Xenorhabdus lircayensis sp. nov., the symbiotic bacterium associated with the entomopathogenic nematode Steinernema unicornum.

Xenorhabdus is a symbiotic group of bacteria associated with entomopathogenic nematodes of the family Steinernematidae. Although the described Steirnernema species list is extensive, not all their symbiotic bacteria have been identified. One single motile, Gram-negative and non-spore-forming rod-shaped symbiotic bacterium, strain VLST, was isolated from the entomopathogenic nematode Steinernema unicornum. Analyses of the 16S rRNA gene determined that the VLST isolate belongs to the genus Xenorhabdus, and its closest related species is Xenorhabdus szentirmaii DSM 16338T (98.2 %). Deeper analyses using the whole genome for phylogenetic reconstruction indicate that VLST exhibits a unique clade in the genus. Genomic comparisons considering digital DNA-DNA hybridization (dDDH) values confirms this result, showing that the VLST values are distant enough from the 70 % threshold suggested for new species, sharing 30.7, 30.5 and 30.3 % dDDH with Xenorhabdus khoisanae MCB, Xenorhabdus koppenhoeferi DSM 18168T and Xenorhabdus miraniensis DSM 18168T, respectively, as the closest species. Detailed physiological, biochemical and chemotaxonomic tests of the VLST isolate reveal consistent differences from previously described Xenorhabdus species. Phylogenetic, physiological, biochemical and chemotaxonomic approaches show that VLST represents a new species of the genus Xenorhabdus, for which the name Xenorhabdus lircayensis sp. nov. (type strain VLST=CCCT 20.04T=DSM 111583T) is proposed.

A study on Xenorhabdus and Photorhabdus isolates from Northeastern Thailand: Identification, antibacterial activity, and association with entomopathogenic nematode hosts.

Xenorhabdus and Photorhabdus are gram negative bacteria that can produce several secondary metabolites, including antimicrobial compounds. They have a symbiotic association with entomopathogenic nematodes (EPNs). The aim of this study was to isolate and identify Xenorhabdus and Photorhabdus species and their associated nematode symbionts from Northeastern region of Thailand. We also evaluated the antibacterial activity of these symbiotic bacteria. The recovery rate of EPNs was 7.82% (113/1445). A total of 62 Xenorhabdus and 51 Photorhabdus strains were isolated from the EPNs. Based on recA sequencing and phylogeny, Xenorhabdus isolates were identified as X. stockiae (n = 60), X. indica (n = 1) and X. eapokensis (n = 1). Photorhabdus isolates were identified as P. luminescens subsp. akhurstii (n = 29), P. luminescens subsp. hainanensis (n = 18), P. luminescens subsp. laumondii (n = 2), and P. asymbiotica subsp. australis (n = 2). The EPNs based on 28S rDNA and internal transcribed spacer (ITS) analysis were identified as Steinernema surkhetense (n = 35), S. sangi (n = 1), unidentified Steinernema (n = 1), Heterorhabditis indica (n = 39), H. baujardi (n = 1), and Heterorhabditis sp. SGmg3 (n = 3). Antibacterial activity showed that X. stockiae (bMSK7.5_TH) extract inhibited several antibiotic-resistant bacterial strains. To the best of our knowledge, this is the first report on mutualistic association between P. luminescens subsp. laumondii and Heterorhabditis sp. SGmg3. This study could act as a platform for future studies focusing on the discovery of novel antimicrobial compounds from these bacterial isolates.

Antibacterial activity of Xenorhabdus and Photorhabdus isolated from entomopathogenic nematodes against antibiotic-resistant bacteria.

Xenorhabdus and Photorhabdus, symbiotically associated with entomopathogenic nematodes (EPNs), produce a range of antimicrobial compounds. The objective of this study is to identify Xenorhabdus and Photorhabdus and their EPNs hosts, which were isolated from soil samples from Saraburi province, and study their antibacterial activity against 15 strains of drug-resistant bacteria. Fourteen isolates (6.1%), consisting of six Xenorhabdus isolates and eight Photorhabdus isolates, were obtained from 230 soil samples. Based on the BLASTN search incorporating the phylogenetic analysis of a partial recA gene, all six isolates of Xenorhabdus were found to be identical and closely related to X. stockiae. Five isolates of Photorhabdus were found to be identical and closely related to P. luminescens subsp. akhurstii. Two isolates of Photorhabdus were found to be identical and closely related to P. luminescens subsp. hainanensis. The remaining isolate of Photorhabdus was found to be identical to P. asymbiotica subsp. australis. The bacterial extracts from P. luminescens subsp. akhurstii showed strong inhibition the growth of S. aureus strain PB36 (MSRA) by disk diffusion, minimal inhibitory concentration, and minimal bactericidal concentration assay. The combination between each extract from Xenorhabdus/Photorhabdus and oxacillin or vancomycin against S. aureus strain PB36 (MRSA) exhibited no interaction on checkerboard assay. Moreover, killing curve assay of P. luminescens subsp. akhurstii extracts against S. aureus strain PB36 exhibited a steady reduction of 105 CFU/ml to 103 CFU/ml within 30 min. This study demonstrates that Xenorhabdus and Photorhabdus, showed antibacterial activity. This finding may be useful for further research on antibiotic production.

Selection of Bacterial Mutants in Late Infections: When Vector Transmission Trades Off against Growth Advantage in Stationary Phase.

Bacterial infections are often composed of cells with distinct phenotypes that can be produced by genetic or epigenetic mechanisms. This phenotypic heterogeneity has proved to be important in many pathogens, because it can alter both pathogenicity and transmission. We studied how and why it can emerge during infection in the bacterium Xenorhabdus nematophila, a pathogen that kills insects and multiplies in the cadaver before being transmitted by the soil nematode vector Steinernema carpocapsae We found that phenotypic variants cluster in three groups, one of which is composed of lrp defective mutants. These mutants, together with variants of another group, have in common that they maintain high survival during late stationary phase. This probably explains why they increase in frequency: variants of X. nematophila with a growth advantage in stationary phase (GASP) are under strong positive selection both in prolonged culture and in late infections. We also found that the within-host advantage of these variants seems to trade off against transmission by nematode vectors: the variants that reach the highest load in insects are those that are the least transmitted.IMPORTANCE Pathogens can evolve inside their host, and the importance of this mutation-fueled process is increasingly recognized. A disease outcome may indeed depend in part on pathogen adaptations that emerge during infection. It is therefore important to document these adaptations and the conditions that drive them. In our study, we took advantage of the possibility to monitor within-host evolution in the insect pathogen X. nematophila We demonstrated that selection occurring in aged infection favors lrp defective mutants, because these metabolic mutants benefit from a growth advantage in stationary phase (GASP). We also demonstrated that these mutants have reduced virulence and impaired transmission, modifying the infection outcome. Beyond the specific case of X. nematophila, we propose that metabolic mutants are to be found in other bacterial pathogens that stay for many generations inside their host.

Cyclo(tetrahydroxybutyrate) production is sufficient to distinguish between Xenorhabdus and Photorhabdus isolates in Thailand.

Bacteria of the genera Photorhabdus and Xenorhabdus produce a plethora of natural products to support their similar symbiotic life cycles. For many of these compounds, the specific bioactivities are unknown. One common challenge in natural product research when trying to prioritize research efforts is the rediscovery of identical (or highly similar) compounds from different strains. Linking genome sequence to metabolite production can help in overcoming this problem. However, sequences are typically not available for entire collections of organisms. Here, we perform a comprehensive metabolic screening using HPLC-MS data associated with a 114-strain collection (58 Photorhabdus and 56 Xenorhabdus) across Thailand and explore the metabolic variation among the strains, matched with several abiotic factors. We utilize machine learning in order to rank the importance of individual metabolites in determining all given metadata. With this approach, we were able to prioritize metabolites in the context of natural product investigations, leading to the identification of previously unknown compounds. The top three highest ranking features were associated with Xenorhabdus and attributed to the same chemical entity, cyclo(tetrahydroxybutyrate). This work also addresses the need for prioritization in high-throughput metabolomic studies and demonstrates the viability of such an approach in future research.

ngrA-dependent natural products are required for interspecies competition and virulence in the insect pathogenic bacterium Xenorhabdus szentirmaii.

Xenorhabdus species are symbionts of entomopathogenic nematodes and pathogens of susceptible insects. Nematodes enter insect hosts and perforate the midgut to invade the haemocoel where Xenorhabdus bacteria are released transitioning to their pathogenic stage. During nematode invasion microbes from the insect gut translocate into the haemocoel. Different species of nematodes carrying specific strains of Xenorhabdus can also invade the same insect. Xenorhabdus species thereby compete for nutrients and space with both related strains and non-related gut microbes. While Xenorhabdus species produce diverse antimicrobial compounds in complex media, their functions in insect hosts are not well understood. We show that Xenorhabdus szentirmaii produced ngrA-dependent antibiotics that were active against both gut-derived microbes and Xenorhabdus nematophila whereas antibiotics of X. nematophila were not active against X. szentirmaii. X. nematophila growth was inhibited in co-cultures with wild-type X. szentirmaii in medium that mimics insect haemolymph. An antibiotic-deficient strain of X. szentirmaii was created by inactivating the ngrA gene that encodes the enzyme that attaches the 4' phosphopantetheinyl moiety to non-ribosomal peptide synthetases involved in antibiotic biosynthesis. X. nematophila growth was not inhibited in co-cultures with the ngrA strain. The growth of X. nematophila was suppressed in Manduca sexta co-injected with wild-type X. szentirmaii and X. nematophila. In contrast, growth of X. nematophila was not suppressed in M. sexta co-injected with the ngrA strain. Two unique compounds were detected by MALDI-TOF MS analysis in haemolymph infected with the wild-type but not with the ngrA strain. Finally, killing of M. sexta was delayed in insects infected with the ngrA strain. These findings indicate that in the insect host X. szentirmaii produces ngrA-dependent products involved in both interspecies competition and virulence.

The first report of Xenorhabdus indica from Steinernema pakistanense: co-phylogenetic study suggests co-speciation between X. indica and its steinernematid nematodes.

During a survey in agricultural fields of the sub-humid region of Meerut district, India, two strains of entomopathogenic nematodes, labelled CS31 and CS32, were isolated using the Galleria baiting technique. Based on morphological and morphometric studies, and molecular data, the nematodes were identified as Steinernema pakistanense, making this finding the first report of this species from India. For the first time, we performed a molecular and biochemical characterization of the bacterial symbiont of S. pakistanense. Furthermore, a co-phylogenetic analysis of the bacteria from the monophyletic clade containing a symbiont of S. pakistanense, together with their nematode hosts, was conducted, to test the degree of nematode-bacteria co-speciation. Both isolates were also tested in a laboratory assay for pathogenicity against two major pests, Helicoverpa armigera and Spodoptera litura. The morphology of the Indian isolates corresponds mainly to the original description, with the only difference being the absence of a mucron in first-generation females and missing epiptygmata in the second generation. The sequences of bacterial recA and gyrB genes have shown that the symbiont of S. pakistanense is closely related to Xenorhabdus indica, which is associated with some other nematodes from the 'bicornutum' group. Co-phylogenetic analysis has shown a remarkable congruence between the nematode and bacterial phylogenies, suggesting that, in some lineages within the Steinernema / Xenorhabdus complex, the nematodes and bacteria have undergone co-speciation. In the virulence assay, both strains caused a 100% mortality of both tested insects after 48 h, even at the lowest doses of 25 infective juveniles per insect, suggesting that S. pakistanense could be considered for use in the biocontrol of these organisms in India.

Steinernema poinari (Nematoda: Steinernematidae): a new symbiotic host of entomopathogenic bacteria Xenorhabdus bovienii.

Three strains of symbiotic bacteria were isolated from an entomopathogenic nematode Steinernema poinari retrieved from soil in eastern Poland. Using 16S rDNA, recA, gltX, gyrB, and dnaN gene sequences for phylogenetic analysis, these strains were shown to belong to the species Xenorhabdus bovienii. The nucleotide identity between the studied S. poinari microsymbionts and other X. bovienii strains calculated for 16S rDNA and concatenated sequences of four protein-coding genes was 98.7-100% and 97.9-99.5%, respectively. The phenotypic properties of the isolates also supported their close phylogenetic relationship with X. bovienii. All three tested X. bovienii strains of different Steinernema clade origin supported the recovery of infective juveniles and subsequent development of the nematode population. However, the colonization degree of new infective juvenile generations was significantly affected by the bacterial host donor/recipient. The colonization degree of infective juveniles reared on bacterial symbionts deriving from a non-cognate clade of nematodes was extremely low, but proved the possible host-switching between non-related Steinernema species.

Characterization of the pixB gene in Xenorhabdus nematophila and discovery of a new gene family.

Xenorhabdus nematophila are Gram-negative bacteria that engage in mutualistic associations with entomopathogenic nematodes. To reproduce, the nematodes invade insects and release X. nematophila into the haemolymph where it functions as an insect pathogen. In complex medium, X. nematophila cells produce two distinct types of intracellular crystalline inclusions, one composed of the methionine-rich PixA protein and the other composed of the PixB protein. Here we show that PixB crystalline inclusions were neither apparent in X. nematophila cells grown in medium that mimics insect haemolymph (Grace's medium) nor in cells grown directly in the insect haemocoel. The identified pixB gene was regulated by a conserved σ70 promoter while the pixA promoter was less well conserved. Expression of pixA and pixB under biological conditions was analysed using GFP promoter reporters. Microplate fluorescence detection and flow cytometry analyses revealed that pixB was expressed at high levels in Grace's medium and in insect haemolymph and at lower levels in complex medium, while pixA was expressed at lower levels under all conditions. Although pixB was highly expressed in Grace's medium, PixB crystalline inclusions were not present, suggesting that under biological conditions PixB production may be controlled post-transcriptionally. Although a pixB-minus strain was constructed, the function of PixB remains unresolved. The pixB gene was present in few Xenorhabdus species and pixB-type genes were identified in some Proteobacteria and Gram-positive species, while pixA was only present in Xenorhabdus species. Two conserved sequences were identified in PixB-type proteins that characterize this previously unrecognized gene family.

The insect pathogenic bacterium Xenorhabdus innexi has attenuated virulence in multiple insect model hosts yet encodes a potent mosquitocidal toxin.

BACKGROUND: Xenorhabdus innexi is a bacterial symbiont of Steinernema scapterisci nematodes, which is a cricket-specialist parasite and together the nematode and bacteria infect and kill crickets. Curiously, X. innexi expresses a potent extracellular mosquitocidal toxin activity in culture supernatants. We sequenced a draft genome of X. innexi and compared it to the genomes of related pathogens to elucidate the nature of specialization. RESULTS: Using green fluorescent protein-expressing X. innexi we confirm previous reports using culture-dependent techniques that X. innexi colonizes its nematode host at low levels (~3-8 cells per nematode), relative to other Xenorhabdus-Steinernema associations. We found that compared to the well-characterized entomopathogenic nematode symbiont X. nematophila, X. innexi fails to suppress the insect phenoloxidase immune pathway and is attenuated for virulence and reproduction in the Lepidoptera Galleria mellonella and Manduca sexta, as well as the dipteran Drosophila melanogaster. To assess if, compared to other Xenorhabdus spp., X. innexi has a reduced capacity to synthesize virulence determinants, we obtained and analyzed a draft genome sequence. We found no evidence for several hallmarks of Xenorhabdus spp. toxicity, including Tc and Mcf toxins. Similar to other Xenorhabdus genomes, we found numerous loci predicted to encode non-ribosomal peptide/polyketide synthetases. Anti-SMASH predictions of these loci revealed one, related to the fcl locus that encodes fabclavines and zmn locus that encodes zeamines, as a likely candidate to encode the X. innexi mosquitocidal toxin biosynthetic machinery, which we designated Xlt. In support of this hypothesis, two mutants each with an insertion in an Xlt biosynthesis gene cluster lacked the mosquitocidal compound based on HPLC/MS analysis and neither produced toxin to the levels of the wild type parent. CONCLUSIONS: The X. innexi genome will be a valuable resource in identifying loci encoding new metabolites of interest, but also in future comparative studies of nematode-bacterial symbiosis and niche partitioning among bacterial pathogens.

Natural product diversity associated with the nematode symbionts Photorhabdus and Xenorhabdus.

Xenorhabdus and Photorhabdus species dedicate a large amount of resources to the production of specialized metabolites derived from non-ribosomal peptide synthetase (NRPS) or polyketide synthase (PKS). Both bacteria undergo symbiosis with nematodes, which is followed by an insect pathogenic phase. So far, the molecular basis of this tripartite relationship and the exact roles that individual metabolites and metabolic pathways play have not been well understood. To close this gap, we have significantly expanded the database for comparative genomics studies in these bacteria. Clustering the genes encoded in the individual genomes into hierarchical orthologous groups reveals a high-resolution picture of functional evolution in this clade. It identifies groups of genes-many of which are involved in secondary metabolite production-that may account for the niche specificity of these bacteria. Photorhabdus and Xenorhabdus appear very similar at the DNA sequence level, which indicates their close evolutionary relationship. Yet, high-resolution mass spectrometry analyses reveal a huge chemical diversity in the two taxa. Molecular network reconstruction identified a large number of previously unidentified metabolite classes, including the xefoampeptides and tilivalline. Here, we apply genomic and metabolomic methods in a complementary manner to identify and elucidate additional classes of natural products. We also highlight the ability to rapidly and simultaneously identify potentially interesting bioactive products from NRPSs and PKSs, thereby augmenting the contribution of molecular biology techniques to the acceleration of natural product discovery.

Isolation and identification of Xenorhabdus and Photorhabdus bacteria associated with entomopathogenic nematodes and their larvicidal activity against Aedes aegypti.

BACKGROUND: Aedes aegypti is a potential vector of West Nile, Japanese encephalitis, chikungunya, dengue and Zika viruses. Alternative control measurements of the vector are needed to overcome the problems of environmental contamination and chemical resistance. Xenorhabdus and Photorhabdus are symbionts in the intestine of entomopathogenic nematodes (EPNs) Steinernema spp. and Heterorhabditis spp. These bacteria are able to produce a broad range of bioactive compounds including antimicrobial, antiparasitic, cytotoxic and insecticidal compounds. The objectives of this study were to identify Xenorhabdus and Photorhabdus isolated from EPNs in upper northern Thailand and to study their larvicidal activity against Ae. aegypti larvae. RESULTS: A total of 60 isolates of symbiotic bacteria isolated from EPNs consisted of Xenorhabdus (32 isolates) and Photorhabdus (28 isolates). Based on recA gene sequencing, BLASTN and phylogenetic analysis, 27 isolates of Xenorhabdus were identical and closely related to X. stockiae, 4 isolates were identical to X. miraniensis, and one isolate was identical to X. ehlersii. Twenty-seven isolates of Photorhabdus were closely related to P. luminescens akhurstii and P. luminescens hainanensis, and only one isolate was identical and closely related to P. luminescens laumondii. Xenorhabdus and Photorhabdus were lethal to Ae aegypti larvae. Xenorhabdus ehlersii bMH9.2_TH showed 100% efficiency for killing larvae of both fed and unfed conditions, the highest for control of Ae. aegypti larvae and X. stockiae (bLPA18.4_TH) was likely to be effective in killing Ae. aegypti larvae given the mortality rates above 60% at 72 h and 96 h. CONCLUSIONS: The common species in the study area are X. stockiae, P. luminescens akhurstii, and P. luminescens hainanensis. Three symbiotic associations identified included P. luminescens akhurstii-H. gerrardi, P. luminescens hainanensis-H. gerrardi and X. ehlersii-S. Scarabaei which are new observations of importance to our knowledge of the biodiversity of, and relationships between, EPNs and their symbiotic bacteria. Based on the biological assay, X. ehlersii bMH9.2_TH begins to kill Ae. aegypti larvae within 48 h and has the most potential as a pathogen to the larvae. These data indicate that X. ehlersii may be an alternative biological control agent for Ae. aegypti and other mosquitoes.

Three Novel Xenorhabdus-Steinernema Associations and Evidence of Strains of X. khoisanae Switching Between Different Clades.

Xenorhabdus species are normally closely associated with entomopathogenic nematodes of the family Steinernematidae. Strain F2, isolated from Steinernema nguyeni, was identified as Xenorhabdus bovienii and strains J194 and SB10, isolated from Steinernema jeffreyense and Steinernema sacchari as Xenorhabdus khoisanae, based on phenotypic characteristics and sequencing of 16S rRNA and housekeeping genes dnaN, gltX, gyrB, infB and recA. All three strains produced antimicrobial compounds that inhibited the growth of Gram-positive and Gram-negative bacteria. This is the first report of associations between strains of the symbiotic bacteria X. bovienii with S. nguyeni, and X. khoisanae with S. jeffreyense and S. sacchari. This provides evidence that strains of Xenorhabdus spp. may switch between nematode species within the same clade and between different clades.

Fitness costs of symbiont switching using entomopathogenic nematodes as a model.

BACKGROUND: Steinernematid nematodes form obligate symbioses with bacteria from the genus Xenorhabdus. Together Steinernema nematodes and their bacterial symbionts successfully infect, kill, utilize, and exit their insect hosts. During this process the nematodes and bacteria disassociate requiring them to re-associate before emerging from the host. This interaction can be complicated when two different nematodes co-infect an insect host. RESULTS: Non-cognate nematode-bacteria pairings result in reductions for multiple measures of success, including total progeny production and virulence. Additionally, nematode infective juveniles carry fewer bacterial cells when colonized by a non-cognate symbiont. Finally, we show that Steinernema nematodes can distinguish heterospecific and some conspecific non-cognate symbionts in behavioral choice assays. CONCLUSIONS: Steinernema-Xenorhabdus symbioses are tightly governed by partner recognition and fidelity. Association with non-cognates resulted in decreased fitness, virulence, and bacterial carriage of the nematode-bacterial pairings. Entomopathogenic nematodes and their bacterial symbionts are a useful, tractable, and reliable model for testing hypotheses regarding the evolution, maintenance, persistence, and fate of mutualisms.

Xenorhabdus thuongxuanensis sp. nov. and Xenorhabdus eapokensis sp. nov., isolated from Steinernema species.

Two slightly yellowish-pigmented, oxidase-negative, rod-shaped and Gram-stain-negative bacterial strains (30TX1T and DL20T), isolated from Steinernema sangi and Steinernema eapokense, respectively, during soil sampling in Vietnam were studied using a polyphasic taxonomic approach. Strain 30TX1T showed highest 16S rRNA gene sequence similarity to the type strain of Xenorhabdus ehlersii (98.9 %) and strain DL20T to that of Xenorhabdus ishibashii (98.7 %). Sequence similarities to all other Xenorhabdus species were lower (<98.4 %). The two strains shared 98 % 16S rRNA gene sequence similarity. Multilocus sequence analysis (MLSA) based on concatenated partial recA, dnaN, gltX, gyrB and infB gene sequences showed a clear distinction of strains 30TX1T and DL20T among each other and to the closest related type strains. DNA-DNA hybridizations between strain DL20T and the type strain of X. ishibashii resulted in a relatedness value of 53 %. Genome-to-genome-based comparisons gave average nucleotide identities of 93.6 % (reciprocal 93.5 %) for strain 30TX1T and X. ehlersii DSM 16337T, of 92.8 % (reciprocal 93 %) for strain DL20T and X. ishibashiiDSM 22670Tand of 93.0 % (reciprocal 93.2 %) for the two novel strains. The fatty acid profile of the strains consisted of the major fatty acids C14 : 0, C16 : 0, C17 : 0 cyclo, C16 : 1ω7c and/or iso-C15 : 0 2-OH, and C18 : 1ω7c. Genome-to-genome comparison and MLSA results together with the differential biochemical and chemotaxonomic properties showed that strains 30TX1T and DL20T represent novel Xenorhabdus species, for which the names Xenorhabdus thuongxuanensis sp. nov. (type strain 30TX1T=CCM 8727T=LMG 29916T) and Xenorhabdus eapokensis sp. nov. (type strain DL20T=CCM 8728T=LMG 29917T) are proposed, respectively.

A New Member of the Growing Family of Contact-Dependent Growth Inhibition Systems in Xenorhabdus doucetiae.

Xenorhabdus is a bacterial symbiont of entomopathogenic Steinernema nematodes and is pathogenic for insects. Its life cycle involves a stage inside the insect cadaver, in which it competes for environmental resources with microorganisms from soil and the insect gut. Xenorhabdus is, thus, a useful model for identifying new interbacterial competition systems. For the first time, in an entomopathogenic bacterium, Xenorhabdus doucetiae strain FRM16, we identified a cdi-like locus. The cdi loci encode contact-dependent inhibition (CDI) systems composed of proteins from the two-partner secretion (TPS) family. CdiB is the outer membrane protein and CdiA is the toxic exoprotein. An immunity protein, CdiI, protects bacteria against inhibition. We describe here the growth inhibition effect of the toxic C-terminus of CdiA from X. doucetiae FRM16, CdiA-CTFRM16, following its production in closely and distantly related enterobacterial species. CdiA-CTFRM16 displayed Mg2+-dependent DNase activity, in vitro. CdiA-CTFRM16-mediated growth inhibition was specifically neutralized by CdiIFRM16. Moreover, the cdi FRM16 locus encodes an ortholog of toxin-activating proteins C that we named CdiCFRM16. In addition to E. coli, the cdiBCAI-type locus was found to be widespread in environmental bacteria interacting with insects, plants, rhizospheres and soils. Phylogenetic tree comparisons for CdiB, CdiA and CdiC suggested that the genes encoding these proteins had co-evolved. By contrast, the considerable variability of CdiI protein sequences suggests that the cdiI gene is an independent evolutionary unit. These findings further characterize the sparsely described cdiBCAI-type locus.

Xenorhabdus bovienii Strain Diversity Impacts Coevolution and Symbiotic Maintenance with Steinernema spp. Nematode Hosts.

UNLABELLED: Microbial symbionts provide benefits that contribute to the ecology and fitness of host plants and animals. Therefore, the evolutionary success of plants and animals fundamentally depends on long-term maintenance of beneficial associations. Most work investigating coevolution and symbiotic maintenance has focused on species-level associations, and studies are lacking that assess the impact of bacterial strain diversity on symbiotic associations within a coevolutionary framework. Here, we demonstrate that fitness in mutualism varies depending on bacterial strain identity, and this is consistent with variation shaping phylogenetic patterns and maintenance through fitness benefits. Through genome sequencing of nine bacterial symbiont strains and cophylogenetic analysis, we demonstrate diversity among Xenorhabdus bovienii bacteria. Further, we identified cocladogenesis between Steinernema feltiae nematode hosts and their corresponding X. bovienii symbiont strains, indicating potential specificity within the association. To test the specificity, we performed laboratory crosses of nematode hosts with native and nonnative symbiont strains, which revealed that combinations with the native bacterial symbiont and closely related strains performed significantly better than those with more divergent symbionts. Through genomic analyses we also defined potential factors contributing to specificity between nematode hosts and bacterial symbionts. These results suggest that strain-level diversity (e.g., subspecies-level differences) in microbial symbionts can drive variation in the success of host-microbe associations, and this suggests that these differences in symbiotic success could contribute to maintenance of the symbiosis over an evolutionary time scale. IMPORTANCE: Beneficial symbioses between microbes and plant or animal hosts are ubiquitous, and in these associations, microbial symbionts provide key benefits to their hosts. As such, host success is fundamentally dependent on long-term maintenance of beneficial associations. Prolonged association between partners in evolutionary time is expected to result in interactions in which only specific partners can fully support symbiosis. The contribution of bacterial strain diversity on specificity and coevolution in a beneficial symbiosis remains unclear. In this study, we demonstrate that strain-level differences in fitness benefits occur in beneficial host-microbe interactions, and this variation likely shapes phylogenetic patterns and symbiotic maintenance. This highlights that symbiont contributions to host biology can vary significantly based on very-fine-scale differences among members of a microbial species. Further, this work emphasizes the need for greater phylogenetic resolution when considering the causes and consequences of host-microbe interactions.

Cabanillasin, a new antifungal metabolite, produced by entomopathogenic Xenorhabdus cabanillasii JM26.

Since the early 1980s, fungi have emerged as a major cause of human disease. Fungal infections are associated with high levels of morbidity and mortality, and are now recognized as an important public health problem. Gram-negative bacterial strains of genus Xenorhabdus are known to form symbiotic associations with soil-dwelling nematodes of the Steinernematidae family. We describe here the discovery of a new antifungal metabolite, cabanillasin, produced by Xenorhabdus cabanillasii. We purified this molecule by cation-exchange chromatography and reverse-phase chromatography. We then determined the chemical structure of cabanillasin by homo- and heteronuclear NMR and MS-MS. Cabanillasin was found to be active against yeasts and filamentous fungi involved in opportunistic infections.

Description of Xenorhabdus khoisanae sp. nov., the symbiont of the entomopathogenic nematode Steinernema khoisanae.

Bacterial strain SF87(T), and additional strains SF80, SF362 and 106-C, isolated from the nematode Steinernema khoisanae, are non-bioluminescent Gram-reaction-negative bacteria that share many of the carbohydrate fermentation reactions recorded for the type strains of recognized Xenorhabdus species. Based on 16S rRNA gene sequence data, strain SF87(T) is shown to be closely related (98% similarity) to Xenorhabdus hominickii DSM 17903(T). Nucleotide sequences of strain SF87 obtained from the recA, dnaN, gltX, gyrB and infB genes showed 96-97% similarity with Xenorhabdus miraniensis DSM 17902(T). However, strain SF87 shares only 52.7% DNA-DNA relatedness with the type strain of X. miraniensis, confirming that it belongs to a different species. Strains SF87(T), SF80, SF362 and 106-C are phenotypically similar to X. miraniensis and X. beddingii, except that they do not produce acid from aesculin. These strains are thus considered to represent a novel species of the genus Xenorhabdus, for which the name Xenorhabdus khoisanae sp. nov. is proposed. The type strain is SF87(T) ( =DSM 25463(T) =ATCC BAA-2406(T)).

Previously unrecognized stages of species-specific colonization in the mutualism between Xenorhabdus bacteria and Steinernema nematodes.

The specificity of a horizontally transmitted microbial symbiosis is often defined by molecular communication between host and microbe during initial engagement, which can occur in discrete stages. In the symbiosis between Steinernema nematodes and Xenorhabdus bacteria, previous investigations focused on bacterial colonization of the intestinal lumen (receptacle) of the nematode infective juvenile (IJ), as this was the only known persistent, intimate and species-specific contact between the two. Here we show that bacteria colonize the anterior intestinal cells of other nematode developmental stages in a species-specific manner. Also, we describe three processes that only occur in juveniles that are destined to become IJs. First, a few bacterial cells colonize the nematode pharyngeal-intestinal valve (PIV) anterior to the intestinal epithelium. Second, the nematode intestine constricts while bacteria initially remain in the PIV. Third, anterior intestinal constriction relaxes and colonizing bacteria occupy the receptacle. At each stage, colonization requires X. nematophila symbiosis region 1 (SR1) genes and is species-specific: X. szentirmaii, which naturally lacks SR1, does not colonize unless SR1 is ectopically expressed. These findings reveal new aspects of Xenorhabdus bacteria interactions with and transmission by theirSteinernema nematode hosts, and demonstrate that bacterial SR1 genes aid in colonizing nematode epithelial surfaces.