Photorhabdus Species Research Articles

Molluscicidal property of symbiotic bacteria associated with entomopathogenic nematodes against Indoplanorbis exustus and Radix rubiginosa, the intermediate hosts of trematode parasites

Indoplanorbis exustus and Radix rubiginosa act as intermediate hosts for veterinary and medical trematode parasites. Snail control is a strategy used to decrease the number of snails and interrupt the life cycle of parasites. The objective of this study was to evaluate the efficacy of Xenorhabdus and Photorhabdus extracts against I. exustus and R. rubiginosa in the laboratory. Ethyl acetate extracts of selected symbiotic bacteria were tested for their molluscicidal activities according to World Health Organization guidelines. Additionally, pathological changes in the snails were observed after treatment with the LC50 values under a light microscope. Indoplanorbis exustus and R. rubiginosa were susceptible to all ethyl acetate extracts of symbiotic bacteria. The lowest LC50 and LC90 at 24 h for I. exustus after exposure to Photorhabdus laumondii subsp. laumondii (bALN18.2_TH) extracts were 81.66 and 151.02 ppm, respectively. Similarly, the lowest LC50 and LC90 at 24 h for R. rubiginosa after exposure to Photorhabdus luminescence subsp. akhurstii (bAPY3.5_TH) extracts were 49.21 and 147.66 ppm, respectively. Photorhabdus species had more substantial molluscicidal effects than Xenorhabdus on these snails. The ethyl acetate extracts of these bacteria are effective when contacting the epithelial cells and foot muscle of the snails. To our knowledge, this is the first report on using Xenorhabdus and Photorhabdus extracts to evaluate molluscicidal activities. These symbiotic bacteria, Xenorhabdus and Photorhabdus, may be useful for controlling snail intermediate hosts.

Manipulation of GameXPeptide synthetase gene expression by a promoter exchange alters the virulence of an entomopathogenic bacterium, Photorhabdus temperata temperata, by modulating insect immune responses.

An entomopathogenic bacterium, Photorhabdus temperata subsp. temperata, is mutualistic to its host nematode, Heterorhabditis megidis. The infective juvenile nematodes enter target insects through natural openings and release the symbiotic bacteria into the insect hemocoel. The released bacteria suppress the insect immune responses and cause septicemia through their secondary metabolites. GameXPeptide (GXP) is one of the common secondary metabolites of most Photorhabdus species and is produced by the catalytic activity of a specific non-ribosomal peptide synthetase called GxpS encoded by the gxpS gene. This study confirmed gxpS to be encoded in the P. temperata temperata genome and analyzed its expression during bacterial growth. LC-MS/MS analysis of the bacterial culture broth contained at least four different GXPs (GXP-A to GXP-D), in which GXP-A was the most abundant. To investigate GXP synthesis following gxpS expression, the gxpS promoter of P. temperata temperata was replaced with an inducible arabinose promoter by homologous recombination. The gxpS transcript levels in the mutant were altered by the addition of l-arabinose. Without the inducer, the gxpS transcript level was significantly lower compared to the wild type and produced significantly lower amounts of the four GXPs. The addition of the inducer to the mutant significantly increased gxpS expression and produced significantly higher levels of the four GXPs compared to the wild type. The metabolite extracts obtained from wild-type and mutant bacteria showed differential immunosuppressive activities according to their GXP contents against the cellular and humoral immune responses of a lepidopteran insect, Spodoptera exigua. Interestingly, the gxpS-mutant bacteria showed less insecticidal activity compared to the wild type, whereas the addition of GXP to the mutant significantly restored insecticidal activity. These results suggest that the gxpS gene encoded in P. temperata temperata is responsible for the production of at least four different GXPs, which play crucial roles in bacterial virulence.

Genome- and metabolome-guided discovery of marine BamA inhibitors revealed a dedicated darobactin halogenase

Darobactins represent a class of ribosomally synthesized and post-translationally modified peptide (RiPP) antibiotics featuring a rare bicyclic structure. They target the Bam-complex of Gram-negative bacteria and exhibit invivo activity against drug-resistant pathogens. First isolated from Photorhabdus species, the corresponding biosynthetic gene clusters (BGCs) are widespread among γ-proteobacteria, including the genera Vibrio, Yersinia, and Pseudoalteromonas (P.). While the organization of the BGC core is highly conserved, a small subset of Pseudoalteromonas carries an extended BGC with additional genes. Here, we report the identification of brominated and dehydrated darobactin derivatives from P.luteoviolacea strains. The marine derivatives are active against multidrug-resistant (MDR) Gram-negative bacteria and showed solubility and plasma protein binding ability different from darobactin A, rendering it more active than darobactin A. The halogenation reaction is catalyzed by DarH, a new class of flavin-dependent halogenases with a novel fold.

Rattus norvegicus (Berkenhout, 1769) (Rodentia: Muridae)’un entomopatojen bakteriler ile enfekte böcek kadavralarına tepkisinin belirlenmesi

Xenorhabdus Thomas & Poinar (Enterobacterales: Morganellaceae) and Photorhabdus Thomas & Poinar (Enterobacterales: Morganellaceae) bacteria are mutualistically associated with Steinernema Travassos, 1927 (Rhabditida: Steinernematidae) and Heterorhabditis Poinar, 1976 (Rhabditida: Heterorhabditidae) nematodes, respectively, and are known to produce several secondary metabolites that protect nematode-killed insects from different competitors. One of these compounds called “the scavenger deterrent factor” (SDF) is known to deter different arthropod, bird, and fish species from feeding on insects killed by Xenorhabdus or Photorhabdus bacteria. The effects of SDF from five different Xenorhabdus and one Photorhabdus species against the Norway rat, Rattus norvegicus (Berkenhout, 1769) (Rodentia: Muridae) were investigated using either a one-choice or two-choice experimental design during 2019-2020 in Aydın Adnan Menderes University. Rats were given four-day-old bacteria-killed Galleria mellonella (L., 1758) (Lepidoptera: Pyralidae) larvae and feeding behavior was observed and recorded. The results demonstrate that the Norway rat is deterred from feeding on insects killed by certain Xenorhabdus and Photorhabdus species and it is likely due to the distastefulness of these cadavers. Ecologically, the data suggest that insects killed by the entomopathogenic nematode/bacterium complex in nature may be protected from attack from insectivorous mammals, especially those that feed on soil-dwelling insects.

Evaluation of the Antifungal Activities of Photorhabdus akhurstii and Its Secondary Metabolites against Phytopathogenic Colletotrichum gloeosporioides.

Colletotrichum gloeosporioides is a phytopathogenic fungus that causes devastating losses in strawberries without effective countermeasures. Members of the genus Photorhabdus exhibit antimicrobial capability and have been found to have the potential for use as biocontrol agents against C. gloeosporioides. Photorhabdus species exhibit two phase variations with a differentiated composition of secondary metabolites designated to each phase. In this study, Photorhabdus akhurstii sp. nov. 0813-124 exhibited phase I (PL1) and phase II (PL2); however, only PL1 displayed distinct inhibition of C. gloeosporioides in the confrontation assay. We identified the bioactive ingredients of P. akhurstii sp. nov. 0813-124 to be glidobactin A and cepafungin I, with MIC values lower than 1.5 and 2.0 µg/mL, respectively. Furthermore, we revealed the biosynthetic gene cluster (BGC) of corresponding bioactive molecules through genomics analysis and determined its expression level in PL1 and PL2. The expression of glidobactin BGC in PL1 increased rapidly within 24 h, while PL2 was eventually stimulated after 60 h. In summary, we demonstrated that P. akhurstii sp. nov. 0813-124 could potentially be used as a biocontrol agent or part of a natural product repertoire for combating C. gloeosporioides.

Colour of heterorhabditis zealandica-infected-Galleria mellonella dependent on the Photorhabdus symbiont, with two new nematode-symbiotic associations reported

Bacterial symbionts associated with entomopathogenic nematodes (EPNs) play an important role in terms of the insecticidal properties of nematodes in pest control. Galleria mellonella larvae, shortly after being infected with three different strains of Heterorhabditis zealandica, which were isolated from South African soil, changed from pale white to steel grey-blue (blue), bright red, and yellow with a green tint (green), respectively. The genetic relatedness of the bacterial symbionts that were isolated from the three strains of H. zealandica was determined by means of comparing the 16S rRNA, recA, gyrB, dnaN, gltX and infB gene sequences. Subsequently, comparing the concatenated sequences revealed the presence of three distinct Photorhabdus species. The H. zealandica strain SF41, associated with Photorhabdus heterorhabditis, produced ‘blue’ G. mellonella larvae. The H. zealandica strain MJ2C, associated with Photorhabdus thracensis, yielded ‘green’ G. mellonella larvae, while the H. zealandica strain LLM associated with Photorhabdus laumondii subsp. laumondii yielded red larvae. The colour changes in G. mellonella larvae were found to have been instigated by a particular Photorhabdus species associated with H. zealandica. The red and ‘green’ phenotypes of G. mellonella larvae were found to represent new combinations of Heterorhabditis and Photorhabdus. In future studies, the colour of infected G. mellonella larvae needs to be reported as a phenotypic character, as it indicates the different bacterial species associated with the same nematode host, as shown in the case of H. zealandica.

Screening and Molecular Identification of Bacteria from the Midgut of Amphimallon solstitiale Larvae Exhibiting Antagonistic Activity against Bacterial Symbionts of Entomopathogenic Nematodes.

Entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) are a group of organisms capable of infecting larvae of insects living in soil, including representatives of the family Scarabaeidae. Their insecticidal activity is related to the presence of symbiotic bacteria Xenorhabdus spp. or Photorhabdus spp. in the alimentary tract, which are released into the insect body, leading to its death caused by bacterial toxins and septicemia. Although the antibacterial activities of symbionts of entomopathogenic nematodes have been well described, there is insufficient knowledge of the interactions between these bacteria and microorganisms that naturally inhabit the alimentary tract of insects infested by nematodes. In this study, 900 bacterial strains isolated from midgut samples of Amphimallon solstitiale larvae were tested for their antagonistic activity against the selected five Xenorhabdus and Photorhabdus species. Cross-streak tests showed significant antibacterial activity of 20 isolates. These bacteria were identified as Bacillus [Brevibacterium] frigoritolerans, Bacillus toyonensis, Bacillus wiedmannii, Chryseobacterium lathyri, Chryseobacterium sp., Citrobacter murliniae, Enterococcus malodoratus, Paenibacillus sp., Serratia marcescens and Serratia sp. Since some representatives of the intestinal microbiota of A. solstitiale are able to inhibit the growth of Xenorhabdus and Photorhrhabdus bacteria in vitro, it can be assumed that this type of bacterial interaction may occur at certain stages of insect infection by Steinernema or Heterorhabditis nematodes.

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.

Antifungal activity of different Xenorhabdus and Photorhabdus species against various fungal phytopathogens and identification of the antifungal compounds from X. szentirmaii.

Xenorhabdus and Photorhabdus spp. are enteric bacterial symbionts of Steinernema and Heterorhabditis nematodes, respectively. These bacteria produce an extensive set of natural products (NPs) with antibacterial, antifungal, antiprotozoal, insecticidal, or other bioactivities when vectored into insect hemocoel by nematodes. We assessed the in vitro activity of different Xenorhabdus and Photorhabdus cell-free supernatants against important fungal phytopathogens, viz., Cryphonectria parasitica, Fusarium oxysporum, Rhizoctonia solani, and Sclerotinia sclerotiorum and identified the bioactive antifungal compound/s present in the most effective bacterial supernatant using the easyPACId (easy promoter-activated compound identification) approach against chestnut blight C. parasitica. Our data showed that supernatants from Xenorhabdus species were comparatively more effective than extracts from Photorhabdus in suppressing the fungal pathogens; among the bacteria assessed, Xenorhabdus szentirmaii was the most effective species against all tested phytopathogens especially against C. parasitica. Subsequent analysis revealed fabclavines as antifungal bioactive compounds in X. szentirmaii, generated by a polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) hybrid system. Fabclavines are broad-spectrum, heat-stable NPs that have great potential as biological control compounds against fungal plant pathogens. More studies are needed to assess the potential phytotoxicity of these compounds and their effects on non-target organisms before commercialization. KEY POINTS: • Chemical fungicides have toxic effects on humans and other non-target organisms. • Alternatives with novel modes of action to supplant current fungicide are needed. • A novel bioactive antifungal compound from Xenorhabdus szentirmaii was identified.

Differential in vitro pathogenicity of Photorhabdus bacterial species against two distinct insect cell lines

Understanding the mode of action of pathogenic bacteria through in vitro studies can provide additional insight into their infection strategies. Here we have characterized the effect of Photorhabdus luminescens and Photorhabdus asymbiotica on two distinct insect cell lines. We report that insect cell survival and metabolism as well as bacterial proliferation differ between infection with two Photorhabdus species. These findings reinforce the notion that P. luminescens and P. asymbiotica deploy diverse tactics to infect insect cells. This knowledge might lead to better appreciation of the interaction between pathogenic bacteria and different types of insect cells.

Symbiosis, virulence and natural-product biosynthesis in entomopathogenic bacteria are regulated by a small RNA.

Photorhabdus and Xenorhabdus species have mutualistic associations with nematodes and an entomopathogenic stage1,2 in their life cycles. In both stages, numerous specialized metabolites are produced that have roles in symbiosis and virulence3,4. Although regulators have been implicated in the regulation of these specialized metabolites3,4, how small regulatory RNAs (sRNAs) are involved in this process is not clear. Here, we show that the Hfq-dependent sRNA, ArcZ, is required for specialized metabolite production in Photorhabdus and Xenorhabdus. We discovered that ArcZ directly base-pairs with the mRNA encoding HexA, which represses the expression of specialized metabolite gene clusters. In addition to specialized metabolite genes, we show that the ArcZ regulon affects approximately 15% of all transcripts in Photorhabdus and Xenorhabdus. Thus, the ArcZ sRNA is crucial for specialized metabolite production in Photorhabdus and Xenorhabdus species and could become a useful tool for metabolic engineering and identification of commercially relevant natural products.

R-type bacteriocins of Xenorhabdus bovienii determine the outcome of interspecies competition in a natural host environment.

Xenorhabdus species are bacterial symbionts of Steinernema nematodes and pathogens of susceptible insects. Different species of Steinernema nematodes carrying specific species of Xenorhabdus can invade the same insect, thereby setting up competition for nutrients within the insect environment. While Xenorhabdus species produce both diverse antibiotic compounds and prophage-derived R-type bacteriocins (xenorhabdicins), the functions of these molecules during competition in a host are not well understood. Xenorhabdus bovienii (Xb-Sj), the symbiont of Steinernema jollieti, possesses a remnant P2-like phage tail cluster, xbp1, that encodes genes for xenorhabdicin production. We show that inactivation of either tail sheath (xbpS1) or tail fibre (xbpH1) genes eliminated xenorhabdicin production. Preparations of Xb-Sj xenorhabdicin displayed a narrow spectrum of activity towards other Xenorhabdus and Photorhabdus species. One species, Xenorhabdus szentirmaii (Xsz-Sr), was highly sensitive to Xb-Sj xenorhabdicin but did not produce xenorhabdicin that was active against Xb-Sj. Instead, Xsz-Sr produced high-level antibiotic activity against Xb-Sj when grown in complex medium and lower levels when grown in defined medium (Grace's medium). Conversely, Xb-Sj did not produce detectable levels of antibiotic activity against Xsz-Sr. To study the relative contributions of Xb-Sj xenorhabdicin and Xsz-Sr antibiotics in interspecies competition in which the respective Xenorhabdus species produce antagonistic activities against each other, we co-inoculated cultures with both Xenorhabdus species. In both types of media Xsz-Sr outcompeted Xb-Sj, suggesting that antibiotics produced by Xsz-Sr determined the outcome of the competition. In contrast, Xb-Sj outcompeted Xsz-Sr in competitions performed by co-injection in the insect Manduca sexta, while in competition with the xenorhabdicin-deficient strain (Xb-Sj:S1), Xsz-Sr was dominant. Thus, xenorhabdicin was required for Xb-Sj to outcompete Xsz-Sr in a natural host environment. These results highlight the importance of studying the role of antagonistic compounds under natural biological conditions.

Competition and Co-existence of Two Photorhabdus Symbionts with a Nematode Host

Photorhabdus spp. (Enterobacteriales: Morganellaceae) occur exclusively as symbionts of Heterorhabditis nematodes for which they provide numerous services, including killing insects and providing nutrition and defence within the cadavers. Unusually, two species (Photorhabdus cinerea and Photorhabdus temperata) associate with a single population of Heterorhabditis downesi at a dune grassland site. Building on previous work, we investigated competition between these two Photorhabdus species both at the regional (between insects) and local (within insect) level by trait comparison and co-culture experiments. There was no difference between the species with respect to supporting nematode reproduction and protection of cadavers against invertebrate scavengers, but P. cinerea was superior to P. temperata in several traits: faster growth rate, greater antibacterial and antifungal activity and colonisation of a higher proportion of nematodes in co-culture. Moreover, where both bacterial symbionts colonised single nematode infective juveniles, P. cinerea tended to dominate in numbers. Differences between Photorhabdus species were detected in the suite of secondary metabolites produced: P. temperata produced several compounds not produced by P. cinerea including anthraquinone pigments. Bioluminescence emitted by P. temperata also tended to be brighter than that from P. cinerea. Bioluminescence and pigmentation may protect cadavers against scavengers that rely on sight. We conclude that while P. cinerea may show greater local level (within-cadaver) competitive success, co-existence of the two Photorhabdus species in the spatially heterogeneous environment of the dunes is favoured by differing specialisations in defence of the cadaver against differing locally important threats.

Making and Breaking Leupeptin Protease Inhibitors in Pathogenic Gammaproteobacteria.

Leupeptin is a bacterial small molecule that is used worldwide as a protease inhibitor. However, its biosynthesis and genetic distribution remain unknown. We identified a family of leupeptins in gammaproteobacterial pathogens, including Photorhabdus, Xenorhabdus, and Klebsiella species, amongst others. Through genetic, metabolomic, and heterologous expression analyses, we established their construction by discretely expressed ligases and accessory enzymes. In Photorhabdus species, a hypothetical protein required for colonizing nematode hosts was established as a new class of proteases. This enzyme cleaved the tripeptide aldehyde protease inhibitors, leading to the formation of "pro-pyrazinones" featuring a hetero-tricyclic architecture. In Klebsiella oxytoca, the pathway was enriched in clinical isolates associated with respiratory tract infections. Thus, the bacterial production and proteolytic degradation of leupeptins can be associated with animal colonization phenotypes.

Lectin PLL3, a Novel Monomeric Member of the Seven-Bladed β-Propeller Lectin Family.

The Photorhabdus species is a Gram-negative bacteria of the family Morganellaceae that is known for its mutualistic relationship with Heterorhabditis nematodes and pathogenicity toward insects. This study is focused on the characterization of the recombinant lectin PLL3 with an origin in P. laumondii subsp. laumondii. PLL3 belongs to the PLL family of lectins with a seven-bladed β-propeller fold. The binding properties of PLL3 were tested by hemagglutination assay, glycan array, isothermal titration calorimetry, and surface plasmon resonance, and its structure was determined by X-ray crystallography. Obtained data revealed that PLL3 binds similar carbohydrates to those that the other PLL family members bind, with some differences in the binding properties. PLL3 exhibited the highest affinity toward l-fucose and its derivatives but was also able to interact with O-methylated glycans and other ligands. Unlike the other members of this family, PLL3 was discovered to be a monomer, which might correspond to a weaker avidity effect compared to homologous lectins. Based on the similarity to the related lectins and their proposed biological function, PLL3 might accompany them during the interaction of P. laumondii with both the nematode partner and the insect host.

Nematode-Associated Bacteria: Production of Antimicrobial Agent as a Presumptive Nominee for Curing Endodontic Infections Caused by Enterococcus faecalis.

Xenorhabdus and/or Photorhabdus bacteria produce antibacterial metabolites to protect insect cadavers against food competitors allowing them to survive in nature with their nematode host. The effects of culture supernatant produced by Xenorhabdus and Photorhabdus spp. were investigated against the multidrug-resistant dental root canal pathogen Enterococcus faecalis. The efficacy of seven different cell-free supernatants of Xenorhabdus and Photorhabdus species against E. faecalis was assessed with overlay bioassay and serial dilution techniques. Additionally, time-dependent inactivation of supernatant was evaluated. Among the seven different bacterial species, X. cabanillasii produced the strongest antibacterial effects. Loss of bioactivity in a phosphopantetheinyl transferase-deficient mutant of X. cabanillasii indicated that this activity is likely based on non-ribosomal peptide synthetases (NRPSs) or polyketide synthases (PKSs). Subsequent in silico analysis revealed multiple possible biosynthetic gene clusters (BGCs) in the genome of X. cabanillasii including a BGC homologous to that of zeamine/fabclavine biosynthesis. Fabclavines are NRPS-derived hexapeptides, which are connected by PKS-derived malonate units to an unusual polyamine, also PKS-derived. Due to the known broad-spectrum bioactivity of the fabclavines, we generated a promoter exchange mutant in front of the fabclavine-like BGC. This leads to over-expression by induction or a knock-out by non-induction which resulted in a bioactive and non-bioactive mutant. Furthermore, MS and MS2 experiments confirmed that X. cabanillasii produces the same derivatives as X. budapestensis. The medicament potential of 10-fold concentrated supernatant of induced fcl promoter exchanged X. cabanillasii was also assessed in dental root canals. Calcium hydroxide paste, or chlorhexidine gel, or fabclavine-rich supernatant was applied to root canals. Fabclavine-rich supernatant exhibited the highest inactivation efficacy of ≥3 log10 steps CFU reduction, followed by calcium hydroxide paste (≤2 log10 step). The mean percentage of E. faecalis-free dental root canals after treatment was 63.6, 45.5, and 18.2% for fabclavine, calcium hydroxide, and chlorhexidine, respectively. Fabclavine in liquid form or preferably as a paste or gel formulation is a promising alternative intracanal medicament.

Symbiotic Bacteria as Potential Agents for Mosquito Control

Xenorhabdus and Photorhabdus species are symbiotic bacteria of the insect-pathogenic soil nematodes that produce insecticidal compounds lethal to prey insects. Recently, there has been much interest in adapting these insecticidals for mosquito control. Here, I advocate the potential of Xenorhabdus/Photorhabdus as natural sources of mosquitocides (larvicides, adulticides) and feeding-deterrents.

Photorhabdus khanii subsp. guanajuatensis subsp. nov., isolated from Heterorhabditis atacamensis, and Photorhabdus luminescens subsp. mexicana subsp. nov., isolated from Heterorhabditis mexicana entomopathogenic nematodes.

Two Gram-negative, rod-shaped, non-spore-forming bacteria, MEX20-17T and MEX47-22T, were isolated from the digestive system of Heterorhabditis atacamensis and Heterorhabditis mexicana entomopathogenic nematodes, respectively. Their 16S rRNA gene sequences suggest that strains MEX20-17T and MEX47-22T belong to the γ-Proteobacteria and to the genus Photorhabdus. Deeper analyses using housekeeping-gene-based and whole-genome-based phylogenetic reconstruction suggest that MEX20-17T is closely related to Photorhabdus khanii and that MEX47-22T is closely related to Photorhabdus luminescens. Sequence similarity scores confirm these observations: MEX20-17T and P. khanii DSM 3369T share 98.9 % nucleotide sequence identity (NSI) of concatenated housekeeping genes, 70.4 % in silico DNA-DNA hybridization (isDDH) and 97 % orthologous average nucleotide identity (orthoANI); and MEX47-22T and P. luminescens ATCC 29999T share 98.9 % NSI, 70.6 % isDDH and 97 % orthoANI. Physiological characterization indicates that both strains differ from all validly described Photorhabdus species and from their more closely related taxa. We therefore propose to classify MEX20-17T and MEXT47-22T as new subspecies within P. khanii and P. luminescens, respectively. Hence, the following names are proposed for these strains: Photorhabdus khanii subsp. guanajuatensis subsp. nov. with the type strain MEX20-17T (=LMG 30372T=CCOS 1191T) and Photorhabdus luminescenssubsp. mexicana subsp. nov. with the type strain MEX47-22T (=LMG 30528T=CCOS 1199T). These propositions automatically create Photorhabdus khanii subsp. khanii subsp. nov. with DSM 3369T as the type strain (currently classified as P. khanii), and Photorhabdus luminescenssubsp. luminescenssubsp. nov. with ATCC 29999T as the type strain (currently classified as P. luminescens).

Secretion Systems and Secreted Proteins in Gram-Negative Entomopathogenic Bacteria: Their Roles in Insect Virulence and Beyond.

Many Gram-negative bacteria have evolved insect pathogenic lifestyles. In all cases, the ability to cause disease in insects involves specific bacterial proteins exported either to the surface, the extracellular environment, or the cytoplasm of the host cell. They also have several distinct mechanisms for secreting such proteins. In this review, we summarize the major protein secretion systems and discuss examples of secreted proteins that contribute to the virulence of a variety of Gram-negative entomopathogenic bacteria, including Photorhabdus, Xenorhabdus, Serratia, Yersinia, and Pseudomonas species. We also briefly summarize two classes of exported protein complexes, the PVC-like elements, and the Tc toxin complexes that were first described in entomopathogenic bacteria.

Whole-genome-based revisit of Photorhabdus phylogeny: proposal for the elevation of most Photorhabdus subspecies to the species level and description of one novel species Photorhabdus bodei sp. nov., and one novel subspecies Photorhabdus laumondii subsp. clarkei subsp. nov.

Bacterial symbionts are crucial for the infectivity and success of entomopathogenic nematodes as biological control agents. The current understanding of the symbiotic relationships is limited by taxonomic uncertainties. Here, we used whole-genome sequencing and traditional techniques to reconstruct the phylogenetic relationships between all described Photorhabdus species and subspecies as well as 11 newly isolated symbiotic bacteria of Heterorhabditis nematodes, including the unreported bacterial partner of H. beicherriana. In silico DNA-DNA hybridization, orthologous average nucleotide identity and nucleotide sequence identity of concatenated housekeeping genes scores were calculated and set into relation with current cut-off values for species delimitation in bacteria. Sequence data were complemented with biochemical and chemotaxonomic markers, and ribosomal protein fingerprinting profiles. This polyphasic approach resolves the ambiguous taxonomy of Photorhabdusand lead to the proposal for the elevation of most of them into a higher taxon and the creation of several new taxa: 15 new species, one of which is newly described: Photorhabdus bodei sp. nov. (type strain LJ24-63T=DSM 105690T=CCOS 1159T) and the other 14 arise through the proposal of elevating already described subspecies to species, and are proposed to be renamed as follows: Photorhabdus asymbioticasubsp. australis as Photorhabdus australis sp. nov., Photorhabdus luminescenssubsp. akhurstii as Photorhabdus akhurstii sp. nov., Photorhabdus luminescenssubsp. caribbeanensis as Photorhabdus caribbeanensis sp. nov., Photorhabdus luminescenssubsp. hainanensis as Photorhabdus hainanensis sp. nov., Photorhabdus luminescenssubsp. kayaii as Photorhabdus kayaii sp. nov., Photorhabdus luminescenssubsp. kleinii as Photorhabdus kleinii sp. nov., Photorhabdus luminescenssubsp. namnaonensis as Photorhabdus namnaonensis sp. nov., Photorhabdus luminescenssubsp. noenieputensis as Photorhabdus noenieputensis sp. nov., Photorhabdus luminescenssubsp.laumondii as Photorhabdus laumondii sp. nov., Photorhabdus temperatasubsp. cinerea as Photorhabdus cinerea sp. nov., Photorhabdus temperatasubsp. khanii as Photorhabdus khanii sp. nov., Photorhabdus temperatasubsp. stackebrandtii as Photorhabdus stackebrandtii sp. nov., Photorhabdus temperatasubsp. tasmaniensis as Photorhabdus tasmaniensis sp. nov., and Photorhabdus temperatasubsp. thracensis as Photorhabdus thracensis sp. nov. In addition, we propose the creation of two new subspecies, one of which arises through the reduction of rank: Photorhabdus laumondii subsp. laumondii comb. nov. (basonym: P. luminescenssubsp. laumondii) and the second one is newly described: Photorhabdus laumondii subsp. clarkei subsp. nov. (type strain BOJ-47T=DSM 105531T=CCOS 1160T). Finally, we propose to emend the description of three species, which results from the proposal of elevating three subspecies to the species status: Photorhabdus asymbiotica, Photorhabdus temperata and Photorhabdus luminescens, formerly classified as Photorhabdus asymbioticasubsp. asymbiotica, Photorhabdus temperatasubsp.temperata and Photorhabdus luminescenssubsp. luminescens, respectively.