Photorhabdus africana sp. nov. isolated from Heterorhabditis entomopathogenic nematodes.

One Gram-negative, rod-shaped bacterial strain, isolated from an undescribed Heterorhabditis entomopathogenic nematode species was characterized to determine its taxonomic position. The 16S rRNA gene sequences indicate that it belongs to the class Gammaproteobacteria, to the family Morganellaceae, to the genus Photorhabdus, and likely represents a novel bacterial species. This strain, designated here as CRI-LCT, was therefore molecularly, biochemically, and morphologically characterized to describe the novel bacterial species. Phylogenetic reconstructions using 16S rRNA gene sequences show that CRI-LCT is closely related to P. laumondii subsp. laumondii TT01T and to P. laumondii subsp. clarkei BOJ-47T. The 16rRNA gene sequences between CRI-LCT and P. laumondii subsp. laumondii TT01T are 99.1% identical, and between CRI-LCT and P. laumondii subsp. clarkei BOJ-47T are 99.2% identical. Phylogenetic reconstructions using whole genome sequences show that CRI-LCT is closely related to P. laumondii subsp. laumondii TT01T and to P. laumondii subsp. clarkei BOJ-47T. Moreover, digital DNA-DNA hybridization (dDDH) values between CRI-LCT and its two relative species P. laumondii subsp. laumondii TT01T and P. laumondii subsp. clarkei BOJ-47T are 65% and 63%, respectively. In addition, we observed that average nucleotide identity (ANI) values between CRI-LCT and its two relative species P. laumondii subsp. laumondii TT01T and P. laumondii subsp. clarkei BOJ-47T are 95.8% and 95.5%, respectively. These values are below the 70% dDDH and the 95-96% ANI divergence thresholds that delimits prokaryotic species. Based on these genomic divergence values, and the phylogenomic separation, we conclude that CRI-LCT represents a novel bacterial species, for which we propose the name Photorhabdus africana sp. nov. with CRI-LCT (= CCM 9390T = CCOS 2112T) as the type strain. The following biochemical tests allow to differentiate P. africana sp. nov. CRI-LCT from other species of the genus, including its more closely related taxa: ß-Galactosidase, citrate utilization, urease and tryptophan deaminase activities, indole and acetoin production, and glucose and inositol oxidation. Our study contributes to a better understanding of the taxonomy and biodiversity of this important bacterial group with great biotechnological and agricultural potential.

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.

Genome assembly and annotation of Photorhabdus heterorhabditis strain ETL reveals genetic features involved in pathogenicity with its associated entomopathogenic nematode and anti-host effectors with biocontrol potential applications.

The genome sequences of entomopathogenic nematode (EPN) bacteria and their functional analyses can lead to the genetic engineering of the bacteria for use as biocontrol agents. The bacterial symbiont Photorhabdus heterorhabditis strain ETL isolated from an insect pathogenic nematode, Heterorhabditis zealandica strain ETL, collected in the northernmost region of South Africa was studied to reveal information that can be useful in the design of improvement strategies for both effective and liquid production method of EPN-based pesticides. The strain ETL genome was found closely related to the type strain genome of P. australis DSM 17,609 (~60 to 99.9% CDSs similarity), but closely related to the not yet genome-sequenced type strain, P. heterorhabditis. It has a genome size of 4,866,148 bp and G + C content of 42.4% similar to other Photorhabdus. It contains 4,351 protein coding genes (CDSs) of which, at least 84% are shared with the de facto type strain P. luminescens subsp. laumondii TTO1, and has 318 unknown CDSs and the genome has a higher degree of plasticity allowing it to adapt to different environmental conditions, and to be virulent against various insects; observed through genes acquired through horizontal gene transfer mechanisms, clustered regularly interspaced short palindromic repeats, non-determined polyketide- and non-ribosomal peptide- synthase gene clusters, and many genes associated with uncharacterized proteins; which also justify the strain ETL's genes differences (quantity and quality) compared to P. luminescens subsp. laumondii TTO1. The protein coding sequences contained genes with both bio-engineering and EPNs mass production importance, of which numerous are uncharacterized.

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.

Photorhabdus heterorhabditis subsp. aluminescens subsp. nov., Photorhabdus heterorhabditis subsp. heterorhabditis subsp. nov., Photorhabdus australis subsp. thailandensis subsp. nov., Photorhabdus australis subsp. australis subsp. nov., and Photorhabdus aegyptia sp. nov. isolated from Heterorhabditis entomopathogenic nematodes.

Three Gram-stain-negative, rod-shaped, non-spore-forming bacteria, BA1T, Q614T and PB68.1T, isolated from the digestive system of Heterorhabditis entomopathogenic nematodes, were biochemically and molecularly characterized to clarify their taxonomic affiliations. The 16S rRNA gene sequences of these strains suggest that they belong to the Gammaproteobacteria, to the family Morganellacea, and to the genus Photorhabdus. Deeper analyses using whole genome-based phylogenetic reconstructions suggest that BA1T is closely related to Photorhabdus akhursti, that Q614T is closely related to Photorhabdus heterorhabditis, and that PB68.1T is closely related to Photorhabdus australis. In silico genomic comparisons confirm these observations: BA1T and P. akhursti 15138T share 68.8 % digital DNA-DNA hybridization (dDDH), Q614T and P. heterorhabditis SF41T share 75.4 % dDDH, and PB68.1T and P. australis DSM 17609T share 76.6  % dDDH. Physiological and biochemical characterizations reveal that these three strains also differ from all validly described Photorhabdus species and from their more closely related taxa, contrary to what was previously suggested. We therefore propose to classify BA1T as a new species within the genus Photorhabdus, Q614T as a new subspecies within P. heterorhabditis, and PB68.1T as a new subspecies within P. australis. Hence, the following names are proposed for these strains: Photorhabdus aegyptia sp. nov. with the type strain BA1T(=DSM 111180T=CCOS 1943T=LMG 31957T), Photorhabdus heterorhabditis subsp. aluminescens subsp. nov. with the type strain Q614T (=DSM 111144T=CCOS 1944T=LMG 31959T) and Photorhabdus australis subsp. thailandensis subsp. nov. with the type strain PB68.1T (=DSM 111145T=CCOS 1942T). These propositions automatically create Photorhabdus heterorhabditis subsp. heterorhabditis subsp. nov. with SF41T as the type strain (currently classified as P. heterorhabditis) and Photorhabdus australis subsp. australis subsp. nov. with DSM17609T as the type strain (currently classified as P. australis).

A survey of entomopathogenic nematodes and their symbiotic bacteria in agricultural areas of northern Thailand.

Entomopathogenic nematodes (EPNs) Steinernema and Heterorhabditis and their symbiotic bacteria, Xenorhabdus and Photorhabdus, have been successfully used for the control of insect pests. The objectives of this study were to survey the EPNs and symbiotic bacteria in the agricultural areas of the Phitsanulok province, Thailand, and to study the association between the soil parameters and presence of EPNs. We collected 200 soil samples from 40 soil sites in agricultural areas (field crops, horticulture crops and forest). The prevalence of EPNs was 8.0% (16/200). Fifteen of the EPN isolates were molecularly identified (based on 28S ribosomal DNA and internal transcribed spacer regions) as Steinernema siamkayai. Seven isolates of Xenorhabdus stockiae were identified using recombinase A sequencing. Phylogenetic analysis revealed that all the Steinernema and Xenorhabdus isolates were closely related to S. siamkayai (Indian strain) and X. stockiae (Thai strain), respectively. Significantly more EPNs were recovered from loam than from clay. Although the association between soil parameters (pH, temperature and moisture) and the presence of EPNs was not statistically significant, the elevation levels of the soil sites with and without EPNs were found to be different. Moreover, statistical comparisons between the agricultural areas revealed no significant differences. Therefore, we concluded that S. siamkayai is associated with X. stockiae in agricultural areas and that there is no association between the soil parameters of agricultural areas and presence of EPNs, except for soil texture and the elevation. Steinernema siamkayai may be applied as a biocontrol agent in agricultural areas.

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.

Identification of Photorhabdus symbionts by MALDI-TOF MS.

Species of the bacterial genus Photorhabus live in a symbiotic relationship with Heterorhabditis entomopathogenic nematodes. Besides their use as biological control agents against agricultural pests, some Photorhabdus species are also a source of natural products and are of medical interest due to their ability to cause tissue infections and subcutaneous lesions in humans. Given the diversity of Photorhabdus species, rapid and reliable methods to resolve this genus to the species level are needed. In this study, we evaluated the potential of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of Photorhabdus species. To this end, we established a collection of 54 isolates consisting of type strains and multiple field strains that belong to each of the validly described species and subspecies of this genus. Reference spectra for the strains were generated and used to complement a currently available database. The extended reference database was then used for identification based on the direct transfer sample preparation method and the protein fingerprint of single colonies. High-level discrimination of distantly related species was observed. However, lower discrimination was observed with some of the most closely related species and subspecies. Our results therefore suggest that MALDI-TOF MS can be used to correctly identify Photorhabdus strains at the genus and species level, but has limited resolution power for closely related species and subspecies. Our study demonstrates the suitability and limitations of MALDI-TOF-based identification methods for assessment of the taxonomic position and identification of Photorhabdus isolates.

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.

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).

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.

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.

Strains of Photorhabdus spp. associated with polish Heterorhabditis isolates: their molecular and phenotypic characterization and symbiont exchange.

The relationships between six bacterial symbionts of the entomopathogenic nematodes Heterorhabditis bacteriophora and Heterorhabditis megidis from Poland to species and subspecies of the genus Photorhabdus were evaluated. This study was based on phylogenetic analysis of sequence data of five genes: 16S rRNA, gyrB, recA, gltX, and dnaN. The bacteria were also characterized phenotypically by biochemical and physiological tests. Our results have revealed that the Photorhabdus strains isolated from H. megidis belong to P. temperata, subsp. temperata and subsp. cinerea. Isolates from H. bacteriophora represent P. luminescens subs. kayaii and P. temperata subs. cinerea. This study for the first time provides evidence for H. bacteriophora and P. temperata subsp. cinerea symbiotic association. In addition, we tested whether the microsymbionts of the Polish H. bacteriophora and H. megidis isolates support the development of non-native nematode host population and colonization of their infective juveniles. It has been shown that the studied Photorhabdus strains can readily swap their nematode host, both at intra- and interspecies level. It supports the hypothesis of different symbiotic associations in the Heterorhabditis-Photorhabdus lineage.

Photorhabdusluminescens subsp. namnaonensis subsp. nov., isolated from Heterorhabditisbaujardi nematodes.

A lightly yellowish-pigmented, oxidase-negative bacterial strain (PB45.5T) isolated from the Nam Nao district of Phetchabun in central Thailand was investigated to determine its taxonomic position. Cells of the isolate showed a rod shaped appearance. The strain stained Gram-negative. Strain PB45.5T shared highest 16S rRNA gene sequence similarity with the type strains of Photorhabdus luminescens subsp. akhurstii (99.2 %) and Photorhabdus luminescens subsp. hainanensis (99.1 %) and lower similarities to all other Photorhabdus luminescens subspecies (<98.0 %). Multilocus sequence analysis (MLSA) based on concatenated partial recA, dnaN, gltX, gyrB and infB gene sequences confirmed the affiliation obtained by 16S rRNA gene sequence analysis but showed a clear distinction of PB45.5T from the closest related type strains. Strain PB45.5T shared only 96.9 % sequence similarity (concatenated nucleotide sequences) with P. luminescens subsp. akhurstii FRG04T and 96.8 % with P. luminescens subsp. hainanensis C8404T. The fatty acid profile of the strain 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. The MLSA results and the differential biochemical and chemotaxonomic properties showed that strain PB45.5T represents a novel P. luminescens subspecies, for which the name Photorhabdus luminescens subsp. namnaonensis subsp. nov. (type strain PB45.5T=LMG 29915T=CCM 8729T) is proposed.

First Report of the Isolation of the Symbiotic Bacterium Photorhabdus luminescens subsp. laumondii Associated with Heterorhabditis safricana from South Africa.

Photorhabdus luminescens subsp. laumondii is closely associated with the entomopathogenic nematode Heterorhabditis bacteriophora and has, to date, not been isolated from other nematode species. This study is the first report of P. luminescens subsp. laumondii from two South African isolates of entomopathogenic nematodes, Heterorhabditis safricana SF281 and H. bacteriophora SF351. Both symbiotic bacterial strains are phenotypically closely related to P. luminescens subsp. laumondii previously isolated and described from H. bacteriophora. The genetic relatedness between P. luminescens subsp. laumondii strains SF281B and SF351B was confirmed by comparing 16S rDNA, recA, gyrB and gltX sequences with sequences of P. luminescens subsp. laumondii, including the type strain (TT01T) and strain E21.

LuxR solos in Photorhabdus species.

Bacteria communicate via small diffusible molecules to mediate group-coordinated behavior, a process designated as quorum sensing. The basic molecular quorum sensing system of Gram-negative bacteria consists of a LuxI-type autoinducer synthase producing acyl-homoserine lactones (AHLs) as signaling molecules, and a LuxR-type receptor detecting the AHLs to control expression of specific genes. However, many proteobacteria possess one or more unpaired LuxR-type receptors that lack a cognate LuxI-like synthase, referred to as LuxR solos. The enteric and insect pathogenic bacteria of the genus Photorhabdus harbor an extraordinarily high number of LuxR solos, more than any other known bacteria, and all lack a LuxI-like synthase. Here, we focus on the presence and the different types of LuxR solos in the three known Photorhabdus species using bioinformatics analyses. Generally, the N-terminal signal-binding domain (SBD) of LuxR-type receptors sensing AHLs have a motif of six conserved amino acids that is important for binding and specificity of the signaling molecule. However, this motif is altered in the majority of the Photorhabdus-specific LuxR solos, suggesting the use of other signaling molecules than AHLs. Furthermore, all Photorhabdus species contain at least one LuxR solo with an intact AHL-binding motif, which might allow the ability to sense AHLs of other bacteria. Moreover, all three species have high AHL-degrading activity caused by the presence of different AHL-lactonases and AHL-acylases, revealing a high quorum quenching activity against other bacteria. However, the majority of the other LuxR solos in Photorhabdus have a N-terminal so-called PAS4-domain instead of an AHL-binding domain, containing different amino acid motifs than the AHL-sensors, which potentially allows the recognition of a highly variable range of signaling molecules that can be sensed apart from AHLs. These PAS4-LuxR solos are proposed to be involved in host sensing, and therefore in inter-kingdom signaling. Overall, Photorhabdus species are perfect model organisms to study bacterial communication via LuxR solos and their role for a symbiotic and pathogenic life style.

Ail and PagC-related proteins in the entomopathogenic bacteria of Photorhabdus genus.

Among pathogenic Enterobacteriaceae, the proteins of the Ail/OmpX/PagC family form a steadily growing family of outer membrane proteins with diverse biological properties, potentially involved in virulence such as human serum resistance, adhesion and entry into eukaryotic culture cells. We studied the proteins Ail/OmpX/PagC in the bacterial Photorhabdus genus. The Photorhabdus bacteria form symbiotic complexes with nematodes of Heterorhabditis species, associations which are pathogenic to insect larvae. Our phylogenetic analysis indicated that in Photorhabdus asymbiotica and Photorhabdus luminescens only Ail and PagC proteins are encoded. The genomic analysis revealed that the Photorhabdus ail and pagC genes were present in a unique copy, except two ail paralogs from P. luminescens. These genes, referred to as ail1Pl and ail2Pl, probably resulted from a recent tandem duplication. Surprisingly, only ail1Pl expression was directly controlled by PhoPQ and low external Mg2+ conditions. In P. luminescens, the magnesium-sensing two-component regulatory system PhoPQ regulates the outer membrane barrier and is required for pathogenicity against insects. In order to characterize Ail functions in Photorhabdus, we showed that only ail2Pl and pagCPl had the ability, when expressed into Escherichia coli, to confer resistance to complement in human serum. However no effect in resistance to antimicrobial peptides was found. Thus, the role of Ail and PagC proteins in Photorhabdus life cycle is discussed.

Photorhabdus heterorhabditis sp. nov., a symbiont of the entomopathogenic nematode Heterorhabditis zealandica.

The bacterial symbionts SF41T and SF783 were isolated from populations of the insect pathogenic nematode Heterorhabditis zealandica collected in South Africa. Both strains were closely related to strain Q614 isolated from a population of Heterorhabditis sp. collected from soil in Australia in the 1980s. Sequence analysis based on a multigene approach, DNA-DNA hybridization data and phenotypic traits showed that strains SF41T, SF783 and Q614 belong to the same species of the genus Photorhabdus with Photorhabdus temperata subsp. cinerea as the most closely related taxon (DNA-DNA hybridization value of 68%). Moreover, the phylogenetic position of Photorhabdus temperata subsp. cinerea DSM 19724T initially determined using the gyrB sequences, was reconsidered in the light of the data obtained by our multigene approach and DNA-DNA hybridization experiments. Strains SF41T, SF783 and Q614 represent a novel species of the genus Photorhabdus, for which the name Photorhabdus heterorhabditis sp. nov. is proposed (type strain SF41T=ATCC BAA-2479T=DSM 25263T).

An unbiased method for clustering bacterial effectors using host cellular phenotypes.

We present a novel method implementing unbiased high-content morphometric cell analysis to classify bacterial effector phenotypes. This clustering methodology represents a significant advance over more qualitative visual approaches and can also be used to classify, and therefore predict the likely function of, unknown effector genes from any microbial genome. As a proof of concept, we use this approach to investigate 23 genetic regions predicted to encode antimacrophage effectors located across the genome of the insect and human pathogen Photorhabdus asymbiotica. Statistical cluster analysis using multiple cellular measures categorized treated macrophage phenotypes into three major groups relating to their putative functionality: (i) adhesins, (ii) cytolethal toxins, and (iii) cytomodulating toxins. Further investigation into their effects on phagocytosis revealed that several effectors also modulate this function and that the nature of this modulation (increased or decreased phagocytosis) is linked to the phenotype cluster group. Categorizing potential functionalities in this way allows rapid functional follow-up of key candidates for more-directed cell biological or biochemical investigation. Such an unbiased approach to the classification of candidate effectors will be useful for describing virulence-related regions in a wide range of genomes and will be useful in assigning putative functions to the growing number of microbial genes whose function remains unclear from homology searching.