Identification of genes involved in the mutualistic colonization of the nematode Heterorhabditis bacteriophora by the bacterium Photorhabdus luminescens
© Easom et al; licensee BioMed Central Ltd. 2010
Received: 19 October 2009
Accepted: 11 February 2010
Published: 11 February 2010
Photorhabdus are Gram negative entomopathogenic bacteria that also have a mutualistic association with nematodes from the family Heterorhabditis. An essential part of this symbiosis is the ability of the bacterium to colonize the gut of the freeliving form of the nematode called the infective juvenile (IJ). Although the colonization process (also called transmission) has been described phenomonologically very little is known about the underlying molecular mechanisms. Therefore, in this study, we were interested in identifying genes in Photorhabdus that are important for IJ colonization.
In this work we genetically tagged P. luminescens TT01 with gfp and constructed a library containing over 3200 mutants using the suicide vector, pUT-Km2. Using a combination of in vitro symbiosis assays and fluorescent microscopy we screened this library for mutants that were affected in their ability to colonize the IJ i.e. with decreased transmission frequencies. In total 8 mutants were identified with transmission frequencies of ≤ 30% compared to wild-type. These mutants were mapped to 6 different genetic loci; the pbgPE operon, galE, galU, proQ, asmA and hdfR. The pbgPE, galE and galU mutants were all predicted to be involved in LPS biosynthesis and, in support of this, we have shown that these mutants are avirulent and sensitive to the cationic antimicriobial peptide, polymyxin B. On the other hand the proQ, asmA and hdfR mutants were not affected in virulence and were either as resistant (proQ) or slightly more sensitive (asmA, hdfR) to polymyxin B than the wild-type (WT).
This is the first report describing the outcome of a comprehensive screen looking for transmission mutants in Photorhabdus. In total 6 genetic loci were identified and we present evidence that all of these loci are involved in the assembly and/or maintenance of LPS and other factors associated with the cell surface. Interestingly several, but not all, of the transmission mutants identified were also avirulent suggesting that there is a significant, but not complete, genetic overlap between pathogenicity and mutualism. Therefore, this study highlights the importance of the cell surface in mediating the symbiotic and pathogenic interactions of Photorhabdus.
Photorhabdus are a genus of bioluminescent, entomopathogenic bacteria that are members of the family Enterobacteriaceae and are thus closely related to Escherichia coli and other important mammalian pathogens. As part of their normal life-cycle Photorhabdus also have a mutualistic interaction with nematodes from the family Heterorhabditis (for a recent review see ). The bacteria are normally found colonizing the gut of the infective juvenile (IJ) stage of the nematode. The IJ is the free-living infective stage of the nematode that is found in the soil and actively searches for potential insect larvae to infect. Once identified the IJ enters the insect through natural openings such as the mouth, anus or spiracles or the IJ can use a small tooth-like appendage to tear the cuticle and gain direct entry into the hemolymph. Once inside the insect the IJ migrates to the hemolymph where unidentified signals stimulate the IJ to regurgitate the bacteria. The bacteria avoid the insect immune response and grow exponentially within the insect until the insect succumbs to septicimeia within 48-72 h of infection . At this point all of the internal organs of the insect have been converted into bacterial biomass. This bioconversion is facilitated by a range of hydrolytic enzymes that are secreted by Photorhabdus, including proteases and lipases. In the presence of high densities of Photorhabdus the IJ is stimulated to recover to a self-fertile adult hermaphrodite and this is the start of nematode reproduction. The hermaphrodite lays eggs and the developing nematode larvae feed on the bacteria present in the insect. As in Caenorhabditis elegans, the Heterorhabditis nematodes develop through 4 juvenile stages (J1-J4) before becoming adults . Nematode reproduction continues for 2-3 generations until unidentified environmental stimuli triggers the formation of an alternative J3 nematode, the IJ, which exits the insect cadaver. Before leaving the insect cadaver the new IJ must be colonized by Photorhabdus and transmission of the bacteria to the IJ is a complex process that has only recently been phenomonologically described . There are 2 striking features associated with the transmission process: 1) the colonization of the rectal gland cells of the adult hermaphrodite by Photorhabdus and 2) the observation that all IJs develop inside the adult hermaphrodite in a process called endotokia matricida. Therefore the bacteria that colonize the adult hermaphrodite are ultimately responsible for the colonization of the IJ .
The molecular mechanisms underlying the transmission process are poorly understood. In the only previous published study that reports a gene involved in transmission it was shown that a mutation in a gene annotated as pbgE1 severely affects the ability of Photorhabdus to colonize the IJ . This mutant was isolated during a screen for genes affecting swimming motility and the pbgE1 mutant was also shown to be severely attenuated in virulence. The pbgE1 gene is predicted to be part of a 7 gene pbgPE operon that is homologous to the arn operon in Salmonella. The arn operon has been shown to be involved in the modification of the lipid A moiety of LPS with L-aminoarabinose in response to the presence of cationic antimicrobial peptides (CAMPs) [6–8]. The pbgE1 mutant did produce altered LPS compared to the wild-type implicating LPS structure as a nematode colonization factor in Photorhabdus. In this study we screened a library of Photorhabdus mutants with the aim of extending our understanding of the transmission process by identifying genes important in the colonization of the H. bacteriophora IJ nematode by P. luminescens TT01.
Construction of a GFP-tagged strain of P. luminescens TT01
Identification of TT01gfp mutants affected in colonization of the IJ
Colonization mutants identified in this study.
Attachment of mutants to abiotic surfaces
Virulence of mutants to insect larvae
Sensitivity of mutants to polymyxin B
In this study we screened over 3000 mutants of a gfp-tagged strain of P. luminescens TT01 for mutants that were reduced in their ability to colonize the guts of the IJ nematode i.e. transmission mutants. In total we identified 8 mutants in 6 different genetic loci: the pbgPE operon, galE, galU, asmA, hdfR and proQ. The transmission frequency of the identified mutants was between 10-30% indicating that none of these genes were required for colonization but, rather, somehow the genes improved the ability of the bacteria to colonize the IJ. Moreover, in those IJs that were colonized, the level of fluorescence observed suggested that the nematodes were carrying the full population of bacteria (data not shown). However we did not test for this directly by crushing and plating individual IJs.
The pbgPE operon is predicted to contain 7 genes, pbgP1234pbgE123, and in this study we identified mutations in both pbgE2 and pbgE3 that were affected in their ability to colonize nematodes. This work confirms an earlier study where we reported that a mutation in pbgE1 was important for both insect virulence and colonization of the IJ . In this previous study we reported that the level of colonization of the pbgE1 mutant was 1% of the wild-type, in contrast to the 10-30% level reported here for the different mutations identified in the pbgPE operon (see Table 1). The reason(s) for this difference is not clear but it is nonetheless evident that the pbgPE operon plays an important role in the colonization of both the insect and the nematode.
In this study we demonstrated that mutations in galU and galE were affected in their ability to colonize the IJ. These genes are predicted to be involved in the biosynthesis of UDP-glucose and UDP-galactose, respectively, important precursors in the production of polysaccharides. The galU gene is predicted to encode glucose-1-phosphate uridyltransferase and is required for the production of UDP-glucose, an important glucosyl donor in the cell. In Salmonella UDP-glucose is required for the production of UDP-arabinose which is used to synthesise L-aminoarabinose for the modification of lipid A in response to CAMPs . We have shown that the galU mutant does phenocopy the pbgE2 mutation suggesting that the galU defect may be explained by the associated defects in L-aminoarabinose biosynthesis. However we have also shown that, in contrast to the pbgE2 mutant, the galU mutant is defective in attachment to abiotic surfaces (see Figure 3) suggesting that the galU mutation is pleitropic. Indeed, in E. coli, a mutation in galU would also be expected to prevent production of the LPS-associated O-antigen . In addition to LPS synthesis, UDPglucose also plays a role in protecting E. coli against thermal and osmotic shocks (through the production of trehalose and membrane-derived oligosaccharides (MDO)) and the negative regulation of σS, the stationary-phase sigma factor [21, 22].
However we have shown that σS is not required for either virulence or IJ colonization by P. luminescens (R. J. Watson and D. J. Clarke, unpublished data) implying that UDP-glucose is important in colonization through its role in polysaccharide biosynthesis. The galE gene is predicted to encode UDP-glucose-4-epimerase, an enzyme responsible for the interconversion of UDP-glucose and UDP-galactose. P. luminescens does not catabolise galactose (our unpublished data) suggesting that the main role of GalE is in the production of UDP-galactose from UDP-glucose. In E. coli both galE and galU are required for the production of LPS O-antigen  and, although the structure of the O-antigen is not known in Photorhabdus, it seems plausible that both UDP-glucose and UDP-galactose will be required for O-antigen biosynthesis. Indeed, given that the galU and galE mutants in P. luminescens are both avirulent to insects, sensitive to CAMPs and defective in colonization of the IJ, it seems likely that these mutants are affected in the same pathway i.e. LPS biosynthesis. Nonetheless it is interesting to note that, in contrast to the galU mutant, the galE mutant is not affected in attachment to an abiotic surface (see Figure 3). However this can be simply explained if, as expected, mutations in galE and galU (i.e. UDP-glucose and UDP-galactose) result in slightly different LPS molecules.
Our identification of mutants in pbgPE, galE and galU clearly implicates LPS as an important player in the colonization of the IJ by Photorhabdus. In this study we also identified mutations in genes that were not directly associated with LPS metabolism; asmA, hdfR and proQ. The asmA gene was originally identified in E. coli as a site for suppressor mutations of an assembly defective porin, OmpF315 . Although the role of AsmA is still not clear it is likley that this protein is involved in organising the outer membrane. In the first instance a mutation in asmA has been shown to result in reduced levels of LPS in the outer membrane of E. coli. In addition a recent study reported that a mutation in asmA in Salmonella enterica serovar Typhimurium resulted in a remodelling of the outer membrane that resulted in an increase in the transcription of marAB, encoding a multi-drug efflux pump . The authors further report that the S. enterica asmA mutant was attenuated in virulence when administered orally to mice and showed a reduced ability to invade epithelial cells thus linking asmA with infection . The hdfR gene was originally annotated as 2 overlapping genes, yifA and pssR, on the E. coli genome but recent analysis confirmed the presence of a sequencing error that resulted in a frameshift and the subsequent mis-annotation [14, 25]. The hdfR gene is predicted to encode a LysR-type regulator that represses the expression of flhDC, and therefore motility, in E. coli. In Proteus mirabilis 2 independent mutations in hdfR were identified in a STM experiment as being important for urinary tract colonization in mice . Motility has been shown to play an important role in P. mirabilis virulence however a role for hdfR in regulating motility in Proteus has not been determined . Interestingly we have shown that the hdfR mutant does not appear to affect swimming motility in P. luminescens (data not shown). Finally we identified a mutation in the proQ gene. This gene is predicted to encode a protein that, in E. coli, is involved in the post-translational activation of ProP, an osmoprotectant/proton symporter that is capable of transporting both proline and glycine betaine in response to increases in osmotic pressure [15, 16]. However the genome of P. luminescens is not predicted to encode a ProP homologue suggesting an alternative role for ProQ in Photorhabdus.
Interestingly the proQ mutant was the most affected in attachment to an abiotic surface suggesting alterations in the cell surface of the mutant (see Figure 3). However the proQ mutant was not sensitive to CAMPs suggesting that the LPS was not affected (see Figure 5). It is also noteworthy that, unlike the other mutants identified in this study, there is the possibility that the mutation in proQ has a polar affect on the downstream gene, prc (see Figure 2). The proQ and prc genes are separated by 20 bp on both the E. coli and P. luminescens genomes and proQ and prc are predicted to be on the same transcription unit in E. colihttp://ecocyc.org. The prc gene encodes a periplamsic protease called Prc or Tsp (tail-specific protease) that processes the C-terminus of FtsI (PBP3) and is required for protection from combined osmotic and thermal stress [28, 29]. Moreover Prc has been shown to interact with NlpI, a lipoprotein that has recently been shown to be involved in the attachment of adherent-invasive E. coli (bacteria associated with Crohns disease) to epithelial cells [30, 31]. In addition, in Pseudomonas aeruginosa, Prc has been implicated in the regulation of alginate production by degrading mutant forms of MucA, the anti-sigma factor that interacts with the alternative sigma factor AlgU . Therefore a decrease in the level of prc transcription may affect the surface of Photorhabdus in a way that prevents colonization of the IJ. However further experimentation is required to determine whether the proQ or prc gene (or both) are responsible for the reported phenotype.
We have identified 5 genetic loci in P. luminescens TT01 that are affected in their ability to colonize IJs of the nematode H. bacteriophora. In order to have a reduced transmission frequency it would be expected that the mutants would be affected in either their ability to infect and replicate within the adult hermpahrodite or in their ability to colonize the IJ. Preliminarly studies, using confocal laser scanning microscopy (CLSM), suggest that all of the mutants are able to infect the adult hermaphrodite (our unpublished data). Therefore the defect in colonization appears to occur at some point later during the transmission process. It has been shown that colonization of the IJ requires binding to the pre-intestinal valve cell in the immature IJ followed by growth and replication of the bacteria in the gut lumen . All of the mutants identified in this study can be implicated in the maintenance of the structure and/or remodelling the bacterial cell surface and it is, therefore, easy to envisage how mutations affecting the cell surface of P. luminescens could affect how the bacteria interact with the IJ. The exact stage and nature of the colonization defect of each mutant is currently under examination.
Bacterial strains and culture conditions
All P. luminescens strains were cultured in LB broth or on LB agar (LB broth plus 1.5% (w/v) agar) at 30°C. Unless otherwise stated all LB agar plates were supplemented with 0.1% (w/v) pyruvate. When required antibiotics were added at the following concentrations: ampicillin (Ap), 100 μg ml-1; chloramphenicol (Cm), 20 μg ml-1; gentamycin (Gm), 20 μg ml-1; kanamycin (Km), 25 μg ml-1and rifampicin (Rif), 50 μg ml-1.
Construction of gfp-tagged P. luminescens TT01
A gfp-tagged strain of P. luminescens TT01 was constructed using the Tn7-based vector, pBKminiTn7-gfp2. Overnight cultures of P. luminescens TT01 (the recipient), E. coli S-17 (λpir) carrying pBKminiTn7-gfp2 (the donor) and E. coli carrying the helper plasmid pUXBF13 (the helper) were prepared in LB broth supplemented with 10 mM MgCl2 (MgLB). The cultures were diluted to OD600 = 0.05 in 20 ml of MgLB and grown, with shaking, at 30°C. The cells were harvested at an OD600= 0.5 and mixed in a 4:1:1 (v/v/v) ratio (recipient:donor:helper) in MgLB. The mixture (total volume = 300 μl) was added to the centre of a MgLB agar plate and incubated at 30°C overnight. The following day the cells were resuspended in MgLB, plated out onto MgLBRif Cm agar plates and incubated at 30°C for 48 h. CmR AmpS KmS exconjugants were selected and colony PCR was used to confirm that the Tn7 and gfp had inserted in the expected position on the TT01 chromosome. The strain successfully tagged with gfp was renamed TT01gfp.
Generating a mutant bank via Tn5 transposon mutagenesis
The Tn5 mutants were generated by conjugating TT01gfp with E. coli S17-1 (λpir) carrying the suicide vector, pUT-Km2, as previously described . In addition to expressing gfp, the Tn7 inserted into the chromosome of TT01gfp also confers resistance to both Cm and Gm. Therefore exconjugants were selected on LB Rif Cm Km and colonies were inoculated into 1.5 mls of LBCm Km in each well of a 96 deep well plate, sealed with a gas permeable seal (Thermo scientific), and incubated overnight at 30°C. A 75 μl aliquot from each well was mixed with 75 μl of 40% (v/v) glycerol in a 96 well plate (Sterilin), sealed with an aluminium seal (Sarstedt), and frozen at -80°C.
Screening for IJ colonization mutants
The nematode is translucent thus enabling visualization of TT01gfp within the gut of the IJ using fluorescence microscopy (see Figure 1). 50 μl of an overnight culture of each TT01gfp::Tn5 mutant was used to inoculate lipid agar supplemented with Rif, Cm and Km. Plates were incubated at 30°C for 48 h before 30 surface-sterilized H. bacteriophora IJs were added to each plate [5, 35]. Symbiosis plates were incubated at 25°C for a minimum of 21 days. Next generation IJs were then washed from the surface of the Petri dish lids using 1 × PBS. An epifluorescent microscope, using blue light to excite gfp and white light to estimate number of IJs present, was used to qualitatively determine the percentage of IJs colonised in each well compared to a TT01gfp control (see Figure 1). Mutants qualitatively determined to have a transmission frequency < 50% were re-tested in triplicate. For a more quantitative estimation of transmission frequency the IJs were washed from the surface of the Petri dish lids and 10 IJ were taken, in quadruplicate, from each symbiosis plate and aliquoted into a 96 well flat-bottomed microtitre plate. Each mutant was therefore represented by 12 wells in the 96 well plate and, using epifluorescence microscope, the percentage colonization (i.e. transmission frequency) was determined per well and an average calculated for each TT01gfp::Tn5 mutant.
Attachment to an abiotic surface
The capacity of P. luminescens to form biofilms was assessed by measuring bacterial attachment to a plastic surface, as previously described . Briefly strains were grown overnight in LB broth, diluted to OD600= 0.05 in fresh LB and 200 μl of the cell suspension was aliquoted in triplicate, into the wells of a Costar® polypropylene (PP) 96-well microtitre plate. The plates were sealed with a gas permeable membrane and incubated, without shaking, at 30°C. At the appropriate time the planktonic cells were removed by aspiration and 250 μl of 0.1% (w/v) crystal violet (CV) was added to each well. The plates were incubated at room temperature for 20 min before rinsing 3 times with 1 × PBS. To quantify biofilm formation the CV was dissolved in 250 μl of 95% ethanol and the CV concentration was determined by measuring the OD595 using a Genios (Tecan) plate reader.
The pathogenicity of P. luminescens was assessed using Galleria mellonella larvae, purchased from Livefood (UK), as the model insect host. Briefly overnight cultures of P. luminescens TT01 were washed 3 times in 1 × PBS before the OD600 was adjusted to 1.0 (equivalent to 4 × 108 cfu ml-1). The culture was diluted with 1 × PBS and 10 μl (equivalent to 200 cfu) was injected into the hemolymph of a G. mellonella larva using a Hamilton syringe and a BD Microlance™ 3 30 G × 1/2" needle.
To test for sensitivity to polymyxin B overnight cultures of each strain were diluted to an OD600 = 0.05 in either fresh LB or LB with 2.5 μg ml-1 of freshly prepared polymyxin B (Sigma). From these dilutions 100 μl of each culture was inoculated, in triplicate, into wells of a 100 well Isotron honeycomb 2 plate. The plates were loaded into the Bioscreen C plate reader programmed to incubate the plates at 30°C and to take an OD600 reading every 15 minutes over a period of 24 hours.
The work outlined in this study was carried out equally in the University of Bath and University College Cork. The research was funded through the Exploiting Genomics initiative of the BBSRC in the UK (86/EGA16183) and Science Foundation Ireland. CAE is supported by a PhD fellowship from the University of Bath.
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