Draft Genome Assembly of the Entomopathogenic Bacterium Photorhabdus luminescens subsp. sonorensis Caborca.

Photorhabdus luminescens subsp. sonorensis strain Caborca is an entomopathogenic bacterium with a dual lifestyle, namely, as a mutualist of the Heterorhabditis sonorensis nematode and a pathogen to a wide range of insect species. The genome assembly, in 231 contigs, is 5.2 Mbp long and includes 25 putative gene clusters for secondary metabolism.

Secondary Metabolites Produced by Heterorhabditis Symbionts and Their Application in Agriculture: What We Know and What to Do Next.

Gram-negative Photorhabdus bacteria have a dual lifestyle: they are mutualists of Heterorhabditis nematodes and are pathogens of insects. Together, this nematode-bacterium partnership has been used to successfully control a wide range of agricultural insect pests. Photorhabdus produce a diverse array of small molecules that play key biological roles in regulating their dual roles. In particular, several secondary metabolites (SM) produced by this bacterium are known to play a critical role in the maintenance of a monoxenic infection in the insect host and are also known to prevent contamination of the cadaver from soil microbes and/or predation by arthropods. A few of the SM this bacteria produce have been isolated and identified, and their biological activities have also been tested in laboratory assays. Over the past two decades, analyses of the genomes of several Photorhabdus spp. have revealed the presence of SM numerous gene clusters that comprise more than 6% of these bacteria genomes. Furthermore, genome mining and characterization of biosynthetic pathways, have uncovered the richness of these compounds, which are predicted to vary across different Photorhabdus spp. and strains. Although progress has been made in the identification and function of SM genes and gene clusters, the targeted testing for the bioactivity of molecules has been scarce or mostly focused on medical applications. In this review, we summarize the current knowledge of Photorhabdus SM, emphasizing on their activity against plant pathogens and parasites. We further discuss their potential in the management of agricultural pests and the steps that need to be taken for the implementation of Photorhabdus SM in pest management.

Bioprospecting for secondary metabolites in the entomopathogenic bacterium Photorhabdus luminescens subsp. sonorensis.

Crude extracts of in vitro and in vivo cultures of two strains of Photorhabdus l. sonorensis (Enterobacteriaceae) were analyzed by TLC, HPLC-UV and LC-MS. Nine unique compounds with mass/charge ratios (m/z) ranging from 331.3 to 713.5 were found in MS analyses. Bioactivity of extracts was assessed on a selection of plant pathogens/pests and non-target species. Caborca strain extracts showed the highest activity against Helicoverpa zea (Lepidoptera: Noctuidae) neonates at all concentrations tested. Mortality ranged from 11% (at 10µg/ml) to 37% (at 40µg/ml). Strain CH35 extracts showed the highest nematicidal activity on Meloidogyne incognita (Tylenchida: Meloidogynidae) at 40µg/ml. Low to no nematicidal activity was observed against the non-target species Steinernema carpocapsae (Rhabditida: Steinernematidae) and Caenorhabditis elegans (Rhabditida: Rhabditidae). Caborca extracts exhibited a strong antibiotic effect on Pseudomonas syringae (Pseudomonadales: Pseudomonadacedae) at 40µg/ml, while both Caborca and CH35 extracts inhibited the growth of Bacillus subitillis (Bacillales: Bacillaceae) at 40µg/ml. All extracts strongly inhibited the growth of the fungus Fusarium oxysporum (Hypocreales: Nectriceae) but not that of Alternaria alternata (Pleosporales: Pleosporaceae). Contrastingly, a moderate to high inhibitory effect was denoted on the non-target biocontrol fungus Beauveria bassiana (Hypocreales: Clavivipitaceae).

Soil sampling and isolation of entomopathogenic nematodes (Steinernematidae, Heterorhabditidae).

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to two families: Steinernematidae and Heterorhabditidae. Until now, more than 70 species have been described in the Steinernematidae and there are about 20 species in the Heterorhabditidae. The nematodes have a mutualistic partnership with Enterobacteriaceae bacteria and together they act as a potent insecticidal complex that kills a wide range of insect species. Herein, we focus on the most common techniques considered for collecting EPN from soil. The second part of this presentation focuses on the insect-baiting technique, a widely used approach for the isolation of EPN from soil samples, and the modified White trap technique which is used for the recovery of these nematodes from infected insects. These methods and techniques are key steps for the successful establishment of EPN cultures in the laboratory and also form the basis for other bioassays that consider these nematodes as model organisms for research in other biological disciplines. The techniques shown in this presentation correspond to those performed and/or designed by members of S. P. Stock laboratory as well as those described by various authors.

Characterization and phylogenetic relationships of Photorhabdus luminescens subsp. sonorensis (γ-Proteobacteria: Enterobacteriaceae), the bacterial symbiont of the entomopathogenic nematode Heterorhabditis sonorensis (Nematoda: Heterorhabditidae).

Photorhabdus are motile Gram-negative bacteria that have a mutualistic association with Heterorhabditis nematodes (Heterorhabditidae). These bacteria possess peculiar biochemical characteristics such as inability to reduce nitrates, and the capacity to ferment only a limited number of carbohydrates. Heterorhabditis nematodes vector the bacteria from one insect host to another and also provide shelter to the bacteria from soil stressors and antagonists. Once inside the insect host, the bacterial symbionts are released and produce toxins and secondary metabolites and broad-spectrum antibiotics, which kill the host by septicemia within 48 h. At present, three Photorhabdus spp. have been identified: P. luminescens, P. temperata, and P. asymbiotica, and many subspecies have also been described. Characterization of new species and subspecies has been based on sequence data, mostly of the 16S rDNA, and also of a selection of protein coding genes. In addition to this, phenotypic traits including temperature growth, colony morphology, color, light production, carbohydrate response, and assimilation, among others, have been considered. In this study, we characterize the bacterial symbiont of Heterorbabditis sonorensis, a recently discovered entomopathogenic nematode species form the Sonoran desert in Arizona, USA. A selection of classic biochemical and molecular methods including sequence data of six genes: 16s rDNA, and four protein coding genes: gyrB, recA, gltX, and dnaN were considered. Evolutionary relationships of this new Photorhabdus subsp. were inferred considering maximum parsimony and Bayesian analyses.

Biosynthesis of the cyclooligomer depsipeptide bassianolide, an insecticidal virulence factor of Beauveria bassiana.

Beauveria bassiana is a facultative entomopathogen with an extremely broad host range that is used as a commercial biopesticide for the control of insects of agricultural, veterinary and medical significance. B. bassiana produces bassianolide, a cyclooligomer depsipeptide secondary metabolite. We have cloned the bbBsls gene of B. bassiana encoding a nonribosomal peptide synthetase (NRPS). Targeted inactivation of the B. bassiana genomic copy of bbBsls abolished bassianolide production, but did not affect the biosynthesis of beauvericin, another cyclodepsipeptide produced by the strain. Comparative sequence analysis of the BbBSLS bassianolide synthetase revealed enzymatic domains for the iterative synthesis of an enzyme-bound dipeptidol monomer intermediate from d-2-hydroxyisovalerate and l-leucine. Further BbBSLS domains are predicted to catalyze the formation of the cyclic tetrameric ester bassianolide by recursive condensations of this monomer. Comparative infection assays against three selected insect hosts established bassianolide as a highly significant virulence factor of B. bassiana.

Biosynthesis of the cyclooligomer depsipeptide beauvericin, a virulence factor of the entomopathogenic fungus Beauveria bassiana.

Beauvericin, a cyclohexadepsipeptide ionophore from the entomopathogen Beauveria bassiana, shows antibiotic, antifungal, insecticidal, and cancer cell antiproliferative and antihaptotactic (cell motility inhibitory) activity in vitro. The bbBeas gene encoding the BbBEAS nonribosomal peptide synthetase was isolated from B. bassiana and confirmed to be responsible for beauvericin biosynthesis by targeted disruption. BbBEAS utilizes D-2-hydroxyisovalerate (D-Hiv) and L-phenylalanine (Phe) for the iterative synthesis of a predicted N-methyl-dipeptidol intermediate, and forms the cyclic trimeric ester beauvericin from this intermediate in an unusual recursive process. Heterologous expression of the bbBeas gene in Escherichia coli to produce the 3189 amino acid, 351.9 kDa BbBEAS enzyme provided a strain proficient in beauvericin biosynthesis. Comparative infection assays with a BbBEAS knockout B. bassiana strain against three insect hosts revealed that beauvericin plays a highly significant but not indispensable role in virulence.