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Abstract Background Photorhabdus are Gram negative bacteria that are pathogenic to insect larvae whilst also having a mutualistic interaction with nematodes from the family Heterorhabditis. Iron is an essential nutrient and bacteria have different mechanisms for obtaining both the ferrous (Fe2+) and ferric (Fe3+) forms of this metal from their environments. In this study we were interested in analyzing the role of Fe3+ and Fe2+ iron uptake systems in the ability of Photorhabdus to interact with its invertebrate hosts. Results We constructed targeted deletion mutants of exbD, feoABC and yfeABCD in P. luminescens TT01. The exbD mutant was predicted to be crippled in its ability to obtain Fe3+ and we show that this mutant does not grow well in iron-limited media. We also show that this mutant was avirulent to the insect but was unaffected in its symbiotic interaction with Heterorhabditis. Furthermore we show that a mutation in feoABC (encoding a predicted Fe2+ permease) was unaffected in both virulence and symbiosis whilst the divalent cation transporter encoded by yfeABCD is required for virulence in the Tobacco Hornworm, Manduca sexta (Lepidoptera) but not in the Greater Wax Moth, Galleria mellonella (Lepidoptera). Moreover the Yfe transporter also appears to have a role during colonization of the IJ stage of the nematode. Conclusion In this study we show that iron uptake (via the TonB complex and the Yfe transporter) is important for the virulence of P. luminescens to insect larvae. Moreover this study also reveals that the Yfe transporter appears to be involved in Mn2+-uptake during growth in the gut lumen of the IJ nematode. Therefore, the Yfe transporter in P. luminescens TT01 is important during colonization of both the insect and nematode and, moreover, the metal ion transported by this pathway is host-dependent.

Abstract Background Photorhabdus are Gram negative entomopathogenic bacteria that also have a mutualistic association with nematodes from the family Heterorhabditis. An essential part of this symbiosis is the ability of the bacterium to colonize the gut of the freeliving form of the nematode called the infective juvenile (IJ). Although the colonization process (also called transmission) has been described phenomonologically very little is known about the underlying molecular mechanisms. Therefore, in this study, we were interested in identifying genes in Photorhabdus that are important for IJ colonization. Results In this work we genetically tagged P. luminescens TT01 with gfp and constructed a library containing over 3200 mutants using the suicide vector, pUT-Km2. Using a combination of in vitro symbiosis assays and fluorescent microscopy we screened this library for mutants that were affected in their ability to colonize the IJ i.e. with decreased transmission frequencies. In total 8 mutants were identified with transmission frequencies of ≤ 30% compared to wild-type. These mutants were mapped to 6 different genetic loci; the pbgPE operon, galE, galU, proQ, asmA and hdfR. The pbgPE, galE and galU mutants were all predicted to be involved in LPS biosynthesis and, in support of this, we have shown that these mutants are avirulent and sensitive to the cationic antimicriobial peptide, polymyxin B. On the other hand the proQ, asmA and hdfR mutants were not affected in virulence and were either as resistant (proQ) or slightly more sensitive (asmA, hdfR) to polymyxin B than the wild-type (WT). Conclusions This is the first report describing the outcome of a comprehensive screen looking for transmission mutants in Photorhabdus. In total 6 genetic loci were identified and we present evidence that all of these loci are involved in the assembly and/or maintenance of LPS and other factors associated with the cell surface. Interestingly several, but not all, of the transmission mutants identified were also avirulent suggesting that there is a significant, but not complete, genetic overlap between pathogenicity and mutualism. Therefore, this study highlights the importance of the cell surface in mediating the symbiotic and pathogenic interactions of Photorhabdus.

Abstract Background Photorhabdus are motile members of the family Enterobactericeae that are pathogenic to insect larvae whilst also maintaining a mutualistic interaction with entomophagous nematodes of the family Heterorhabditiae. The interactions between Photorhabdus and its hosts are thought to be an obligate part of the bacteria's life-cycle in the environment. Motility often plays a key role in mediating bacteria-host interactions and, in this study, we were interested in characterising the role of motility in the Photorhabdus-nematode-insect tripartite association. Results We constructed deletion mutants of flgG (blocking flagella production) and motAB (blocking flagella rotation) in P. luminescens TT01. Using these mutants we show that both the ΔflgG and ΔmotAB mutants are equally as good as the wild-type (WT) bacteria in killing insects and supporting nematode growth and development suggesting that flagella production and motility are not required for pathogenicity or mutualism. However we show that the production of flagella is associated with a significant metabolic cost during growth on agar plates suggesting that, although not required for pathogenicity or mutualism, there must be a strong selective pressure to retain flagella production (and motility) during the interactions between Photorhabdus and its different hosts. To this end we show that both the ΔflgG and ΔmotAB mutants are out-competed by WT Photorhabdus during prolonged incubation in the insect revealing that motile bacteria do have a fitness advantage during colonisation of the insect larva. Conclusion This is the first report of a role for motility in Photorhabdus and we show that, although not required for either pathogenicity or mutualism, motility does contribute to the competitive fitness of Photorhabdus during infection of the insect (and, to a lesser extent, the nematode). This adaptive function is similar to the role ascribed to motility in mammalian pathogens such as uropathogenic Escherichia coli (UPEC). Therefore, in addition to describing a role for motility in Photorhabdus, this study reinforces the relevance and utility of this bacterium as a model for studying bacteria-host interactions.