Microbiome composition is frequently studied by the amplification and high-throughput sequencing of specific molecular markers (metabarcoding). Various hypervariable regions of the 16S rRNA gene are ...classically used to estimate bacterial diversity, but other universal bacterial markers with a finer taxonomic resolution could be employed. We compared specificity and sensitivity between a portion of the rpoB gene and the V3 V4 hypervariable region of the 16S rRNA gene.
We first designed universal primers for rpoB suitable for use with Illumina sequencing-based technology and constructed a reference rpoB database of 45,000 sequences. The rpoB and V3 V4 markers were amplified and sequenced from (i) a mock community of 19 bacterial strains from both Gram-negative and Gram-positive lineages; (ii) bacterial assemblages associated with entomopathogenic nematodes. In metabarcoding analyses of mock communities with two analytical pipelines (FROGS and DADA2), the estimated diversity captured with the rpoB marker resembled the expected composition of these mock communities more closely than that captured with V3 V4. The rpoB marker had a higher level of taxonomic affiliation, a higher sensitivity (detection of all the species present in the mock communities), and a higher specificity (low rates of spurious OTU detection) than V3 V4. We compared the performance of the rpoB and V3 V4 markers in an animal ecosystem model, the infective juveniles of the entomopathogenic nematode Steinernema glaseri carrying the symbiotic bacteria Xenorhabdus poinarii. Both markers showed the bacterial community associated with this nematode to be of low diversity (< 50 OTUs), but only rpoB reliably detected the symbiotic bacterium X. poinarii.
Our results confirm that different microbiota composition data may be obtained with different markers. We found that rpoB was a highly appropriate marker for assessing the taxonomic structure of mock communities and the nematode microbiota. Further studies on other ecosystems should be considered to evaluate the universal usefulness of the rpoB marker. Our data highlight two crucial elements that should be taken into account to ensure more reliable and accurate descriptions of microbial diversity in high-throughput amplicon sequencing analyses: i) the need to include mock communities as controls; ii) the advantages of using a multigenic approach including at least one housekeeping gene (rpoB is a good candidate) and one variable region of the 16S rRNA gene. This study will be useful to the growing scientific community describing bacterial communities by metabarcoding in diverse ecosystems.
The holistic view of bacterial symbiosis, incorporating both host and microbial environment, constitutes a major conceptual shift in studies deciphering host-microbe interactions. Interactions ...between Steinernema entomopathogenic nematodes and their bacterial symbionts, Xenorhabdus, have long been considered monoxenic two partner associations responsible for the killing of the insects and therefore widely used in insect pest biocontrol. We investigated this "monoxenic paradigm" by profiling the microbiota of infective juveniles (IJs), the soil-dwelling form responsible for transmitting Steinernema-Xenorhabdus between insect hosts in the parasitic lifecycle.
Multigenic metabarcoding (16S and rpoB markers) showed that the bacterial community associated with laboratory-reared IJs from Steinernema carpocapsae, S. feltiae, S. glaseri and S. weiseri species consisted of several Proteobacteria. The association with Xenorhabdus was never monoxenic. We showed that the laboratory-reared IJs of S. carpocapsae bore a bacterial community composed of the core symbiont (Xenorhabdus nematophila) together with a frequently associated microbiota (FAM) consisting of about a dozen of Proteobacteria (Pseudomonas, Stenotrophomonas, Alcaligenes, Achromobacter, Pseudochrobactrum, Ochrobactrum, Brevundimonas, Deftia, etc.). We validated this set of bacteria by metabarcoding analysis on freshly sampled IJs from natural conditions. We isolated diverse bacterial taxa, validating the profile of the Steinernema FAM. We explored the functions of the FAM members potentially involved in the parasitic lifecycle of Steinernema. Two species, Pseudomonas protegens and P. chlororaphis, displayed entomopathogenic properties suggestive of a role in Steinernema virulence and membership of the Steinernema pathobiome.
Our study validates a shift from monoxenic paradigm to pathobiome view in the case of the Steinernema ecology. The microbial communities of low complexity associated with EPNs will permit future microbiota manipulation experiments to decipher overall microbiota functioning in the infectious process triggered by EPN in insects and, more generally, in EPN ecology.
Pseudomonas protegens is a rhizosphere pseudomonad with a high agronomical potential (entomopathogenic and beneficial to plants) and bio-catalytic activities, but no selective medium has been ...described for its isolation. We developed a semi-selective minimum agar medium for the specific isolation and growth of P. protegens. We searched for both (i) a carbon source allowing the growth of P. protegens but potentially inhibiting the growth of other pseudomonads and (ii) an antimicrobial agent suppressing other members of the bacterial rhizosphere community. The M9-PP-agar medium consists of M9 base agar with adipic acid as the only carbon source and Irgasan® as an anti-bacterial agent. We tested the selectivity and sensitivity of M9-PP-agar by measuring the growth of 68 bacterial strains from 36 different species on this medium. Ten of the species tested were able to grow on M9-PP-agar medium: four species from the Pseudomonadaceae (Pseudomonas aeruginosa, Pseudomonas protegens, Pseudomonas putida, Stenotrophomonas maltophilia) as well as Achromobacter xylosoxidans, Agrobacterium tumefaciens, Brevundimonas sp., Serratia liquefaciens, Serratia marcescens and Variovorax paradoxus. All colonies were white, except for those of P. protegens (12 strains), which were typically brown. We demonstrated the efficiency of the M9-PP agar medium for P. protegens isolation, by inoculating two soils with the reference strain P. protegens CHAOT and then reisolating them. We also developed a fitF-PCR test targeting a regulator gene of the insecticidal P. protegens fit locus, for the rapid molecular detection of P. protegens colonies. We, therefore, developed a highly specific process for the routine isolation of new P. protegens strains from the soil environment, based on the use of a semi-selective medium and the specific color of colonies.
•The M9-PP-agar medium consists of M9 base agar with adipic acid and Irgasan®.•The M9-PP-agar medium is a semi-selective minimum medium for the specific isolation and growth of Pseudomonas protegens.•Only P. protegens displays brown pigmentation of the colonies.•We demonstrated the efficacy of M9-PP agar medium for the isolation of P. protegens from soil matrices.•We developed a fitF-PCR test for the rapid molecular detection of P. protegens colonies.
We used the information from a set of concatenated sequences from four genes (recA, gyrB, dnaN and gltX) to investigate the phylogeny of the genera Photorhabdus and Xenorhabdus (entomopathogenic ...bacteria associated with nematodes of the genera Heterorhabditis and Steinernema, respectively). The robustness of the phylogenetic tree obtained by this multigene approach was significantly better than that of the tree obtained by a single gene approach. The comparison of the topologies of single gene phylogenetic trees highlighted discrepancies which have implications for the classification of strains and new isolates; in particular, we propose the transfer of Photorhabdus luminescens subsp. thracensis to Photorhabdus temperata subsp. thracensis comb. nov. (type strain CIP 108426T =DSM 15199T). We found that, within the genus Xenorhabdus, strains or isolates that shared less than 97 % nucleotide identity (NI), calculated on the concatenated sequences of the four gene fragments (recA, gyrB, dnaN and gltX) encompassing 3395 nucleotides, did not belong to the same species. Thus, at the 97% NI cutoff, we confirm the current 20 species of the genus Xenorhabdus and propose the description of a novel species, Xenorhabdus vietnamensis sp. nov. (type strain VN01T =CIP 109945T =DSM 22392T). Within each of the three current species of the genus Photorhabdus, P. asymbiotica, P. luminescens and P. temperata, strains or isolates which shared less than 97% NI did not belong to the same subspecies. Comparisons of the four gene fragments plus the rplB gene fragment analysed separately led us to propose four novel subspecies: Photorhabdus luminescens subsp. caribbeanensis subsp. nov. (type strain HG29T =CIP 109949T =DSM 22391T), P. luminescens subsp. hainanensis subsp. nov. (type strain C8404T = CIP 109946T =DSM 22397T), P. temperata subsp. khanii subsp. nov. (type strain C1T =NC19(T) =CIP 109947T =DSM 3369T), and P. temperata subsp. tasmaniensis subsp. nov. (type strain T327T =CIP 109948T =DSM 22387T).
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•Spodoptera frugiperda genome contains two genes encoding prophenoloxidases.•SfPPOs complement one another for hemolymph phenoloxidase activity.•SfPPO1Δ,PPO2Δ double mutant presents ...morphological defects during pupal stage.•SfPPO1Δ,PPO2Δ double mutant pupae are not able to develop into adults.
Upon infection, the phenoloxidase system in arthropods is rapidly mobilized and constitutes a major defense system against invaders. The activation of the key enzymes prophenoloxidase (PPO) and their action in immunity through melanization and encapsulation of foreign bodies in hemolymph has been described in many insects. On the other hand, little is known about PPOs involvement in other essential functions related to insect development.
In this paper, we investigated the function of the two PPOs of the crop pest, Spodoptera frugiperda (PPO1 and PPO2). We show that PPOs are mainly expressed in hemocytes with the PPO2 expressed at higher levels than the PPO1. In addition, these two genes are expressed in the same tissue and at the same stages of insect development. Through the generation of loss-of-function mutants by CRISPR/Cas9 method, we show that the presence of PPOs is essential for the normal development of the pupa and the survival of the insect.
Institut National de la Recherche Agronomique, Unité d'Ecologie Microbienne des Insectes et Interactions hôte-Pathogène, Université Montpellier II, Place Eugène Bataillon, Case courrier 54, Bâtiment ...24, 34095 Montpellier CEDEX 5, France
Correspondence Patrick Tailliez tailliez{at}univ-montp2.fr
We investigated the diversity of a collection of 76 Xenorhabdus strains, isolated from at least 27 species of Steinernema nematodes and collected in 32 countries, using three complementary approaches: 16S rRNA gene sequencing, molecular typing and phenotypic characterization. The 16S rRNA gene sequences of the Xenorhabdus strains were highly conserved (similarity coefficient >95 %), suggesting that the common ancestor of the genus probably emerged between 250 and 500 million years ago. Based on comparisons of the 16S rRNA gene sequences, we identified 13 groups and seven unique sequences. This classification was confirmed by analysis of molecular typing profiles of the strains, leading to the classification of new isolates into the Xenorhabdus species described previously and the description of ten novel Xenorhabdus species: Xenorhabdus cabanillasii sp. nov. (type strain USTX62 T =CIP 109066 T =DSM 17905 T ), Xenorhabdus doucetiae sp. nov. (type strain FRM16 T =CIP 109074 T =DSM 17909 T ), Xenorhabdus griffiniae sp. nov. (type strain ID10 T =CIP 109073 T =DSM 17911 T ), Xenorhabdus hominickii sp. nov. (type strain KE01 T =CIP 109072 T =DSM 17903 T ), Xenorhabdus koppenhoeferi sp. nov. (type strain USNJ01 T =CIP 109199 T =DSM 18168 T ), Xenorhabdus kozodoii sp. nov. (type strain SaV T =CIP 109068 T =DSM 17907 T ), Xenorhabdus mauleonii sp. nov. (type strain VC01 T =CIP 109075 T =DSM 17908 T ), Xenorhabdus miraniensis sp. nov. (type strain Q1 T =CIP 109069 T =DSM 17902 T ), Xenorhabdus romanii sp. nov. (type strain PR06-A T =CIP 109070 T =DSM 17910 T ) and Xenorhabdus stockiae sp. nov. (type strain TH01 T =CIP 109067 T =DSM 17904 T ). The Xenorhabdus strains studied here had very similar phenotypic patterns, but phenotypic features nonetheless differentiated the following species: X. bovienii , X. cabanillasii , X. hominickii , X. kozodoii , X. nematophila , X. poinarii and X. szentirmaii . Based on phenotypic analysis, we identified two major groups of strains. Phenotypic group G A comprised strains able to grow at temperatures of 3542 °C, whereas phenotypic group G B comprised strains that grew at temperatures below 35 °C, suggesting that some Xenorhabdus species may be adapted to tropical or temperate regions and/or influenced by the growth and development temperature of their nematode host.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences obtained in this study are listed in Table 1.
In bacteria, DNA-methyltransferase are responsible for DNA methylation of specific motifs in the genome. This methylation usually occurs at a very high rate. In the present study, we studied the ...MTases encoding genes found in the entomopathogenic bacteria Xenorhabdus. Only one persistent MTase was identified in the various species of this genus. This MTase, also broadly conserved in numerous Gram-negative bacteria, is called Dam: DNA-adenine MTase. Methylome analysis confirmed that the GATC motifs recognized by Dam were methylated at a rate of >99% in the studied strains. The observed enrichment of unmethylated motifs in putative promoter regions of the X. nematophila F1 strain suggests the possibility of epigenetic regulations. The overexpression of the Dam MTase responsible for additional motifs to be methylated was associated with impairment of two major phenotypes: motility, caused by a downregulation of flagellar genes, and hemolysis. However, our results suggest that dam overexpression did not modify the virulence properties of X. nematophila. This study increases the knowledge on the diverse roles played by MTases in bacteria.
The Steinernema carpocapsae-Xenorhabdus nematophila association is a nematobacterial complex used in biological control of insect crop pests. The infection success of this dual pathogen strongly ...depends on its interactions with the host's immune system. Here, we used the lepidopteran pest Spodoptera frugiperda to analyze the respective impact of each partner in the induction of its immune responses. First, we used previously obtained RNAseq data to construct the immunome of S. frugiperda and analyze its induction. We then selected representative genes to study by RT-qPCR their induction kinetics and specificity after independent injections of each partner. We showed that both X. nematophila and S. carpocapsae participate in the induction of stable immune responses to the complex. While X. nematophila mainly induces genes classically involved in antibacterial responses, S. carpocapsae induces lectins and genes involved in melanization and encapsulation. We discuss putative relationships between these differential inductions and the pathogen immunosuppressive strategies.
•We describe a comprehensive immunome of the lepidopteran pest Spodoptera frugiperda.•We show the induction of the different immune pathways by the nematobacterial complex S. carpocapsae – X. nematophila.•Each pathogen partner induced a strong stable response of the immunome.•The bacterial partner induces the expression of genes involved in antibacterial response.•The nematode partner induces the expression of genes involved in melanization and encapsulation.
Xenorhabdus bovienii bacteria have a dual lifestyle: they are mutualistic symbionts to many species of Steinernema nematodes and are pathogens to a wide array of insects. Previous studies have shown ...that virulence of X.bovienii-Steinernema spp. pairs decreases when the nematodes associate with non-cognate bacterial strains. However, the virulence of the X. bovienii strains alone has not been fully investigated. In this study, we characterized the virulence of nine X. bovienii strains in Galleria mellonella and Spodoptera littoralis and performed a comparative genomic analysis to correlate observed phenotypes with strain genotypes. Two X. bovienii strains were found to be highly virulent against the tested insect hosts, while three strains displayed attenuated insect virulence. Comparative genomic analyses revealed the presence of several clusters present only in virulent strains, including a predicted type VI secretion system (T6SS). We performed intra-species-competition assays, and showed that the virulent T6SS+ strains generally outcompeted the less virulent T6SS- strains. Thus, we speculate that the T6SS in X. bovienii may be another addition to the arsenal of antibacterial mechanisms expressed by these bacteria in an insect, where it could potentially play three key roles: (1) competition against the insect host microbiota; (2) protection of the insect cadaver from necrotrophic microbial competitors; and (3) outcompeting other Xenorhabdus species and/or strains when co-infections occur.
A symbiotic bacterium, strain IMI 397775T, was isolated from the insect-pathogenic nematode Steinernema australe. On the basis of 16S rRNA gene sequence similarity, this bacterial isolate was shown ...to belong to the genus Xenorhabdus, in agreement with the genus of its nematode host. The accurate phylogenetic position of this new isolate was defined using a multigene approach and showed that isolate IMI 397775T shares a common ancestor with Xenorhabdus doucetiae FRM16T and Xenorhabdus romanii PR06-AT, the symbiotic bacteria associated with Steinernema diaprepesi and Steinernema puertoricense, respectively. The nucleotide identity (less than 97 %) between isolate IMI 397775T, X. doucetiae FRM16T and X. romanii PR06-AT calculated for the concatenated sequences of five gene fragments encompassing 4275 nt, several phenotypic traits and the difference between the upper temperatures that limit growth of these three bacteria allowed genetic and phenotypic differentiation of isolate IMI 397775T from the two closely related species. Strain IMI 397775T therefore represents a novel species, for which the name Xenorhabdus magdalenensis sp. nov. is proposed, with the type strain IMI 397775T ( = DSM 24915T).
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