BACKGROUND
photorhabdus bacteria are pathogens of insects, and obligate symbionts with insect-pathogenic heterorhabditid nematodes  <cit> .
these host nematodes invade an insect and regurgitate the bacteria from their gut  <cit> .
the bacteria then colonize the infected insect and release both insecticides that kill the insect host and antibiotics to kill any invading and competing microbes  <cit> .
following several rounds of nematode and bacterial replication, a new generation of infective juvenile  nematodes re-uptake the bacteria and exit the cadaver to find new hosts  <cit> .
this dual requirement for symbiosis and virulence makes photorhabdus an excellent model organism for studying bacterial colonization and developmental behaviour in addition to a potential source of potent new insecticidal proteins and antibiotics  <cit> .
the genus photorhabdus comprises three distinct species: p. temperata, p. luminescens and p. asymbiotica.
although all three are highly pathogenic to insects, p. asymbiotica was originally isolated from human wounds and its nematode vector has only recently been identified  <cit> .
little is known about transmission into human patients, but p. asymbiotica is unique in the genus in being able to grow at 37°c and is considered an emerging human pathogen  <cit> .
in an attempt to find potential host-interacting proteins that are relevant to either human or insect infections we used two-dimensional  gel electrophoresis to compare supernatant proteins secreted at 28°c and 37°c.
we identified a number of proteins that were differentially produced at these temperatures.
two small proteins were of particular interest, because they were secreted at a very high level at 28°c but were not detectable at the clinically relevant temperature of 37°c.
one of these proteins was encoded by a gene on a plasmid found only in p. asymbiotica strains.
the other was encoded by a chromosomal gene previously identified in a proteomic study of p. luminescens tt <dig>  <cit> .
we present here the first detailed investigation into the role of this second highly secreted protein present in both p. luminescens and p. asymbiotica.
RESULTS
identification of pam by two-dimensional electrophoretic analysis of the p. asymbiotica atcc <dig> secreted proteins
given the availability of p. asymbiotica atcc <dig> genomic sequence and the ability of this strain to grow at both clinically relevant  and insect relevant  temperatures, we used proteomics to identify secreted proteins that may be important for the two different hosts.
two-dimensional gel electrophoresis of supernatant proteins revealed two small highly abundant proteins  that were secreted at 28°c but not at 37°c .
we compared the maldi-tof profiles of these proteins with a database of all the predicted proteins from the finished p. asymbiotica genome sequencing project  <cit>  for their identification.
one of these proteins, s <dig>  was found to be encoded by a gene present on the plasmids of clinical p. asymbiotica strains but absent from all p. temperata and p. luminescens strains so far examined.
this plasmid, ppau <dig>  has homology to the yersinia pestis pmt <dig> plasmid, which is essential for vectoring by the flea host.
the small s <dig> protein is similar to the ypmt <dig> c hypothetical protein which has a bacterial ig-like domain  although its function is not known.
the second protein, s <dig> , matched plu <dig> previously identified in proteomic studies of p. luminescens tt <dig>  <cit> .
in strain tt <dig>  the product of the plu <dig> gene is the most highly secreted protein, accounting for more than 30% of the total extracellular proteins.
the p. asymbiotica atcc <dig> homologue is a protein of  <dig> amino acids with a predicted mass of  <dig>  kda and a calculated isoelectric point of  <dig> .
searches of current protein databases show limited similarity to known proteins.
the best sequence match is seen between amino acids 19- <dig> of pam which show 31% identity to amino acids 10- <dig> of the  <dig>  kda component of a bacillus thuringiensis binary toxin  <cit> .
injectable insecticidal activity has been reported for pit, a protein encoded by the homologous gene of pam in p. luminescens subsp.
akhurstii strain ynd <dig>  <cit> .
we used pcr to elucidate the distribution of the s <dig> and pam genes in the genus photorhabdus .
as predicted, the gene encoding s <dig> was only seen in the plasmid-carrying p. asymbiotica isolates and is presumably of relevance only to these strains  <cit> .
an alignment of pam sequences from p. asymbiotica atcc <dig> and p. luminescens tt <dig> revealed a high level of dna homology .
we amplified and sequenced pam from  <dig> other strains of the genus photorhabdus.
sequence comparison of the predicted proteins revealed very high amino acid conservation, with  <dig> % similarity between even the most diverse sequences.
in addition, the inferred phylogeny of the pam genes from different members of the genus follows the same clade-groupings as multi-locus sequence typing data  <cit>  suggesting that pam is ancestral to the genus.
in order to facilitate further analysis of the pam protein an antibody was raised to a peptide  conserved in the pam protein family.
in vivo and in vitro production of pam
as the identification of highly-secreted pam occurred at 28°c, a temperature relevant to the infection of insect hosts, we monitored pam production over time in galleria mellonella larvae injected with either p. luminescens tt <dig>  or p. asymbiotica atcc <dig> .
we observed high levels of production in the insect host at  <dig> h post-injection which continued for a further  <dig> days, suggesting a possible role of this secreted protein in the occupation of the insect cadaver.
it is also possible that pam is produced in the insect before  <dig> h and has not been detected with our methods.
we were unable to isolate tissues within the insect for pam-specific production patterns due to internal disruption of the cadaver  <dig> h after infection.
in vitro production of pam was monitored in p. asymbiotica atcc <dig> liquid cultures, and it was first detected in supernatants by western blot after  <dig> h  <dig> min of growth in lb medium at 28°c, corresponding to the exponential phase of the culture .
pam continued to be produced throughout growth into stationary phase  and up to  <dig> day-old cultures .
as expected, no pam was released at 37°c although cell-associated pam could be detected, indicating it is synthesized but not released into the surrounding milieu.
the fact that pam protein is released only at insect-relevant temperatures and the difficulties with genetic manipulation and transformation of p. asymbiotica strain atcc <dig>  led us to make a pam knock-out strain in the well-characterized p. luminescens tt <dig>  figure 3b shows a western blot demonstrating the absence of pam in the mutant strain tt01pam.
for heterologous expression in e. coli, pam was amplified from p. asymbiotica atcc <dig> and cloned in the arabinose-inducible vector pbad <dig>  under translational control of its native shine-dalgarno region.
heterologous production of pam was confirmed by western blot .
the recombinant protein was purified using ion-exchange chromatography for further analysis .
pam does not influence insect virulence or nematode symbiosis
given pam's similarity to a part of the b. thuringiensis  <dig>  kda cry toxin and the previous insecticidal studies on the homologous pit from strain ynd <dig>  <cit> , we tested pam for toxicity to insects.
first, we compared the virulence of the tt01pam strain with the tt01rif parental strain by injection into g. mellonella using standard lt <dig> assays, where approx  <dig> cells from a diluted overnight culture were injected per insect, and  <dig> insect larvae were used per treatment.
no significant delay in insect death of the tt01pam strain  relative to the tt01rif  was observed, indicating that pam does not play a major role in insect pathogenicity.
we also injected g. mellonella and m. sexta larvae with a range of dilutions from suspensions of sonicated e. coli cells producing pam, but we saw no toxicity .
finally, to assess oral toxicity, we fed m. sexta neonate larvae with suspensions of sonicated cells producing pam.
we observed no significant differences in larval weight gain after one week  between e. coli expressing pam , e. coli control carrying the empty vector  and pbs buffer as control , indicating that pam does not cause oral toxicity or delay in feeding in m. sexta.
our data suggest no role of pam in insect virulence under the conditions tested.
we examined the ability of tt01pam to form an effective symbiosis with the host nematode heterorhabditis bacteriophora.
we saw no defect in transmission efficiencies  of tt01pam  when compared to tt <dig> wild type .
we also observed no significant differences between nematodes carrying tt01pam and those carrying tt <dig> wild type when we assessed other traits relevant for symbiosis such as: recovery from infective juveniles  to hermaphrodites  and development to second generation in vitro, repackaging of the bacteria and infection of g. mellonella with re-coupled epn-complex and emergence yield .
the results from all the aspects investigated suggest that pam is not essential either for the symbiotic stage of the photorhabdus lifecycle or for pathogenic activity in the insect.
pam binds to eps in the extracellular matrix and modifies cell attachment
to investigate the localization of pam in p. luminescens tt <dig> cells, sections of bacterial colonies were observed under transmission electron microscopy  revealing large amounts of exopolysaccharide -like matrix filling the spaces between cells .
we used immunogold localization of pam in these sections and found that the protein is associated with this extracellular material that is distributed surrounding the cells .
in tt01pam the eps-like material was still present but we did not see specific binding of the antibody , suggesting that although pam binds to the extracellular matrix, it does not significantly alter its production or general structure.
furthermore, western-blot analysis using the anti-pam antibody revealed that pam could be detected in crude eps preparations , confirming that from all the extracellular matrix components pam binds at least to eps.
our studies revealed that eps-bound pam can be released by the action of sds and salt  but not by mechanical disruption  .
as pam binds to eps and eps has been shown to be important in biofilm formation  <cit> , we investigated the possibility that pam influences the different stages of biofilm formation.
pellicle assays and biofilm growth in microscopy chambers did not show differences in mature biofilm formation between tt01rif and tt01pam .
to analyze the influence of pam on the early steps of biofilm formation, namely initial attachment, we looked at attachment of the two strains to glass coverslips when cultured ex vitro in hemolymph plasma.
as shown in figure  <dig>  the parental tt01rif cells attached in greater numbers than tt01pam to the glass surface in hemolymph, but not in lb medium or schneiders insect growth medium .
importantly, we were also able to detect pam in cell and supernatant fractions in bacteria grown in hemolymph plasma at  <dig> hours.
to study in more detail the role of pam in attachment and its adhesive properties, we used surface plasmon resonance  to measure binding to an abiotic gold surface.
first, we used washed cells in an attempt to assess the role of pam when it is bound to the eps surrounding the bacterium: tt01pam showed increased binding to the surface compared to the parental tt01rif , indicating that the presence of the protein reduces adhesion to the surface in these conditions.
similarly, in pam-expressing e. coli we observed a decrease in adhesion compared to e. coli control .
using spr to assess the effect of pam secreted into the medium, we analyzed the supernatants of cultures.
in this case we found the opposite effect: when pam, either from tt01rif or recombinant e. coli cultures, was secreted in the supernatant we observed a greater change in spr angle, indicating that in the presence of pam more material bound to the gold surface than from the supernatant of cells lacking pam, tt01pam and control e. coli .
we checked that this effect was due specifically to the presence of pam in the supernatant by blocking pam binding with addition of the anti-pam antibody .
these results suggest that secreted pam binds to surfaces, while when pam is bound to the cell surface it makes these cells less able to attach.
structural studies of pam
in order to better understand the physicochemical properties that confer on pam the ability to bind eps and influence cell attachment, we investigated the structural properties of the protein by circular dichroism  spectroscopy and differential scanning calorimetry .
cd spectra at near-uv and far-uv wavelengths were obtained for purified heterologously produced pam.
weak spectra were recorded in the near-uv, but a strong signal was obtained between  <dig> nm and  <dig> nm in the far-uv range.
a large positive maximum at  <dig> nm and negative maxima at  <dig> nm and  <dig> nm indicate that the secondary structure of the protein is largely α-helical  <cit> .
analysis of the spectra using the cdsstr variable selection method gave secondary structure estimates of 58% helix, 8% strand, 16% turns and 18% unordered structure.
the normalized root mean standard deviation  for the estimates provides a goodness-of-fit measure of the correspondence between the experimental and calculated spectra ; we obtained a nrmsd value of  <dig> , which suggests a very accurate prediction of the secondary structure.
however, this prediction depends ultimately on how closely the reference dataset proteins used to derive the calculated spectra share structural similarity to pam  <cit> .
the temperature stability of pam was measured using dsc.
energy changes in purified recombinant protein were recorded as the sample was heated at a constant rate from 20°c to 95°c.
the sample was then allowed to cool before the analysis was repeated.
the thermal transition curve measured for pam reveals two things: firstly, the protein is relatively thermostable, not undergoing a change in enthalpy until the temperature of the system was above 60°c, and reaching a transition midpoint at  <dig> °c.
above this midpoint, energy is released and the thermal profile drops toward the baseline .
secondly, upon reheating pam follows a similar profile, except for a slight shoulder between approximately 60°c and 70°c.
this shoulder is indicative of misfolding, with the protein not making all of its native contacts, but its magnitude suggests that the protein was largely able to refold to its original conformation and unfold at a rate identical to that measured in the first scan.
discussion
we have studied a previously identified protein  which in p. asymbiotica atcc <dig> is secreted in a temperature-dependent manner, suggestive of a host-specific role in insects.
in the closely related insect-only pathogen p. luminescens tt <dig>  pam has been estimated to constitute more than 30% of total secreted protein  <cit> , potentially indicating an important role in the lifestyle of this bacterium.
further sequence alignment and the inferred phylogeny of the pam genes from different photorhabdus species suggest that pam is both ancestral and conserved throughout the genus.
where variable regions in amino acid sequence do exist, they could therefore be responsible for determining functional specificity of the protein within strains.
given the characteristic dual lifecycle of photorhabdus, with both a nematode-symbiotic and a insect-pathogenic stage, the limited similarity of pam with b. thuringiensis cry <dig> insecticidal protein, and the previous insecticidal studies with pit  <cit> , the first phenotypes tested with the pam mutant were toxicity to insects and symbiotic efficiency with the bacterium's partner nematode h. bacteriophora.
interestingly, the deletion of the pam gene did not affect the ability of p. luminescens tt <dig> to support nematode growth, the production of infective juveniles, re-association of the bacteria with the worm or their ability to re-infect an insect.
similarly, we were not able to demonstrate any difference in insect survival  when g. mellonella were injected with wild-type or pam mutant strains, but this could result from the high redundancy of virulence factors in photorhabdus  <cit> .
in the case of pam recombinant protein, which did not cause toxicity either by injection or feeding assays, it is possible that pam is not toxic by itself but requires a second, as yet unidentified, protein partner that operates in a binary toxin-type system.
the closest known homolog of pam is the  <dig>  kda cry <dig> protein from b. thuringiensis, which only exerts effective mortality when coupled with its partner cry <dig>  <cit> .
the precise mode of action of cry <dig> toxins remains unclear, but susceptible insects show histopathological symptoms in the midgut epithelium, characterized by cell blebbing and vacuolation  <cit> .
we have not found any genes in photorhabdus that are predicted to encode a component similar to cry <dig>  it should be noted that our findings are contrary to reports of toxicity of purified pam protein by li and co-workers  <cit> .
it is possible that the pam variant they produced  as a gst-fusion from p. luminescens subsp.
akhurstii ynd <dig>  either has a much greater inherent toxicity to g. mellonella, or that the different method of purification used by these authors preserved pam's toxic phenotype.
the fact that we did not find any toxic effect of pam towards insects, or any decrease in the efficiency of interaction with the symbiotic nematode, led us to investigate whether it was expressed during insect infection at all.
western blots with anti-pam antibody against proteins isolated from infected insects suggested that pam was first produced at  <dig> h and not earlier during the infection process, and that it was continuously produced for at least  <dig> days after insect death.
although the possibility exists that earlier production was below the detection limit of our assay, we note that  <dig> h coincides with death of the insect as determined by lt <dig> assays.
these results indicate that pam may play a role in occupancy of the insect cadaver rather than killing of the host and are consistent with a previous study of p. luminescens genes upregulated upon insect infection, in which pam  was not present among the identified genes encoding several toxins and metabolic enzymes  <cit> .
we have detected pam both as secreted protein in the extracellular medium and bound to the eps decorating the extracellular matrix surrounding cells.
however, the observable structure of eps/matrix is not significantly altered by the presence or absence of pam.
although we observed no differences in mature biofilm, we found that pam influences the early stages of bacterial attachment in hemolymph.
spr data from e. coli and p. luminescens cultures showed that membrane-bound pam reduces the ability of cells to bind to the abiotic surface of the metallic gold of the probe, and that the secreted protein itself is able to bind to this surface.
the observation that pam expression increases binding to an abiotic surface in insect blood is in contrast to the findings from the spr analysis which suggest pam lowers the adhesive properties of the cell.
however these observed differences in attachment between the wild type and pam mutant in the hemolymph are not directly comparable with the spr data.
in the first case the cells are grown in the media where attachment is assessed and the combination of secreted and cell-bound pam contributes to the phenotype, while for spr we analyzed washed cells and supernatant separately.
furthermore, insect blood is a far more complex environment than the pbs used to resuspend the cells in the spr study, so potential interactions of pam and the bacterium with components of the insect immune system must be considered.
together, these data indicate that pam is a secreted adhesive factor that modifies the surface properties of the cell, affecting the attachment process, specifically cell-to-cell and cell-to-surface attachment.
although it is important to note that attachment to abiotic substrata is not the same as attachment to living or devitalized tissue, we believe that this modification of adhesion by pam may be involved in one or several processes key to the biology of the bacterium.
for instance, once photorhabdus has been regurgitated by ij nematodes, it must colonize and invade the midgut  <cit>  and this establishment of a biofilm, following attachment, is recognized as an important step in many microbial infections  <cit> .
since the effect of deleting pam does not result in a complete gain or loss of attachment, the protein may allow some plasticity in colonization during the infection.
perhaps pam allows adhesion to specific tissues or a transient attachment, or even to facilitate the release of cells from biofilms to colonize other tissues within the host, acting in an analogous manner to glycanases described other biofilm-forming bacteria  <cit> .
the predicted amino acid sequence of pam gives little clue to its role or of the potential structure that mediates its adhesive properties.
to get an insight into the structure of pam, we analyzed the protein with circular dichroism spectroscopy.
our far-uv cd data strongly indicate that pam is a helical protein, with  <dig>  helix segments per  <dig> residues and an average helix length of  <dig>  residues.
by contrast, only 8% of residues are expected to form β-strands.
we obtained only very weak spectra for pam in the near-uv wavelengths, but 1d 1h and 2d 1h-15n hsqc nmr spectra  and high melting temperature from differential scanning calorimetry experiments confirm that the protein has well defined tertiary structure.
a degree of tertiary structural prediction is available from the far-uv spectra, specifically the position of the spectral cross-over from positive to negative, and the magnitude of the negative maximum at  <dig> nm  <cit> .
these both suggest that pam is a α+β protein.
rather than having intermixed segments, such proteins have separate α-helix and β-sheet-rich regions  <cit> .
interestingly, although pam is not secreted at 37°c in p. asymbiotica, it shows thermal stability far beyond this.
differential scanning calorimetry revealed that the protein does not begin to thermally denature until heated to temperatures above 60°c.
the transition midpoint is  <dig> °c, suggesting that pam is particularly thermostable for a protein produced by an organism considered to be psychrophilic  <cit> .
in fact, this midpoint is approaching that seen in thermophilic bacteria and archaea  <cit> .
without high resolution structural analyses we are unable to explore precise contributions to the thermal stability of pam, but the high α-helix content is likely to be significant; thermostable proteins are richer in α-helices than mesophilic proteins  <cit> .
the observed ability of pam to refold to its native conformation following denaturation may be biologically significant; this folding indicates that the protein can form its native structure in the absence of molecular chaperones, outside of the cell if it is secreted as an unfolded polypeptide.
it is as yet not clear how pam is secreted from the cell as we can detect no recognizable signal motifs, neither were found in pit  <cit> .
finally, although the role of this highly secreted protein in photorhabdus biology has not yet been completely elucidated, we have shown its possible relevance in cell attachment.
our findings indicate that pam is a secreted adhesive factor of photorhabdus that modifies attachment of cells to surfaces in biotic  and abiotic  conditions.
thus, it might be involved at different cell-cell and cell-surface adhesion stages during the insect host colonization, such as the production of a biofilm-like matrix on the insect gut, the spread of bacteria within the insect cadaver and potentially a resource protection role, binding to the insect tissues and preventing other saprophites from taking advantage of the biomass.
the high levels of secretion and the degree of conservation within the genus are congruent with pam modulating these important activities.
very little is known about photorhabdus infections in humans, but a recent study has found that, unlike the extracellular growth of p. luminescens in insects  <cit> , a clinical isolate of p. asymbiotica is a facultative intracellular pathogen when incubated with human macrophage-like cells  <cit> .
future studies may investigate what role if any pam has in the infection of mammalian cells.
CONCLUSIONS
in this study we show that the highly abundant pam protein is able to bind to exopolysaccharides and change the attachment properties of photorhabdus.
deletion of pam altered bacterial adhesion to surfaces but did not cause a decrease in virulence towards galleria mellonella larvae.
however, pam is produced during insect infection suggesting a role for this protein in the insect cadaver, possibly in the colonization of the insect body.
sequence analysis of pam in multiple isolates showed that it is ancestral and conserved in the genus photorhabdus and thus deserves further investigations to clarify its role in the complex cycle of photorhabdus biology.

 photorhabdus bacteria are pathogens of insects, and obligate symbionts with insect-pathogenic heterorhabditid nematodes. these host nematodes invade an insect and regurgitate the bacteria from their gut. the bacteria then colonize the infected insect and release both insecticides that kill the insect host and antibiotics to kill any invading and competing microbes. following several rounds of nematode and bacterial replication, a new generation of infective juvenile nematodes re-uptake the bacteria and exit the cadaver to find new hosts. this dual requirement for symbiosis and virulence makes photorhabdus an excellent model organism for studying bacterial colonization and developmental behaviour in addition to a potential source of potent new insecticidal proteins and antibiotics. the genus photorhabdus comprises three distinct species: p. temperata, p. luminescens and p. asymbiotica. although all three are highly pathogenic to insects, p. asymbiotica was originally isolated from human wounds and its nematode vector has only recently been identified. little is known about transmission into human patients, but p. asymbiotica is unique in the genus in being able to grow at 37°c and is considered an emerging human pathogen. in an attempt to find potential host-interacting proteins that are relevant to either human or insect infections we used two-dimensional gel electrophoresis to compare supernatant proteins secreted at 28°c and 37°c. we identified a number of proteins that were differentially produced at these temperatures. two small proteins were of particular interest, because they were secreted at a very high level at 28°c but were not detectable at the clinically relevant temperature of 37°c. one of these proteins was encoded by a gene on a plasmid found only in p. asymbiotica strains. the other was encoded by a chromosomal gene previously identified in a proteomic study of p. luminescens tt. we present here the first detailed investigation into the role of this second highly secreted protein present in both p. luminescens and p. asymbiotica.  identification of pam by two-dimensional electrophoretic analysis of the p. asymbiotica atcc secreted proteins given the availability of p. asymbiotica atcc genomic sequence and the ability of this strain to grow at both clinically relevant and insect relevant temperatures, we used proteomics to identify secreted proteins that may be important for the two different hosts. two-dimensional gel electrophoresis of supernatant proteins revealed two small highly abundant proteins that were secreted at 28°c but not at 37°c. we compared the maldi-tof profiles of these proteins with a database of all the predicted proteins from the finished p. asymbiotica genome sequencing project for their identification. one of these proteins, s was found to be encoded by a gene present on the plasmids of clinical p. asymbiotica strains but absent from all p. temperata and p. luminescens strains so far examined. this plasmid, ppau has homology to the yersinia pestis pmt plasmid, which is essential for vectoring by the flea host. the small s protein is similar to the ypmt c hypothetical protein which has a bacterial ig-like domain although its function is not known. the second protein, s, matched plu previously identified in proteomic studies of p. luminescens tt. in strain tt the product of the plu gene is the most highly secreted protein, accounting for more than 30% of the total extracellular proteins. the p. asymbiotica atcc homologue is a protein of amino acids with a predicted mass of kda and a calculated isoelectric point of. searches of current protein databases show limited similarity to known proteins. the best sequence match is seen between amino acids 19- of pam which show 31% identity to amino acids 10- of the kda component of a bacillus thuringiensis binary toxin. injectable insecticidal activity has been reported for pit, a protein encoded by the homologous gene of pam in p. luminescens subsp. akhurstii strain ynd. we used pcr to elucidate the distribution of the s and pam genes in the genus photorhabdus. as predicted, the gene encoding s was only seen in the plasmid-carrying p. asymbiotica isolates and is presumably of relevance only to these strains. an alignment of pam sequences from p. asymbiotica atcc and p. luminescens tt revealed a high level of dna homology. we amplified and sequenced pam from other strains of the genus photorhabdus. sequence comparison of the predicted proteins revealed very high amino acid conservation, with % similarity between even the most diverse sequences. in addition, the inferred phylogeny of the pam genes from different members of the genus follows the same clade-groupings as multi-locus sequence typing data suggesting that pam is ancestral to the genus. in order to facilitate further analysis of the pam protein an antibody was raised to a peptide conserved in the pam protein family. in vivo and in vitro production of pam as the identification of highly-secreted pam occurred at 28°c, a temperature relevant to the infection of insect hosts, we monitored pam production over time in galleria mellonella larvae injected with either p. luminescens tt or p. asymbiotica atcc. we observed high levels of production in the insect host at h post-injection which continued for a further days, suggesting a possible role of this secreted protein in the occupation of the insect cadaver. it is also possible that pam is produced in the insect before h and has not been detected with our methods. we were unable to isolate tissues within the insect for pam-specific production patterns due to internal disruption of the cadaver h after infection. in vitro production of pam was monitored in p. asymbiotica atcc liquid cultures, and it was first detected in supernatants by western blot after h min of growth in lb medium at 28°c, corresponding to the exponential phase of the culture. pam continued to be produced throughout growth into stationary phase and up to day-old cultures. as expected, no pam was released at 37°c although cell-associated pam could be detected, indicating it is synthesized but not released into the surrounding milieu. the fact that pam protein is released only at insect-relevant temperatures and the difficulties with genetic manipulation and transformation of p. asymbiotica strain atcc led us to make a pam knock-out strain in the well-characterized p. luminescens tt figure 3b shows a western blot demonstrating the absence of pam in the mutant strain tt01pam. for heterologous expression in e. coli, pam was amplified from p. asymbiotica atcc and cloned in the arabinose-inducible vector pbad under translational control of its native shine-dalgarno region. heterologous production of pam was confirmed by western blot. the recombinant protein was purified using ion-exchange chromatography for further analysis. pam does not influence insect virulence or nematode symbiosis given pam's similarity to a part of the b. thuringiensis kda cry toxin and the previous insecticidal studies on the homologous pit from strain ynd, we tested pam for toxicity to insects. first, we compared the virulence of the tt01pam strain with the tt01rif parental strain by injection into g. mellonella using standard lt assays, where approx cells from a diluted overnight culture were injected per insect, and insect larvae were used per treatment. no significant delay in insect death of the tt01pam strain relative to the tt01rif was observed, indicating that pam does not play a major role in insect pathogenicity. we also injected g. mellonella and m. sexta larvae with a range of dilutions from suspensions of sonicated e. coli cells producing pam, but we saw no toxicity. finally, to assess oral toxicity, we fed m. sexta neonate larvae with suspensions of sonicated cells producing pam. we observed no significant differences in larval weight gain after one week between e. coli expressing pam, e. coli control carrying the empty vector and pbs buffer as control, indicating that pam does not cause oral toxicity or delay in feeding in m. sexta. our data suggest no role of pam in insect virulence under the conditions tested. we examined the ability of tt01pam to form an effective symbiosis with the host nematode heterorhabditis bacteriophora. we saw no defect in transmission efficiencies of tt01pam when compared to tt wild type. we also observed no significant differences between nematodes carrying tt01pam and those carrying tt wild type when we assessed other traits relevant for symbiosis such as: recovery from infective juveniles to hermaphrodites and development to second generation in vitro, repackaging of the bacteria and infection of g. mellonella with re-coupled epn-complex and emergence yield. the results from all the aspects investigated suggest that pam is not essential either for the symbiotic stage of the photorhabdus lifecycle or for pathogenic activity in the insect. pam binds to eps in the extracellular matrix and modifies cell attachment to investigate the localization of pam in p. luminescens tt cells, sections of bacterial colonies were observed under transmission electron microscopy revealing large amounts of exopolysaccharide -like matrix filling the spaces between cells. we used immunogold localization of pam in these sections and found that the protein is associated with this extracellular material that is distributed surrounding the cells. in tt01pam the eps-like material was still present but we did not see specific binding of the antibody, suggesting that although pam binds to the extracellular matrix, it does not significantly alter its production or general structure. furthermore, western-blot analysis using the anti-pam antibody revealed that pam could be detected in crude eps preparations, confirming that from all the extracellular matrix components pam binds at least to eps. our studies revealed that eps-bound pam can be released by the action of sds and salt but not by mechanical disruption. as pam binds to eps and eps has been shown to be important in biofilm formation, we investigated the possibility that pam influences the different stages of biofilm formation. pellicle assays and biofilm growth in microscopy chambers did not show differences in mature biofilm formation between tt01rif and tt01pam. to analyze the influence of pam on the early steps of biofilm formation, namely initial attachment, we looked at attachment of the two strains to glass coverslips when cultured ex vitro in hemolymph plasma. as shown in figure the parental tt01rif cells attached in greater numbers than tt01pam to the glass surface in hemolymph, but not in lb medium or schneiders insect growth medium. importantly, we were also able to detect pam in cell and supernatant fractions in bacteria grown in hemolymph plasma at hours. to study in more detail the role of pam in attachment and its adhesive properties, we used surface plasmon resonance to measure binding to an abiotic gold surface. first, we used washed cells in an attempt to assess the role of pam when it is bound to the eps surrounding the bacterium: tt01pam showed increased binding to the surface compared to the parental tt01rif, indicating that the presence of the protein reduces adhesion to the surface in these conditions. similarly, in pam-expressing e. coli we observed a decrease in adhesion compared to e. coli control. using spr to assess the effect of pam secreted into the medium, we analyzed the supernatants of cultures. in this case we found the opposite effect: when pam, either from tt01rif or recombinant e. coli cultures, was secreted in the supernatant we observed a greater change in spr angle, indicating that in the presence of pam more material bound to the gold surface than from the supernatant of cells lacking pam, tt01pam and control e. coli. we checked that this effect was due specifically to the presence of pam in the supernatant by blocking pam binding with addition of the anti-pam antibody. these results suggest that secreted pam binds to surfaces, while when pam is bound to the cell surface it makes these cells less able to attach. structural studies of pam in order to better understand the physicochemical properties that confer on pam the ability to bind eps and influence cell attachment, we investigated the structural properties of the protein by circular dichroism spectroscopy and differential scanning calorimetry. cd spectra at near-uv and far-uv wavelengths were obtained for purified heterologously produced pam. weak spectra were recorded in the near-uv, but a strong signal was obtained between nm and nm in the far-uv range. a large positive maximum at nm and negative maxima at nm and nm indicate that the secondary structure of the protein is largely α-helical. analysis of the spectra using the cdsstr variable selection method gave secondary structure estimates of 58% helix, 8% strand, 16% turns and 18% unordered structure. the normalized root mean standard deviation for the estimates provides a goodness-of-fit measure of the correspondence between the experimental and calculated spectra; we obtained a nrmsd value of, which suggests a very accurate prediction of the secondary structure. however, this prediction depends ultimately on how closely the reference dataset proteins used to derive the calculated spectra share structural similarity to pam. the temperature stability of pam was measured using dsc. energy changes in purified recombinant protein were recorded as the sample was heated at a constant rate from 20°c to 95°c. the sample was then allowed to cool before the analysis was repeated. the thermal transition curve measured for pam reveals two things: firstly, the protein is relatively thermostable, not undergoing a change in enthalpy until the temperature of the system was above 60°c, and reaching a transition midpoint at °c. above this midpoint, energy is released and the thermal profile drops toward the baseline. secondly, upon reheating pam follows a similar profile, except for a slight shoulder between approximately 60°c and 70°c. this shoulder is indicative of misfolding, with the protein not making all of its native contacts, but its magnitude suggests that the protein was largely able to refold to its original conformation and unfold at a rate identical to that measured in the first scan. discussion we have studied a previously identified protein which in p. asymbiotica atcc is secreted in a temperature-dependent manner, suggestive of a host-specific role in insects. in the closely related insect-only pathogen p. luminescens tt pam has been estimated to constitute more than 30% of total secreted protein, potentially indicating an important role in the lifestyle of this bacterium. further sequence alignment and the inferred phylogeny of the pam genes from different photorhabdus species suggest that pam is both ancestral and conserved throughout the genus. where variable regions in amino acid sequence do exist, they could therefore be responsible for determining functional specificity of the protein within strains. given the characteristic dual lifecycle of photorhabdus, with both a nematode-symbiotic and a insect-pathogenic stage, the limited similarity of pam with b. thuringiensis cry insecticidal protein, and the previous insecticidal studies with pit, the first phenotypes tested with the pam mutant were toxicity to insects and symbiotic efficiency with the bacterium's partner nematode h. bacteriophora. interestingly, the deletion of the pam gene did not affect the ability of p. luminescens tt to support nematode growth, the production of infective juveniles, re-association of the bacteria with the worm or their ability to re-infect an insect. similarly, we were not able to demonstrate any difference in insect survival when g. mellonella were injected with wild-type or pam mutant strains, but this could result from the high redundancy of virulence factors in photorhabdus. in the case of pam recombinant protein, which did not cause toxicity either by injection or feeding assays, it is possible that pam is not toxic by itself but requires a second, as yet unidentified, protein partner that operates in a binary toxin-type system. the closest known homolog of pam is the kda cry protein from b. thuringiensis, which only exerts effective mortality when coupled with its partner cry. the precise mode of action of cry toxins remains unclear, but susceptible insects show histopathological symptoms in the midgut epithelium, characterized by cell blebbing and vacuolation. we have not found any genes in photorhabdus that are predicted to encode a component similar to cry it should be noted that our findings are contrary to reports of toxicity of purified pam protein by li and co-workers. it is possible that the pam variant they produced as a gst-fusion from p. luminescens subsp. akhurstii ynd either has a much greater inherent toxicity to g. mellonella, or that the different method of purification used by these authors preserved pam's toxic phenotype. the fact that we did not find any toxic effect of pam towards insects, or any decrease in the efficiency of interaction with the symbiotic nematode, led us to investigate whether it was expressed during insect infection at all. western blots with anti-pam antibody against proteins isolated from infected insects suggested that pam was first produced at h and not earlier during the infection process, and that it was continuously produced for at least days after insect death. although the possibility exists that earlier production was below the detection limit of our assay, we note that h coincides with death of the insect as determined by lt assays. these results indicate that pam may play a role in occupancy of the insect cadaver rather than killing of the host and are consistent with a previous study of p. luminescens genes upregulated upon insect infection, in which pam was not present among the identified genes encoding several toxins and metabolic enzymes. we have detected pam both as secreted protein in the extracellular medium and bound to the eps decorating the extracellular matrix surrounding cells. however, the observable structure of eps/matrix is not significantly altered by the presence or absence of pam. although we observed no differences in mature biofilm, we found that pam influences the early stages of bacterial attachment in hemolymph. spr data from e. coli and p. luminescens cultures showed that membrane-bound pam reduces the ability of cells to bind to the abiotic surface of the metallic gold of the probe, and that the secreted protein itself is able to bind to this surface. the observation that pam expression increases binding to an abiotic surface in insect blood is in contrast to the findings from the spr analysis which suggest pam lowers the adhesive properties of the cell. however these observed differences in attachment between the wild type and pam mutant in the hemolymph are not directly comparable with the spr data. in the first case the cells are grown in the media where attachment is assessed and the combination of secreted and cell-bound pam contributes to the phenotype, while for spr we analyzed washed cells and supernatant separately. furthermore, insect blood is a far more complex environment than the pbs used to resuspend the cells in the spr study, so potential interactions of pam and the bacterium with components of the insect immune system must be considered. together, these data indicate that pam is a secreted adhesive factor that modifies the surface properties of the cell, affecting the attachment process, specifically cell-to-cell and cell-to-surface attachment. although it is important to note that attachment to abiotic substrata is not the same as attachment to living or devitalized tissue, we believe that this modification of adhesion by pam may be involved in one or several processes key to the biology of the bacterium. for instance, once photorhabdus has been regurgitated by ij nematodes, it must colonize and invade the midgut and this establishment of a biofilm, following attachment, is recognized as an important step in many microbial infections. since the effect of deleting pam does not result in a complete gain or loss of attachment, the protein may allow some plasticity in colonization during the infection. perhaps pam allows adhesion to specific tissues or a transient attachment, or even to facilitate the release of cells from biofilms to colonize other tissues within the host, acting in an analogous manner to glycanases described other biofilm-forming bacteria. the predicted amino acid sequence of pam gives little clue to its role or of the potential structure that mediates its adhesive properties. to get an insight into the structure of pam, we analyzed the protein with circular dichroism spectroscopy. our far-uv cd data strongly indicate that pam is a helical protein, with helix segments per residues and an average helix length of residues. by contrast, only 8% of residues are expected to form β-strands. we obtained only very weak spectra for pam in the near-uv wavelengths, but 1d 1h and 2d 1h-15n hsqc nmr spectra and high melting temperature from differential scanning calorimetry experiments confirm that the protein has well defined tertiary structure. a degree of tertiary structural prediction is available from the far-uv spectra, specifically the position of the spectral cross-over from positive to negative, and the magnitude of the negative maximum at nm. these both suggest that pam is a α+β protein. rather than having intermixed segments, such proteins have separate α-helix and β-sheet-rich regions. interestingly, although pam is not secreted at 37°c in p. asymbiotica, it shows thermal stability far beyond this. differential scanning calorimetry revealed that the protein does not begin to thermally denature until heated to temperatures above 60°c. the transition midpoint is °c, suggesting that pam is particularly thermostable for a protein produced by an organism considered to be psychrophilic. in fact, this midpoint is approaching that seen in thermophilic bacteria and archaea. without high resolution structural analyses we are unable to explore precise contributions to the thermal stability of pam, but the high α-helix content is likely to be significant; thermostable proteins are richer in α-helices than mesophilic proteins. the observed ability of pam to refold to its native conformation following denaturation may be biologically significant; this folding indicates that the protein can form its native structure in the absence of molecular chaperones, outside of the cell if it is secreted as an unfolded polypeptide. it is as yet not clear how pam is secreted from the cell as we can detect no recognizable signal motifs, neither were found in pit. finally, although the role of this highly secreted protein in photorhabdus biology has not yet been completely elucidated, we have shown its possible relevance in cell attachment. our findings indicate that pam is a secreted adhesive factor of photorhabdus that modifies attachment of cells to surfaces in biotic and abiotic conditions. thus, it might be involved at different cell-cell and cell-surface adhesion stages during the insect host colonization, such as the production of a biofilm-like matrix on the insect gut, the spread of bacteria within the insect cadaver and potentially a resource protection role, binding to the insect tissues and preventing other saprophites from taking advantage of the biomass. the high levels of secretion and the degree of conservation within the genus are congruent with pam modulating these important activities. very little is known about photorhabdus infections in humans, but a recent study has found that, unlike the extracellular growth of p. luminescens in insects, a clinical isolate of p. asymbiotica is a facultative intracellular pathogen when incubated with human macrophage-like cells. future studies may investigate what role if any pam has in the infection of mammalian cells.  in this study we show that the highly abundant pam protein is able to bind to exopolysaccharides and change the attachment properties of photorhabdus. deletion of pam altered bacterial adhesion to surfaces but did not cause a decrease in virulence towards galleria mellonella larvae. however, pam is produced during insect infection suggesting a role for this protein in the insect cadaver, possibly in the colonization of the insect body. sequence analysis of pam in multiple isolates showed that it is ancestral and conserved in the genus photorhabdus and thus deserves further investigations to clarify its role in the complex cycle of photorhabdus biology. 
our findings indicate that pam plays an important role in enhancing surface attachment in insect blood. 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. interestingly, surface plasmon resonance binding studies revealed that the pam protein itself has adhesive properties. the pam protein shows limited sequence similarity to the kda component of a binary toxin of bacillus thuringiensis. 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. in bacterial colonies, pam is associated with an extracellular polysaccharide -like matrix, and modifies the ability of wild-type cells to attach to an artificial surface. 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. a comparison of the proteins secreted by a clinical isolate of p. asymbiotica at simulated insect and human temperatures led to the identification of a small and highly abundant protein, designated pam, that is only secreted at the lower temperature. we studied a highly abundant protein, pam, which is secreted in a temperature-dependent manner in p. asymbiotica.
 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.  a comparison of the proteins secreted by a clinical isolate of p. asymbiotica at simulated insect and human 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 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 -like matrix, and modifies the ability of wild-type cells to attach to an artificial surface. interestingly, surface plasmon resonance 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.  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. 