Identification of Xenorhabdus nematophila genes required for mutualistic colonization of Steinernema carpocapsae nematodes

One stage in the symbiotic interaction between the bacterium Xenorhabdus nematophila and its nematode host, Steinernema carpocapsae, involves the species-specific colonization of the nematode intestinal vesicle by the bacterium. To characterize the bacterial molecular determinants that are essential for vesicle colonization, we adapted and applied a signature-tagged mutagenesis (STM) screen to this system. We identified 15 out of 3000 transposon mutants of X. nematophila with at least a 15-fold reduction in average vesicle colonization. These 15 mutants harbour disruptions in nine separate loci. Three of these loci have predicted open reading frames (ORFs) with similarity to genes (rpoS, rpoE, lrp) encoding regulatory proteins; two have predicted ORFs with similarity to genes (aroA, serC) encoding amino acid biosynthetic enzymes; one, designated nilB (nematode intestine localization), has an ORF with similarity to a gene encoding a putative outer membrane protein (OmpU) in Neisseria; and three, nilA, nilC and nilD, have no apparent homologues in the public database. nilA, nilB and nilC are linked on a single 4 kb locus. nilB and nilC are > 104-fold reduced in their ability to colonize the nematode vesicle and are predicted to encode membrane-localized proteins. The nilD locus contains an extensive repeat region and several small putative ORFs. Other than reduced colonization, the nilB, nilC and nilD mutants did not display alterations in any other phenotype tested, suggesting a specific role for these genes in allowing X. nematophila to associate with the nematode host.     

Mutational analyses reveal overall topology and functional regions of NilB, a bacterial outer membrane protein required for host association in a model of animal-microbe mutualism

The gammaproteobacterium Xenorhabdus nematophila is a mutualistic symbiont that colonizes the intestine of the nematode Steinernema carpocapsae. nilB (nematode intestine localization) is essential for X. nematophila colonization of nematodes and is predicted to encode an integral outer membrane beta-barrel protein, but evidence supporting this prediction has not been reported. The function of NilB is not known, but when expressed with two other factors encoded by nilA and nilC, it confers upon noncognate Xenorhabdus spp. the ability to colonize S. carpocapsae nematodes. We present evidence that NilB is a surface-exposed outer membrane protein whose expression is repressed by NilR and growth in nutrient-rich medium. Bioinformatic analyses reveal that NilB is the only characterized member of a family of proteins distinguished by N-terminal region tetratricopeptide repeats (TPR) and a conserved C-terminal domain of unknown function (DUF560). Members of this family occur in diverse bacteria and are prevalent in the genomes of mucosal pathogens. Insertion and deletion mutational analyses support a beta-barrel structure model with an N-terminal globular domain, 14 transmembrane strands, and seven extracellular surface loops and reveal critical roles for the globular domain and surface loop 6 in nematode colonization. Epifluorescence microscopy of these mutants demonstrates that NilB is necessary at early stages of colonization. These findings are an important step in understanding the function of NilB and, by extension, its homologs in mucosal pathogens.     

The global regulator Lrp contributes to mutualism, pathogenesis and phenotypic variation in the bacterium Xenorhabdus nematophila

Xenorhabdus nematophila is a Gram-negative bacterium that leads both pathogenic and mutualistic lifestyles. In this study, we examine the role of Lrp, the leucine-responsive regulatory protein, in regulating both of these lifestyles. lrp mutants have attenuated virulence towards Manduca sexta insects and are defective in suppression of both cellular and humoral insect immunity. In addition, an lrp mutant is deficient in initiating colonization of and growth within mutualistic host nematodes. Furthermore, nematodes reared on lrp mutant lawns exhibit decreased overall numbers of nematode progeny. To our knowledge, this is the first demonstration of virulence attenuation associated with an lrp mutation in any bacterium, as well as the first report of a factor involved in both X. nematophila symbioses. Protein profiles of wild-type and mutant cells indicate that Lrp is a global regulator of expression in X. nematophila, affecting approximately 65% of 290 proteins. We show that Lrp binds to the promoter regions of genes known to be involved in basic metabolism, mutualism and pathogenesis, demonstrating that the regulation of at least some host interaction factors is likely direct. Finally, we demonstrate that Lrp influences aspects of X. nematophila phenotypic variation, a spontaneous process that occurs during prolonged growth in stationary phase.     

The Steinernema carpocapsae intestinal vesicle contains a subcellular structure with which Xenorhabdus nematophila associates during colonization initiation

Steinernema carpocapsae infective juvenile (IJ) nematodes are intestinally colonized by mutualistic Xenorhabdus nematophila bacteria. During IJ development, a small number of ingested X. nematophila cells initiate colonization in an anterior region of the intestine termed the vesicle and subsequently multiply within this host niche. We hypothesize that efficient colonization of a high percentage of S. carpocapsae individuals (typically>85%) is facilitated by bacterial adherence to a site(s) in the nematode intestine. We provide evidence that the adherence site is a structure in the lumen of the IJ vesicle that we have termed the intravesicular structure (IVS). The IVS is an untethered cluster of anucleate spherical bodies that co-localizes with colonizing X. nematophila cells, but does not require X. nematophila for its formation. Colocalization with the IVS is readily apparent in IJs colonized by X. nematophila mutants that initiate intestinal colonization but fail to proliferate normally, suggesting that bacterial-IVS interaction occurs early in the colonization process. Treatment with insect haemolymph induces anal release of X. nematophila from colonized IJs and induces release of the IVS from uncolonized S. carpocapsae IJs. Released IVS were probed with several carbohydrate-specific lectins. One lectin, wheat-germ agglutinin, reacts strongly with a mucus-like substance that is present around individual spheres in the aggregate IVS. Potential roles for the IVS in mediating X. nematophila colonization of the nematode intestine are discussed.     

The Xenorhabdus nematophila nilABC genes confer the ability of Xenorhabdus spp. to colonize Steinernema carpocapsae nematodes

Members of the Steinernema genus of nematodes are colonized mutualistically by members of the Xenorhabdus genus of bacteria. In nature, Steinernema carpocapsae nematodes are always found in association with Xenorhabdus nematophila bacteria. Thus, this interaction, like many microbe-host associations, appears to be species specific. X. nematophila requires the nilA, nilB, and nilC genes to colonize S. carpocapsae. In this work, we showed that of all the Xenorhabdus species examined, only X. nematophila has the nilA, nilB, and nilC genes. By exposing S. carpocapsae to other Xenorhabdus spp., we established that only X. nematophila is able to colonize S. carpocapsae; therefore, the S. carpocapsae-X. nematophila interaction is species specific. Further, we showed that introduction of the nilA, nilB, and nilC genes into other Xenorhabdus species enables them to colonize the same S. carpocapsae host tissue that is normally colonized by X. nematophila. Finally, sequence analysis supported the idea that the nil genes were horizontally acquired. Our findings indicate that a single genetic locus determines host specificity in this bacteria-animal mutualism and that host range expansion can occur through the acquisition of a small genetic element.     

The hmsHFRS operon of Xenorhabdus nematophila is required for biofilm attachment to Caenorhabditis elegans

The bacterium Xenorhabdus nematophila is an insect pathogen and an obligate symbiont of the nematode Steinernema carpocapsae. X. nematophila makes a biofilm that adheres to the head of the model nematode Caenorhabditis elegans, a capability X. nematophila shares with the biofilms made by Yersinia pestis and Yersinia pseudotuberculosis. As in Yersinia spp., the X. nematophila biofilm requires a 4-gene operon, hmsHFRS. Also like its Yersinia counterparts, the X. nematophila biofilm is bound by the lectin wheat germ agglutinin, suggesting that beta-linked N-acetyl-D-glucosamine or N-acetylneuraminic acid is a component of the extracellular matrix. C. elegans mutants with aberrant surfaces that do not permit Yersinia biofilm attachment also are resistant to X. nematophila biofilms. An X. nematophila hmsH mutant that failed to make biofilms on C. elegans had no detectable defect in symbiotic association with S. carpocapsae, nor was virulence reduced against the insect Manduca sexta.     

Analysis of Xenorhabdus nematophila metabolic mutants yields insight into stages of Steinernema carpocapsae nematode intestinal colonization

Xenorhabdus nematophila colonizes the intestinal tract of infective-juvenile (IJ) stage Steinernema carpocapsae nematodes. During colonization, X. nematophila multiplies within the lumen of a discrete region of the IJ intestine termed the vesicle. To begin to understand bacterial nutritional requirements during multiplication in the IJ vesicle, we analysed the colonization behaviour of several X. nematophila metabolic mutants, including amino acid and vitamin auxotrophs. X. nematophila mutants defective for para-aminobenzoate, pyridoxine or l-threonine biosynthesis exhibit substantially decreased colonization of IJs (0.1-50% of wild-type colonization). Analysis of gfp-labelled variants revealed that those mutant cells that can colonize the IJ vesicle differ noticeably from wild-type X. nematophila. One aberrant colonization phenotype exhibited by the metabolic mutants tested, but not wild-type X. nematophila, is a spherical shape indicative of apparently non-viable X. nematophila cells within the vesicle. Because these spherical cells appear to have initiated colonization but failed to proliferate, we term this type of colonization 'abortive'. In a portion of IJs grown on para-aminobenzoate auxotrophs, X. nematophila does not exhibit abortive colonization but rather reduced growth and filamentous cell morphology. Several mutants with defects in other amino acid, vitamin and nutrient metabolism pathways colonize IJs to wild-type levels suggesting that the IJ vesicle is replete with respect to a number of nutrients.     

Pyrimidine nucleoside salvage confers an advantage to Xenorhabdus nematophila in its host interactions

Xenorhabdus nematophila is a mutualist of entomopathogenic nematodes and a pathogen of insects. To begin to examine the role of pyrimidine salvage in nutrient exchange between X. nematophila and its hosts, we identified and mutated an X. nematophila tdk homologue. X. nematophila tdk mutant strains had reduced virulence toward Manduca sexta insects and a competitive defect for nematode colonization in plate-based assays. Provision of a wild-type tdk allele in trans corrected the defects of the mutant strain. As in Escherichia coli, X. nematophila tdk encodes a deoxythymidine kinase, which converts salvaged deoxythymidine and deoxyuridine nucleosides to their respective nucleotide forms. Thus, nucleoside salvage may confer a competitive advantage to X. nematophila in the nematode intestine and be important for normal entomopathogenicity.     

They've got a ticket to ride: Xenorhabdus nematophila-Steinernema carpocapsae symbiosis

The association between the bacterium Xenorhabdus nematophila and the nematode Steinernema carpocapsae is emerging as a model system to understand mutually beneficial symbioses. X. nematophila, but not other Xenorhabdus species, colonize a discrete region of a specific developmental stage of S. carpocapsae nematodes. Recent progress has led to the identification of bacterial genes necessary for colonization. Furthermore, new details have been elucidated regarding the morphology and physiology of the colonization site and the bacteria within it. A deeper understanding of the molecular mechanisms underlying the association of X. nematophila will undoubtedly yield insights into fundamental processes underlying the ubiquitous association of microbes with animals.     

The xnp1 P2-like tail synthesis gene cluster encodes xenorhabdicin and is required for interspecies competition

Xenorhabdus nematophila, the mutualistic bacterium of the nematode Steinernema carpocapsae, produces the R-type bacteriocin called xenorhabdicin, which is thought to confer a competitive advantage for growth in the insect host. We have identified a P2-like tail synthesis gene cluster (xnp1) that is required for xenorhabdicin production. The xnp1 genes were expressed constitutively during growth and were induced by mitomycin C. Deletion of either the sheath (xnpS1) or fiber (xnpH1) genes eliminated xenorhabdicin production. Production of R-type bacteriocins in a host organism had not been shown previously. We show that xenorhabdicin is produced in the hemocoel of insects infected with the wild type but not with the ΔxnpS1 deletion strain. Xenorhabdicin prepared from the wild-type strain killed the potential competitor Photorhabdus luminescens TT01. P. luminescens was eliminated during coculture with wild-type X. nematophila but not with the ΔxnpS1 strain. Furthermore, P. luminescens inhibited reproduction of S. carpocapsae in insect larvae, while coinjection with wild-type X. nematophila, but not the ΔxnpS1, strain restored normal reproduction, demonstrating that xenorhabdicin was required for killing P. luminescens and protecting the nematode partner. Xenorhabdicin killed X. nematophila from Steinernema anatoliense, demonstrating for the first time that it possesses intraspecies activity. In addition, activity was variable against diverse strains of Xenorhabdus and Photorhabdus and was not correlated with phylogenetic distance. These findings are discussed in the context of the role of xenorhabdicin in the life cycle of the mutualistic bacterium X. nematophila.     

Pyrimidine Nucleoside Salvage Confers an Advantage to Xenorhabdus nematophila in Its Host Interactions

Xenorhabdus nematophila is a mutualist of entomopathogenic nematodes and a pathogen of insects. To begin to examine the role of pyrimidine salvage in nutrient exchange between X. nematophila and its hosts, we identified and mutated an X. nematophila tdk homologue. X. nematophila tdk mutant strains had reduced virulence toward Manduca sexta insects and a competitive defect for nematode colonization in plate-based assays. Provision of a wild-type tdk allele in trans corrected the defects of the mutant strain. As in Escherichia coli, X. nematophila tdk encodes a deoxythymidine kinase, which converts salvaged deoxythymidine and deoxyuridine nucleosides to their respective nucleotide forms. Thus, nucleoside salvage may confer a competitive advantage to X. nematophila in the nematode intestine and be important for normal entomopathogenicity.     

Role of Mrx fimbriae of Xenorhabdus nematophila in competitive colonization of the nematode host

Xenorhabdus nematophila engages in mutualistic associations with the infective juvenile (IJ) stage of specific entomopathogenic nematodes. Mannose-resistant (Mrx) chaperone-usher-type fimbriae are produced when the bacteria are grown on nutrient broth agar (NB agar). The role of Mrx fimbriae in the colonization of the nematode host has remained unresolved. We show that X. nematophila grown on LB agar produced flagella rather than fimbriae. IJs propagated on X. nematophila grown on LB agar were colonized to the same extent as those propagated on NB agar. Further, progeny IJs were normally colonized by mrx mutant strains that lacked fimbriae both when bacteria were grown on NB agar and when coinjected into the insect host with aposymbiotic nematodes. The mrx strains were not competitively defective for colonization when grown in the presence of wild-type cells on NB agar. In addition, a phenotypic variant strain that lacked fimbriae colonized as well as the wild-type strain. In contrast, the mrx strains displayed a competitive colonization defect in vivo. IJ progeny obtained from insects injected with comixtures of nematodes carrying either the wild-type or the mrx strain were colonized almost exclusively with the wild-type strain. Likewise, when insects were coinjected with aposymbiotic IJs together with a comixture of the wild-type and mrx strains, the resulting IJ progeny were predominantly colonized with the wild-type strain. These results revealed that Mrx fimbriae confer a competitive advantage during colonization in vivo and provide new insights into the role of chaperone-usher fimbriae in the life cycle of X. nematophila.     

Potentiating effect of Bacillus thuringiensis subsp. kurstaki on pathogenicity of entomopathogenic bacterium Xenorhabdus nematophila K1 against diamondback moth (Lepidoptera: Plutellidae)

Xenorhabdus nematophila is the symbiotic bacterium of an entomopathogenic nematode, Steinernema carpocapsae. When the nematode enters a target insect, the symbiotic bacteria are released into the hemocoel. After inducing host immunosuppression, the bacteria multiply in the hemocoel and cause fatal septicemia. For optimal field application to control insect pests, culturing mass numbers of the nematodes would be costly. In this study, Bacillus thuringiensis (Bt) was chosen as an alternative natural vector, which would be relatively economical for field application. Bt infection of gut epithelium would form a bacterial passage between the gut lumen and hemocoel, which facilitates the orally fed X. nematophila to infect the hemocoel. Diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), used in this study was tolerant to Bt because only 10% mortality was noted in response to 2 times higher concentration than recommended for commercial B. t. kurstaki, although this species was susceptible only during early instars. The orally fed X. nematophila caused significant mortality to early instars of P. xylostella, but not late instars. When both X. nematophila and Bt were fed to late instars of P. xylostella, they showed significantly enhanced mortality, in which X. nematophila cells were recovered from the hemocoel of the treated P. xylostella. However, when only X. nematophila was fed, no cells were recovered from the hemolymph. This study suggests that X. nematophila can be applied to control P. xylostella in a mixture with Bt in the field without its nematode host.     

Bactericidal activity of colicin V is mediated by an inner membrane protein, SdaC, of Escherichia coli

Colicin V (ColV) is a peptide antibiotic that kills sensitive cells by disrupting their membrane potential once it gains access to the inner membrane from the periplasmic face. Recently, we constructed a translocation suicide probe, RR-ColV, that is translocated into the periplasm via the TAT pathway and thus kills the host cells. In this study, we obtained an RR-ColV-resistant mutant by using random Tn10 transposition mutagenesis. Sequencing analysis revealed that the mutant carried a Tn10 insertion in the sdaC (also called dcrA) gene, which is involved in serine uptake and is required for C1 phage adsorption. ColV activity was detected both in the cytoplasm and in the periplasm of this mutant, indicating that RR-ColV was translocated into the periplasm but failed to interact with the inner membrane. The sdaC::Tn10 mutant was resistant only to ColV and remained sensitive to colicins Ia, E3, and A. Most importantly, the sdaC::Tn10 mutant was killed when ColV was anchored to the periplasmic face of the inner membrane by fusion to EtpM, a type II integral membrane protein. Taken together, these results suggest that the SdaC/DcrA protein serves as a specific inner membrane receptor for ColV.     

Analysis of the PixA inclusion body protein of Xenorhabdus nematophila

The symbiotic pathogenic bacterium Xenorhabdus nematophila produces two distinct intracellular inclusion bodies. The pixA gene, which encodes the 185-residue methionine-rich PixA inclusion body protein, was analyzed in the present study. The pixA gene was optimally expressed under stationary-phase conditions but its expression did not require RpoS. Analysis of a pixA mutant strain showed that PixA was not required for virulence towards the insect host or for colonization of or survival within the nematode host, and was not essential for nematode reproduction. The pixA gene was not present in the genome of Xenorhabdus bovienii, which also produces proteinaceous inclusions, indicating that PixA is specifically produced in X. nematophila.     

Monoxenic liquid culture of the entomopathogenic nematode Steinernema carpocapsae using a culture medium containing whey kinetics and modeling

The submerged culture of the entomopathogenic nematode Steinernema carpocapsae and its symbiotic bacterium, Xenorhabdus nematophila, was carried out in orbitally agitated bottles using a culture medium containing whey (in grams per litre: 500 whey, 20 yeast extract, 10 dried egg yolk-food grade, 3 sodium chloride, 37 corn oil-food grade). Maximum total viable nematode concentrations of 198,333ml(-1) were achieved within fermentations of 24 days with 64% of the nematode population within the infective juvenile stage (IJ) (126,666ml(-1)) at the end. The kinetics of the bioprocess was well modelled using the four-parameter Sigmoidal model and the corresponding maximum specific rates of nematode production (0.47 day(-1)), carbohydrates consumption (0.0008g(carbohydrates)g(nematodes)(-1)day(-1)) and nitrogen consumption (4.44g(nitrogen)g(nematodes)(-1)day(-1)) are first proposed. Besides, X. nematophila appears to have the capacity of lactose hydrolysis.     

Identification and functional characterization of a Xenorhabdus nematophila oligopeptide permease

The bacterium Xenorhabdus nematophila is a mutualist of Steinernema carpocapsae nematodes and a pathogen of insects. Presently, it is not known what nutrients the bacterium uses to thrive in these host environments. In other symbiotic bacteria, oligopeptide permeases have been shown to be important in host interactions, and we therefore sought to determine if oligopeptide uptake is essential for growth or symbiotic functions of X. nematophila in laboratory or host environments. We identified an X. nematophila oligopeptide permease (opp) operon of two sequential oppA genes, predicted to encode oligopeptide-binding proteins, and putative permease-encoding genes oppB, oppC, oppD, and oppF. Peptide-feeding studies indicated that this opp operon encodes a functional oligopeptide permease. We constructed strains with mutations in oppA(1), oppA(2), or oppB and examined the ability of each mutant strain to grow in a peptide-rich laboratory medium and to interact with the two hosts. We found that the opp mutant strains had altered growth phenotypes in the laboratory medium and in hemolymph isolated from larval insects. However, the opp mutant strains were capable of initiating and maintaining both mutualistic and pathogenic host interactions. These data demonstrate that the opp genes allow X. nematophila to utilize peptides as a nutrient source but that this function is not essential for the existence of X. nematophila in either of its host niches. To our knowledge, this study represents the first experimental analysis of the role of oligopeptide transport in mediating a mutualistic invertebrate-bacterium interaction.