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

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

Specificity and putative mode of action of a mosquito larvicidal toxin from the bacterium Xenorhabdus innexi.

Reduction of mosquito-borne diseases relies, in part, on the use of synthetic pesticides to control pest mosquitoes. This reliance has led to genetic resistance, environmental contamination and the nondiscriminatory elimination of both pest and non-pest species. To expand our options for control, we screened entomopathogenic bacteria for potential larvicidal activity. A lipopeptide from the bacterium, Xenorhabdus innexi, was discovered that displayed potent larvicidal activity. The LC50s of the lipopeptide towards Aedes aegypti, Culex pipiens and Anopheles gambiae larvae were 1.81, 1.25 and 1.86 parts-per-million, respectively. No mortality was observed in other insect species tested. The putative mode of action of the lipopeptide suggested that after orally ingestion, it bound to the apical membrane of anterior midgut cells and created pores in the cellular membranes. The rapid neutralization of midgut pH suggested the pores disabled the H+-V-ATPase on the basal membrane and led to epithelial cell death. Specificity and toxicity towards mosquito larvae and the unique mode of action makes this lipopeptide a potentially attractive bacterial insecticide for control of mosquitoes.

Photobactin: a catechol siderophore produced by Photorhabdus luminescens, an entomopathogen mutually associated with Heterorhabditis bacteriophora NC1 nematodes.

The nematode Heterorhabditis bacteriophora transmits a monoculture of Photorhabdus luminescens bacteria to insect hosts, where it requires the bacteria for efficient insect pathogenicity and as a substrate for growth and reproduction. Siderophore production was implicated as being involved in the symbiosis because an ngrA mutant inadequate for supporting nematode growth and reproduction was also deficient in producing siderophore activity and ngrA is homologous to a siderophore biosynthetic gene, entD. The role of the siderophore in the symbiosis with the nematode was determined by isolating and characterizing a mini-Tn5-induced mutant, NS414, producing no detectable siderophore activity. This mutant, being defective for growth in iron-depleted medium, was normal in supporting nematode growth and reproduction, in transmission by the dauer juvenile nematode, and in insect pathogenicity. The mini-Tn5 transposon was inserted into phbH; whose protein product is a putative peptidyl carrier protein homologous to the nonribosomal peptide synthetase VibF of Vibrio cholerae. Other putative siderophore biosynthetic and transport genes flanking phbH were characterized. The catecholate siderophore was purified, its structure was determined to be 2-(2,3-dihydroxyphenyl)-5-methyl-4,5-dihydro-oxazole-4-carboxylic acid [4-(2,3-dihydroxybenzoylamino)-butyl]-amide, and it was given the generic name photobactin. Antibiotic activity was detected with purified photobactin, indicating that the siderophore may contribute to antibiosis of the insect cadaver. These results eliminate the lack of siderophore activity as the cause for the inadequacy of the ngrA mutant in supporting nematode growth and reproduction.

For the insect pathogen Photorhabdus luminescens, which end of a nematode is out?

The nematode Heterorhabditis bacteriophora is the vector for transmitting the entomopathogenic bacterium Photorhabdus luminescens between insect larvae. The dauer juvenile (DJ) stage nematode selectively retains P. luminescens in its intestine until it releases the bacteria into the hemocoel of an insect host. We report the results of studying the transmission of the bacteria by its nematode vector. Cells of P. luminescens labeled with green fluorescent protein preferentially colonized a region of the DJ intestine immediately behind the basal bulb, extending for various distances toward the anus. Incubation of DJ nematodes in vitro in insect hemolymph induced regurgitation of the bacteria. Following a 30-min lag, the bacteria migrated in a gradual and staggered movement toward and ultimately exited the mouth. This regurgitation reaction was induced by a low-molecular-weight, heat- and protease-stable, anionic component present in arthropod hemolymph and in supernatants from insect cell cultures. Nematodes anesthetized with levamisole or treated with the antihelmenthic agent ivermectin did not release their bacteria into hemolymph. The ability to visualize P. luminescens in the DJ nematode intestine provides the first clues to the mechanism of release of the bacteria during infection of insect larvae. This and the partial characterization of a component of hemolymph triggering release of the bacteria render this fascinating example of both a mutualistic symbiosis and disease transmission amenable to future genetic and molecular study.

Expression analysis of the ssgA gene product, associated with sporulation and cell division in Streptomyces griseus.

The ssgA gene of Streptomyces griseus B2682, when present in high copy number, results in both suppression of sporulation and fragmented growth of mycelia. Western analysis with polyclonal antibodies against the gene product (SsgA) revealed a close correlation between SsgA accumulation and the onset of sporulation in wild-type cells. The protein was only detected in the cytoplasm. Certain developmental mutants of S. griseus (afs, reIC and brgA) which are defective in aerial mycelium formation in solid culture and submerged spore formation in liquid culture failed to accumulate SsgA. The SsgA protein appeared shortly (1 h) after nutritional shift-down of strain B2682 cells. afs mutant cells sporulated and expressed SsgA only when A-factor was present both before and after nutritional shift-down. Introduction of the ssgA gene in a low-copy-number vector into strain B2682 resulted in fivefold overexpression of SsgA, and was accompanied by fragmented growth of mycelia and suppression of submerged spore formation (in liquid culture) and aerial mycelium formation (in solid culture). Streptomycin production was not inhibited. In a control experiment, a nonfunctional ssgA gene possessing a frameshift mutation near its N-terminus had no effect on either growth or sporulation. It is proposed that the ssgA gene product plays a role in promoting the developmental process of S. griseus.