Photorhabdus luminescens is an enterobacterium that is symbiotic with soil entomopathogenic nematodes and pathogenic to a wide range of insects. P. luminescens promotes its own transmission among susceptible insect populations using its nematode host as vector 1 . Its life cycle comprises a symbiotic stage in the nematode's gut and a virulent stage in the insect larvae, which it kills through toxemia and septicemia. After the nematode attacks a prey insect and P. luminescens is released, the bacterium produces a wide variety of virulence factors ensuring rapid insect killing. Bioconversion of the insect cadaver by exoenzymes produced by the bacteria allows the bacteria to multiply and the nematode to reproduce. During this process P. luminescens produces antibiotics to prevent invasion of the insect cadaver by bacterial or fungal competitors. Finally, elimination of competitors allows P. luminescens and the nematode to reassociate specifically before leaving the insect cadaver 2,3 .To better understand this complex life style, we determined the genome sequence of P. luminescens subspecies laumondii strain TT01 4 , a symbiont of the nematode Heterorhabditis bacteriophora isolated on Trinidad and Tobago. RESULTS General featuresStrain TT01 possesses a single circular chromosome of 5,688,987 bp with an average GC content of 42.8%. No plasmid replicon was found.A total of 4,839 protein-coding genes, including 157 pseudogenes, seven complete sets (23S, 5S and 16S) of ribosomal RNA operons and 85 tRNA genes, were predicted ( Fig. 1; Supplementary Table 1 online). Toxins against insectsMore toxin genes were predicted in the P. luminescens genome than in any other bacterial genome sequenced yet. A large number of these toxins may be involved in the killing of a wide variety of insects. Some may act synergistically or use redundancy for 'overkill' 5 , ensuring a quick death of the host. In addition, some may kill insects by interfering with their development. In the TT01 genome, two paralogs, plu4092 and plu4436, encode proteins similar to juvenile hormone esterases (JHEs) of the insect Leptinotarsa decemlineata 6 . Juvenile hormone maintains the insect in a larval state. Its inactivation by JHE allows metamorphosis to proceed. JHEs may be used to trigger the insect endocrine machinery at an inappropriate time and thus represents a promising approach for insect control 7 . These genes are located downstream of highly related orphan genes (plu4093 and plu4437), suggesting a locus duplication.The toxicity of the proteins encoded by these two loci was verified experimentally. Two Escherichia coli clones, containing the recombinant BAC1A02 and BAC8C11, were shown to be toxic toward insects. BAC1A02, which contains the locus plu4093-plu4092, exhibited substantial oral toxicity toward three mosquito species, Aedes aegypti,
The luminescent entomopathogenic bacterium Photorhabdus luminescens produces several yet-uncharacterized broad-spectrum antibiotics. We report the identification and characterization of a cluster of eight genes (named cpmA to cpmH) responsible for the production of a carbapenem-like antibiotic in strain TT01 of P. luminescens. The cpm cluster differs in several crucial aspects from other car operons. The level of cpm mRNA peaks during exponential phase and is regulated by a Rap/Hor homolog identified in the P. luminescens genome. Marker-exchange mutagenesis of this gene in the entomopathogen decreased antibiotic production. The luxS-like signaling mechanism of quorum sensing also plays a role in the regulation of the cpm operon. Indeed, luxS, which is involved in the production of a newly identified autoinducer, is responsible for repression of cpm gene expression at the end of the exponential growth phase. The importance of this carbapenem production in the ecology of P. luminescens is discussed.Photorhabdus luminescens, a gram-negative luminescent gamma-proteobacterium, forms an entomopathogenic symbiosis with a soil nematode of the genus Heterorhabditis. This bacterium undergoes a complex life cycle that involves a symbiotic stage, in which bacteria colonize the intestine of the nematodes, and a pathogenic stage, in which susceptible insects are killed by the combined action of the nematode and the bacteria. After entering the insect host, the nematodes expel their bacterial symbionts into the insect hemocoel. Once released, the bacteria proliferate, kill the insect within 24 h to 48 h, and enhance conditions for nematode reproduction by providing nutrients and other growth factors. Remarkably, the insect carcass does not putrefy over a period of several weeks, because P. luminescens produces several broad-spectrum antibiotics that prevent contamination of the insect cadaver by other microorganisms. These products are believed to be secreted into the insect hemolymph when the bacteria enter stationary-phase conditions. Two P. luminescens antibiotic molecules have now been identified. They consist of a red pigment with antimicrobial activity, identified as an anthraquinone derivative, and a hydroxystilbene derivative, 1,3-dihydroxy-4-isopropylstilbene, which showed strong fungicidal activity in previous studies (20,30).Carbapenems are a class of -lactam antibiotics synthesized by a biochemical route distinct from that found in the biosynthetic pathways of the sulfur-ring -lactams, namely, penicillins, cephamycins, and cephalosporins (5, 43). As naturally produced metabolites, they were originally isolated in the late 1970s from Streptomyces species. The first carbapenem antibiotic discovered was thienamycin, produced in small quantities
Photorhabdus luminescens is a symbiont of entomopathogenic nematodes. Analysis of the genome sequence of this organism revealed a homologue of PhoP-PhoQ, a two-component system associated with virulence in intracellular bacterial pathogens. This organism was shown to respond to the availability of environmental magnesium. A mutant with a knockout mutation in the regulatory component of this system (phoP) had no obvious growth defect. It was, however, more motile and more sensitive to antimicrobial peptides than its wild-type parent. Remarkably, the mutation eliminated virulence in an insect model. No insect mortality was observed after injection of a large number of the phoP bacteria, while very small amounts of parental cells killed insect larvae in less than 48 h. At the molecular level, the PhoPQ system mediated Mg 2؉ -dependent modifications in lipopolysaccharides and controlled a locus (pbgPE) required for incorporation of 4-aminoarabinose into lipid A. Mg 2؉ -regulated gene expression of pbgP1 was absent in the mutant and was restored when phoPQ was complemented in trans. This finding highlights the essential role played by PhoPQ in the virulence of an entomopathogen.
Bacterial virulence is an integrative process that may involve quorum sensing. In this work, we compared by global expression profiling the wild-type entomopathogenic Photorhabdus luminescens subsp. laumondii TT01 to a luxS-deficient mutant unable to synthesize the type 2 quorum-sensing inducer AI-2. AI-2 was shown to regulate more than 300 targets involved in most compartments and metabolic pathways of the cell. AI-2 is located high in the hierarchy, as it controls the expression of several transcriptional regulators. The regulatory effect of AI-2 appeared to be dose dependent. The luxS-deficient strain exhibited decreased biofilm formation and increased type IV/V pilus-dependent twitching motility. AI-2 activated its own synthesis and transport. It also modulated bioluminescence by regulating the synthesis of spermidine. AI-2 was further shown to increase oxidative stress resistance, which is necessary to overcome part of the innate immune response of the host insect involving reactive oxygen species. Finally, we showed that the luxS-deficient strain had attenuated virulence against the lepidopteran Spodoptera littoralis. We concluded that AI-2 is involved mainly in early steps of insect invasion in P. luminescens.
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