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.

Extracellular polysaccharide (EPS) is produced by diverse bacterial pathogens and fulfills assorted roles, including providing a structural matrix for biofilm formation and more specific functions in virulence, such as protection against immune defenses. We report here the first investigation of some of the genes important for biofilm formation inPhotorhabdus luminescensand demonstrate the key role of the phosphomannose isomerase gene,manA, in the structure of functional EPS. Phenotypic analyses of amanA-deficient mutant showed the importance of EPS in motility, insect virulence, and biofilm formation on abiotic surfaces as well as the requirement of this gene for the use of mannose as the sole carbon source. Conversely, this defect had no apparent impact on symbiosis with the heterorhabditid nematode vector. A more detailed analysis of biofilm formation revealed that themanAmutant was able to attach to surfaces with the same efficiency as that of the wild-type strain but could not develop the more extended biofilm matrix structures. A compositional analysis ofP. luminescensEPS reveals how themanAmutation has a major effect on the formation of a complete, branched EPS.

Background: Photorhabdus are Gram-negative nematode-symbiotic and insect-pathogenic bacteria. The species Photorhabdus asymbiotica is able to infect humans as well as insects. We investigated the secreted proteome of a clinical isolate of P. asymbiotica at different temperatures in order to identify proteins relevant to the infection of the two different hosts. Results: A comparison of the proteins secreted by a clinical isolate of P. asymbiotica at simulated insect (28°C) and human (37°C) temperatures led to the identification of a small and highly abundant protein, designated Pam, that is only secreted at the lower temperature. The pam gene is present in all Photorhabdus strains tested and shows a high level of conservation across the whole genus, suggesting it is both ancestral to the genus and probably important to the biology of the bacterium. The Pam protein shows limited sequence similarity to the 13.6 kDa component of a binary toxin of Bacillus thuringiensis. Nevertheless, injection or feeding of heterologously produced Pam showed no insecticidal activity to either Galleria mellonella or Manduca sexta larvae. In bacterial colonies, Pam is associated with an extracellular polysaccharide (EPS)-like matrix, and modifies the ability of wild-type cells to attach to an artificial surface. Interestingly, Surface Plasmon Resonance (SPR) binding studies revealed that the Pam protein itself has adhesive properties. Although Pam is produced throughout insect infection, genetic knockout does not affect either insect virulence or the ability of P. luminescens to form a symbiotic association with its host nematode, Heterorhabditis bacteriophora. Conclusions: We studied a highly abundant protein, Pam, which is secreted in a temperature-dependent manner in P. asymbiotica. Our findings indicate that Pam plays an important role in enhancing surface attachment in insect blood. Its association with exopolysaccharide suggests it may exert its effect through mediation of EPS properties. Despite its abundance and conservation in the genus, we find no evidence for a role of Pam in either virulence or symbiosis.

A PCR identification method in which four primers that recognize homologous conserved regions in the Sinorhizobium melilotigenome are used was developed and tested. The regions used for identification were the nodbox 4 locus, which is located in one of the symbiotic megaplasmids, and the mucR gene, which is located in the chromosome. The new method was used to establish a collection ofS. meliloti strains from polluted soils.

Walnut milk residues (WMR) were investigated for the first time through their phenolic characterization including soluble (free, esterified, and etherified) phenolics and those released from their insoluble-bound form (insoluble-bound phenolic hydrolysates, IBPHs) and their antioxidant properties. Free phenolics were recovered and alkaline or acid hydrolysis were used to recover the remaining phenolic fractions. Total phenolic compounds (TPCs) and their antioxidant activity were analyzed by Folin–Ciocalteu, FRAP, and ORAC methods, respectively. Soluble phenolics (free + esterified + etherified fractions) showed a higher TPC (275.3 mg GAE 100 g⁻¹ dw) and antioxidant activity (FRAP: 138.13 µmol TE g⁻¹ dw; ORAC: 45.41 µmol TE g⁻¹ dw) with respect to the IBPH. There was a significant correlation between TPC and FRAP and ORAC values regardless of the fraction and tested sample. Phenolic acids and flavonoids were identified and quantified by ultra-performance liquid chromatography–electrospray tandem mass spectrometry (UPLC-ESI-MS/MS). Gallic acid, mainly in the free form (3061.0 µg 100 g⁻¹), was the most representative, followed by biochanin A, identified for the first time in a walnut product and mostly present in the fraction released from the esterified form (593.75 µg 100 g⁻¹). No detrimental cytotoxic impact on Caco-2 cells was observed. Hence, WMR could be considered a potential source for the development of nutraceutical and/or antioxidant food additives.