Project description:Dam, the most described bacterial DNA-methyltransferase, is widespread in gamma-proteobacteria. Dam DNA methylation can play a role in various genes expression and is involved in pathogenicity of several bacterial species. In the entomopathogenic bacterium Photorhabdus luminescens, a dam ortholog was identified. Overexpression of dam in P. luminescens did not impair growth ability in vitro. In contrast, compared to a control strain harboring an empty plasmid, a significant decrease in motility was observed in the dam-overexpressing strain. In addition, the dam-overexpressing P. luminescens strain showed a delayed virulence compared to that of the control strain after injection in larvae of the lepidopteran Spodoptera littoralis. These results reveal that Dam plays a major role during P. luminescens insect infection.
Project description:Photorhabdus luminescens is a bioluminescent entomopathogenic bacterium that undergoes phenotypic variation and lives in mutualistic association with nematodes of the family Heterorhabditidae. The pair infects and kills insects, and during their coordinated lifecycle, the bacteria produce an assortment of specialized metabolites to regulate its mutualistic and pathogenic roles. As part of our search for new specialized metabolites from the Photorhabdus genus, we examined organic extracts from P. luminescens grown in an amino-acid-rich medium based on the free amino-acid levels found in the circulatory fluid of its common insect prey, the Galleria mellonella larva. Reversed-phase HPLC/UV/MS-guided fractionation of the culture extracts led to the identification of two new pyrazinone metabolites, lumizinones A (1) and B (2), together with two N-acetyl dipeptides (3 and 4). The lumizinones were produced only in the phenotypic variant associated with nematode development and insect pathogenesis. Their chemical structures were elucidated by analysis of 1D and 2D NMR and high-resolution ESI-QTOF-MS spectral data. The absolute configurations of the amino acids in 3 and 4 were determined by Marfey's analysis. Compounds 1-4 were evaluated for their calpain protease inhibitory activity, and lumizinone A (1) showed inhibition with an IC50 (half-maximal inhibitory concentration) value of 3.9??m.
Project description:Photorhabdus luminescens is a nematode-symbiotic, gram negative, bioluminescent bacterium, belonging to the family of Enterobacteriaceae. Recent studies show the importance of this bacterium as an alternative source of insecticides, as well as an emerging human pathogen. Various toxins have been identified and characterized in this bacterium. These toxins are classified into four major groups: the toxin complexes (Tcs), the Photorhabdus insect related (Pir) proteins, the "makes caterpillars floppy" (Mcf) toxins and the Photorhabdus virulence cassettes (PVC); the mechanisms however of toxin secretion are not fully elucidated. Using bioinformatics analysis and comparison against the components of known secretion systems, multiple copies of components of all known secretion systems, except the ones composing a type IV secretion system, were identified throughout the entire genome of the bacterium. This indicates that Photorhabdus luminescens has all the necessary means for the secretion of virulence factors, thus it is capable of establishing a microbial infection.
Project description:BackgroundPhotorhabdus luminescens is an enteric bacterium, which lives in mutualistic association with soil nematodes and is highly pathogenic for a broad spectrum of insects. A complete genome sequence for the type strain P. luminescens subsp. laumondii TT01, which was originally isolated in Trinidad and Tobago, has been described earlier. Subsequently, a rifampicin resistant P. luminescens strain has been generated with superior possibilities for experimental characterization. This strain, which is widely used in research, was described as a spontaneous rifampicin resistant mutant of TT01 and is known as TT01-RifR.ResultsUnexpectedly, upon phenotypic comparison between the rifampicin resistant strain and its presumed parent TT01, major differences were found with respect to bioluminescence, pigmentation, biofilm formation, haemolysis as well as growth. Therefore, we renamed the strain TT01-RifR to DJC. To unravel the genomic basis of the observed differences, we generated a complete genome sequence for strain DJC using the PacBio long read technology. As strain DJC was supposed to be a spontaneous mutant, only few sequence differences were expected. In order to distinguish these from potential sequencing errors in the published TT01 genome, we re-sequenced a derivative of strain TT01 in parallel, also using the PacBio technology. The two TT01 genomes differed at only 30 positions. In contrast, the genome of strain DJC varied extensively from TT01, showing 13,000 point mutations, 330 frameshifts, and 220 strain-specific regions with a total length of more than 300 kb in each of the compared genomes.ConclusionsAccording to the major phenotypic and genotypic differences, the rifampicin resistant P. luminescens strain, now named strain DJC, has to be considered as an independent isolate rather than a derivative of strain TT01. Strains TT01 and DJC both belong to P. luminescens subsp. laumondii.
Project description:A phoP mutant of the entomopathogenic bacterium P. luminescens is attenuated in virulence in insects and susceptible to antimicrobial peptides such as Polymyxin B. The first goal of this study is to compare transcriptomes of the phoP mutant and wild type strain to identify the PhoP regulon. (i) We first compared both strains grown in LB medium. (ii) As we know that low Mg conditions induce expression of PhoP-dependent genes, we also compared transcriptomes of the phoP and wild-type strain after growth in M9 low Mg and in M9 High Mg. To decipher the polymyxin B regulon, comparisons of transcriptomes of the wild type strain grown in LB medium with or without addition of polymyxin B were also performed
Project description:BACKGROUND:The Gram-negative bacterium Photorhabdus asymbiotica (Pa) has been recovered from human infections in both North America and Australia. Recently, Pa has been shown to have a nematode vector that can also infect insects, like its sister species the insect pathogen P. luminescens (Pl). To understand the relationship between pathogenicity to insects and humans in Photorhabdus we have sequenced the complete genome of Pa strain ATCC43949 from North America. This strain (formerly referred to as Xenorhabdus luminescens strain 2) was isolated in 1977 from the blood of an 80 year old female patient with endocarditis, in Maryland, USA. Here we compare the complete genome of Pa ATCC43949 with that of the previously sequenced insect pathogen P. luminescens strain TT01 which was isolated from its entomopathogenic nematode vector collected from soil in Trinidad and Tobago. RESULTS:We found that the human pathogen Pa had a smaller genome (5,064,808 bp) than that of the insect pathogen Pl (5,688,987 bp) but that each pathogen carries approximately one megabase of DNA that is unique to each strain. The reduced size of the Pa genome is associated with a smaller diversity in insecticidal genes such as those encoding the Toxin complexes (Tc's), Makes caterpillars floppy (Mcf) toxins and the Photorhabdus Virulence Cassettes (PVCs). The Pa genome, however, also shows the addition of a plasmid related to pMT1 from Yersinia pestis and several novel pathogenicity islands including a novel Type Three Secretion System (TTSS) encoding island. Together these data suggest that Pa may show virulence against man via the acquisition of the pMT1-like plasmid and specific effectors, such as SopB, that promote its persistence inside human macrophages. Interestingly the loss of insecticidal genes in Pa is not reflected by a loss of pathogenicity towards insects. CONCLUSION:Our results suggest that North American isolates of Pa have acquired virulence against man via the acquisition of a plasmid and specific virulence factors with similarity to those shown to play roles in pathogenicity against humans in other bacteria.
Project description: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.
Project description:Lumiquinone A (1), an unusual aminobenzoquinone member within the phenylpropanoid class of natural products, together with the known compound 3,5-dihydroxy-4-isopropyl-trans-stilbene (2), was isolated from the entomopathogenic bacterium Photorhabdus luminescens TT01. On the basis of the analysis of extensive 2D NMR and high-resolution ESI-QTOF-MS spectral data, the structure of 1 was determined to be a 2-amino-5-hydroxy-1,4-benzoquinone substituted with (E)-2-phenylvinyl and isopropyl functional groups. Free ?-aminomalonate medium supplementation significantly enhanced production of 1 relative to 2 in a dose-dependent manner, suggesting that promiscuous polyketide synthase processing of malonate- versus ?-aminomalonate-derived substrates represents a competitive route for polyketide structural diversification. Metabolites 1 and 2 were active against Bacillus subtilis and Saccharomyces cerevisiae.
Project description:Dam, the most described bacterial DNA-methyltransferase, is widespread in gamma-proteobacteria. Dam DNA methylation can play a role in various genes expression and is involved in pathogenicity of several bacterial species. The purpose of this study was to determine the role played by the dam ortholog identified in the entomopathogenic bacterium Photorhabdus luminescens. Complementation assays of an Escherichia coli dam mutant showed the restoration of the DNA methylation state of the parental strain. Overexpression of dam in P. luminescens did not impair growth ability in vitro. In contrast, compared to a control strain harboring an empty plasmid, a significant decrease in motility was observed in the dam-overexpressing strain. A transcriptome analysis revealed the differential expression of 208 genes between the two strains. In particular, the downregulation of flagellar genes was observed in the dam-overexpressing strain. In the closely related bacterium Xenorhabdus nematophila, dam overexpression also impaired motility. In addition, the dam-overexpressing P. luminescens strain showed a delayed virulence compared to that of the control strain after injection in larvae of the lepidopteran Spodoptera littoralis. These results reveal that Dam plays a major role during P. luminescens insect infection.
Project description:Bacterially produced small molecules demonstrate a remarkable range of structural and functional diversity and include some of our most useful biological probes and therapeutic agents. Annotations of bacterial genomes reveal a large gap between the number of known small molecules and the number of biosynthetic genes/loci that could produce such small molecules, a gap that most likely originates from tight regulatory control by the producing organism. This study coupled a global transcriptional regulator, HexA, to secondary metabolite production in Photorhabdus luminescens, a member of the Gammaproteobacteria that participates in a complex symbiosis with nematode worms and insect larvae. HexA is a LysR-type transcriptional repressor, and knocking it out to create a P. luminescens DeltahexA mutant led to dramatic upregulation of biosynthesized small molecules. Use of this mutant expanded a family of stilbene-derived small molecules, which were known to play important roles in the symbiosis, from three members to at least nine members.