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Photorhabdus and a Host of Hosts
Nick R. Waterfield,1 Todd Ciche,2 and David Clarke3
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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

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Annu. Rev. Microbiol. 2009. 63:557–74 First published online as a Review in Advance on June 23, 2009 The Annual Review of Microbiology is online at micro.annualreviews.org This article’s 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 efficient 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 identified 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

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

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 insect’s open blood system, the hemocoel, the nematodes regurgitate 50–200 bacterial cells directly into the blood, or hemolymph (12). These few bacteria are sufficient to overcome the insect’s 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
Waterfield

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 10–14 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 insect’s 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) classified 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 confirmed this classification. 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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asymbiotica contains at least two subclades.org by Pontificia Universidad Javeriana on 09/17/13. luminescens (insect) Hm/Hb HIT/JUN ATCC43949 (USA) Annu. 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. They also show a change in the pattern of protein. or even (c) act a lure Prey W14 TT01 P. and Australian isolates. most Photorhabdus species resist phagocytic uptake www. and it has also been suggested that the primary role for bioluminescence is as a sink for molecular oxygen. although the precise mechanism still remains elusive (31. The primary variant (P1) is the symbiotically competent form that is isolated from EPNs (45). (b) provide a visual aposematic warning to nocturnal scavenging animals. where it peaks at the end of the exponential phase.org • Photorhabdus and a Host of Hosts Mutualism: a beneficial relationship between different species 559 . suggesting it is a key player in the regulatory network controlling pathogenicity and mutualism (45. For personal use only. representing U. 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). Rev. Recent work has shown that although motility is not required for either pathogenicity or symbiosis.S. where they uniquely produce a thin line of annular hemolysis. Critically. light emission is seen only after the insect is dead and tissue degradation is at an advanced stage (15). The luciferase reaction is highly efficient at utilizing molecular oxygen. Nevertheless. two studies aimed at elucidating the mechanisms behind this P1/P2 switch used microarray and proteomic approaches. facultatively anaerobic rods (54). 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. bioluminescent. light and metabolite production. Downloaded from www. the clinical isolates also grow at temperatures ranging from 37 to 42◦ C. 46). This bioluminescence has been hypothesized to (a) represent a signal from bacteria to bacteria or to the nematode. it remains to be seen if all members of this species are capable of causing clinical infections. to attract further insect prey to the infected insect cadaver. 2009. Second. Photorhabdus species are highly motile. Several observations argue against this role. asymbiotica (human + insect) Kingscliff (Australia) K122 P. 64). Microbiol. P.annualreviews. All species grow well at 28◦ C on blood agar. With this in mind Joyce & Clarke (45) used P1 and P2 variants to provide clues regarding the molecular basis behind symbiosis and pathogenicity. synchronizing symbiosis. Model type strains of three different species are indicated in their different clades. it does confer a competitive advantage during colonization of the insect (21). In vivo. Bioluminescence occurs in all strains on agar plates and in liquid medium. More recently.becoming clear that they are not restricted to the continents of North America and Australia (50). A third clade comprising the HIT and JUN isolates from mainland Europe confirms these genotypic forms are not restricted to the two continents.annualreviews. however. A unidirectional phenotypic variation has been described for Pl and Pt (4). They identified a gene with homology to hexA from Erwinia that represses symbiosis factors in P2. First. P.63:557-574. temperata (insect) Figure 1 A schematic representation of the multilocus sequence typing phylogeny of the genus Photorhabdus.

although the bacteria can be readily cultured in vitro using standard peptone-based media. One interesting possibility is that Photorhabdus may have taken on the responsibility of producing BCFAs required by their nematode partner. Most bacteria-host interactions involve some level of metabolic interaction. Transmission of Photorhabdus by H. Downloaded from www. the nematodes switch to feeding behavior and ingest symbiont bacteria. Pulse-chase experiments revealed that the adherent cells formed a persistent infection as a biofilm while other symbiont cells were transiently present in the intestine. for nematode growth. Photorhabdus is required.org by Pontificia Universidad Javeriana on 09/17/13. bacteriophora Nematodes Photorhabdus bacteria are transmitted by the specialized IJ stage of the nematode. rather it usually requires its specific cognate bacterial strain. The transmission cycle starts when an IJ enters an insect and regurgitates the Photorhabdus into the insect hemocoel (13). The molecular basis of this nutritional specificity remains to be elucidated but some recent work has implicated a role for iron (77). In this regard. NEMATODE SYMBIONT Metabolic Interactions Annu. The IJ is an alternative third larval stage of the nematode that is developmentally arrested and resistant to environmental stresses. Exogenous sterols are important for the development of the bacteriophorous nematode Caenorhabditis elegans. Every bacterial cell is released into the hemocoel. a few of which adhere to the posterior nematode intestine (Figure 2). Microbiol. After persisting for up to 38 h. yet they grow readily on the same bacteria cultured on lipid agar (nutrient agar supplemented with cod liver oil and corn syrup). Rev. Furthermore. After regurgitation. where they multiply · · Ciche Clarke . elegans. 12). An interesting aspect of the metabolic interaction between Photorhabdus and the nematode is that Heterorhabditis will not grow on just any strain of Photorhabdus. The production of BCFA is not widespread in the Enterobacteriaceae and the presence of BCFA in Photorhabdus may represent an adaptation to symbiosis. which harbors them in the intestinal lumen as it disperses from an insect cadaver in search of a new insect host (9. most bioluminescent bacteria known to date are associated with animal symbiosis. BCFAs play an important role in the development of C. and indicating that Photorhabdus is maternally transmitted by an elaborate sequence of selective events (13). perhaps reflecting the challenge they face from the insect immune system (12). the Heterorhabditis-Photorhabdus interaction appears to be obligate. the adherent bacteria invade three rectal gland cells.anyway. 12). then it is unlikely to have been restricted to Photorhabdus only and we would expect to see many more bioluminescent terrestrial pathogens.annualreviews. 49). in which the nematode itself encodes the genes required for its production (48. 560 Waterfield Photorhabdus also produces large quantities of crystalline inclusion proteins (CipA and CipB) that have a potential role in nematode nutrition (3). IJs are formed inside the maternal nematode body cavities. Photorhabdus produces a significant amount of iso-branched fatty acids (BCFAs). Moreover. and development is also dependent on factors present in the insect. making it difficult to definitively define an exact role in symbiosis for these proteins (3).63:557-574. a process termed endotokia matricida (9. Finally. Mutants in cipA and cipB are unable to support nematode growth and development. In the environment. and it is likely that Heterorhabditis will have the same requirement (8). suggesting a cryptic underlying selective force. but not sufficient. the nematodes can be cultured in vitro by inoculating Photorhabdus biomass grown on agar plates with either IJs or eggs. causing matricide. The nematodes will not grow or develop on Photorhabdus cultured on Luria-Bertani agar. although they are pleiotropic. Therefore. 2009. with isoC15:0 being the dominant fatty acid found in Photorhabdus phospholipids produced by the bkdABC operon (44). For personal use only. if this represented a general mechanism to evade phagocytic destruction.

Step 8: Lysis of the apical side of the rectal gland cells releasing symbionts to pre-IJs developing in the maternal body cavity.) Step 3: Nematodes ingest bacteria and a few adhere to the posterior intestine.org • Photorhabdus and a Host of Hosts 561 . t = hours post-infective juvenile (IJ) addition to symbiont lawns. www. F 1) g( in 2 t=6h Complete symbionts release Off sp r 3 5 µm t = 120 h Symibont adherence to the anterior IJ. For personal use only. Step 4: Symbionts grow as a biofilm while additional symbionts adhere. Step 10: Invasion of the pharyngeal-intestinal valve cells. green color is autofluorescence of the nematode intestine. (In Step 2 only. Step 5: Invasion of the rectal gland cells by adherent symbionts. Downloaded from www. Nematodes in steps 3–8 were chased with unlabeled symbionts so that only persistent bacteria are visible. 2009. Step 1: Symbionts are regurgitated from the intestine until (Step 2) no symbionts remain.org by Pontificia Universidad Javeriana on 09/17/13. visible because the exposure of the epifluorescent micrographs is 10 times greater than the other micrographs. Step 9: Symbiont adherence to the pharyngeal-intestinal valve cell. laumondii in a nematode host. IJs develop inside the maternal body 8 Photorhabdus transmission cycle 0) 10 µm t=8h Symbiont adherence and worm feeding 4 10 µm al er n t a M 10 µm t = 112 h Symbionts released into pseudocoelom (P t = 36 h Biofilm formation and adherence 7 6 5 µm 5 5 µm 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. Microbiol.annualreviews. Steps 6 and 7: Intracellular growth of symbiont bacteria and vacuole replication.11 10 µm 10 t = 136 h Pharyngeal-intestinal valve cell invasion t = 136–240 h Exit and symbiont growth in the intestinal lumen t=0h Infective juvenile (IJ) 1 Nematode Bacteria 10 µm t=4h Regurgitation of intestinal symbionts 9 Annu. Step 11: Exit and growth of symbiont cells in the IJ intestinal lumen.63:557-574.annualreviews. Rev.

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.g. This presumably indicates to the nematode that there are enough bacteria present to support its growth and development. In Pt NC19.annualreviews. it is not sufficient by itself and viable Photorhabdus must also be present (44). Importantly. highlighting the · · Ciche Clarke . suggesting that additional secondary metabolites are required for normal nematode growth and reproduction (1. Thus. Interkingdom Signal Production Recovery is the first step in nematode development. 10). where they persist in a presumably semidormant state before a new insect host is found. A fimbrial operon essential for Photorhabdus to colonize the maternal intestine and the stringent starvation protein A regulator. elegans (39). recovered IJs that reached adulthood also failed to reproduce efficiently on the ngrA mutant. Recently. although ST is required for IJ recovery. Less than 24 h later. 62). STs are polyketide molecules often produced by plants in response to stress and infection. IJ recovery is independent of insect signals but requires a food signal produced by the bacteria (1. IJ recovery is normally stimulated by uncharacterized molecule(s) present in the insect hemolymph (12.Virulence factor: a molecule produced by a pathogen that damages the host ST: stilbene molecule Annu. 18). The recovery of IJs inoculated onto a lawn of Pl TT01 cells mutated in ST production is only 5–15% of that observed with wild-type Pl TT01 bacteria (44). The biochemical pathway responsible for ST production in Photorhabdus has been characterized and is significantly different from that observed in plants (44. For personal use only. bacteriophora nematodes involves many events shared with pathogens that cause disease (e. usually a single symbiont cell adheres to the pharyngeal intestinal valve cells of the IJ offspring. 2009. During in vitro culturing on Photorhabdus. 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. invasion. and it refers to the postinfection recovery of the IJ into a self-fertile adult hermaphrodite. and Photorhabdus is the only nonplant organism known to produce a stilbene. The adherent cell(s) divides and appears to invade the valve cells before exiting and colonizing the intestinal lumen. In the insect. The siderophore was determined not to be required for nematode growth (11). In addition we have recently described the characterization of a locus ( plu4185-plu4195) involved in the production of a polyketide molecule. Photorhabdus transmission by H. Laser ablation of the ASJ chemosensory neuron in Heterorhabditis IJs and treatment with known 562 Waterfield inhibitors of muscarinic acetylcholine receptors and cGMP signaling pathways prevented IJ recovery in vitro (39). It is likely that Photorhabdus employs effectors related to virulence factors in pathogens for adhesion. Microbiol. Intracellular symbionts are released to the IJs by apical lysis of the rectal gland cells. the phosphopantethienyl (PPANT) transferase gene ngrA is required to support nematode growth reproduction (10).org by Pontificia Universidad Javeriana on 09/17/13. Nevertheless. required for postadhesion survival in the IJ have recently been identified (T. and intracellular growth in animal cells. Ciche. anthraquinone. transmission of Photorhabdus by Heterorhabditis is an elaborate process involving cell-specific infection of maternal and offspring nematodes. cell-specific adhesion/invasion). this is only the second report of Type II PKS activity in gram-negative bacteria. However. Determination of these and other factors for transmission and pathogenesis is likely to illuminate common and distinct processes involved in mutualism and infectious disease. inside membrane-enclosed vacuoles for 48 h as the nematode progeny hatch and develop in the maternal body cavity. while the intestine remains intact and is relatively devoid of other intestinal symbionts (13). suggesting again the importance of the ST molecule. sspA. Downloaded from www.5-dihydroxy-4-isopropylstilbene. a major secondary metabolite produced by Photorhabdus (44). Rev.63:557-574. a component of this food signal was identified as a stilbene (ST) called 3. responsible for the yellow/orange pigment produced by Pl TT01 (5).. 78). unpublished data).

there is significant potential in Photorhabdus for the production of novel bioactive molecules. potential for novel and/or unusual PKS-related biochemistry in Photorhabdus. 47.annualreviews. melanization typically occurs around groups of hyperphagocytic hemocytes. the model organism for secondary metabolite production (and the source of >90% of clinically important antibiotics).annualreviews. such as phagocytic destruction using free radicals and killer proteases. which we discuss below. and it is possible that they share a common evolutionary origin (30. Upon release into the insect hemocoel. has provided the first step in isolating and characterizing a number of these secondary metabolites (75).Annu. 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). the release of at most only 50–200 bacteria in a normal infection means that the bacteria are adapted to be highly virulent and can resist immune systems from diverse genetic backgrounds. sexta larvae with nonpathogenic Escherichia coli elicits effective immunity against subsequent infection with the otherwise lethal Pl TT01. which is both an insect and human pathogen. PAMP: pathogenassociated molecular pattern PO: phenol oxidase Hemocyte: an insect immune cell INSECT PREY Immune Challenge The EPN complex shows little specificity in its choice of insect prey species. encapsulating them and inflicting free radical damage in the process. but that Photorhabdus can nevertheless overcome this response (24). the insect immune system is the melanization and encapsulation response mediated by phenoloxidase (PO) (37).org by Pontificia Universidad Javeriana on 09/17/13. 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). In Manduca sexta. immulection-2. Preventing the upregulation of these PAMP molecules with RNAi abolished this protection. as seen by an increase in mRNA for PAMPs. including recognition of pathogenassociated molecular patterns (PAMPs) by receptors and the production of antimicrobial peptides (AMPs) and lysozyme. The 5. The innate immune systems of insects and mammals share many common features (47). 2009. This enzyme deposits melanin onto invading organisms. Photorhabdus has evolved multiple virulence factors and strategies to deal with immediate immune threats and to kill the host. which has the advantage of a fully sequenced genome and welldeveloped genetic tools (39.5 Mb genome of Pl TT01 has been sequenced (http://genolist. Prior infection of M. Rev. whereas suppression of specific AMP production had little effect (23). This proportion is greater than the 3. 65.pasteur. the implications of which become obvious when considering the evolution of Pa. which contained a greater number and diversity of toxin genes than any other sequenced genome at the time (20). Innate immune factors include cellular responses. 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. For personal use only. 38). In many ways the PO system is reminiscent of the complement cascade of vertebrates. and also AMPs including attacin and cecropin. and peptidoglycan recognition protein. Furthermore. This protection correlates with the upregulation of PAMP molecules such as hemolin. A novel virulence mapping technique termed RVA. Downloaded from www. More recent studies have used the dipteran host Drosophila melanogaster. Therefore.8% observed in Streptomyces. 67).org • Photorhabdus and a Host of Hosts 563 . Microbiol. sexta recognizes a Photorhabdus infection.63:557-574.fr/PhotoList/). Other studies have confirmed that M. sexta can in fact fight off a Photorhabdus infection if given a head start. These requirements appear to be reflected in the genome of Pl TT01. discussed below. Photorhabdus bacteria face the fast-acting innate immune system (41). www. This suggests that M. forming a nodule on the surface of tissues (17). and humoral responses.

61). The TTSS operon of the recently sequenced clinical strain Pa ATCC 43949 does not encode the lopT gene. sexta larvae. Electron microscopy revealed that the bacteria occupy a specific niche between the extracellular matrix and basal membrane of the midgut epithelium. Several toxin systems that disable or kill the hemocytes have been characterized. All strains of Photorhabdus encode multiple homologues of the Tc genes. Like many pathogenic gram-negative bacteria. Another gut-active toxin produced by all strains of Photorhabdus is the multidomain Mcf1 toxin. Colonization of the blood side of the midgut is initiated at the anterior and then proceeds posteriorly along its length as infection time continued. At least two of the Pl strains. these are associated with an alternative secretion system called Photorhabdus virulence cassettes (PVCs). reminiscent of TTSS effectors (74. the highly secreted gut-active toxin complex A (Tca) and a metalloprotease. preventing their phagocytic uptake. suggesting multiple roles in infection. For personal use only. PrtA (15. and secretion within such an enclosed niche. suggesting independent acquisi564 Waterfield tion (71). A detailed study by Silva et al. which is a prominent feature of Photorhabdus infection. into these cells. and all are open to hemocyte attack. Downloaded from www. a powerful TTSS-delivered Pseudomonas cytotoxin (26).annualreviews. SopB in Salmonella is a TTSS-delivered effector involved in intracellular persistence in human macrophages (80). although the functions of these homologues remains obscure in most cases (27. The mcf1 gene is present in all strains so far examined but appears to be encoded at different genomic loci in each case. Following injection the bacteria multiplied rapidly both on the midgut and in the hemolymph (61).org by Pontificia Universidad Javeriana on 09/17/13.63:557-574. at least during transit to the gut. coli. the TTSS is involved in attachment to hemocytes and in delivery of at least one effector molecule. 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. 19). also have a homologue called Mcf2. Other TTSS effector molecules are yet to be identified in Pl TT01. LopT is a close homolog of YopT from Yersinia pestis. in tight grooves made by infolding of the epithelium. This potent toxin is taken up into target cells by endocytosis. There are multiple. When heterologously expressed from cosmid clones in E. in which it also serves to inhibit phagocytosis by macrophages.Avoiding the Cellular Response TTSS: Type three secretion system Macrophage: a mammalian professional phagocytic immune cell PVC: Photorhabdus virulence cassette Annu. not all the bacteria reside here. 68. PVC genomic islands superficially resemble prophage and consist of around 15 genes. Close association of bacteria with the epithelium. In Pl TT01. Rev. 2009. Although this site may be difficult for the hemocytes to enter because of their size. (61) followed the fate of Pl W14 after injection into M. 69. Furthermore. This shows a striking conservation of immune avoidance mechanisms in a mammalian or insect pathogen (7). conserved between all PVC elements. W14 and TT01. instead it encodes a close homologue of ExoU. Microbiol. a subtractive DNA hybridization study between Pa and Pl TT01 identified a sopB gene homologue in the clinical strain (63). suggesting they play an important role in the biology of the organism. homologous to either phage or other macromolecular assembly genes. This protein toxin has little cell specificity and can also induce apoptosis in hemocytes and a range of cultured human cells. where it induces apoptosis by a novel BH3-domain-mediated effect (16. HrmA (73). the PVC elements · · Ciche Clarke . however. diverse PVC elements in all Photorhabdus genomes so far examined. may facilitate the rapid destruction of the gut. Photorhabdus encodes a type three secretion system (TTSS). 79) (Figure 3a). 72). A range of TTSS effector-like molecules can be seen in the genomes of Photorhabdus strains. suggesting they are mobile and acquired by horizontal transfer (Figure 3b). 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. Toxins Immunohistochemistry revealed that within the gut niche the bacteria express at least two virulence factors. LopT.

asymbiotica (Pa) ATCC 43949. 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. 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). presumably ancestral. For personal use only. The thick red arrows below the afp diagram indicate where insertional mutagenesis abolished toxic activity. Note several elements are common. The names or closest homologues of payload genes are indicated. 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.annualreviews. Rev. The homologous Serratia entomophila (Se) afp element is shown. 2009.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 Annu. In Pa ATCC 43949 there is only one. (b) A diagram showing the range of PVC elements in Photorhabdus luminescens (Pl ) TT01 and P. Downloaded from www. b P. while the thin blue arrows are mutants that retained function. element in this locus. The conserved regions are shown as green boxes and the toxin genes in red.org • Photorhabdus and a Host of Hosts 565 . www.63:557-574. Microbiol.annualreviews.org by Pontificia Universidad Javeriana on 09/17/13. whereas others are unique to each species. 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 ). and similar ORFs have been color coded.

it is not known whether the bacterium is resistant to AMPs specifically or whether it blocks translation or secretion of the peptides. Other humoral factors that Photorhabdus must resist include circulating lysozyme and AMPs. Pl TT01 cells carrying a mutation in the pbgPE operon (predicted to be responsible for the modification of LPS with l-aminoarabinose in response to AMP) are avirulent. Microbiol. 60) and proteins homologous to XaxA and XaxB. Photorhabdus has several iron acquisition systems including the production of the siderophore photobactin (11). Like R-type pyocins. These include the PirA and PirB proteins (20.R. 70). first identified in the closely related entomopathogen Xenorhabdus nematophila (66. Serratia entomophila. No antibacterial activity has been associated with PVC elements. a mutation in the exbD gene of Pt K122 resulted in a strain that was severely limited in its ability to acquire iron. suggesting that iron is indeed limiting in the insect (77).63:557-574. The attenuation could be rescued by preloading the insect with iron. Culex pipiens. For personal use only. can also be seen in the two completed Photorhabdus genomes that have cytotoxic effects (6. hemolytic. Annu. Rev. 75). Finally. The PirAB toxins exhibit a range of activity including potent injectable activity against Galleria larvae. Strains of Pl and Pa encode binary toxin systems. all animals attempt to limit bacterial replication through the tight binding and compartmentalization of iron. reminiscent of a released TTSS. which comprise two proteins required for toxicity. such as PhlAB. This mutant was attenuated in virulence and this attenuation was correlated with the growth rate of the bacteria in the insect (77). In Photorhabdus the PVC elements are tightly regulated at the translational level and not expressed at 37◦ C. which causes larvae of Costelytra zealandica (New Zealand grass grub) to cease feeding (Figure 3a). XaxAB has cytolytic. Waterfield and proapoptotic effects on insect and mammalian cells. Like many animal pathogens.org by Pontificia Universidad Javeriana on 09/17/13. although premature and nonlocalized activation of the PO system could also represent a virulence strategy. Conversely. Finally. Larvae injected with the purified PVCpnf element melanize and die within minutes. The significance of this is not yet clear. In Serratia this is known as the antifeeding prophage.annualreviews. 75). and Anopheles gambiae (20. the only other bacteria so far seen to encode this system is another insect pathogen. with a reduction in circulating hemocytes showing extensive actin rearrangement (79). which are bacteriophage tails adapted to kill related strains of bacteria. but they do have potent toxicity when injected into Galleria larvae (79). Furthermore. Although Photorhabdus induces the transcription of AMP mRNA. Waterfield. suggesting that Photorhabdus does in fact adapt to AMP production during infection (2). Downloaded from www. Indeed the LT50 (time taken to kill 50% of the insects) of wildtype Pt K122 was reduced in insects preloaded with iron. oral activity against the caterpillar pest Plutella xylostella. Recent work in our lab has confirmed that PVC element toxins are expressed during insect infection and that the PVC structures can attach to the surface of hemocytes using SEM (N. 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. suggesting they may be inappropriate in vertebrate infections. Despite the significant numbers of pathogen genomes now sequenced. a range of homologues of two partner hemolysin genes. allowing Photorhabdus to avoid the melanization response (22). the PVC elements consist of an inner stylet and an outer sheath that can be seen in a relaxed or contracted injection form. PrtS. and oral toxicity against three different species of mosquito. Avoiding the Humoral Responses The secondary metabolite ST is also a potent inhibitor of the PO enzyme. 2009. Aedes aegypti. 566 · · Ciche Clarke . during infection that strongly induces melanization (40). In this case the AFP element is presumably active against gut cells rather than hemocytes (43).Entomopathogen: an insect pathogen produce a large 250-nm-long structure similar to R-type pyocins. Pl TT01 also secretes a protease.

57). as Pa is not yet included on the standard clinical bacteriology databases (34). in the United States. elegans. 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). which is later shed during secretion. This finding suggests that the orally toxic Tca may be adapted for deterring scavengers such as ants rather than killing the insect host. however. coli (59). Genomic loci in the completed sequences of both Pl TT01 (32) and Pa ATCC 43949 (N. Finally. We recently confirmed that a clinical www. unpublished data) have been identified. In vivo expression studies in M. elegans. 35. although it is not clear how the nematode makes this distinction (60). However. Downloaded from www. Photorhabdus expresses a range of antimicrobial factors. time-lapse microscopy revealed that the amoebae are not directly killed by the bacterium under the conditions tested. suggesting that Photorhabdus prevents uptake as it does in insect hemocytes (7) (Figure 4b). confirming they are related to the phage P2 family of R-type pyocins. ST also repels some Steinernema species (42).63:557-574. Microbiol. It is not yet clear.annualreviews. Recently we have been studying the interaction between Photorhabdus strains and the free-living soil amoeba Acanthamoeba polyphaga.org • Photorhabdus and a Host of Hosts 567 .R. Both strains encode a DNA invertase and have regions bounded by inverted repeats containing different tail fiber genes. (60) demonstrated that two strains of Pl reduced survival rates of C. To date little work has addressed how Photorhabdus combats scavenging organisms such as nematodes and amoeba.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. These cases appear to form geographical clusters—Victoria and Southeast Queensland in Australia and San Antonio. 34. Rev. Furthermore. Photorhabdus produces a range of proteinaceous antibacterial molecules including S. Texas. 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).org by Pontificia Universidad Javeriana on 09/17/13. If grown at 28◦ C. suggesting a phase variation system evolved to increase the target host range. For personal use only. Annu. R-type pyocins are much larger. Further. both species of Photorhabdus swim up to the amoeba and attach as a small microcolony (Figure 4a). modified 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. They showed that C. 2009. including C. The ST molecule also constitutes a broad-spectrum antibiotic showing strong antibacterial and antifungal properties (51. A range of S-type pyocins characterized in Pl W14 called lumicins show activity against other strains of Photorhabdus and even E. it is possible that Photorhabdus may also attempt to protect the insect carcass from larger scavenging animals.annualreviews. S-type pyocins constitute toxic proteins capable of degrading macromolecules in target cells. but not on Heterorhabditis. In addition to small-molecule antibiotics. For this reason. whether this means that there is a specific environmental source for the infection in these areas or simply that laboratories in these areas have become familiar with the identification of the pathogen. Waterfield. we see no phagocytic uptake or invasion of the amoeba by Pl TT01. The true incidence of human infection is likely much higher than these reports would suggest. 54). THE HUMAN HOST Pa was first isolated only from clinical infections.and R-type pyocins. the purified ST and an indole produced by Pl HD have strong nematicidal effects on a range of nematodes. A small cognate immunity protein protects the cell during biosynthesis. elegans avoided the two pathogenic strains but not a third nonpathogenic strain of Pl. with 14 cases reported in the literature from the United States and Australia (25. Sicard et al.

Pl TT01 neither invades the amoeba nor is it taken up by phagocytosis. while 0. Waterfield. A ratio of 5 ml water in 50 g of sand gave a 100% infection rate. Our findings (N. or other common disease states. At least in Australia the incidence of infection appears to correlate with warm.R. Downloaded from www.a b Figure 4 Images on the left are bright-field illumination and those on the right are under fluorescence illumination. isolate of Pa from a patient in Kingscliff. 55). This is supported by the presence of a homologue of the sopB effector in the clinical isolates that is absent from Pl. (a) GFP-labeled Pl TT01 attached to an Acanthamoeba polyphaga cell. not an opportunistic secondary colonizer of existing wounds. Note no GFP-labeled bacteria can be seen inside the amoeba (arrow). personal communication). 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. gender. Although no mammalian models yet exist. a phenotype Annu. The infection also can exhibit a relapsing course extending over several weeks even when appropriate antibiotics are administered (33). Most cases present with either locally invasive or disseminated soft tissue infection with a typical clinical descriptor of a painful necrotic ulcer. Most clinical cases are associated with infection during outdoor activity. many of the patients showed the appearance of soft tissue infection at sites remote from the initial infection. Microbiol. Vlisidou. unpublished data) and those of Costa et al. indicating bacteremic spread. Costa et al. wet conditions ( J. 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. Waterfield & I. and 20 ml of water gave no productive infections (N. and the primary foci of infection on limb extremities support contact with soil as the primary route of infection. we recently showed an absolute correlation between infection rate of G. (b) Despite prolonged and intimate exposure. Gerrard.R. also exists in a symbiotic partnership with a Heterorhabditis nematode. so the exact mechanics of infection remains elusive. In support of this. unpublished data). We do not know how the H.63:557-574. Pa infections represent a primary pathogen. 568 Waterfield · · Ciche Clarke . confirmed that Pa invades insect hemocytes during infection. we have used tissue culture to investigate the interaction between Pa and macrophage cell lines. 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). We suggest this finding provides an excellent experimental system to model the effects of climate change on emerging infections. The Kingscliff Pa strain exhibited only weak bioluminescence in liquid culture at 30◦ C but strong light emission at 37◦ C. 10. In addition. 2.annualreviews. gerrardi (34. H. suggesting the intriguing yet not investigated possibility that the soft tissue lesions could in fact glow (55). There are no apparent predisposing factors to infection such as age. Rev. Australia. The microcolony shown (arrow) is not the result of bacterial growth.org by Pontificia Universidad Javeriana on 09/17/13. gerrardi– EPN penetrated the patient’s skin or if the nematode also entered the patient’s body. (14) confirm that Pa is a facultative intracellular pathogen that replicates inside mouse and human macrophage-like cells. For personal use only. mellonella and moisture content of sand that had been inoculated with the Kingscliff EPN complex. 2009.

This approach is particularly useful in Photorhabdus because of the high level of toxin redundancy. polyphaga. screens large insertion genomic libraries for toxicity against four different taxa: the protist A. Waterfield.63:557-574. the nematode C. In the future. 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. The identification 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. In addition both strains possess a range of different genomic islands. and presented an obvious route for novel drug discovery. C.org by Pontificia Universidad Javeriana on 09/17/13. In an attempt to map genes induced upon ex¨ posure to insect homogenates. Microbiol. RVA successfully identified a large number of virulence loci through their gain of function in E. www.. Finally. This approach. et al.S. pestis. (53) identified 24 Pl TT01 promoters that were induced in G. silences inflammatory reactions. unpublished data). This parallels the related pathogen. Corton. suggesting that a defensive mechanism adopted by the insect-only pathogens may be equally effective against human-complement-mediated killing (N. it also provides a superb opportunity to understand the role of invertebrates in the emergence of human diseases. Finally. the mcf1 toxin. isolates (14). Wilkinson. and promoters for a range of metabolic operons (53).R. coli (75). PVC payload genes plu2400. with Pa now recognized as both an insect and human pathogen.org • Photorhabdus and a Host of Hosts 569 . suggestive of significant levels of horizontal gene transfer and differential host ranges (20). not seen in the other two members of the genus. Downloaded from www. unpublished data). which is also a facultative intracellular pathogen that induces apoptosis in macrophages. we have recently demonstrated that both Pa and Pl TT01 are inherently resistant to killing by commercially available human serum. The Australian isolates of Pa also invade nonphagocytic cell lines and induce apoptosis in cells faster than the U.annualreviews. plu1672. termed rapid virulence annotation (RVA). CONCLUSIONS Photorhabdus provides an excellent model for the study of both symbiosis and pathogenicity. Crossman. These included promoters that drive expression of the tc toxin components tccC1 and tcdA1. P. the human pathogenic strain has a smaller genome with reduced toxin diversity. Furthermore. L. and allows dissemination through the lymphatic system (56). mellonella infection.Annu. Y. which will greatly assist the study of the symbiotic interaction.R. We have now completed a full in silico genomic comparison between Pa ATCC 43949 (United States) and Pl TT01 that awaits publication (N. For personal use only. secondary metabolite genes. Waterfield. Munch et al. the availability of genome sequences and the ability to genetically manipulate the bacteria. In addition. supporting the hypothesis that virulence factors evolved against insect hosts may be readily redeployed against humans. unmasking virulence factors that would otherwise be hard to detect. We saw no obvious mammalian-only toxins. and a mouse macrophage cell line ( J774-2). a Heterorhabditis genome is also currently being sequenced. the nematode partner. 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. sexta. and plu1645. Rev. Sanchez Contreras. Briefly. the moth M. and the host insects will ultimately enable us to understand the molecular interplay between pathogenicity and symbiosis. 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.annualreviews. M. 2009. elegans.

Gen. J. Annu. Insertional inactivation of genes encoding the crystalline inclusion proteins of Photorhabdus luminescens results in mutants with pleiotropic phenotypes. 2009. 2001. Boemare RJ. 1998. and the insect hosts provide an unparalleled model system for understanding animal-bacteria interactions. Clarke DJ. or financial holdings that might be perceived as affecting the objectivity of this review. and SFI for support. The PhlA hemolysin from the entomopathogenic bacterium Photorhabdus luminescens belongs to the two-partner secretion family of hemolysins. 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. TAC thanks the Center for Microbial Pathogenesis at Michigan State University and the USDA for support. Biochemical and physiological characterization of colony form variants in Xenorhabdus spp. Boemare N. 3. BBSRC. 180(5):1261–69 4. Microbiol. This symbiosis is highly evolved and is highly strain specific. A type II polyketide synthase is responsible for anthraquinone biosynthesis in Photorhabdus luminescens. Nematology 3:849–53 2.annualreviews. J. Givaudan A. Jenke-Kodama H. Genome sequences have confirmed a large repertoire of genes involved in host interactions. For personal use only.63:557-574. Postgenomic techniques and the ability to genetically manipulate the bacterium. providing an excellent model of an emerging human pathogen. 2005. the symbiont nematode. Bode HB. asymbiotica is capable of causing infections in humans. 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.SUMMARY POINTS 1. especially genes encoding putative toxins. 4. Bacteriol. 134:751–61 5. Clarke DJ. DJC and NRW thanks the Leverhulme Trust. Akhurst NE. Bacteriol. 7.org by Pontificia Universidad Javeriana on 09/17/13. Chembiochem 8:1721–28 6. 187(1):77–84 3. The large repertoire of secondary metabolite gene clusters suggests these bacteria represent an excellent source of novel drug candidates. ACKNOWLEDGMENTS The authors would like to thank all past and present members of their research groups. funding. providing further selection for virulence. 184(14):3871–78 570 Waterfield · · Ciche Clarke . Ehlers R-U. Bacteriol. Bennett HP. 2. P. (Enterobacteriaceae). Downloaded from www. 2002. 1988. All strains of Photorhabdus live in a mutualistic relationship with insect pathogenic nematodes of the genus Heterorhabditis. Kunst F. Brachmann AO. Photorhabdus must also compete with a wide range of saprophytic microorganisms from the soil. J. Ensign JC. The pbgPE operon in Photorhabdus luminescens is required for pathogenicity and symbiosis. Bintrim SB. J. Duchaud E. LITERATURE CITED 1. Microbiol. Rev. Aumann J. 5. Schw¨ ar G. DISCLOSURE STATEMENT The authors are not aware of any affiliations. memberships. Brillard J. 6. 2007. Joyce SA.

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Contents Annu. 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. 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.I. Downloaded from www. Baker. Thermophiles.63:557-574. 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. Noah Reynolds. Ashley M. Aly.org by Pontificia Universidad Javeriana on 09/17/13. 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. For personal use only. 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. Carol S. 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. 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 v . and Stefan H. 2009 Frontispiece Lars G. 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 Diversification of Microbial Eukaryotes Jan O. Vaughan. 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 Influenza Viruses Terrence M.I. 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. and Cellulolytic Anaerobes Lars G. Rev. Microbiol. 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. Annual Review of Microbiology Volume 63. 2009.annualreviews. Tumpey and Jessica A.

and Matthew S.63:557-574. and Ole Andreas Økstad p p p 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. Lidstrom p p p p p p p p p p p p p p 477 Genomics. Mycorrhizal Fungi. Time. James Galagan. Michael Oberholzer. Marina G. Daniel E. Neafsey. Nicolas J. For personal use only. Zakayi P. 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. Tourasse. Hood.Mitochondrial Evolution and Functions in Malaria Parasites Akhil B. and Cell Biology of Magnetosome Formation Christian Jogler and Dirk Schüler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 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 vi Contents . Garner. Vaidya and Michael W. 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. 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. 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. Fisher. Microbiol. 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 Bifidobacteria: Molecular Approaches to Study Diversity and Activity Michiel Kleerebezem and Elaine E. 2009. and Susan F. 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. Kalyuzhnaya. and Host Matthew C.J. Kabututu. 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. Genetics. Downloaded from www.org by Pontificia Universidad Javeriana on 09/17/13.annualreviews. Gorbushina and William J. Rev. 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 Annu. Ralston. Jason H. Trenton W. and Kent L. Melehani. and Mary E.

annualreviews. 2009. Waterfield.annualreviews. 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. Microbiol.63:557-574.org/ Contents vii . 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 Annu. Todd Ciche.org by Pontificia Universidad Javeriana on 09/17/13. For personal use only. Cumulative Index of Contributing Authors. Downloaded from www. Rev. Volumes 59–63 p p p 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.Photorhabdus and a Host of Hosts Nick R.