The role of iron uptake in pathogenicity and symbiosis in Photorhabdus luminescens TT01

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.

Figure 1

The exbD mutant of P. luminescens TT01. A) The exbD locus on the genome of P. luminescens TT01 (taken from Colibase at http://xbase.bham.ac.uk/colibase). B) Siderophore production by P. temperata K122, P. temperata K122 exbD::Km, P. luminescens TT01 and P. luminescens TT01 ΔexbD. The bacteria were cultured overnight at 30°C in LB broth and the OD600 of the culture was adjusted to 1. An aliquot of 10 μl of each cell suspension was inoculated onto the surface of an LB agar plate containing CAS solution and the plate was incubated at 30°C for 48 h. Siderophore production is observed as the orange halo surrounding the growing colony. C) The growth of P. luminescens TT01 ΔexbD is sensitive to the levels of iron in the medium. TT01 (diamonds) and the ΔexbD mutant (circles) were grown in fresh LB (open symbols) or LB broth supplemented with 50 μM 2'2'-dipyridyl (filled symbols). Growth curves were done in triplicate and a representative curve is shown.

Figure 2

The feoABC and yfeABCD loci in P. luminescens TT01. The genetic loci predicted to encode the FeoABC permease and YfeABCD transporter (taken from Colibase at http://xbase.bham.ac.uk/colibase). The genes deleted in this study are highlighted with the dashed line boxes.

Figure 3

The growth of P. luminescens in the presence of 2'2'-dipyridyl. The sensitivity of each mutant to iron levels was assayed by determining the ability of each mutant to grow in the presence of increasing concentrations of 2'2'-dipyridyl. The OD₆₀₀ of overnight cultures of each strain was adjusted to 1 and 10 μl of the cell suspension was spotted onto the surface of an LB agar plate supplemented with the indicated concentration of 2'2'-diyridyl. The plates were incubated at 30°C for 48 h before a digital photograph of each agar plate was taken. The final image was assembled by cutting and pasting the appropriate colony from each photograph using Adobe Photoshop 7. It is important to highlight that the photographs were not manipulated in any other way. The data shown is a representative example and the experiment was repeated in triplicate.

Figure 4

Virulence of the ΔexbD and ΔyfeABCD mutants can be rescued by FeCl₃. Overnight cultures were prepared and 1000 CFU of WT (diamonds), ΔexbD (squares) and ΔyfeABCD (triangles) were injected into 5^th instar M. sexta larvae (open symbols) or larvae pre-injected with 10 μl of a solution containing 5 mM FeCl₃ (filled symbols). The data shown is the mean of at least 2 independent experiments (with n = 10 insects/experiment). For clarity the standard deviations are not shown but these values were within expected limits (0-35%).

Figure 5

The IJ yield after growth on P. luminescens. The different bacterial strains were inoculated onto lipid agar plates, incubated for 3-4 days at 30°C and 40 surface-sterilised H. bacteriophora IJs were added to the biomass. The plates were incubated for 21 days at 25°C and the IJ yields were determined (i.e. total number of IJs/50). For each experiment 5 plates were analyzed for each strain and the experiment was repeated 3 times. Therefore the data shown is the mean ± standard deviation of n = 15 plates for each strain. Statistical significance was determined using a T-test and IJ yields significantly different (P < 0.01) to those obtained using TT01 are indicated with an asterisk.

Figure 6

Colonization of IJ nematodes with TT01 and mutant derivatives. A) Individual IJ nematodes (n = 10), grown on the different bacterial strains (as indicated), were crushed and the lysate was plated on LB agar to enumerate the CFU within the nematode. The data is shown as a boxplot where the horizontal line within the box represents the median value. The box extends to the first and third quartiles and the whiskers show the upper and lower limits of the data (as defined by the statistical test). Asterisks represent outliers. The level of colonization of strains carrying the ΔyfeABCD allele was significantly lower than TT01 (P < 0.0001, Mann-Whitney). B) As above except that the lysate from each crushed IJ was plated on LB agar with or without added 0.1% (w/v) pyruvate, as indicated.