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J Bacteriol. Jul 2004; 186(13): 4376–4381.
PMCID: PMC421592

Variation in the Effectors of the Type III Secretion System among Photorhabdus Species as Revealed by Genomic Analysis

Abstract

Entomopathogenic bacteria of the genus Photorhabdus harbor a type III secretion system. This system was probably acquired prior to the separation of the species within this genus. Furthermore, the core components of the secretion machinery are highly conserved but the predicted effectors differ between Photorhabdus luminescens and P. asymbiotica, two highly related species with different hosts.

Bacterial pathogens have evolved complex mechanisms to invade hosts, to escape host defenses, to multiply, and, finally, to cause harm to their hosts (6, 20). According to Wassenaar and Gaastra (27), virulence genes are those directly responsible for pathological damage caused by pathogens and are normally absent from their nonpathogenic relatives. These genes can be organized in so-called pathogenicity islands that are large genomic regions, often unstable and probably acquired during evolution via horizontal genetic transfer (14, 17). This feature became particularly apparent for a set of approximately 20 genes that together encode a pathogenicity device called the type III secretion system (TTSS) (29) whose central function is the delivery of bacterial proteins into eukaryotic cells (3). More than 20 TTSSs have been discovered so far in gram-negative bacteria pathogenic for mammals and plants (3, 15) but also in bacterial symbionts of plants and insects (4, 5, 25).

Photorhabdus and Xenorhabdus (members of the Enterobacteriaceae family) (2, 12) are bacterial symbionts of entomopathogenic nematodes belonging to the families Heterorhabditidae and Steinernematidae, respectively. These bacteria are transported by their nematode vectors into the hemocoel of the insect host, which is quickly killed by a combination of toxin action and septicemia (12). Three species were defined in the genus Photorhabdus: Photorhabdus luminescens, P. temperata, and P. asymbiotica (11). In addition, P. luminescens and P. temperata are subdivided into subspecies as follows: P. luminescens subsp. luminescens, P. luminescens subsp. akhurstii, and P. luminescens subsp. laumondii and P. temperata subsp. temperata (11). P. asymbiotica was never found associated with entomopathogenic nematodes but is isolated from human infections (8, 19). Recently, Akhurst et al. have proposed two subspecies for P. asymbiotica: P. asymbiotica for an American clinical strain and P. australis for an Australian clinical strain (1).

The recent identification of TTSSs in P. luminescens (7, 28, 31) prompted us to analyze the genomic diversity of TTSS within the genus Photorhabdus.

Comparative genomics of TTSS organization in Photorhabdus.

Comparisons of the genomic organization of the Photorhabdus TTSSs (Fig. (Fig.1)1) were performed with three strains: P. luminescens subsp. laumondii (strain TT01) (http://genolist.pasteur.fr/PhotoList/) (7), P. luminescens subsp. akhurstii (strain W14; accession number AY144116) (28), and P. asymbiotica (strain ATCC43949), for which the genome sequence is being determined at the Sanger Center (http://www.sanger.ac.uk/Projects/P_asymbiotica/). We identified a TTSS-encoding locus in all three strains analyzed. Our analyses revealed, first, identical TTSS backbones, including all the genes predicted to encode the injectisome (Sct/Lss proteins): i.e., the basal body, the needle-like structure (sctF), and the translocator (lopB-, lopD-, and lcrV-like genes). As previously reported (28, 31), Photorhabdus TTSSs display many striking similarities to the Yersinia pestis and Pseudomonas aeruginosa TTSSs. P. luminescens subsp. laumondii and P. luminescens subsp. akhurstii as well as P. asymbiotica possess the same genetic organization required for the complete assembly of a functional TTSS machinery. Moreover, these three Photorhabdus species harbor genes similar to the P. aeruginosa exsC and exsD genes encoding transcriptional regulators, which are absent from Yersinia spp., suggesting that the regulation of this system is more related to that performing regulation in P. aeruginosa.

FIG. 1.
Comparative genomics of TTSS organization in Photorhabdus species. Positions of the primer pairs are indicated. ERIC, enterobacterial repetitive intergenic consensus.

Second, the locations of the TTSS are identical in all these strains and are downstream of the Enterobacteriaceae housekeeping gene cspI encoding a cold shock protein (26). This finding suggests that prior to speciation, a Photorhabdus ancestor had acquired a TTSS as a block. As they are often plasmid encoded or located on pathogenicity islands, their mobile character has been suggested (29). In the case of the Photorhabdus TTSS, we did not detect the classical characteristics of a pathogenicity island (i.e., insertion into a tRNA gene and different GC content). However, four genes (plu3747 through plu3750) encoding proteins similar to bacteriophage proteins were detected in the close vicinity of the Photorhabdus TTSS, suggesting that the Photorhabdus TTSS may have been acquired via an integrative bacteriophage.

Third, despite the highly conserved organization and protein sequences of the core components of the secretion machinery (TTSS backbone in Fig. Fig.1),1), P. luminescens is predicted to encode a protein similar to the Yersinia YopT effector whereas P. asymbiotica harbors a gene encoding a protein homologous to the P. aeruginosa ExoU effector (see below).

Distribution of the TTSS among different strains of Photorhabdus and Xenorhabdus species.

To determine whether all different Photorhabdus species and subspecies (Table (Table1)1) harbor similar TTSSs, PCR amplification was performed on genomic DNA of 11 Photorhabdus strains (Table (Table1).1). In addition, seven strains of the more distantly related Xenorhabdus species were included. The oligonucleotide primer sets used (Table (Table2)2) were designed in the conserved regions of the known TTSS sequences (Fig. (Fig.1).1). Genes of the delivery system were named according to the nomenclature proposed by Hueck (15). For this study, the sctC/lssC, sctV/lssV/lcrD, and lopB genes were chosen because they are representative of the different parts of the injectisome located in different operons (15). Standard PCR with each primer set was performed in a 50-μl reaction volume with a Gene Amp 2400 thermocycler system (Perkin Elmer), and PCR products were subjected to 0.7% agarose gel electrophoresis for analysis. Genomic DNA from the 11 Photorhabdus strains was successfully amplified using specific primers for these three genes (Table (Table3).3). However, amplification results were negative for the seven Xenorhabdus strains, even under lower-level annealing conditions.

TABLE 1.
Bacterial strains used in this study
TABLE 2.
Oligonucleotide primer pairs used
TABLE 3.
PCR assays for sctC, sctV, lopB, lopT-spcT and lopU-spcU in Photorhabdus and Xenorhabdus species

As a second control for the presence of the TTSS, Southern blot analysis was performed using the sctC, sctV, and lopB genes as probes. P. luminescens TT01 genomic DNA was labeled using a PCR digoxigenin DNA labeling kit, and hybridization was revealed using a digoxigenin detection kit (Roche) according to the manufacturer's instructions. Southern blot hybridizations were performed on BglII,- EcoRI-, and HindIII-digested DNA from the previously mentioned Photorhabdus and Xenorhabdus strains (Table (Table1).1). No signal was detected using Xenorhabdus chromosomal DNA as a template even under low-stringency conditions, whereas a clear band of the expected size was visible when Photorhabdus chromosomal DNA was used as a template (data not shown). Thus, our results using PCR and Southern blotting data indicate that sctC, sctV, and lopB genes are conserved in all Photorhabdus strains. This is in agreement with a recent finding determined on the basis of a limited microarray analysis (18). Furthermore, our data suggest that species of the phylogenetically related genus Xenorhabdus (12) lack a TTSS or harbor a highly divergent system.

Phylogenetic analysis.

To analyze the relatedness of the Photorhabdus TTSS and to compare it to the taxonomic position of the organism, a phylogenetic study was undertaken. We used the sctV gene (homologous to the lcrD gene of Yersinia spp.) encoding an inner-membrane protein, because sctV is among the best-conserved members of the TTSS. PCR products were isolated using a High Pure PCR purification product kit (Roche), and sequencing was performed on an ABI 3700 sequencer. Sequences were aligned using ClustalW (24), and phylogenetic trees were constructed by using the neighbor-joining method and Kimura distance values (21). A bootstrap confidence analysis was replicated 500 times (9).

The resulting trees are shown in Fig. Fig.2.2. The various taxa could be divided into five distinct groups (Fig. (Fig.2A):2A): Ysc, Hrp1, Hrp2, Inv/Mxi/Spa, and EscC/Ssa (13); the Photorhabdus TTSS falls into the Ysc group. We extended this phylogenetic analysis to species of the genus Photorhabdus (Fig. (Fig.2B).2B). Both clinical subspecies grouped in a single cluster. The three P. luminescens subspecies P. luminescens subsp. laumondii, P. luminescens subsp. akhurstii, and P. luminescens subsp. luminescens formed a second cluster, and the P. temperata strains formed a third cluster. Remarkably, the resulting sctV tree is similar to the 16S tree (1, 11), indicating that the TTSS phylogeny reflects the phylogeny of the group. Because the same clusters and subclusters describing genetic heterogeneity among strains belonging to the genus Photorhabdus can be distinguished, we therefore suggest that the sctV gene and probably the entire TTSS backbone were present in the Photorhabdus ancestor and were not recently independently acquired.

FIG. 2.
Phylogenetic trees of bacterial sctV genes (A) and Photorhabdus species sctV genes (B). The ClustalW program with default parameters was used for aligning sequences and generating trees (branch strength values after bootstrapping 500 times are shown for ...

Analysis of the diversity of the Photorhabdus TTSS effectors.

As the most remarkable elements are the genes predicted to encode the effectors, we therefore examined the two loci in which they lie in the 11 Photorhabdus strains previously mentioned. Oligonucleotide primers were selected in the flanking genes (i.e., sctL and cspI for the lopT/slcT locus and sctU and lscA for the lopU/slcU locus) and designed in conserved regions. PCR products were obtained for all 11 strains tested, and their sizes are reported in Table Table3.3. PCR fragments were sequenced for strains Hb, DO04, Meg, 3265-86, and AU9800946, which are representatives of each subspecies. Sequences were aligned with the previously available sequences (i.e., those of strains TT01, W14, and P. asymbiotica).

For the sctL-cspI locus, a 2,020-bp fragment containing the lopT and the slcT genes was amplified for all the P. luminescens strains and a 450-bp intergenic region was amplified for the P. asymbiotica and P. temperata strains. For the sctU-lscA region, we amplified a 4.5-kb fragment containing lopU and slcU genes for all the P. asymbiotica strains and a 1,000- to 1,100-bp intergenic region for P. luminescens and P. temperata strains. Intriguingly, no effector-encoding genes were detected in the DNA sequences at these locations in the P. temperata strains Meg and XlNach (Table (Table33 and Fig. Fig.1);1); however, we could not rule out the possibility that they were elsewhere on the genome.

P. luminescens lopT is predicted to encode a protein similar to the Yersinia YopT effector. YopT is a cysteine protease (22, 23) that causes cytoskeletal disruption and contributes to the antiphagocytic effect of Yersinia (16, 30). The P. luminescens lopT gene is located at the 3′ end of the TTSS and, as in Yersinia, forms a bicistronic operon with a gene predicted to encode a LopT chaperone, namely, SlcT. These similarities with Yersinia Yop/SycT proteins are highly suggestive of a similar role for their P. luminescens counterpart, which is in agreement with the observation of LopT expression concomitant with the in vivo TTSS-dependent inhibition of phagocytosis in the orthopteran Locusta migratoria (Brugirard-Ricaud et al., submitted for publication).

Remarkably, the P. asymbiotica locus does not harbor yopT homologues. However, it contains lopU, a gene predicted to encode a protein similar to the P. aeruginosa ExoU effector. ExoU displays a potent phospholipase activity inducing disruption of epithelial and macrophage cell lines (10). The P. asymbiotica lopU gene is located between sctU and exsA-like genes and forms a probable operon with a gene predicted to encode a LopU chaperone, namely, SlcU. This location corresponds to the inversion point between the Photorhabdus and P. aeruginosa TTSS backbones (15).

In this report we show that all Photorhabdus species contain a remarkably conserved TTSS backbone but that the effectors seem to belong to the flexible gene pool, as they differ considerably among the different species. Moreover, enterobacterial repetitive intergenic consensus sequences were occasionally found in the vicinity of the effector loci, suggesting that they may be recombination hot spots and may account for genome plasticity. It is therefore tempting to speculate that in contrast to the TTSS backbone, the genes encoding the effectors may have been acquired at different steps of the evolution and then have been selected according to the ecological niches and the host ranges of the different Photorhabdus species.

Acknowledgments

This work received financial support from the Institut National de la Recherche Agronomique and the Ministère de l'Industrie et des Finances (Après séquençage des Génomes). K.B.-R. was funded by a MENRT grant (2052.2001).

We wish to thank Isabelle Gonçalves for help with bioinformatics and Carmen Buchrieser for critical reading of the manuscript.

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