Genome comparisons provide insights into the role of secondary metabolites in the pathogenic phase of the Photorhabdus life cycle
© The Author(s). 2016
Received: 6 February 2016
Accepted: 29 June 2016
Published: 3 August 2016
Bacteria within the genus Photorhabdus maintain mutualistic symbioses with nematodes in complicated lifecycles that also involves insect pathogenic phases. Intriguingly, these bacteria are rich in biosynthetic gene clusters that produce compounds with diverse biological activities. As a basis to better understand the life cycles of Photorhabdus we sequenced the genomes of two recently discovered representative species and performed detailed genomic comparisons with five publically available genomes.
Here we report the genomic details of two new reference Photorhabdus species. By then conducting genomic comparisons across the genus, we show that there are several highly conserved biosynthetic gene clusters. These clusters produce a range of bioactive small molecules that support the pathogenic phase of the integral relationship that Photorhabdus maintain with nematodes.
Photorhabdus contain several genetic loci that allow them to become specialist insect pathogens by efficiently evading insect immune responses and killing the insect host.
Members of the genus Photorhabdus include both insect and human pathogens. Despite only three distinct species described to date (P. luminescens, P. temperata and P. asymbiotica), significant sequence divergence within each species has led to the identification of several subspecies [1–7]. All three species maintain complex life cycles that include a nematode mutualistic symbiont as well as a pathogenic phase. During the symbiotic phase, the bacteria colonize nematodes of the genus Heterorhabditis during the infective juvenile (IJ) stage. The nematodes are generally free living in soil and seek out insects to infect so as to utilize the nutrients for growth and perpetuation of their progeny . This is the dominant life cycle of the Photorhabdus however, occasional human infections by P. asymbiotica do occur . During the infective stage, nematodes enter the insect and release the bacteria directly into the hemolymph where the bacteria also proliferate and eventually kill the insect. The insect cadaver provides a rich source of nutrients for both the nematode and the bacteria. Following proliferation of both, the bacteria re-colonize the nematode IJs before re-entering the soil in search of a new host .
Throughout this existence, the nematodes provide the bacteria with a means of transport while the bacteria supply a variety of secondary metabolites produced by biosynthetic gene clusters (BGCs). Products of these BGCs are small molecules, frequently polyketides (PK), or non-ribosomal peptides (NRP) and can additionally include bacteriocins, siderophores and fatty acids among others. While there are common themes in their biosynthesis, each class of small molecule has a different mechanism of production and probably varying functions, with the majority of currently known metabolites reported as having some antimicrobial role [10–16]. Not all of these metabolites are required for symbiosis  so secondary metabolite biosynthesis alone - while important - does not explain the conservation of their corresponding genetic loci among closely related Photorhabdus or other entomopathogens of the genus Xenorhabdus .
The conservation of these general types of molecules led us to investigate whether there was a more generally conserved function. Through genome mining and using representative genomes from each species (and subspecies) of Photorhabdus, we compare seven different genomes in order to better understand the differences between the specific niche of each bacterium and the key analogous functions among the shared protein-coding DNA sequences (CDS).
Significant research has been conducted on Xenorhabdus species and their response to infection of insects. The role of some compounds produced by members of both genera has firmly been established as symbiotic factors [17, 19, 20] while others are predicted to be involved in this process. A role for a small number of secondary metabolites has been proposed in nematode development, however the majority of the BGCs appear to have little effect on this process (unpublished data). Following insect infection by nematodes, the bacteria are released into the insect hemolymph, quickly activating the cellular and humoral immune responses against the causative pathogens via one of two pathways, the Toll or immunodeficiency (IMD) pathways. The Toll pathway is activated in response to infection by Gram-positive bacteria and fungi using pattern-recognition receptors that respond to pathogen-associated molecular patterns [21–23]. On the other hand, Gram-negative pathogens activate the IMD pathway. This differential activation results in expression of a distinct set of genes for each in response to the type of infection occurring. However, subsets of overlapping sequences that are activated in both pathways have been identified in Drosophila and act synergistically in order to more efficiently deal with invading organisms [24, 25]. Alternatively, prophenoloxidase (proPO) pathways can be activated by exposure to lipopolysaccharides, peptidoglycan, amphiphilic lipids or even damaged cells [26, 27]. ProPO is activated through cleavage by a serine protease resulting in active phenoloxidase (PO) that assists in pathogen isolation and lysis . Several different serine protease inhibitors heavily regulate this system, as excess PO can be detrimental to the host [27, 29]. Some compounds from P. luminescens have recently been given defined roles in suppressing some parts of this insect immune response [30, 31].
One previous study has examined the similarities between P. luminescens and Yersinia enterocolitica in order to draw conclusions regarding key factors involved in insect pathogenesis . In order to determine the conserved features of members of Photorhabdus and draw more specific conclusions with respect to the essential roles of proteins in the Photorhabdus lifecycle, we sequenced two novel isolates that, together with the already sequenced genomes, provide a broad geographical and genomic perspective of the genus. Using a comparative genomic approach, we highlight mechanisms that are conserved across the genus and predict possible functions of the products of the numerous BGCs and conserved signaling pathways.
Genome composition of Photorhabdus spp. collected from Thailand
In order to establish a broad collection of Photorhabdus strains, we sequenced two additional isolates collected from Thailand . However, Thanwisai et al. did note that the bacteria grouped into five distinct clades with Group 3 still lacking a reference strain. Sequencing of Photorhabdus PB45.5 and PB68.1 now provide reference sequences for Groups 3 and 5, respectively . These Whole Genome Shotgun projects have been deposited at GenBank under the accession numbers LOIC00000000 and LOMY00000000, respectively.
Following sequencing and assembly (statistics available in Additional file 1), we performed an average nucleotide identity analysis on the genomes in order to determine the species. Photorhabdus PB68.1 was closely related to P. asymbiotica subsp. australis (ANI = 97.0 %) while Photorhabdus PB45.5 is most closely related to P. luminescens subsp. laumondii TTO1 (ANI = 91.4 %) and may represent a novel subspecies. The genomes consist of 4,918,001 and 5,425,505 bp with GC contents of 42.0 and 42.7 % respectively. P. asymbiotica PB68.1 is predicted to contain 4600 CDS whilst Photorhabdus PB45.5 contains only 4353 CDS.
Together with P. luminescens TTO1 (NC_005126) , P. temperata subsp. khanii NC19 (NZ_AYSJ00000000) [4, 34], P. temperata subsp. temperata M1021 (NZ_AUXQ00000000) , P. temperata subsp. thracencis DSM 15199 (NZ_CP011104)  and P. asymbiotica ATCC 43949 (NC_012962)  we identified ortholog families across the seven strains. During ortholog identification, all protein singletons were removed from further analysis. This analysis suggests that the core Photorhabdus genome consists of a total of 2101 CDS, 520 of which are absent in E. coli K12 (Additional file 2). Using the KAAS server , KEGG orthology numbers were assigned to the fully assembled genomes (Additional file 3) and mapping to KEGG pathways was performed (Additional file 4). No obvious differences were apparent except for a much greater number of two-component systems present in P. luminescens TTO1 (96) compared to either P. asymbiotica ATCC 43949 (87) or P. temperata subsp. thracensis DSM 15199 (84).
Biosynthetic gene clusters are numerous and diverse
Summary of Photorhabdus BGCs
P. luminescens TTO1
P. luminescens subsp. PB45.5
P. asymbiotica ATCC 43949
P. asymbiotica subsp. australis PB68.1
P. temperata subsp. thracensis
P. temperata subsp. temperata M1021
P. temperata subsp. khanii NC19
P. luminescens TTO1 contains 23 predicted BGCs with several of the products already described, many of which have reported antimicrobial activity [17, 24, 36–44]. Ten of these BGCs correlate with a core set of secondary metabolites that exists within the genus (Fig. 1). Some of these natural products are involved in development of the nematode while strains completely deficient in secondary metabolite production fail to support nematode development (Tobias, Heinrich, Eresmann, Neubacher and Bode, unpublished results). Structural similarities, compound class comparisons and proven structure-function relationships suggests that many of these remaining products have one of two main functions; cell-cell signaling or immune evasion. We suggest that the reported antimicrobial activities of some natural products may merely be a coincidental side effect of the actual compound function similar to some antibiotics . Another possibility is that the same compound might have different functions in different biological contexts as exemplified by isopropylstilbene from Photorhabdus acting as an antibiotic against fungi and bacteria , shows cytotoxic activity against insect and other eukaryotic cells  while also required for proper nematode development .
Several regions in the genomes appear to contain multiple adjacent BGCs (clusters 25, 33, 41 and 43), deduced from the presence of multiple terminal thioesterase (TE) domains that usually define the endpoint of a NRPS pathway, with three of the four present in P. temperata strains (Additional file 5). This may indicate a complementary function of the products of the BGC as seen for pristinamycin, a synergistically acting two-component antibiotic . Identifying the products and functions of those BGCs that are species-specific (Additional files 5 and 6) may provide insights into the different niches occupied by these bacteria.
Immune evasion mechanisms
Many of the remaining compounds have yet to have a definitive function assigned to them. However, the extensive research performed in Xenorhabdus and similar compounds from other species, suggests that many have immune evasion functions. There is the distinct possibility that Photorhabdus BGCs are essential for supporting the nematode development, perhaps helping to distinguish them from closely related species that also infect insects, without nematodal assistance, such as Serratia or Yersinia . While this may be true for some compounds, we suggest that the mutualistic symbiosis has been made more successful by acquisition of new BGCs by the bacteria enabling them to more efficiently overcome the host defense and consequently, killing the host more efficiently so that both bacteria and nematode benefit.
Siderophores are often essential in causing virulence in a range of bacteria (recently reviewed in [51–53]). One conserved cluster in Photorhabdus is predicted to produce a myxochelin-like siderophore (cluster 5, Fig. 1). Myxochelins have been shown to target and suppress the activity of 5-lipooxygenase , a key enzyme in the insect innate immune response (reviewed in ). Additionally, P. luminescens contains a further cluster with predicted siderophore function, a hydroxymate-like siderophore (cluster 74). Hydroxymate siderophores are potent histone deacetylase inhibitors. Histone deacetylases are involved in transcriptional reprogramming during wounding and infection and have been shown to repress antimicrobial peptide (AMP) production in Galleria mellonella, a common insect model for Photorhabdus virulence . In addition to these specific roles, we cannot rule out the possibility that these siderophores also play a more general iron-scavenging role within the insect or nematode.
Phospholipase-2 (PLA-2) is a part of the eicosanoid biosynthesis pathway that is activated in response to recognition of pathogens by the insect. The eicosanoids are essential in mediating activation of phagocytosis and proPO production in the insect hemolymph . Seo et al. (2012) have recently found that several Photorhabdus species are capable of inhibiting this by production of benzylideneacetone thereby preventing the recruitment of hemocytes and activation of phagocytosis [37, 58]. Benzylideneacetone is likely derived from the IPS biosynthetic pathway (extension of the phenylalanine derived cinnamoyl-CoA), which is a BGC conserved in all strains (cluster 9, Fig. 1) . A further mechanism of insect immune suppression is via proteasome inhibition. Recently, glidobactin A and its iso-branched acyl derivative cepafungin, products of an NRPS-PKS hybrid gene cluster that is highly conserved (Fig. 1), were reported to be produced by Photorhabdus and are potent proteasome inhibitors [39, 59]. An overview of possible immune evasive and suppression mechanisms as they relate to natural products in P. luminescens is provided in Fig. 2.
Two-component signal transduction systems
Six two-component systems (TCS) were conserved in all Photorhabdus species as well as E. coli, with a further system present only in all Photorhabdus strains. Among the conserved two-component systems is the well described CpxRA TCS, which is involved in a range of cellular processes from synthesis and translocation of cell membrane proteins [60–63] to resistance to AMPs  and various other virulence phenotypes [65–67]. BaeRS was also implicated in regulating multidrug resistance in E. coli  while TctED is involved in tricarboxylic acid transport  and UhpAB in involved in sugar transport pathways, responding to extracellular glucose . The OmpR/EnvZ TCS is also well described in E. coli and is central in regulating the Omp locus in response to external osmolarity alterations [71, 72]. The final TCS is the PhoPQ system, which is post-translationally controlled by sRNAs  and responds to magnesium concentrations or AMPs in the environment . However, the single TCS unique to Photorhabdus is the AstSR that was previously identified as being important in Photorhabdus phase switching  and identified as a likely component involved in insect infection .
We have recently reported two new classes of bacterial signaling molecules in Photorhabdus, namely the photopyrones (PPY) and dialkylresorcinols (DAR) [36, 40]. The DAR and PPY signaling pathways represent new methods of cell-cell communication and were discovered through the analysis of LuxR orphans (reviewed in ). While the DAR locus was identified in all strains (a part of the IPS biosynthesis shown in cluster 9, Fig. 1), the PPYs were only found in P. luminescens TTO1 and P. temperata subsp. thracencis suggesting a far less important role for PPYs (cluster 72, Additional file 5). Additionally, there are several other LuxR orphans in these bacteria with unidentified signals. One possibility is that some of the unknown clusters produce compounds can be sensed by these receptors. Another possibility that has been raised is the promiscuous activation of these receptors through compounds produced by either the nematode or insect prey, representing a form of cross-kingdom communication .
Only three additionally conserved regulatory proteins are present in all Photorhabdus examined. Two of these candidates are from the class of aforementioned LuxR orphans while the remaining is the HpaA regulator involved in the degradation of 4-hydroxyphenylacetic acid, which while absent in E. coli K12, is present in several other E. coli strains [77, 78]. It is also important to note that 872 (409 absent in E. coli K12) hypothetical proteins are additionally conserved with several potentially having undefined regulatory roles (Additional file 2).
Other conserved virulence factors
Other predicted virulence factors conserved across the genus include a number of different protein toxins, a fli locus for flagellar assembly, a secretion system as well as various other insect associated proteins. PrtA, a protein known to be involved in insect colonization is present in all Photorhabdus strains . Additionally, the genus contains a particularly large repertoire of protein toxins. The insecticidal toxin complex (Tc) proteins are over-represented in the total number. The Tc toxins consist of four sub-types, predicted to have different host targets , each of which is represented in the P. luminescens genomes. In total there are 16 annotated Tc protein families, all of which are present in both P. luminescens strains while the P. temperata strains have between eight and 11 proteins and the P. asymbiotica strains have only eight. Additionally, the repeats-in-toxin (Rtx)-like toxin are cytotoxins conserved in many Gram-negative pathogens  and similarly in all Photorhabdus. The mcf (makes caterpillars floppy) toxin now has an established role in insect pathogenicity in P. luminescens TTO1 with its presence enough to allow E. coli to kill insects [82–84]. Interestingly, this protein is present in all strains except for P. temperata M1021. However, the absence of this and a disproportionate number of other CDS that are present in all other Photorhabdus may just be indicative of the highly fragmented nature of this assembly in comparison to the others. Re-sequencing of this strain using long-read technology will provide more conclusive answers.
Photorhabdus contains only a single Type III secretion system (T3SS) that is absent in E. coli K12. Most strains have maintained the entire system while P. temperata M1021 has lost three genes (sctC, sctV and sctP), while P. asymbiotica and P. temperata subsp. thracencis are missing sctE and sctP, respectively. Of these missing homologs, only SctC and SctV are described as core proteins in this T3SS [85, 86] suggesting that P. temperata M1021 contains a non-functional T3SS. Additionally, this strain is the only strain lacking a full flagellar assembly locus (Additional file 7). Since there is significant evidence that this T3SS has a role in exporting insecticidal toxins , it is possible that this is merely an assembly artifact or that these bacteria instead kill insects via a different mechanism than that predicted by other Photorhabdus.
In terms of other host-associated proteins, each strain contains at least one predicted bacteriocin (Table 1), presumably to protect the insect cadaver from scavenging competitors. A total of five different bacteriocins were identified of which only one homolog is conserved in all P. luminescens and P. temperata strains but absent from both P. asymbiotica isolates (cluster 10, Fig. 1). Elucidation of the mechanism of this bacteriocin or a specific target may provide some insight into the competitors encountered by the respective species.
Probably virulence associated
Cell wall and cell processes
Phage and insertion sequence
The identification of conserved protein families across Photorhabdus has helped to shed light on possible pathways essential to the intricate lifecycle of the genus. Given the roles assigned to known compounds as well as those that have yet to be confirmed but share similarities with known compounds, we suggest that many of these BGCs have been acquired as virulence factors early during speciation of the Photorhabdus, with one of two main functions; cell-cell communication, or modulating the insect immune response. The common belief is that many of these specialized metabolites are essential for differing antimicrobial roles. However, given the relatively low biological activity of these compounds we propose that, although they appear to have these activities, this is merely a side effect of their true function. Deconstructing the novel regulatory pathways will go a long way towards understanding each individual environment. Furthermore, the elucidation of the functions of products of the BGCs as well as whole genome comparisons to the Xenorhabdus species will be important areas of future research to fully understand the ecological niche occupied by these bacteria.
Strains and culture conditions
Strains used in this study and their accession numbers
Photorhabdus luminescens TTO1
Photorhabdus luminescens subsp. PB45.5
Photorhabdus asymbiotica ATCC 43949
Photorhabdus asymbiotica subsp. australis PB68.1
Photorhabdus temperata subsp. thracensis DSM 15199
Photorhabdus temperata subsp. temperata M1021
Photorhabdus temperata subsp. khanii NC19
DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer’s instructions. Photorhabdus PB68.1 and PB45.5 were sequenced at Eurofins Genomics (Ebersberg, Germany) using an Illumina HiSeq2500 instrument with 150 bp paired end reads.
Genome assembly and annotation
Raw reads were processed to trim the attached adapters and low-quality bases from both ends using Trimmomatic (v 0.32)  with the parameters “ILLUMINACLIP:<path to adapter sequences>:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15”. Further, an in-house perl script (Additional file 11) was used to discard read pairs having an average base quality less than 30, having Ns in the sequence or less than 90 bases long. After cleaning reads using the above criteria, cleaned read pairs with a minimum 90 bases in both forward and reverse reads were used for assembly. De novo assemblies were carried out using Velvet (v 1.2.10) . To obtain optimal assemblies for both genomes, 12 assemblies for each genome were generated using odd k-mer lengths between 71 and 89, with default parameters and “-exp_cov auto”. Optimal assembly was chosen on the basis of assembled genome size, longest scaffold size, number of scaffolds, N50, N90, percentage of N in the assembly. For both genomes, the optimal assembly was obtained with a k-mer length of 89. Scaffolds longer than 300 bases were considered for gene prediction and further analyses. Following assembly, all genomes were annotated using prokka (v1.12) with default settings and --addgenes, −-compliant and --gram neg options activated . Protein orthologs among the seven Photorhabdus strains and E. coli K12 were determined using proteinortho5 . ANI calculations were performed using EzGenome (available at http://www.ezbiocloud.net/ezgenome). Assigning of KEGG orthology numbers and mapping to KEGG pathways was performed using the KEGG automatic annotation server . QUAST was used to assess assembly quality .
Secondary metabolite cluster identification
BGCs were identified using antiSMASH v3.0  together with the optional ClusterFinder algorithm using the annotated genomes as input. DNA sequences of clusters identified by antiSMASH were used in Mauve alignments to identify homologous regions to gene clusters from the already available, fully assembled genomes, enabling in silico reconstruction of some BGCs that were heavily fragmented. Presence of isnA and isnB, genes known to produce rhabduscin, an important immunomodulatory compound in related species, was performed manually using BLASTp (v2.2.29) as a part of the BLAST+ suite  with the IsnA and IsnB sequences from Xenorhabdus nematophila  used as input.
AMP, antimicrobial peptide; ANI, average nucleotide identity; BGC, biosynthetic gene cluster; DAR, dialkylresorcinol; FAS, fatty acid synthase; IJ, infective juvenile; IMD, immunodeficiency; IPS, isopropylstilbene; NRP, non-ribosomal peptide; NRPS, non-ribosomal peptide synthetase; PK, polyketide; PKS, polyketide synthase; PLA-2, phospholipase-A2; PO, phenoloxidase; PPY, photopyrone; proPO, pro-phenoloxidase; T3SS, type III secretion system; Tc, toxin complex; TCS, two-component system
Research in the Bode Laboratory is supported by European research starting grant under grant agreement no. 311477. A Postdoctoral Fellowship from the Alexander von Humboldt Foundation supports NJT. Research in the Thines and Bode labs are supported by the LOEWE funding initiative of the Government of Hessen in the framework of IPF and the Thines lab is also supported by BiK-F.
Availability of data and materials
The datasets supporting the conclusions of this article are available in the Genbank repository, under the accession numbers LOIC00000000 (Photorhabdus PB45.5) and LOMY00000000 (Photorhabdus PB68.1).
NJT extracted the DNA. BM, DKG, RS and MT designed and performed the genome assemblies. NJT and TS participated in genome annotation. NJT analysed the data. NJT and HBB conceived of the study. NJT, TS and HBB helped to design the experiment. NJT and HBB drafted the manuscript. MT, TS and HBB provided computational infrastructure. All authors have read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
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