotic showing strong antibacterial and antifungal properties (51, 57). In addition to small-molecule antibiotics, Photorhabdus produces a range of proteinaceous antibacterial molecules including S- and R-type pyocins. S-type pyocins constitute toxic proteins capable of degrading macromolecules in target cells. A small cognate immunity protein protects the cell during biosynthesis, which is later shed during secretion. A range of S-type pyocins characterized in Pl W14 called lumicins show activity against other strains of Photorhabdus and even E. coli (59). R-type pyocins are much larger, modied bacteriophage tail structures used to kill closely related strains that can also be seen in transmission electron micrographs of particulate material from Photorhabdus culture supernatants. Genomic loci in the completed sequences of both Pl TT01 (32) and Pa ATCC 43949 (N.R. Watereld, unpublished data) have been identied, conrming they are related to the phage P2 family of R-type pyocins. Both strains encode a DNA invertase and have regions bounded by inverted repeats containing different tail ber genes, suggesting a phase variation system evolved to increase the target host range. To date little work has addressed how Photorhabdus combats scavenging organisms such as nematodes and amoeba. Sicard et al. (60) demonstrated that two strains of Pl reduced survival rates of C. elegans. They showed that C. elegans avoided the two pathogenic strains but not a third nonpathogenic strain of Pl, although it is not clear how the nematode makes this distinction (60). Further, the puried ST and an indole produced by Pl HD have strong nematicidal effects on a range of nematodes,

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including C. elegans, but not on Heterorhabditis. ST also repels some Steinernema species (42). Recently we have been studying the interaction between Photorhabdus strains and the free-living soil amoeba Acanthamoeba polyphaga. We have demonstrated that the amoebae cannot reproduce and grow on either Pl TT01 or Pa ATCC 43939 even when on suitable rich agar medium (PYG). However, time-lapse microscopy revealed that the amoebae are not directly killed by the bacterium under the conditions tested. If grown at 28 C, both species of Photorhabdus swim up to the amoeba and attach as a small microcolony (Figure 4a). Furthermore, we see no phagocytic uptake or invasion of the amoeba by Pl TT01, suggesting that Photorhabdus prevents uptake as it does in insect hemocytes (7) (Figure 4b). Finally, it is possible that Photorhabdus may also attempt to protect the insect carcass from larger scavenging animals. In vivo expression studies in M. sexta showed that the highly toxic Tcd complex is expressed by Pl W14 early in infection in low amounts and that the highly expressed Tca is produced only after death (15). This nding suggests that the orally toxic Tca may be adapted for deterring scavengers such as ants rather than killing the insect host.

THE HUMAN HOST

Pa was rst isolated only from clinical infections, with 14 cases reported in the literature from the United States and Australia (25, 34, 35, 54). These cases appear to form geographical clustersVictoria and Southeast Queensland in Australia and San Antonio, Texas, in the United States. It is not yet clear, however, whether this means that there is a specic environmental source for the infection in these areas or simply that laboratories in these areas have become familiar with the identication of the pathogen. The true incidence of human infection is likely much higher than these reports would suggest, as Pa is not yet included on the standard clinical bacteriology databases (34). We recently conrmed that a clinical

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Figure 4 Images on the left are bright-eld illumination and those on the right are under uorescence illumination. (a) GFP-labeled Pl TT01 attached to an Acanthamoeba polyphaga cell. The microcolony shown (arrow) is not the result of bacterial growth, rather the bacteria rapidly swim up to the motile amoeba cells and attach at the rear of the amoeba (which is moving upward in the image shown). This was imaged as a real-time movie using an inverted microscope with the bacteria and amoeba on a thin layer of agar on a glass slide. (b) Despite prolonged and intimate exposure, Pl TT01 neither invades the amoeba nor is it taken up by phagocytosis. Note no GFP-labeled bacteria can be seen inside the amoeba (arrow).

isolate of Pa from a patient in Kingscliff, Australia, also exists in a symbiotic partnership with a Heterorhabditis nematode, H. gerrardi (34, 55). We do not know how the H. gerrardi EPN penetrated the patients skin or if the nematode also entered the patients body, so the exact mechanics of infection remains elusive. Most clinical cases are associated with infection during outdoor activity, and the primary foci of infection on limb extremities support contact with soil as the primary route of infection. At least in Australia the incidence of infection appears to correlate with warm, wet conditions

( J. Gerrard, personal communication). In support of this, we recently showed an absolute correlation between infection rate of G. mellonella and moisture content of sand that had been inoculated with the Kingscliff EPN complex. A ratio of 5 ml water in 50 g of sand gave a 100% infection rate, while 0, 2, 10, and 20 ml of water gave no productive infections (N.R. Watereld, unpublished data). We suggest this nding provides an excellent experimental system to model the effects of climate change on emerging infections. Pa infections represent a primary pathogen, not an opportunistic secondary colonizer of existing wounds. There are no apparent predisposing factors to infection such as age, gender, or other common disease states. Most cases present with either locally invasive or disseminated soft tissue infection with a typical clinical descriptor of a painful necrotic ulcer. In addition, many of the patients showed the appearance of soft tissue infection at sites remote from the initial infection, indicating bacteremic spread. Consistent with this is the observation that Pa has been isolated not only from pus at the initial foci of infection but also from blood and even sputum in one case. The infection also can exhibit a relapsing course extending over several weeks even when appropriate antibiotics are administered (33). The Kingscliff Pa strain exhibited only weak bioluminescence in liquid culture at 30 C but strong light emission at 37 C, suggesting the intriguing yet not investigated possibility that the soft tissue lesions could in fact glow (55). Although no mammalian models yet exist, we have used tissue culture to investigate the interaction between Pa and macrophage cell lines. Our ndings (N.R. Watereld & I. Vlisidou, unpublished data) and those of Costa et al. (14) conrm that Pa is a facultative intracellular pathogen that replicates inside mouse and human macrophage-like cells. This is supported by the presence of a homologue of the sopB effector in the clinical isolates that is absent from Pl. Costa et al. conrmed that Pa invades insect hemocytes during infection, a phenotype

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not seen in the other two members of the genus. The Australian isolates of Pa also invade nonphagocytic cell lines and induce apoptosis in cells faster than the U.S. isolates (14). This ability to invade immune cells suggests a route of bacteremia spread in the clinical cases and would provide the bacteria with a niche inaccessible to many of the systemic humoral immune responses of the mammalian host. This parallels the related pathogen, Y. pestis, which is also a facultative intracellular pathogen that induces apoptosis in macrophages, silences inammatory reactions, and allows dissemination through the lymphatic system (56). Finally, we have recently demonstrated that both Pa and Pl TT01 are inherently resistant to killing by commercially available human serum, suggesting that a defensive mechanism adopted by the insect-only pathogens may be equally effective against human-complement-mediated killing (N.R. Watereld, unpublished data).

THE POSTGENOMIC ERA

The availability of completed genome sequences for Pl TT01 (20) and Pa ATCC 43949 (United States) (58) has ushered in a new era in the study of Photorhabdus. Furthermore, a Heterorhabditis genome is also currently being sequenced, which will greatly assist the study of the symbiotic interaction. We have now completed a full in silico genomic comparison between Pa ATCC 43949 (United States) and Pl TT01 that awaits publication (N.R. Watereld, P. Wilkinson, L. Crossman, C. Corton, M. Sanchez Contreras, et al., unpublished data). Briey, the human pathogenic strain has a smaller genome with reduced toxin diversity. In addition both strains possess a range of different genomic islands, suggestive of signicant levels of horizontal gene transfer and differential host ranges (20). In an attempt to map genes induced upon ex posure to insect homogenates, Munch et al. (53) identied 24 Pl TT01 promoters that were induced in G. mellonella infection. These

included promoters that drive expression of the tc toxin components tccC1 and tcdA1; PVC payload genes plu2400, plu1672, and plu1645; the mcf1 toxin; secondary metabolite genes; and promoters for a range of metabolic operons (53). Finally, we recently used Pa as a model of an emerging human pathogen to develop an assumption-free approach for mapping virulence factors across whole pathogen genomes. This approach, termed rapid virulence annotation (RVA), screens large insertion genomic libraries for toxicity against four different taxa: the protist A. polyphaga, the nematode C. elegans, the moth M. sexta, and a mouse macrophage cell line ( J774-2). RVA successfully identied a large number of virulence loci through their gain 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 hard to detect. The identication of a range of potent secondary metabolite biosynthetic operons in these toxicity screens has also provided an excellent way to understand the role of these small molecules in infection, and presented an obvious route for novel drug discovery. We saw no obvious mammalian-only toxins, supporting the hypothesis that virulence factors evolved against insect hosts may be readily redeployed against humans.

CONCLUSIONS
Photorhabdus provides an excellent model for the study of both symbiosis and pathogenicity. In addition, with Pa now recognized as both an insect and human pathogen, it also provides a superb opportunity to understand the role of invertebrates in the emergence of human diseases. In the future, the availability of genome sequences and the ability to genetically manipulate the bacteria, the nematode partner, and the host insects will ultimately enable us to understand the molecular interplay between pathogenicity 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 strain specic. 2. Pl and Pt have not been found outside either the nematode or the insect hosts and so are adapted only for survival in these biotic environments. 3. Genome sequences have conrmed a large repertoire of genes involved in host interactions, especially genes encoding putative toxins. 4. P. asymbiotica is capable of causing infections in humans, providing an excellent model of an emerging human pathogen.
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5. Photorhabdus must also compete with a wide range of saprophytic microorganisms from the 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, the symbiont 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 holdings that might 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. DJC and NRW thanks the Leverhulme Trust, BBSRC, and SFI for support. TAC thanks the Center for Microbial Pathogenesis at Michigan State University and the USDA for support. LITERATURE CITED
1. Aumann J, Ehlers R-U. 2001. Physico-chemical properties and mode of action of a signal from the symbiotic bacterium Photorhabdus luminescens inducing dauer juvenile recovery in the entomopathogenic nematode Heterorhabditis bacteriophora. Nematology 3:84953 2. Bennett HP, Clarke DJ. 2005. The pbgPE operon in Photorhabdus luminescens is required for pathogenicity and symbiosis. J. Bacteriol. 187(1):7784 3. Bintrim SB, Ensign JC. 1998. Insertional inactivation of genes encoding the crystalline inclusion proteins of Photorhabdus luminescens results in mutants with pleiotropic phenotypes. J. Bacteriol. 180(5):126169 4. Boemare RJ, Akhurst NE. 1988. Biochemical and physiological characterization of colony form variants in Xenorhabdus spp. (Enterobacteriaceae). J. Gen. Microbiol. 134:75161 5. Brachmann AO, Joyce SA, Jenke-Kodama H, Schw ar G, Clarke DJ, Bode HB. 2007. A type II polyketide synthase is responsible for anthraquinone biosynthesis in Photorhabdus luminescens. Chembiochem 8:172128 6. Brillard J, Duchaud E, Boemare N, Kunst F, Givaudan A. 2002. The PhlA hemolysin from the entomopathogenic bacterium Photorhabdus luminescens belongs to the two-partner secretion family of hemolysins. J. Bacteriol. 184(14):387178
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7. Brugirard-Ricaud K, Duchaud E, Givaudan A, Girard PA, Kunst F, et al. 2005. Site-specic antiphagocytic function of the Photorhabdus luminescens type III secretion system 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. 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 is essential for Photorhabdus luminescens to support growth and reproduction of the entomopathogenic nematode 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 NC1 nematodes. Appl. Environ. Microbiol. 69(8):470613 12. Ciche TA, Ensign JC. 2003. For the insect pathogen Photorhabdus luminescens, which end of a nematode is out? Appl. Environ. Microbiol. 69(4):189097 13. Ciche TA, Kim KS, Kaufmann-Daszczuk B, Nguyen KC, Hall DH. 2008. Cell invasion and matricide 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. The emerging human pathogen Photorhabdus asymbiotica is a facultative intracellular bacterium and induces apoptosis of macrophage-like cells. Infect. Immun. 77:102230 15. 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 makes caterpillars oppy (mcf ) allows Escherichia coli to persist within and kill insects. Proc. Natl. Acad. Sci. USA 99:1074247 17. Dean P, Potter U, Richards EH, Edwards JP, Charnley AK, Reynolds SE. 2004. Hyperphagocytic haemocytes 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 dauer stage 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 Makes caterpillars oppy (Mcf ) promotes apoptosis in mammalian cells. Cell Microbiol. 6(4):34553 20. Duchaud E, Rusniok C, Frangeul L, Buchrieser C, Givaudan A, et al. 2003. The genome sequence of 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 Photorhabdus 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 insectpathogenic 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 recognition protein mediated immune responses in the tobacco hornworm Manduca sexta causes increased susceptibility 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 a functional 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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9. Discusses the similarities between the genomics of the well known model C. elegans and Heterorhabditis.

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13. Describes the complex process of symbiotic transmission of Photorhabdus by the nematode host and reveals the similarities with pathogenic interactions.

20. Describes the genome sequence of Pl TT01, revealing the huge number of genes involved in host interactions.

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34. Describe the discovery and characterization of the heterorhabditid symbiont for P. asymbiotica and conrms its status as a duel human/insect pathogen (also see Reference 55).

39. Discusses how the well-characterized insect model Drosophila may be used to dissect the Photorhabdus infection process.

44. Describes the rst nonplant biochemical pathway for the production of STs and identies ST as an interkingdom signaling molecule that controls nematode growth and development.

28. Fischer-Le Saux M, Viallard V, Brunel B, Normand P, Boemare NE. 1999. Polyphasic classication of the genus Photorhabdus and proposal of new taxa: P. luminescens subsp. luminescens subsp. nov., P. luminescens subsp. akhurstii subsp. nov., P. luminescens subsp. laumondii subsp. nov., P. temperata sp. nov., P. temperata subsp. 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 that kill bugs. Annu. Rev. Microbiol. 51:4772 30. Fujita T, Matsushita M, Endo Y. 2004. The lectin-complement pathwayits role in innate immunity and evolution. Immunol. Rev. 198:185202 31. Gaudriault S, Pages S, Lanois A, Laroui C, Teyssier C, et al. 2008. Plastic architecture of bacterial genome revealed 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 a P2related prophage remnant locus of Photorhabdus luminescens encoding an R-type phage tail-like particle. FEMS Microbiol. Lett. 233(2):22331 33. Gerrard J, Watereld N, Vohra R, ffrench-Constant R. 2004. Human infection with Photorhabdus asymbiotica: an emerging bacterial pathogen. Microbes Infect. 6(2):22937 34. Gerrard JG, Joyce SA, Clarke DJ, ffrench-Constant RH, Nimmo GR, et al. 2006. Nematode symbiont for Photorhabdus asymbiotica. Emerg. Infect. Dis. 12(10):156264 35. Gerrard JG, McNevin S, Alfredson D, Forgan-Smith R, Fraser N. 2003. Photorhabdus species: bioluminescent 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 human infection. Commun. Dis. Intell. 27(4):54041 37. Gillespie JP, Kanost MR, Trenczek T. 1997. Biological mediators of insect immunity. Annu. Rev. Entomol. 42:61143 38. Hall M, Scott T, Sugumaran M, Soderhall K, Law JH. 1995. Proenzyme of Manduca sexta phenol oxidase: purication, activation, substrate specicity of the active enzyme, and molecular cloning. Proc. Natl. Acad. Sci. USA 92(17):776468 39. Hallem EA, Rengarajan M, Ciche TA, Sternberg PW. 2007. Nematodes, bacteria, and ies: a tripartite model for nematode parasitism. Curr. Biol. 17(10):898904 40. Held KG, LaRock CN, DArgenio DA, Berg CA, Collins CM. 2007. A metalloprotease secreted by the insect pathogen Photorhabdus luminescens induces melanization. Appl. Environ. Microbiol. 73(23):762228 41. Hoffmann D, Hoffmann JA. 1990. Cellular and molecular aspects of insect immunity. Res. Immunol. 141:89596 42. Hu K, Li J, Webster JM. 1999. Nematicidal metabolites produced by Photorhabdus luminescens (Enterobacteriaceae), bacterial symbiont of entomopathogenic nematodes. Nematology 1(5):45769 43. Hurst MR, Glare TR, Jackson TA. 2004. Cloning Serratia entomophila antifeeding genesa putative defective prophage active against the grass grub Costelytra zealandica. J. Bacteriol. 186(15):511628 G, et al. 2008. Bacterial biosynthesis of a 44. Joyce SA, Brachmann AO, Glazer I, Lango L, Schwar multipotent stilbene. Angew. Chem. 47:194245 45. Joyce SA, Clarke DJ. 2003. A hexA homologue from Photorhabdus regulates pathogenicity, symbiosis and phenotypic variation. Mol. Microbiol. 47(5):144557 46. Joyce SA, Watson RJ, Clarke DJ. 2006. The regulation of pathogenicity and mutualism in Photorhabdus. Curr. Opin. Microbiol. 9(2):12732 47. Khush RS, Lemaitre B. 2000. Genes that ght infection: What the Drosophila genome says about animal immunity. Trends Genet. 16(10):44249 48. Kniazeva M, Crawford QT, Seiber M, Wang CY, Han M. 2004. Monomethyl branched-chain fatty acids play 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 growth control in parallel to the insulin receptor pathway and its biosynthesis is feedback-regulated in C. elegans. Genes Dev. 22(15):210210 50. Kuwata R, Yoshiga T, Yoshida M, Kondo E. 2008. Mutualistic association of Photorhabdus asymbiotica with Japanese heterorhabditid entomopathogenic nematodes. Microbes Infect. 10(7):73441 51. Li J, Chen G, Webster JM, Czyzewska E. 1995. Antimicrobial metabolites from a bacterial symbiont. J. Nat. Prod. 58(7):108186
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52. Marokhazi J, Watereld N, LeGoff G, Feil F, Stabler R, et al. 2003. Using a DNA microarray to investigate the distribution of insect virulence factors in strains of Photorhabdus bacteria. J. Bacteriol. 185(15):464856 53. Munch 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, and molecular characterization of strains of Photorhabdus luminescens from infected humans in Australia. J. Clin. Microbiol. 37(11):364753 55. Plichta KL, Joyce SA, Clarke D, Watereld N, Stock SP. 2009. Heterorhabditis gerrardi n. sp. (Nematoda: Heterorhabditidae): the hidden host of Photorhabdus asymbiotica (Enterobacteriaceae: gammaProteobacteria). J. Helminthol. 16:112 56. Pujol C, Bliska JB. 2005. Turning Yersinia pathogenesis outside in: subversion of macrophage function by intracellular yersiniae. Clin. Immunol. 114(3):21626 57. Richardson WH, Schmidt TM, Nealson KH. 1988. Identication of an anthraquinone pigment and a hydroxystilbene antibiotic from Xenorhabdus luminescens. Appl. Environ. Microbiol. 54(6):16025 58. 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. Sicard M, Hering S, Schulte R, Gaudriault S, Schulenburg H. 2007. The effect of 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 signal production of Photorhabdus luminescens inducing the recovery 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 human pathogen 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 to study innate immunity and host-pathogen interactions. Curr. Opin. Microbiol. 5(1):10210 66. Vigneux R, Zumbihl R, Jubelin G, Ribeiro C, Poncet J, et al. 2007. The xaxAB genes encoding a new apoptotic 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 hostpathogen 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 phenotypes 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 are similar to a developmentally regulated insect protein but show no juvenile hormone esterase activity. FEMS Microbiol. Lett. 245(1):4752 71. Watereld NR. 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 insecticidal toxin Makes caterpillars oppy 2 (Mcf2) shows similarity to HrmA, an avirulence protein from a plant pathogen. FEMS Microbiol. Lett. 229(2):26570 74. Watereld NR, Daborn PJ, ffrench-Constant RH. 2002. Genomic islands in Photorhabdus. Trends Microbiol. 10(12):54145
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75. Describes a powerful postgenomic technique for mapping virulence genes in bacterial pathogens.

76. Highlights the idea that insect pathogens provide a reservoir of potential human pathogens, including the agents of plague, anthrax and Photorhabdus.

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75. Watereld NR, Sanchez-Contreras M, Eleftherianos I, Dowling A, 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. 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, Clarke DJ. 2005. The exbD gene of Photorhabdus temperata is required for full virulence in insects and symbiosis with the nematode Heterorhabditis. Mol. Microbiol. 56(3):76373 78. Williams JS, Thomas M, Clarke DJ. 2005. The gene stlA encodes a phenylalanine ammonia-lyase that is involved in the production of a stilbene antibiotic in Photorhabdus luminescens TT01. Microbiology 151(Pt. 8):254350 79. Yang G, Dowling AJ, Gerike U, ffrench-Constant RH, Watereld NR. 2006. Photorhabdus virulence cassettes 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 acts in conjunction with other bacterial effectors to promote host cell actin cytoskeleton rearrangements and bacterial internalization. Mol. Microbiol. 39(2):24859

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Annual Review of Microbiology Volume 63, 2009

Frontispiece Lars G. Ljungdahl p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p xii A Life with Acetogens, Thermophiles, and Cellulolytic Anaerobes Lars G. Ljungdahl p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1 Regulation of Translation Initiation by RNA Binding Proteins Paul Babitzke, Carol S. Baker, and Tony Romeo p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 27 Chemotaxis-Like Regulatory Systems: Unique Roles in Diverse Bacteria John R. Kirby p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 45 Aminoacyl-tRNA Synthesis and Translational Quality Control Jiqiang Ling, Noah Reynolds, and Michael Ibba p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 61 Resurrected Pandemic Inuenza Viruses Terrence M. Tumpey and Jessica A. Belser p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Interspecies Chemical Communication in Bacterial Development Paul D. Straight and Roberto Kolter p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 99 Lipid Signaling in Pathogenic Fungi Ryan Rhome and Maurizio Del Poeta p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 119 Biological Insights from Structures of Two-Component Proteins Rong Gao and Ann M. Stock p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 133 Role of GTPases in Bacterial Ribosome Assembly Robert A. Britton p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 155 Gene Transfer and Diversication of Microbial Eukaryotes Jan O. Andersson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 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 p p p p p p p p p p p p p p p p p p p p p p p p p p p p 195 How Sweet it is! Cell Wall Biogenesis and Polysaccharide Capsule Formation in Cryptococcus neoformans Tamara Lea Doering p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 223
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Mitochondrial Evolution and Functions in Malaria Parasites Akhil B. Vaidya and Michael W. Mather p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 249 Probiotic and Gut Lactobacilli and Bidobacteria: Molecular Approaches to Study Diversity and Activity Michiel Kleerebezem and Elaine E. Vaughan p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 269 Global Emergence of Batrachochytrium dendrobatidis and Amphibian Chytridiomycosis in Space, Time, and Host Matthew C. Fisher, Trenton W.J. Garner, and Susan F. Walker p p p p p p p p p p p p p p p p p p p p p p p p p 291
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Anaerobic Oxidation of Methane: Progress with an Unknown Process Katrin Knittel and Antje Boetius p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 311 The Trypanosoma brucei Flagellum: Moving Parasites in New Directions Katherine S. Ralston, Zakayi P. Kabututu, Jason H. Melehani, Michael Oberholzer, and Kent L. Hill p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 335 Plants, Mycorrhizal Fungi, and Bacteria: A Network of Interactions Paola Bonfante and Iulia-Andra Anca p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 363 Evolutionary Roles of Upstream Open Reading Frames in Mediating Gene Regulation in Fungi Heather M. Hood, Daniel E. Neafsey, James Galagan, and Matthew S. Sachs p p p p p p p p p 385 Single-Cell Ecophysiology of Microbes as Revealed by Raman Microspectroscopy or Secondary Ion Mass Spectrometry Imaging Michael Wagner p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 411 Microbiology of the Atmosphere-Rock Interface: How Biological Interactions and Physical Stresses Modulate a Sophisticated Microbial Ecosystem Anna A. Gorbushina and William J. Broughton p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 431 What Sets Bacillus anthracis Apart from Other Bacillus Species? Anne-Brit Kolst, Nicolas J. Tourasse, and Ole Andreas kstad p p p p p p p p p p p p p p p p p p p p p p p p p p p 451 The Expanding World of Methylotrophic Metabolism Ludmila Chistoserdova, Marina G. Kalyuzhnaya, and Mary E. Lidstrom p p p p p p p p p p p p p p 477 Genomics, Genetics, and Cell Biology of Magnetosome Formation Christian Jogler and Dirk Schler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 501 Predatory Lifestyle of Bdellovibrio bacteriovorus Renee Elizabeth Sockett p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 523 Plant-Growth-Promoting Rhizobacteria Ben Lugtenberg and Faina Kamilova p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 541

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Photorhabdus and a Host of Hosts Nick R. Watereld, Todd Ciche, and David Clarke p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 557 Management of Oxidative Stress in Bacillus Peter Zuber p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 575 Sociobiology of the Myxobacteria Gregory J. Velicer and Michiel Vos p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 599 Index
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Cumulative Index of Contributing Authors, Volumes 5963 p p p p p p p p p p p p p p p p p p p p p p p p p p p 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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