Xenorhabdus Species Research Articles

Insecticidal Toxin Complexes from Photorhabdus luminescens.

Various bacterial toxins have potent insecticidal activity. Recently, the Toxin complexes (Tc's) of Photorhabdus and Xenorhabdus species have become an increased focus of current research. These large tripartite toxins with molecular masses >1.4megadaltons consist of three components termed A, B, and C (or TcA, TcB, and TcC). While TcA is involved in receptor binding and toxin translocation, TcC possesses the specific toxin enzyme activity and TcB is a linker between components TcA and TcC. Here, a structure function analysis of the toxins is described and the application of Tc toxins as potential insecticides is discussed.

Xenorhabdus bovienii CS03, the bacterial symbiont of the entomopathogenic nematode Steinernema weiseri, is a non-virulent strain against lepidopteran insects

Xenorhabdus bacteria (γ-proteobacteria: Enterobacteriaceae) have dual lifestyles. They have a mutualistic relationship with Steinernema nematodes (Nematoda: Steinernematidae) and are pathogenic to a wide range of insects. Each Steinernema nematode associates with a specific Xenorhabdus species. However, a Xenorhabdus species can have multiple nematode hosts. For example, Xenorhabdus bovienii (Xb) colonizes at least nine Steinernema species from two different phylogenetic clades. The Steinernema–Xb partnership has been found in association with different insect hosts. Biological and molecular data on the Steinernema jollieti–Xb strain SS-2004 pair have recently been described. In particular, the Xb SS-2004 bacteria are virulent alone after direct injection into insect, making this strain a model for studying Xb virulence. In this study, we searched for Xb strains attenuated in virulence. For this purpose, we underwent infection assays with five Steinernema spp.–Xb pairs with two insects, Galleria mellonella (Lepidoptera: Pyralidae) and Spodoptera littoralis (Lepidoptera: Noctuidae). The S. weiseri–Xb CS03 pair showed attenuated virulence and lower fitness in S. littoralis in comparison to the other nematode-bacteria pairs. Furthermore, when injected alone into the hemolymph of G. mellonella or S. littoralis, the Xb CS03 bacterial strain was the only non-virulent strain. By comparison with the virulent Xb SS-2004 strain, Xb CS03 showed an increased sensitivity to the insect antimicrobial peptides, suggesting an attenuated response to the insect humoral immunity. To our current knowledge, Xb CS03 is the first non-virulent Xb strain identified. We propose this strain as a new model for studying the Xenorhabdus virulence.

Attenuated virulence and genomic reductive evolution in the entomopathogenic bacterial symbiont species, Xenorhabdus poinarii.

Bacteria of the genus Xenorhabdus are symbionts of soil entomopathogenic nematodes of the genus Steinernema. This symbiotic association constitutes an insecticidal complex active against a wide range of insect pests. Unlike other Xenorhabdus species, Xenorhabdus poinarii is avirulent when injected into insects in the absence of its nematode host. We sequenced the genome of the X. poinarii strain G6 and the closely related but virulent X. doucetiae strain FRM16. G6 had a smaller genome (500–700 kb smaller) than virulent Xenorhabdus strains and lacked genes encoding potential virulence factors (hemolysins, type 5 secretion systems, enzymes involved in the synthesis of secondary metabolites, and toxin–antitoxin systems). The genomes of all the X. poinarii strains analyzed here had a similar small size. We did not observe the accumulation of pseudogenes, insertion sequences or decrease in coding density usually seen as a sign of genomic erosion driven by genetic drift in host-adapted bacteria. Instead, genome reduction of X. poinarii seems to have been mediated by the excision of genomic blocks from the flexible genome, as reported for the genomes of attenuated free pathogenic bacteria and some facultative mutualistic bacteria growing exclusively within hosts. This evolutionary pathway probably reflects the adaptation of X. poinarii to specific host.

Putative toxins from the entomopathogenic bacterium Photorhabdus luminescens kill Armadillidium vulgare (Terrestrial isopod)

Terrestrial isopods can be killed by some entomopathogenic bacteria among Xenorhabdus and Photorhabdus species even with no or very limited multiplication. This suggests that toxemia and not septicemia is the major cause of entomopathogenic bacteria pathogenicity against these crustaceans. In this paper, we revealed that the injection of stationary phase culture supernatant of P. luminescens TT01, in which toxins can be accumulated, led alone to a rapid decrease in the number of host immune cells and killed most of the Armadillidium vulgare individuals within 48h. The pathogenicity was strongly attenuated when supernatant was heated and totally suppressed after 100-kDa filtration suggesting that the toxin responsible for killing A. vulgare would be a protein above this size. Additionally, we tested the culture supernatant of two TT01 mutants that have been previously shown as being altered in their pathogenicity against lepidopteran insects one of them being known as exhibiting lower expression of some toxins. However, the supernatants of the mutants was as pathogenic for A. vulgare as the wild type strains suggesting that the toxins involved in killing A. vulgare may be different than previously described ones.

Rearing and Injection of <em>Manduca sexta</em> Larvae to Assess Bacterial Virulence

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and tomato. The susceptibility of M. sexta larvae to a variety of entomopathogenic bacterial species(1-5), as well as the wealth of information available regarding the insect's immune system(6-8), and the pending genome sequence(9) make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species. Their large size also facilitates efficient tissue/hemolymph extraction for analysis of the host response to infection. The method presented here describes the direct injection of bacteria into the hemocoel (blood cavity) of M. sexta larvae. This approach can be used to analyze and compare the virulence characteristics of various bacterial species, strains, or mutants by simply monitoring the time to insect death after injection. This method was developed to study the pathogenicity of Xenorhabdus and Photorhabdus species, which typically associate with nematode vectors as a means to gain entry into the insect. Entomopathogenic nematodes typically infect larvae via natural digestive or respiratory openings, and release their symbiotic bacterial contents into the insect hemolymph (blood) shortly thereafter(10). The injection method described here bypasses the need for a nematode vector, thus uncoupling the effects of bacteria and nematode on the insect. This method allows for accurate enumeration of infectious material (cells or protein) within the inoculum, which is not possible using other existing methods for analyzing entomopathogenesis, including nicking(11) and oral toxicity assays(12). Also, oral toxicity assays address the virulence of secreted toxins introduced into the digestive system of larvae, whereas the direct injection method addresses the virulence of whole-cell inocula. The utility of the direct injection method as described here is to analyze bacterial pathogenesis by monitoring insect mortality. However, this method can easily be expanded for use in studying the effects of infection on the M. sexta immune system. The insect responds to infection via both humoral and cellular responses. The humoral response includes recognition of bacterial-associated patterns and subsequent production of various antimicrobial peptides(7); the expression of genes encoding these peptides can be monitored subsequent to direct infection via RNA extraction and quantitative PCR(13). The cellular response to infection involves nodulation, encapsulation, and phagocytosis of infectious agents by hemocytes(6). To analyze these responses, injected insects can be dissected and visualized by microscopy(13, 14).

Comparative analysis of P2-type remnant prophage loci in Xenorhabdus bovienii and Xenorhabdus nematophila required for xenorhabdicin production

The xnp1 remnant P2-type prophage of Xenorhabdus nematophila produces xenorhabdicin that is active against closely related species. Xenorhabdicin had not been characterized previously in other Xenorhabdus species. Here, we show xenorhabdicin production in six different strains of Xenorhabdus bovienii. The sequenced genome of X. bovienii SS-2004 was found to possess a highly conserved remnant P2-type cluster (xbp1). Inactivation of the xbpS1 sheath gene resulted in loss of bacteriocin activity, indicating that the xbp1 locus was required for xenorhabdicin production. xbp1 and xnp1 contain a CI-type repressor, a dinI gene involved in stabilization of ssDNA-RecA complexes and are inducible with mitomycin C, suggesting that both loci are regulated by cleavage of the CI repressor. Both xnp1 and xbp1 lack typical P2-type lysis genes but contain a predicted endolysin gene (enp) that may be involved in cell lysis. The main tail fibers of xnp1 and xbp1 are mosaic structures with divergent C-terminal regions suggesting they differ in host specificity. Several genes encoding C-terminal tail fiber fragments are present in the same position in xnp1 and xbp1. Recombination between the main fiber genes and the C-terminal fragments could potentially expand the host range specificity of xenorhabdicin in the respective strains.

Virulence and competitive ability in an obligately killing parasite

Mixed infections are thought to have a major influence on the evolution of parasite virulence. During a mixed infection, higher within-host parasite growth is favored under the assumption that it is critical to the competitive success of the parasite. As within-host parasite growth may also increase damage to the host, a positive correlation is predicted between virulence and competitive success. However, when parasites must kill their hosts in order be transmitted, parasites may spend energy on directly attacking their host, even at the cost of their within-host growth. In such systems, a negative correlation between virulence and competitive success may arise. We examined virulence and competitive ability in three sympatric species of obligately killing nematode parasites in the genus Steinernema. These nematodes exist in a mutualistic symbiosis with bacteria in the genus Xenorhabdus. Together the nematodes and their bacteria kill the insect host soon after infection, with reproduction of both species occurring mainly after host death. We found significant differences among the three nematode species in the speed of host killing. The nematode species with the lowest and highest levels of virulence were associated with the same species of Xenorhabdus, indicating that nematode traits, rather than the bacterial symbionts, may be responsible for the differences in virulence. In mixed infections, host mortality rate closely matched that associated with the more virulent species, and the more virulent species was found to be exclusively transmitted from the majority of coinfected hosts. Thus, despite the requirement of rapid host death, virulence appears to be positively correlated with competitive success in this system. These findings support a mechanistic link between parasite growth and both anti-competitor and anti-host factors.

A multigene approach for assessing evolutionary relationships of Xenorhabdus spp. (γ-Proteobacteria), the bacterial symbionts of entomopathogenic Steinernema nematodes

Xenorhabdus spp., are gram-negative bacterial symbionts of entomopathogenic nematodes in the genus Steinernema. A specialized and intimate relationship exists between nematode and bacteria, affecting many of their life history traits, such as nutrition, dispersal, host-finding, foraging and defense from biotic and abiotic factors. Xenorhabdus currently comprises more than 20 species isolated from Steinernema spp. with diverse host range, host foraging behavior, reproductive modes and environmental tolerance. Xenorhabdus phylogenies have historically been based on 16s rDNA sequence analyses, and only recently has data from housekeeping genes been employed. The prevalence of lateral gene transfer among bacteria calls for a wider perspective when considering their phylogeny. With the increasing number of Xenorhabdus species and strains, various perspectives need to be considered for investigating the evolutionary history of these nematode bacterial symbionts, In this study, we reconstruct the evolutionary histories of 30 species of Xenorhabdus considering the traditional 16s rDNA gene region as well as the housekeeping genes recA and serC. Datasets were analyzed individually and then combined, using a variety of phylogenetic criteria.

Isolation and activity ofXenorhabdusantimicrobial compounds against the plant pathogensErwinia amylovoraandPhytophthora nicotianae

Broad-spectrum antibiotics produced by symbiotic bacteria [entomopathogenic bacterium (EPB)] of entomopathogenic nematodes keep monoxenic conditions in insect cadavers in soil. This study evaluated antibiotics produced by EPB for their potential to control plant pathogenic bacteria and oomycetes. Entomopathogenic bacterium produce antibiotics effective against the fire blight bacterium Erwinia amylovora, including streptomycin resistant strains, and were as effective in phytotron experiments as kasugamycin or streptomycin. Xenorhabdus budapestensis and X. szentirmaii antibiotics inhibited colony formation and mycelial growth of Phytophthora nicotianae. From X. budapestensis, an arginine-rich fraction (bicornutin) was adsorbed by Amberlite((R)) XAD 1180, and eluted with methanol : 1 n HCI (99 : 1). Bicornutin inactivated zoospores, and inhibited germination and colony formation of cystospores at <<25 ppm. An UV-active molecule (bicornutin-A, MW = 826), separated by HPLC and thin-layer chromatography, was identified as a novel hexa-peptide : RLRRRX. Xenorhabdus budapestensis produces metabolites with strong antibacterial and cytotoxic activity. Individual compounds can be isolated, identified and patented, but their full antimicrobial potential may be multiplied by synergic interactions. Active compounds of two new Xenorhabdus species might control plant diseases caused by pathogens of great importance to agriculture such as Erw. amylovora and P. nicotianae.

The bacterium associated with the entomopathogenic nematode Steinernema abbasi (Nematoda: Steinernematidae) isolated from Taiwan

A symbiotic bacterium of the entomopathogenic nematode, Steinernema abbasi, isolated from Taiwan, determined to be a species of Xenorhabdus based on its physiological and biochemical characteristics has been determined to be similar to Xenorhabdus indica of S. abbasi Oman isolate as based on sequence analyses of 16S rDNA.

Biochemical and Molecular Profiling of Indigenous Xenorhabdus Isolates Associated with Steinernema Spp

Bacterial symbionts were obtained from entomopathogenic nematodes Steinernema carpocapsae, S, riobrave, S. feltiae and S. tami, and identified as belonging to Xenorhabdus species by subjecting to biochemical characterization. The Xenorhabdus isolates were further characterized for their interspecific variation by protein profiling and RFLP analysis of 16S rDNA. The protein profiles recorded discernible differences with protein distribution ranging from 97Kda to 14Kda, but most of the fragments were common to all isolates. RFLP analysis of 16S rDNA of the isolates using eight restriction enzymes showed the distinctness of isolates and based on the restriction enzyme patterns, the isolates were grouped into two clusters. The study showed that a combination of biochemical and molecular techniques could be used for the identification and characterization of Xenorhabdus isolates.

Biochemical and molecular profiling of indigenous Xenorhabdus isolates associated with Steinernema spp.

Bacterial symbionts were obtained from entomopathogenic nematodes Steinernema carpocapsae, S, riobrave, S. feltiae and S. tami, and identified as belonging to Xenorhabdus species by subjecting to biochemical characterization. The Xenorhabdus isolates were further characterized for their interspecific variation by protein profiling and RFLP analysis of 16S rDNA. The protein profiles recorded discernible differences with protein distribution ranging from 97Kda to 14Kda, but most of the fragments were common to all isolates. RFLP analysis of 16S rDNA of the isolates using eight restriction enzymes showed the distinctness of isolates and based on the restriction enzyme patterns, the isolates were grouped into two clusters. The study showed that a combination of biochemical and molecular techniques could be used for the identification and characterization of Xenorhabdus isolates.

Xenorhabdus antibiotics: a comparative analysis and potential utility for controlling mastitis caused by bacteria

The role of antibiotics produced by bacterial symbionts of entomopathogenic nematodes is to suppress growth of microbes in the soil environment. These antibiotics are active against Gram-positive and Gram-negative bacteria, and were tested against mastitis isolates from dairy cows. Two bioassays were adapted for Xenorhabdus antibiotics; an overlay method on agar plates, and serially diluted, cell-free, Xenorhabdus cultures. The antimicrobial activities of the liquid cultures of 13 strains from five Xenorhabdus species were further evaluated. Antimicrobial activities of the type strains of X. nematophila, X. budapestensis and X. szentirmaii were tested on mastitis isolates of Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae with both bioassays. A previously reported antibiotic from X. nematophila, nematophin, was synthesized in three steps from tryptamine and 4-methyl-2-oxovaleric acid sodium salt. The antibiotics of all three Xenorhabdus strains were powerful in either bioassay, but the sensitivity of the isolates differed from each other. While Kl. pneumoniae was the least susceptible, Staph. aureus had the highest sensitivity to each Xenorhabdus strain. Xenorhabdus szentirmaii and X. budapestensis were more potent antibiotic producers than X. nematophila, and raceme nematophin was ineffective against all mastitis isolates. These results indicate that Xenorhabdus antibiotics are effective against mastitis isolates and should be further evaluated for their potential in mastitis control or prevention.

Optical mapping as a routine tool for bacterial genome sequence finishing.

BackgroundIn sequencing the genomes of two Xenorhabdus species, we encountered a large number of sequence repeats and assembly anomalies that stalled finishing efforts. This included a stretch of about 12 Kb that is over 99.9% identical between the plasmid and chromosome of X. nematophila.ResultsWhole genome restriction maps of the sequenced strains were produced through optical mapping technology. These maps allowed rapid resolution of sequence assembly problems, permitted closing of the genome, and allowed correction of a large inversion in a genome assembly that we had considered finished.ConclusionOur experience suggests that routine use of optical mapping in bacterial genome sequence finishing is warranted. When combined with data produced through 454 sequencing, an optical map can rapidly and inexpensively generate an ordered and oriented set of contigs to produce a nearly complete genome sequence assembly.

Symbiosis

Symbiosis

Comparative bacterial genomics and its use in undergraduate education

Abstract Photorhabdus spp. and Xenorhabdus spp. both form mutualistic associations with entomopathogenic nematodes and function as potent pathogenic agents towards a variety of insects. The availability of genetic approaches and the ability to grow the bacteria in standard media make Photorhabdus and Xenorhabdus promising models for the study of host–microbe interactions. A particularly attractive aspect of this system is that both harmful and beneficial interactions can be studied in a single microbe. The genome sequence of Photorhabdus luminescens subspecies laumondii strain TT01 has been completed and a genome project for Photorhabdus asymbiotica has been initiated. Likewise, a collaborative project to compare the genomes of two species of Xenorhabdus is in progress. The availability of genomic sequences for Photorhabdus and Xenorhabdus will open new avenues of investigation and enhance our understanding of the molecular mechanism by which a bacterium can function as both symbionts and pathogens. In this paper, the genomic information from the Photorhabdus species are compared with the genome of the related species, Yersinia pestis, and the usefulness of genomics in undergraduate education will provide excellent training for future scientists and assist in a better understanding of the nematode–bacterium complex.

Taxonomic Identification of Bacteria, Associated with Bulgarian Populations of Entomopathogenic Nematodes from Genus Steinernema (Rhabditida, Steinernematidae) II

ABSTRACTThe entomopathogenic nematode from the genera Steinernema and Heterorhabditis are symbiotically associated with bacteria from the genera Xenorhabdus and Photorhabdus. The bacteria belong to the family Enterobacteriaceae; pathogenic when injected into the hemocoel of Galleria mellonella (Lepidopthera) larvae. All Xenorhabdus and Photorhabdus isolates have been shown to produce two forms when cultured in vitro. These colony forms indicate two phases that differ in their morphological and biochemical characteristics. Each nematode species has a specific association with one species of bacterium. Xenorhabdus or Photorhabdus species may be associated with more than one species (1, 2). Entomopathogenic nematodes represent one important part of the spectrum of biocontrol agents. They are used to control insect pests in high-value crops and potentially they could be used in integrated pest management, organic farming and sustainable agriculture systems to control soil-borne insect pests. During our study we found bacteria belonging not only to the family Enterobacteriaceae. The results of an investigation on taxonomic identification of bacteria, isolated from three different entomopathogenic populations are presented in this paper.

Description of four novel species of Xenorhabdus, family Enterobacteriaceae: Xenorhabdus budapestensis sp. nov., Xenorhabdus ehlersii sp. nov., Xenorhabdus innexi sp. nov., and Xenorhabdus szentirmaii sp. nov.

The taxonomic affiliation was determined for four Xenorhabdus strains isolated from four Steinernema hosts from different countries. As compared to the five validly described Xenorhabdus species, i.e., X. nematophila, X. japonica, X. beddingii, X. bovienii and X. poinarii, these isolates represented novel species on the basis of 16S rRNA gene sequences and riboprint patterns, as well as by physiological and metabolic properties. They were named Xenorhabdus budapestensis sp. nov., type strain DSM 16342 T, isolated from Steinernema bicornutum; Xenorhabdus ehlersii sp. nov., type strain DSM 16337 T, isolated from Steinernema serratum; Xenorhabdus innexi sp. nov., type strain DSM 16336 T isolated from Steinernema scapterisci; and Xenorhabdus szentirmaii sp. nov., type strain DSM 16338 T, isolated from Steinernema rarum.

Invasion of insect blood tissue by Xenorhabdus bovienii

Xenorhabdus species are entomopathogenic Gram-negative bacteria that belong to the family Enterobacteriaceae. They exist in two main phenotypic variations referred to as phase I and phase II. Invasion studies showed that Xenorhabdus bovienii phase I, unlike phase II, adhered to and were phagocytosed by Drosophila melanogaster malignant blood neoplasm, mbn-2. The ingested bacteria within the phagosome multiplied and escaped into the mbn-2 cytoplasm. Further bacterial multiplication occurred within the cytoplasm with a longer postinvasion incubation period, resulting in mbn-2 lysis. X. bovienii multiplied more rapidly in artificial media than in mbn-2. The generation time of phase I X. bovienii in the artificial media was 2.5- to 3-fold that in mbn-2. Cell free extract of X. bovienii phase I fluoresced under UV light, unlike that of phase II.

Entomopathogenic symbiotic bacteria, Xenorhabdus and Photorhabdus of nematodes

Xenorhabdus and Photorhabdus species are entomopathogenic bacteria with a wide insect host range, that belong to the family Enterobacteriaceae. Xenorhabdus and Photorhabdus species symbiotically associate with nematodes of the families Steinernematidae and Heterorhabditidae respectively. The factor(s) determining the symbiotic interaction between nematodes and bacteria are yet to be identified. Xenorhabdus and Photorhabdus species exist in two main phenotypic forms, a phenomenon known as phase variation. The phase I (or primary form) varies from phase II (or secondary form) in certain physiological and morphological characteristics. There is no variation in the DNA integrity of phase I and phase II and this supports epigenetic regulatory mechanism in phase variation. Certain pathogenic determinants such as pili, lipopolysaccharides and toxins contribute to the pathogenicity of Xenorhabdus and Photorhabdus species, and both appear to be equally pathogenic to insects. The observed similarity in their virulence to insect hosts may reflect possible in vivo conversion of phase II to phase I, however the host cellular invasion and virulence is yet to be properly understood. The virulence of Xenorhabdus variants varies among insects apparently due to factors which include the feeding habits of the insects. The molecular mechanism and biological significance of phase variation are presently unknown.