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J Bacteriol. Jan 2005; 187(1): 77–84.
PMCID: PMC538804

The pbgPE operon in Photorhabdus luminescens Is Required for Pathogenicity and Symbiosis

Abstract

Photorhabdus is a genus of gram-negative Enterobacteriaceae that is pathogenic to insect larvae while also maintaining a mutualistic relationship with nematodes from the family Heterorhabditis, where the bacteria occupy the gut of the infective juvenile (IJ) stage of the nematode. In this study we describe the identification and characterization of a mutation in the pbgE1 gene of Photorhabdus luminescens TT01, predicted to be the fifth gene in the pbgPE operon. We show that this mutant, BMM305, is strongly attenuated in virulence against larvae of the greater wax moth, Galleria mellonella, and we report that BMM305 is more sensitive to the cationic antimicrobial peptide, polymyxin B, and growth in mildly acidic pH than the parental strain of P. luminescens. Moreover, we also show that the lipopolysaccharide (LPS) present on the surface of BMM305 does not appear to contain any O antigen. Complementation studies reveal that the increased sensitivity to polymyxin B and growth in mildly acidic pH can be rescued by the in trans expression of pbgE1, while the defects in O-antigen assembly and pathogenicity require the in trans expression of pbgE1 and the downstream genes pbgE2 and pbgE3. Finally, we show that BMM305 is defective in symbiosis as this mutant is unable to colonize the gut of the IJ stage of the nematode. Therefore, we conclude that the pbgPE operon is required for both pathogenicity and symbiosis in P. luminescens.

Photorhabdus is a genus of gram-negative, bioluminescent insect pathogenic bacteria that also has a mutualistic relationship with entomophagous nematodes from the family Heterorhabditis (8). The bacteria colonize the gut of the infective juvenile (IJ) stage of the nematode, a free-living, infectious stage that lives in the soil and actively seeks out insect larvae. The IJ enters the larva and regurgitates the bacteria into the hemolymph, where the bacteria multiply and kill the insect within 24 to 48 h of infection. The bacteria also produce a wide range of extracellular hydrolytic enzymes that function to convert the internal organs and tissues of the insect into a nutrient soup that can support nematode growth and development. After two to three generations within the insect, the nematodes develop into IJs, the bacteria recolonize the intestinal tract, and the IJs emerge from the cadaver in search of a new host (for a recent review, see reference 19).

It has been suggested that pathogenicity and symbiosis share common molecular mechanisms and that the outcome of a bacterium-host interaction is the result of a negotiation between the organisms involved in the interaction (28). The Photorhabdus-nematode-insect tripartite system provides a useful model system in which this question can be directly addressed. DNA sequencing has revealed that Photorhabdus contains many genes predicted to encode proteins with homology to virulence factors characterized in other bacterial pathogens (16, 20). Recent work has identified the Tc toxin, a large protein complex that is orally active against insect larvae, and the Mcf-1 and Mcf-2 toxins that permit nonpathogenic Escherichia coli to persist in and kill insect larvae (9, 14, 43). The PhoPQ two-component pathway has been shown to be required for the virulence of many pathogens (3, 23, 25, 39), and it has recently been shown that the PhoPQ two-component pathway is also required for pathogenicity in Photorhabdus (15). Therefore, it appears that the infection of insect larvae by Photorhabdus shares common pathways that control pathogenicity in well-characterized mammalian pathogens such as Salmonella enterica. Other studies have also identified several Photorhabdus genes that are required for the symbiosis between the bacteria and the nematode (7, 11). These genes encode proteins that are required for the normal growth and development of the nematode in vivo and in vitro. However, as of yet, no gene has been identified with a role in both symbiosis and pathogenicity, although we have recently identified a protein, HexA, that appears to play an important role in the regulation of both pathogenicity and symbiosis, suggesting that there is a link between these two contrasting lifestyles (32).

In this study we have used transposon mutagenesis to identify a gene in Photorhabdus luminescens TT01 that is required for both pathogenicity and symbiosis. We show that the transposon has inserted into pbgE1, a gene with homology to pmrK from S. enterica. In addition to the defects in both symbiosis and pathogenicity, we show that the mutation in pbgE1 renders strain TT01 more sensitive to growth in mildly acidic conditions and the presence of the cationic antimicrobial peptide (CAMP), polymyxin B. This is the first report of a gene in Photorhabdus that is required for both symbiosis and pathogenicity and, therefore, highlights the potential genetic overlap between these interactions and the utility of the tripartite Photorthabdus-nematode-insect association as a model system for studying and comparing these bacterium-host interactions.

MATERIALS AND METHODS

Strains, plasmids, and growth conditions.

Strains and plasmids used in this study are listed in Table Table1.1. We have used a spontaneous rifampin-resistant mutant of P. luminescens TT01 as the wild type in all experiments. Bacteria were routinely cultured in Luria-Bertani (LB) broth or on LB agar (LB broth plus 1.5% [wt/vol] agar) at 28°C for P. luminescens and 37°C for E. coli, unless otherwise stated. The nematode strain used was Heterorhabditis bacteriophora TT01, and this is the normal partner of P. luminescens TT01. When required, antibiotics were added at the following concentrations: ampicillin, 100 μg/ml; kanamycin, 30 μg/ml; and rifampin, 50 μg/ml.

TABLE 1.
Strains and plasmids

Nematode cultures.

Nematode stocks were maintained by passage through larvae of the greater wax moth, Galleria mellonella (supplied by Livefood, Rooks Bridge, Somerset, United Kingdom). Before infections, IJs were surface sterilized by incubating the nematodes in 0.4% (wt/vol) hyamine (Sigma) before washing in several volumes of phosphate-buffered saline (PBS). Approximately 1,000 surface-sterilized IJs in 1 ml of PBS were transferred to filter paper in a 9-cm petri dish. Ten G. mellonella larvae were added to each dish and placed at 25°C to allow infection of the insect hosts. After 9 days the insect cadavers were transferred to White traps for collection of the emerging infective juveniles (44).

DNA manipulations.

Chromosomal DNA was isolated from BMM305 by using standard procedures (41). The chromosomal DNA flanking the transposon was cloned and sequenced as previously described (32).

Transposon mutagenesis.

A spontaneous rifampin-resistant mutant of P. luminescens TT01 was mated with E. coli S17-1 carrying pLOF-Kn (29). Both strains were grown to an optical density at 600 nm (OD600) of 0.6, and the donor and recipient bacteria were mixed at a ratio of 1:4. The cells were left overnight at room temperature and Rifr Knr exconjugants were selected by plating the conjugation mix onto selective LB agar plates. Individual mutants were then tested for motility by picking colonies onto swim agar plates, and the plates were incubated at 28°C and checked at 24 and 48 h.

Cloning of pbgE1, pbgE2, and pbgE3.

The pbgE1 gene was amplified from P. luminescens TT01 chromosomal DNA by using Pfu polymerase and the primers pbgE1F (5′-TCGAGCCATGGTTGAATAACCGGGCGTGTAAGG-3′) and pbgE1R (5′-ACGGCTCTAGATCATGGTTGCTTCTCATAAACC). The pbgE1F primer contains a restriction site for NcoI that overlaps with the ATG start codon of pbgE1, and the pbgE1R primer contains a restriction site for XbaI. The incorporation of the NcoI site resulted in the second codon of pbgE1 being mutated from TTG (encoding Leu) to GTG (encoding Val). These restriction sites facilitated the cloning of the pbgE1 gene into pTRC99a, resulting in plasmid pBMM500. In the same way, a DNA fragment containing pbgE1E2E3 was amplified by using Pfu polymerase and the primers pbgE1F and pbgE3R (5′-ACGGCTCTAGATCATTCTGGACGGCTAATC-3′), and the PCR product was cloned into pTRC99a, resulting in plasmid pBMM503. The integrity of all plasmids was confirmed by DNA sequencing.

Electroporation.

Electrocompetent Photorhabdus cells were prepared by inoculating 100 ml of LB with 200 μl from an overnight culture. The culture was incubated at 28°C with shaking and grown to early exponential phase (OD600 of 0.2 to 0.3). The cell suspension was immediately placed on ice for 90 min, and the cells were harvested by centrifugation at 4,000 rpm (Jovan BR4i centrifuge) for 10 min at 4°C. The supernatant was discarded, and the pellet was resuspended in 100 ml of ice-cold SH buffer (5% [wt/vol] sucrose, 1 mM HEPES). The cells were harvested and sequentially washed in 50 ml of SH buffer and 1.6 ml of SH buffer, and the cell pellet was finally resuspended in 160 μl of SH buffer and stored on ice. Under these conditions we observed that BMM305 lysed, and, therefore, for this strain all washes were carried out by using a modified SH buffer that contained 500 mM HEPES. Plasmid DNA was mixed with 40 μl of freshly prepared electrocompetent cells in a 0.2-cm electroporation cuvette (Bio-Rad), and the cells were subjected to a 2.5-kV pulse from a MicroPulser (Bio-Rad).

Polymyxin resistance assay.

Standard overnight cultures were grown, and 20 μl of the overnight culture was inoculated into 3 ml of sterile LB with freshly prepared polymyxin B added to the required concentration. The culture was incubated in a shaking water bath at 28°C overnight, and the OD600 was taken, and relative OD600 values were calculated as described in the text.

Acid tolerance.

Cultures were grown overnight, and 20 μl was inoculated into 3 ml of sterile LB containing organic or inorganic acid. For inorganic acid tolerance, LB was supplemented with 50 mM MES (morpholineethanesulfonic acid), and the pH was adjusted by using concentrated HCl and/or NaOH. For organic acid tolerance LB was buffered with 50 mM MES, and various concentrations of acetic acid were added before the pH of the medium was adjusted to 7 or 6.5, as above. Cultures were incubated in a shaking water bath at 28°C overnight, and the OD600 was taken and relative OD600 values were calculated as described in the text.

SDS-PAGE analysis of LPS.

Lipopolysaccahride (LPS) was isolated from P. luminescens as previously described (17). Briefly, 25 ml of an overnight culture was pelleted by centrifugation at 4,000 rpm (Jovan BR4i) for 15 min at room temperature, and the supernatant was discarded. The pellet was then resuspended in 5 ml of 0.5 M EDTA (pH 8.0), and the cell suspension was agitated on a Bibby Stuart Science Blood Tube Rotator SB1 for 5 h. The bacteria were pelleted by centrifugation at 13,000 rpm for 3 min, and the LPS-containing supernatant was retained and removed to a fresh Eppendorf tube and centrifuged at 13,000 rpm (Jovan BR4i) for 20 min at room temperature. The LPS-containing pellet was resuspended in 10 μl of proteinase K (10 mg/ml) and incubated at 37°C for 1 h before 10 μl of 2× loading dye (125 mM Tris [pH 6.8], 10% [vol/vol] β-mercaptoethanol, 4% [wt/vol] sodium dodecyl sulfate [SDS], 20% [vol/vol] glycerol, 0.05% [wt/vol] bromophenol blue) was added, and the samples were heated to 100°C for 10 min and placed immediately on ice. The LPS was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) by using a 12.5% polyacrylamide gel and visualized as previously described (12).

Pathogenicity assay.

Pathogenicity against insect larvae was determined as previously described (32). For competition assays, strain TT01 and mutant bacteria were grown overnight at 28°C in LB broth, and equal numbers of cells from each culture were mixed to give a final cell density of 104 CFU/ml. A sample of 10 μl of this mixture (100 CFU in total) was injected directly into the hemolymph of G. mellonella larvae, and the infected larvae were incubated at 25°C for 48 h, at which time the insect larvae were dead. Individual larvae were then surface sterilized (by dipping in ethanol and passing the larva through a bunsen flame), immersed, and homogenized in LB broth. Aliquots were then plated on LB agar and, after incubation at 28°C for 48 h, 100 individual colonies were picked and checked for resistance to kanamycin. In this way the ratio of TT01 (Kns) to mutant (Knr) bacteria present in the insect at the end of the infection could be calculated, and this ratio is the competitive index.

Symbiosis assay.

In vitro symbiosis assays were performed by using lipid agar plates (18), inoculated with the appropriate bacteria and grown at 28°C for 48 h. Approximately 20 surface-sterilized H. bacteriophora TT01 IJs were added to each plate, and the plates were further incubated at 25°C until the new generation of IJs emerged (normally, 2 to 3 weeks). These IJs were surface sterilized, and the number of bacteria colonizing the nematode gut was determined by crushing the nematodes and plating the lysate on LB agar. To determine the competitive index for symbiosis, we performed symbiosis assays as described, except that equal numbers of colonies of strains TT01 and BMM305 were mixed before inoculation onto lipid agar plates. After the new generation of IJs was recovered, the fraction of wild-type to mutant bacteria present in the nematode gut was calculated by crushing the nematodes and replica plating the lysate on selective LB agar plates. In addition, the ratio of TT01 to BMM305 bacteria on the lipid agar plate at the end of the symbiosis assay was also determined to ensure that there was no significant deviation from the original inoculation.

RESULTS

Identification of a mutant in the pbgE1 gene of P. luminescens TT01.

In the course of carrying out a screen to identify mutants of P. luminescens TT01 that were defective in motility, we identified one mutant, strain BMM305, with approximately 50% of the motility of the wild-type strain when tested in swim agar (data not shown). Preliminary pathogenicity tests showed that this mutant appeared avirulent when injected into insect larvae (data not shown), and, therefore, BMM305 was selected for further analysis. Southern blot analysis, with the kanamycin resistance gene of the transposon used as a probe, revealed that BMM305 contained a single transposon insertion in the chromosome (data not shown).

The site of insertion of the transposon in BMM305 was identified by subcloning the chromosomal EcoRV fragment containing the gene for kanamycin resistance into pBR322 (see Materials and Methods), resulting in plasmid pBMM305. As EcoRV does not cut within the transposon, any resulting kanamycin-resistant clones will contain the transposon and some flanking chromosomal DNA. Sequencing of this flanking DNA revealed that the transposon had inserted into a gene annotated as pbgE1 (plu2656) in the recently published P. luminescens TT01 genome (16). The gene pbgE1 is predicted to be part of the pbgPE operon (pbgP1P2P3P4E1E2E3), and in silico analyses predict that this operon is homologous to the pmrHFIJKLM operon in S. enterica (Fig. (Fig.1).1). In S. enterica the pmrHFIJKLM operon has been shown to encode the proteins responsible for the biosynthesis of l-aminoarabinose and the subsequent ligation of this amino sugar onto the lipid A moiety of LPS (5, 26, 27).

FIG. 1.
Genomic organization of the pbgPE operon in P. luminescens and the pmrHFIJKLM operon in S. enterica. The predicted amino acid identity between homologues and the position at which the transposon has inserted in BMM305 are indicated.

BMM305 is highly attenuated in insect virulence.

We had originally observed that BMM305 was unable to kill larvae of the greater wax moth, Galleria mellonella, when 100 bacteria were injected directly into the hemolymph of the larva (data not shown). P. luminescens is normally virulent to G. mellonella, and the injection of <5 bacterial cells into the insect hemolymph is normally sufficient to result in larval death within 48 to 72 h. Therefore, to determine the full extent of the pathogenicity defect associated with BMM305, we injected the larvae of the G. mellonella with different doses of TT01 or BMM305, and we calculated the 50% lethal dose (LD50) as the number of bacteria required to kill 50% of the insects in 48 h. As expected, the LD50 of wild-type bacteria was estimated to be <5 bacteria/larva (data not shown). On the other hand, the LD50 of BMM305 was calculated as being 102,000 ± 20,000 CFU/insect, indicating that this mutant is not completely avirulent. Therefore, it appears that strain BMM305 is significantly attenuated in virulence to G. mellonella larvae. In support of this, in each case when we injected a mixture containing equal numbers of strain TT01 and BMM305 cells into G. mellonella larvae, we recovered only TT01 bacteria from the insect cadaver, suggesting that the BMM305 mutant is unable to compete with the wild-type bacteria in vivo.

In BMM305, the transposon was inserted into pbgE1, the fifth of seven genes in the pbgPE operon. Therefore, it was possible that the observed attenuation may be due to either the interruption of the pbgE1 gene and/or polar effects on the expression of the pbgE2 and pbgE3 genes. To test this we cloned pbgE1 and pbgE1E2E3 into pTRC99a for complementation studies, resulting in plasmids pBMM500 and pBMM503, respectively. These plasmids were transformed into BMM305, and 100, 1,000 and 10,000 CFU were injected into G. mellonella larvae, and insect mortality was recorded after 96 h. As expected, TT01 killed all of the larvae, even at the lowest injected dose, and virulence was unaffected by the presence of the pTRC99a vector (Fig. (Fig.2).2). On the other hand, BMM305 killed only between 10 to 20% of insect larvae at the highest injected dose. Interestingly, the in trans expression of pbgE1 alone in BMM305 was not sufficient to restore full virulence, suggesting that the insertion in pbgE1 affected the expression of pbgE2 and pbgE3 (Fig. (Fig.2).2). Indeed, in support of this, the expression of pbgE1E2E3 from pBMM503 restored the virulence of BMM305 to TT01 levels, suggesting that virulence requires the expression of pbgE1, pbgE2, and pbgE3 (Fig. (Fig.22).

FIG. 2.
Pathogenicity of BMM305. Cells of TT01 and BMM305 bacteria, containing pTRC99a (vector), pBMM500 (pbgE1), or pBMM503 (pbgE1E2E3), were grown overnight at 28°C in LB broth; the cells were harvested, resuspended in PBS, and 100 (white columns), ...

BMM305 has increased sensitivity to polymyxin B.

In S. enterica serovar Typhimurium, the pmrHFIJKLM operon is regulated in response to the presence of CAMPs such as polymyxin B, and mutations in this operon render the cells more sensitive to the presence of CAMPs (26, 27). Insects produce CAMPs as part of their innate immune response against invading bacteria. To test whether BMM305 was more susceptible to the presence of these peptides, bacteria were grown overnight in LB and diluted into fresh LB supplemented with different concentrations of polymyxin B (see Materials and Methods). These cultures were incubated overnight at 28°C, and the level of sensitivity to polymyxin B was assessed by calculating the relative OD600 values. Therefore, the level of growth at a particular concentration of polymyxin B (as assessed by OD600 values) was divided by the OD600 value obtained for the culture grown in the absence of polymyxin B. In this way we observed that, even in the presence of very low concentrations of polymyxin B, the growth of TT01 decreased by approximately 50% compared to growth in the untreated culture, and this decrease was constant over the range of drug concentrations used in this study (Fig. (Fig.3A).3A). However, it is clear that the BMM305 mutant is more susceptible to the presence of polymyxin B, and growth is completely inhibited at a concentration of 0.4 μg/ml (Fig. (Fig.3A).3A). Therefore, mutations in the pbgPE operon increase the sensitivity of P. luminescens to polymyxin B.

FIG. 3.
Sensitivity of P. luminescens to polymyxin B. (A) Cells of TT01 (white columns) and BMM305 (gray columns) were grown overnight at 28°C in LB broth supplemented with the indicated amount of polymyxin B. Growth was then measured and compared to ...

Plasmids pBMM500 and pBMM503 were transformed into BMM305, and transformants were tested for their sensitivity to polymyxin B. When either pbgE1 or pbgE1E2E3 is supplied in trans, we can clearly see that growth of BMM305 in the presence of 0.4 μg of polymyxin B per ml is restored to wild-type levels, although complementation appears to be more complete in the presence of pBMM503 (Fig. (Fig.3B).3B). This suggests that the increased sensitivity to polymyxin B observed in BMM305 is largely due to the mutation in pbgE1 and is not due to significant polar effects on the expression of either pbgE2 or pbgE3. However, as the attenuation of BMM305 is only complemented by the in trans expression of pbgE1E2E3, we can conclude that the increased sensitivity to CAMPs does not account for the observed decrease in virulence.

The LPS of BMM305 lacks O antigen.

It has been shown that, in S. enterica, mutations in pmrK result in subtle changes in the structure of the LPS, and we wanted to know if the lesion in pbgE1 affected the structure of LPS in P. luminescens. Therefore, we cultured strains TT01 and BMM305 overnight in LB at 28°C, and the LPS was extracted, separated by SDS-PAGE, and visualized by silver staining following incubation with periodic acid (see Materials and Methods). The wild-type LPS exhibits the characteristic ladder profile associated with smooth LPS containing modally distributed O-antigen chain lengths (Fig. (Fig.4,4, lane 1). However, the LPS from BMM305 did not exhibit the ladder profile, indicating that the LPS on the surface of BMM305 is lacking O antigen (Fig. (Fig.4).4). In addition to the EDTA-based protocol that we used in this study, we also extracted the LPS from TT01 and BMM305 by using hot phenol, and we obtained the same results as presented here (data not shown). This suggests that, in contrast to the situation in S. enterica, the pbgPE operon in P. luminescens is required for the biosynthesis and/or the correct assembly of the O antigen. Interestingly, this O-antigen-defective phenotype was not complemented by pBMM500 but was complemented by pBMM503, indicating that the expression of pbgE1, pbgE2, and pbgE3 is required for the correct assembly of the O antigen (Fig. (Fig.4).4). Therefore, the lack of O antigen correlates with the decrease in virulence observed in BMM305.

FIG. 4.
The LPS of BMM305 does not contain any O antigen. Strains (as indicated) were grown overnight in LB broth at 28°C, the cells were harvested, and LPS was isolated as described in Materials and Methods. The LPS from equal numbers of cells (estimated ...

BMM305 shows increased sensitivity to mildly acidic pH.

The absence of O antigen has been shown to increase the sensitivity of enteropathogenic E. coli to acid stress (6). The great majority of insects have hemolymph that is mildly acidic (38). Therefore, to test whether BMM305 was more sensitive to mildly acidic pH, we cultured TT01 and BMM305 cells overnight in LB and inoculated the bacteria into fresh LB that had been buffered to pH values between 6 and 7 with MES (see Materials and Methods). The cultures were then incubated overnight at 28°C, and the ability of the bacteria to grow was assessed by calculating the relative OD600 values; i.e., the level of growth at a particular pH (as assessed by OD600 values) was divided by the OD600 value obtained for the culture grown at pH 7.0. In this way we showed that at pH 6.5 BMM305 grows only to approximately 35% of the level observed at pH 7.0 compared to a level of 95% achieved by the parental strain. At pH 6 the mutant bacteria grew only to approximately 5% of the level observed at pH 7, while the wild-type bacteria were still largely unaffected in growth (Fig. (Fig.5A).5A). Therefore, BMM305 is more sensitive to mildly acidic pH.

FIG. 5.
Sensitivity of P. luminescens to acidic pH. (A) Cells of TT01 (white columns) and BMM305 (gray columns) cells were grown overnight in LB broth buffered to the indicated pH. Growth was then compared to growth at a pH 7, and the data are presented as the ...

To test whether this acid sensitivity was due to the insertion in pbgE1 or a polar effect on the expression of pbgE2 and pbgE3, we transformed BMM305 with either pBMM500 or pBMM503 and measured the growth in LB broth buffered to a pH of 7, 6.5, and 6. Our results clearly show that the in trans expression of either pbgE1 or pbgE1E2E3 restores the ability of BMM305 to grow at a pH of 6.0 (Fig. (Fig.5B).5B). This suggests that the observed acid sensitivity of BMM305 is due to the insertion in pbgE1 and not due to any polar effects on the expression of pbgE2 or pbgE3. Therefore, the observed increase in sensitivity to mildly acidic pH is not due to the lack of O antigen, and, in addition, acid sensitivity cannot explain the decrease in virulence observed with BMM305.

The pbgE1 gene is required for bacterial colonization of the nematode gut.

We have shown that the virulence of BMM305 to larvae of G. mellonella is significantly attenuated and that this attenuation is complemented by the addition of the pbgE1E2E3 genes in trans but not by pbgE1 alone. We were also interested to see what effect, if any, the insertion in pbgE1 has on the mutualistic interaction between the bacteria and the nematode. There are two stages in the symbiosis between the bacteria and the nematode: (i) the ability of the bacteria to support nematode growth and development and (ii) the ability of the bacteria to colonize the gut of the IJ. Both of these stages can be assayed in vitro by growing the mutant bacteria on lipid agar plates for 48 h at 28°C before the plates are seeded with surface-sterilized IJs of the nematode. The ability of the bacteria to support nematode growth can then be assessed both qualitatively (by observing nematode development on plates) and quantitatively (by counting the number of IJs that develop from the agar plate). Therefore, lipid agar plates were inoculated with either TT01 or BMM305, and after a 48-h incubation the plates were seeded with IJs of the H. bacteriophora TT01 nematode (see Materials and Methods). No quantitative or qualitative differences were observed when the nematodes growing on TT01 bacteria were compared to those growing on the BMM305 mutant bacteria, suggesting that the mutation in pbgE1 does not affect the ability of the bacteria to support nematode growth and development (data not shown). To determine the level of bacterial colonization, the new generation of IJs was harvested and crushed, and the lysate was spread on LB agar. We observed that the level of colonization (number of CFU per IJ) achieved by BMM305 was always approximately 1% that achieved by wild-type bacteria (retention levels were 1.1 ± 1.4 CFU/IJ for BMM305 [means ± standard deviation; n = 11] compared to 154.1 ± 161.8 CFU/IJ for TT01 [n = 20]). Moreover, in each case when we inoculated IJs onto lipid agar plates that had been seeded with a 1:1 mixture of TT01 to BMM305, we detected only the presence of TT01 in the gut of the IJs that developed from these plates, indicating that BMM305 is unable to cocolonize the nematode with TT01 (data not shown). It is important to note that the insertion in pbgE1 did not affect the ability of BMM305 to grow and survive on lipid agar plates, and plates that were originally seeded with a 1:1 mixture of TT01 and BMM305 still retained the 1:1 ratio after 14 days of incubation at 28°C. In addition, we have also shown that the presence of the transposon per se does not affect bacterial retention in the nematode gut (data not shown). Unfortunately, we were unable to perform complementation studies on this phenotype as the long incubation times on lipid agar plates (in excess of 2 weeks) resulted in the loss of the plasmid from the bacteria. Nonetheless, it is clear that BMM305 is severely affected in its ability to colonize the nematode gut, suggesting that the pbgPE operon in Photorhabdus is required for colonization of both the insect and the nematode.

DISCUSSION

P. luminescens TT01 has a pathogenic relationship with insect larvae and a mutualistic relationship with nematodes from the family Heterorhabditis, where the bacteria colonize the gut of the IJ stage of the nematode. We have shown that a mutant strain of TT01, BMM305, which is disrupted in the pbgE1 gene, is attenuated in virulence to insect larvae and is unable to colonize the gut of the nematode. BMM305 is severely affected in the production and assembly of O antigen, and this defect correlates with the decrease in pathogenicity observed with this mutant.

The pbgE1 gene from P. luminescens is the fifth gene in the pbgP1P2P3P4E1E2E3 operon, and it has strong homology with the pmrK gene in S. enterica, with the predicted proteins encoded by these genes having 45% amino acid identity. The pmrK gene is the fifth gene of the pmrHFIJKLM operon, and it is predicted to encode a protein with mannosyltransferase activity. It has been suggested that PmrK is involved in the ligation of the l-aminoarabinose moiety to the lipid A (42). Mutations in pmrK (pqaB) have been shown to affect S. enterica virulence and resistance to polymyxin B (5, 27, 42). We have shown that mutations in pbgE1 also affect the virulence of P. luminescens and the sensitivity of this bacteria to polymyxin B, implying that pbgE1 and pmrK are orthologous genes. Therefore, it is likely that pbgE1 is involved in the decoration of the lipid A of P. luminescens with l-aminoarabinose. A recent genetic analysis of the role of the pmrHFIJKLM operon in the resistance of S. enterica to polymyxin B has shown that all of the genes, with the exception of pmrM, appear to be required for normal resistance to the CAMP (27). Although we have not undertaken a comprehensive functional analysis of each gene in the pbgPE operon, our studies do indicate that both pbgE2 and pbgE3 (homologous to pmrL and pmrM, respectively) are not required for resistance to polymyxin B in TT01. Therefore, in Photorhabdus, these genes are probably not required for the modification of the lipid A with l-aminoarabinose.

In addition to the defect in l-aminoarabinose production, a mutation in pmrK (pqaB) in S. enterica serovar Typhi also resulted in a small decrease in the chain length of O antigen (5). Recent work in Salmonella and Shigella has shown that the length of the O antigen plays a significant role in the virulence of the bacteria (36, 37). In this study we have shown that a mutation in the pmrK homologue in P. luminescens TT01, pbgE1, resulted in the production of LPS with no O antigen, as determined by SDS-PAGE. This lack of O antigen was complemented by the in trans expression of pbgE1E2E3 but not pbgE1 alone, suggesting that pbgE2 and pbgE3 are involved in the production and/or export of the O antigen. In Salmonella, the O antigen is assembled on the periplasmic side of the cytoplasmic membrane through the action of a membrane-localized complex containing the proteins Wzx, Wzy, and Wzz (for a review, see reference 40). Genetic and biochemical evidence suggests that Wzx is the O-unit transporter, Wzy is the O-antigen polymerase, and Wzz is the chain-length regulator (40). Although PbgE2 and PbgE3 do not have any homology to Wzx, Wzy, or Wzz, in silico analysis does reveal homology (41% identity in both cases) to proteins that are implicated in metabolite export (see http://www.ncbi.nlm.nih.gov/COG/new/release/cow.cgi?cog = COG0697). Therefore, it is possible that PbgE2 and PbgE3 can interact with the Wzx-Wzy-Wzz complex and affect either the transport of the O antigen across the cytoplasmic membrane or the ligation of the O antigen onto the lipid A-core molecule.

In S. enterica serovar Typhi, mutations in the pmrK (pqaB) gene have also been shown to decrease the bacterial intracellular growth rate in PMA-differentiated U937 cells (5). Moreover, the pmrHFIJKLM operon was shown to be important for S. enterica serovar Typhimurium infection via the oral route but not the intraperitoneal route, suggesting that this operon was important for resistance to host innate responses within intestinal cells (27). We have shown that the pbgPE operon in P. luminescens is important for pathogenicity and that the virulence of BMM305 is reduced by several orders of magnitude in comparison to pathogenicity of the parental strain. Therefore, the pmrHFIJKLM/pbgPE operon has an important role in the virulence of both mammalian and insect pathogens.

Insects have an innate immune response that has both humoral and cellular components (34), and a major part of the humoral response is the production of CAMPs (24, 30, 31). We have shown that BMM305 has increased sensitivity to the CAMP polymyxin B and, in addition, that this mutant does not grow at mildly acidic pH. Therefore, as the pH of the hemolymph of insects is generally around 6.5, either or both of these phenotypes could explain the decreased virulence of BMM305 against G. mellonella larvae. We have reported that the increased sensitivity to polymyxin B and to pH levels can be complemented by the in trans expression of pbgPE1 alone. As the restoration of virulence in BMM305 requires the in trans expression of pbgE1, pbgE2, and pbgE3, it is clear that neither the increased sensitivity to antimicrobial peptides nor the increased sensitivity to acidic pH can explain the attenuation observed in BMM305.

The cell-mediated innate immune response in insects involves hemocytes that recognize invading organisms and respond by both phagocytosis and encapsulation (for a review, see reference 34). It is conceivable that alterations in the surface of the bacteria might affect the surface-surface interactions between the bacteria and the immune cell, and there is evidence suggesting that Photorhabdus organisms survive and proliferate in the insect hemolymph by killing the insect hemocytes (4, 14). In preliminary tests it has been shown that BMM305 can adhere to insect immune cells equally as well as TT01 (P. Dean and S. Reynolds, personal communication). We are currently undertaking more exhaustive tests on the role of the innate response of G. mellonella in the attenuation of BMM305 and the interaction of the O antigen with the components of this immune response.

It has been shown that the expression of the pmrHFIJKLM operon in S. enterica is controlled by the PmrAB two-component pathway (26, 27, 45). PmrB is a sensor kinase that responds to high levels of Fe3+ in the environment and activates PmrA by phosphorylation, and phosphorylated PmrA binds to DNA and increases the expression of the pmrHFIJKLM operon (45, 46). In addition, PmrD (encoded by the pmrD gene) can activate signaling through the PmrAB pathway in a posttranslational manner, and the expression of pmrD is under the control of the PhoPQ two-component pathway (33). PhoPQ responds to the level of Mg2+ in the environment, and, therefore, the expression of the pmrHFIJKLM operon can also be regulated by the level of Mg2+ in an Fe3+-independent manner (10, 33). In silico analysis of the genome sequence of TT01 does reveal the presence of homologues of phoP and phoQ (plu2807 and plu2808, respectively), but there is no homologue of pmrA, pmrB, or pmrD. Nonetheless, it has recently been shown that the expression of the pbgPE operon in P. luminescens is regulated by the PhoPQ pathway in a Mg2+-dependent manner, suggesting that operon might be regulated differently in Photorhabdus than in S. enterica (15).

We have shown that BMM305 is unable to colonize the nematode gut, suggesting that the pbgPE operon is required for both pathogenicity and symbiosis in Photorhabdus. Xenorhabdus is a closely related species of bacteria that associates with nematodes from the family Steinernema, and the bacteria colonize a specialized sac in the gut of the IJ stage of the nematode (for reviews, see references 21 and 22). Recent work has revealed that colonization of this sac by Xenorhabdus occurs in two steps: initial attachment by a small number of bacteria, followed by bacterial growth to fill the sac (35). Although it is not yet known whether Photorhabdus colonizes the Heterorhabditis IJ in the same way, we can speculate that the colonization defect of BMM305 is the result of either the inability of the bacteria to attach to the gut wall or the inability of the bacteria to grow and/or persist in the gut after attachment.

In Photorhabdus the PhoPQ pathway has been shown to be required for pathogenicity, and PhoPQ was shown to control the expression of the pbgPE operon in response to Mg2+ levels (15). In this study we show that the pbgPE operon is required for virulence, suggesting that the observed avirulence of the phoPQ mutant might be due to downstream effects on the expression of the pbgPE operon, although this remains to be confirmed. In addition we have shown that the pbgPE operon is also required for normal colonization of the gut of the Heterorhabditis IJ, although the role of PhoPQ regulation in this interaction has not been tested. The closely related rhabtidid nematode Caenorhabditis elegans has been used recently as a model for the study of bacterial virulence (1, 13). By using this model it has been shown that S. enterica serovar Typhimurium establishes a persistent infection in the gut of C. elegans that kills the nematode after 3 to 5 days (3). Interestingly, mutations in both phoP and phoQ rendered S. enterica serovar Typhimurium significantly less virulent to the C. elegans nematode (3). Therefore, as in P. luminescens, the PhoPQ pathway in S. enterica also controls the expression of genes that are involved in the colonization of the gut of a nematode. In this study we have also shown that the pbgPE operon is involved in the biosynthesis and assembly of O antigen in P. luminescens. It has also been shown that the O antigen of S. enterica serovar Typhimurium is required for the bacteria to establish persistent infections in the intestines of C. elegans (2). These striking parallels in nematode colonization by a pathogen (S. enterica) and a symbiont (P. luminescens) highlight the potential overlap in the molecular mechanisms controlling these bacterium-host interactions. In addition, as it appears that PhoPQ-regulated genes are required for both pathogenicity and symbiosis in P. luminescens, it will be interesting to further characterize the PhoPQ regulon in Photorhabdus in order to further analyze the role of the genes controlled by this pathway in these contrasting interactions.

Acknowledgments

This work was supported by a grant to D.J.C. from the Exploiting Genomics Initiative of the Biotechnology and Biological Sciences Research Council (BBSRC) of the United Kingdom. We also acknowledge the BBSRC for their award of a Ph.D. studentship to H.P.J.B.

We thank the laboratories of Richard ffrench-Constant and Stuart Reynolds and other members of the D.J.C. laboratory for useful discussion.

REFERENCES

1. Aballay, A., and F. M. Ausubel. 2002. Caenorhabditis elegans as a host for the study of host-pathogen interactions. Curr. Opin. Microbiol. 5:97-101. [PubMed]
2. Aballay, A., E. Drenkard, L. R. Hilbun, and F. M. Ausubel. 2003. Caenorhabditis elegans innate immune response triggered by Salmonella enterica requires intact LPS and is mediated by a MAPK signaling pathway. Curr. Biol. 13:47-52. [PubMed]
3. Aballay, A., P. Yorgey, and F. M. Ausubel. 2000. Salmonella typhimurium proliferates and establishes a persistent infection in the intestine of Caenorhabditis elegans. Curr. Biol. 10:1539-1542. [PubMed]
4. Au, C., P. Dean, S. E. Reynolds, and R. H. ffrench-Constant. 2004. Effect of the insect pathogenic bacterium Photorhabdus on insect phagocytes. Cell Microbiol. 6:89-95. [PubMed]
5. Baker, S. J., J. S. Gunn, and R. Morona. 1999. The Salmonella typhi melittin resistance gene pqaB affects intracellular growth in PMA-differentiated U937 cells, polymyxin B resistance and lipopolysaccharide. Microbiology 145:367-378. [PubMed]
6. Barua, S., T. Yamashino, T. Hasegawa, K. Yokoyama, K. Torii, and M. Ohta. 2002. Involvement of surface polysaccharides in the organic acid resistance of Shiga toxin-producing Escherichia coli O157:H7. Mol. Microbiol. 43:629-640. [PubMed]
7. Bintrim, S. B., and J. C. Ensign. 1998. Insertional inactivation of genes encoding the crystalline inclusion proteins of Photorhabdus luminescens results in mutants with pleiotropic phenotypes. J. Bacteriol. 180:1261-1269. [PMC free article] [PubMed]
8. Boemare, N. E. 2002. Biology, taxonomy and systematics of Photorhabdus and Xenorhabdus., p. 35-56. In R. Gaugler (ed.), Entomopathogenic nematology. CABI Publishing, Wallingford, United Kingdom.
9. Bowen, D., T. A. Rocheleau, M. Blackburn, O. Andreev, E. Golubeva, R. Bhartia, and R. H. ffrench-Constant. 1998. Insecticidal toxins from the bacterium Photorhabdus luminescens. Science 280:2129-2132. [PubMed]
10. Chamnongpol, S., M. Cromie, and E. A. Groisman. 2003. Mg2+ sensing by the Mg2+ sensor PhoQ of Salmonella enterica. J. Mol. Biol. 325:795-807. [PubMed]
11. Ciche, T. A., S. B. Bintrim, A. R. Horswill, and J. C. Ensign. 2001. A phosphopantetheinyl transferase homolog is essential for Photorhabdus luminescens to support growth and reproduction of the entomopathogenic nematode Heterorhabditis bacteriophora. J. Bacteriol. 183:3117-3126. [PMC free article] [PubMed]
12. Clarke, D. J., and B. C. A. Dowds. 1995. Virulence mechanisms of Photorhabdus sp. strain K122 toward wax moth larvae. J. Invertebr Pathol. 66:149-155.
13. Couillault, C., and J. J. Ewbank. 2002. Diverse bacteria are pathogens of Caenorhabditis elegans. Infect. Immun. 70:4705-4707. [PMC free article] [PubMed]
14. Daborn, P. J., N. Waterfield, C. P. Silva, C. P. Au, S. Sharma, and R. H. ffrench-Constant. 2002. A single Photorhabdus gene makes caterpillars floppy (mcf), allows Escherichia coli to persist within and kill insects. Proc. Natl. Acad. Sci. USA 99:10742-10747. [PMC free article] [PubMed]
15. Derzelle, S., E. Turlin, E. Duchaud, S. Pages, F. Kunst, A. Givaudan, and A. Danchin. 2004. The PhoP-PhoQ two-component regulatory system of Photorhabdus luminescens is essential for virulence in insects. J. Bacteriol. 186:1270-1279. [PMC free article] [PubMed]
16. Duchaud, E., C. Rusniok, L. Frangeul, C. Buchrieser, A. Givaudan, S. Taourit, S. Bocs, C. Boursaux-Eude, M. Chandler, J. F. Charles, E. Dassa, R. Derose, S. Derzelle, G. Freyssinet, S. Gaudriault, C. Medigue, A. Lanois, K. Powell, P. Siguier, R. Vincent, V. Wingate, M. Zouine, P. Glaser, N. Boemare, A. Danchin, and F. Kunst. 2003. The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens. Nat. Biotechnol. 21:1307-1313. [PubMed]
17. Dunphy, G. B., and J. M. Webster. 1991. Antihemocytic surface components of Xenorhabdus nematophilus var. dutki and their modification by serum of nonimmune larvae of Galleria mellonella. J. Invertebr. Pathol. 58:40-51.
18. Dunphy, G. B., and J. M. Webster. 1989. The monoxenic culture of Neoplectana carpocapsae DD136 and Heterorhabditis heliothidis. Rev. Nematol. 12:113-123.
19. ffrench-Constant, R., N. Waterfield, P. Daborn, S. Joyce, H. Bennett, C. Au, A. Dowling, S. Boundy, S. Reynolds, and D. Clarke. 2003. Photorhabdus: towards a functional genomic analysis of a symbiont and pathogen. FEMS Microbiol. Rev. 26:433-456. [PubMed]
20. Ffrench-Constant, R. H., N. Waterfield, V. Burland, N. T. Perna, P. J. Daborn, D. Bowen, and F. R. Blattner. 2000. A genomic sample sequence of the entomopathogenic bacterium Photorhabdus luminescens W14: potential implications for virulence. Appl. Environ. Microbiol. 66:3310-3329. [PMC free article] [PubMed]
21. Forst, S., and D. J. Clarke. 2002. Nematode-bacterium symbiosis, p. 57-77. In R. Gaugler (ed.), Entomopathogenic nematology. CABI Publishing, Wallingford, United Kingdom.
22. Forst, S., B. Dowds, N. Boemare, and E. Stackebrandt. 1997. Xenorhabdus and Photorhabdus spp.: bugs that kill bugs. Annu. Rev. Microbiol. 51:47-72. [PubMed]
23. Garcia Vescovi, E., F. C. Soncini, and E. A. Groisman. 1994. The role of the PhoP/PhoQ regulon in Salmonella virulence. Res. Microbiol. 145:473-480. [PubMed]
24. Gillespie, J. P., M. R. Kanost, and T. Trenczek. 1997. Biological mediators of insect immunity. Annu. Rev. Entomol. 42:611-643. [PubMed]
25. Groisman, E. A. 2001. The pleiotropic two-component regulatory system PhoP-PhoQ. J. Bacteriol. 183:1835-1842. [PMC free article] [PubMed]
26. Gunn, J. S., K. B. Lim, J. Krueger, K. Kim, L. Guo, M. Hackett, and S. I. Miller. 1998. PmrA-PmrB-regulated genes necessary for 4-aminoarabinose lipid A modification and polymyxin resistance. Mol. Microbiol. 27:1171-1182. [PubMed]
27. Gunn, J. S., S. S. Ryan, J. C. Van Velkinburgh, R. K. Ernst, and S. I. Miller. 2000. Genetic and functional analysis of a PmrA-PmrB-regulated locus necessary for lipopolysaccharide modification, antimicrobial peptide resistance, and oral virulence of Salmonella enterica serovar Typhimurium. Infect. Immun. 68:6139-6146. [PMC free article] [PubMed]
28. Hentschel, U., M. Steinert, and J. Hacker. 2000. Common molecular mechanisms of symbiosis and pathogenesis. Trends Microbiol. 8:226-231. [PubMed]
29. Herrero, M., V. DeLorenzo, and K. N. Timmis. 1990. Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. J. Bacteriol. 172:6557-6567. [PMC free article] [PubMed]
30. Hoffmann, J. A. 2003. The immune response of Drosophila. Nature 426:33-38. [PubMed]
31. Hoffmann, J. A., and J. M. Reichhart. 2002. Drosophila innate immunity: an evolutionary perspective. Nat. Immunol. 3:121-126. [PubMed]
32. Joyce, S. A., and D. J. Clarke. 2003. A hexA homologue from Photorhabdus regulates pathogenicity, symbiosis and phenotypic variation. Mol. Microbiol. 47:1445-1457. [PubMed]
33. Kox, L. F., M. M. Wosten, and E. A. Groisman. 2000. A small protein that mediates the activation of a two-component system by another two-component system. EMBO J. 19:1861-1872. [PMC free article] [PubMed]
34. Lavine, M. D., and M. R. Strand. 2002. Insect hemocytes and their role in immunity. Insect Biochem. Mol. Biol. 32:1295-1309. [PubMed]
35. Martens, E. C., K. Heungens, and H. Goodrich-Blair. 2003. Early colonization events in the mutualistic association between Steinernema carpocapsae nematodes and Xenorhabdus nematophila bacteria. J. Bacteriol. 185:3147-3154. [PMC free article] [PubMed]
36. Morona, R., C. Daniels, and L. Van Den Bosch. 2003. Genetic modulation of Shigella flexneri 2a lipopolysaccharide O antigen modal chain length reveals that it has been optimized for virulence. Microbiology 149:925-939. [PubMed]
37. Murray, G. L., S. R. Attridge, and R. Morona. 2003. Regulation of Salmonella typhimurium lipopolysaccharide O antigen chain length is required for virulence; identification of FepE as a second Wzz. Mol. Microbiol. 47:1395-1406. [PubMed]
38. Nation, J. L. 2002. Insect physiology and biochemistry. CRC Press, Boca Raton, Fla.
39. Perez, E., S. Samper, Y. Bordas, C. Guilhot, B. Gicquel, and C. Martin. 2001. An essential role for phoP in Mycobacterium tuberculosis virulence. Mol. Microbiol. 41:179-187. [PubMed]
40. Raetz, C. R., and C. Whitfield. 2002. Lipopolysaccharide endotoxins. Annu. Rev. Biochem. 71:635-700. [PMC free article] [PubMed]
41. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
42. Trent, M. S., A. A. Ribeiro, S. Lin, R. J. Cotter, and C. R. Raetz. 2001. An inner membrane enzyme in Salmonella and Escherichia coli that transfers 4-amino-4-deoxy-l-arabinose to lipid A: induction on polymyxin-resistant mutants and role of a novel lipid-linked donor. J. Biol. Chem. 276:43122-43131. [PubMed]
43. Waterfield, N. R., P. J. Daborn, A. J. Dowling, G. Yang, M. Hares, and R. H. ffrench-Constant. 2003. The insecticidal toxin Makes caterpillars floppy 2 (Mcf2) shows similarity to HrmA, an avirulence protein from a plant pathogen. FEMS Microbiol. Lett. 229:265-270. [PubMed]
44. White, G. F. 1927. A method for obtaining infective nematode larva from cultures. Science 66:302-303. [PubMed]
45. Wosten, M. M., and E. A. Groisman. 1999. Molecular characterization of the PmrA regulon. J. Biol. Chem. 274:27185-27190. [PubMed]
46. Wosten, M. M., L. F. Kox, S. Chamnongpol, F. C. Soncini, and E. A. Groisman. 2000. A signal transduction system that responds to extracellular iron. Cell 103:113-125. [PubMed]

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