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Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 2000 Aug;66(8):3310–3329. doi: 10.1128/aem.66.8.3310-3329.2000

A Genomic Sample Sequence of the Entomopathogenic Bacterium Photorhabdus luminescens W14: Potential Implications for Virulence

Richard H Ffrench-Constant 1,*, Nicholas Waterfield 1, Valerie Burland 2, Nicole T Perna 2, Phillip J Daborn 1, David Bowen 3, Frederick R Blattner 2
PMCID: PMC92150  PMID: 10919786

Abstract

Photorhabdus luminescens is a pathogenic bacterium that lives in the guts of insect-pathogenic nematodes. After invasion of an insect host by a nematode, bacteria are released from the nematode gut and help kill the insect, in which both the bacteria and the nematodes subsequently replicate. However, the bacterial virulence factors associated with this “symbiosis of pathogens” remain largely obscure. In order to identify genes encoding potential virulence factors, we performed ∼2,000 random sequencing reads from a P. luminescens W14 genomic library. We then compared the sequences obtained to sequences in existing gene databases and to the Escherichia coli K-12 genome sequence. Here we describe the different classes of potential virulence factors found. These factors include genes that putatively encode Tc insecticidal toxin complexes, Rtx-like toxins, proteases and lipases, colicin and pyocins, and various antibiotics. They also include a diverse array of secretion (e.g., type III), iron uptake, and lipopolysaccharide production systems. We speculate on the potential functions of each of these gene classes in insect infection and also examine the extent to which the invertebrate pathogen P. luminescens shares potential antivertebrate virulence factors. The implications for understanding both the biology of this insect pathogen and links between the evolution of vertebrate virulence factors and the evolution of invertebrate virulence factors are discussed.


Photorhabdus luminescens is an insect-pathogenic gram-negative proteobacterium that forms a “symbiosis of pathogens” with insect-pathogenic nematodes (52). In this symbiosis the bacteria are carried in the guts of entomopathogenic nematodes belonging to the family Heterorhabditidae (members of a different group of bacteria, Xenorhabdus spp., are carried in the guts of members of a different group of nematodes, the Steinernematidae). Upon invasion of an insect host by a nematode, the bacteria are released from the gut directly into the open blood circulatory system of the insect, the hemocoel (52). Here the bacteria are thought to release a wide variety of potential virulence factors, including high-molecular-weight toxin complexes (Tc), lipopolysaccharide (LPS), proteases, lipases, and a range of different antibiotics (52). Inferences concerning the involvement of these factors in killing of the insect or in overcoming the insect immune system, however, often result merely from documentation of secretion of the factors into bacterial culture supernatants. Studies examining the precise role of virulence factors during the infection process in insects have not been performed, and studies of Photorhabdus mutants are rare. As a prelude to genetic analysis of potential virulence factors in P. luminescens, we were interested in obtaining a sample sequence of strain W14 in order to document the classes of genes present and to begin to design suitable experiments for analysis of the genes based on a likely idea of their functions.

The relative advantages of sample sequence analysis versus full-scale analysis of a finished bacterial genome have been discussed elsewhere (119). However, there are several points relevant to the current discussion, as discussed briefly below. First, a sample sequence can be completed at a fraction of the cost of completion of a full genome. Second, a surprisingly high percentage of the genome can be captured even with a 1× sample sequence. Given the current uncertainty concerning the exact genome size of P. luminescens, the percent coverage obtained in this study is hard to estimate; however, McClelland and Wilson (119) suggested that a 1× genome equivalent for the 4.78-Mbp Salmonella typhi genome would require only 12,000 reads of 400 bases. Such coverage would ensure that almost every cistron was represented in the sample sequence. The 2,000 reads reported here obviously do not give this level of coverage, but, as shown below, even the limited sample sequence obtained revealed ample evidence concerning the types of virulence systems that P. luminescens may employ in its complex life cycle.

Although few potential P. luminescens virulence factors have been examined in detail (either biochemically or genetically), we can attempt to predict the likely role of bacterial virulence systems in killing an insect, in overcoming an insect immune system, or in facilitating bacterial and/or nematode growth. It is thought that once P. luminescens is released from the nematode gut into the insect hemocoel, it plays multiple roles in helping the nematode overcome its host (52). To do this, the bacteria need to overcome both the cellular (hemocytic) and peptide-mediated (antibacterial polypeptide) components of the insect immune system. Furthermore, the bacteria stop the insect from feeding and probably render its tissues suitable for consumption by both the bacteria and the nematodes. Anti-insect virulence mechanisms might, therefore, include, but not be limited to, toxins active against the insect gut and/or hemocytes and enzymes (such as proteases) capable of both degrading insect tissue and disabling the antibacterial peptides also associated with the insect immune system. Equally important in its role in overcoming an insect host, P. luminescens must ensure that the insect cadaver does not act as a breeding ground for opportunistic soil bacteria, fungi, and/or other species of nematodes. We might, therefore, expect P. luminescens to secrete a wide range of antimicrobial, antifungal, and nematicidal compounds, as previously documented by other workers (52). The aim of the present study was, therefore, to identify genes that encode likely virulence factors as a prelude to a functional analysis of the genes via targeted knockout and assay of the resulting mutants in the host infection process. Not only should such an analysis allow us to elucidate how the virulence factors act on the insect, but the gene sequences may also provide an indication of the evolution and potential origins of the virulence factors.

MATERIALS AND METHODS

Genomic library construction and sequencing.

Genomic DNA from P. luminescens W14 was size selected to obtain 1- to 2-kb fragments and then cloned into M13 Janus as previously described (28, 115). DNA templates were purified from library clones and sequenced by using dye terminator-labeled fluorescent cycle sequencing (model ABI377 automated sequencer; Applied Biosystems Division, Perkin-Elmer). Single sequencing reads (average length, ∼400 bp) were obtained for one end of 2,122 clones. Sequences were truncated to exclude the phage arms and multiple cloning site and were then submitted to the BLASTX servers at the National Center for Biotechnology Information. Clones giving hits to either Tc-, protease-, or Rtx-like-encoding genes were then sequenced from the other end or “flipped.”

Comparison with Escherichia coli K-12.

Trimmed (vector removed and high-quality trim with SeqManII) P. luminescens sequence reads were searched against the DNA and protein sequences of E. coli MG1655 by using BLASTN and BLASTX with a local server. The output was parsed and sorted to give three subsets of data with different levels of identity. No alignment length criteria were imposed on the output. The results, therefore, included short alignments and multiple hits for many sequences, all of which were legitimate similarities.

Nucleotide sequence accession number.

The nucleotide sequence determined in this study has been deposited in the DDBJ/EMBL/GenBank database under accession no. AQ989457AQ991805.

RESULTS AND DISCUSSION

Comparison with E. coli K-12.

The results of the comparison of P. luminescens sequences with E. coli K-12 sequences are shown in Fig. 1. Even though the number of query sequences was relatively small (<0.5× genome coverage), we clearly observed that there is significant conservation of sequences, particularly protein sequences, between the two genomes, which is consistent with the relatively close phylogenetic relationship of the two organisms (both are members of the family Enterobacteriaceae). Regions of the genome conserved in Escherichia and Photorhabdus strains begin to define the components of the putative ancestral chromosome of members of the gamma subdivision of the class Proteobacteria. The large excess of hits at the protein level compared to the DNA level at all stringencies suggests that the divergence between orthologous sequences is sufficient to obscure true matches at the nucleotide level. Even the number of protein hits changed extensively as we varied the criteria for a significant match. Thus, we were reluctant to choose an arbitrary cutoff for determining orthology (as has been done for other sample sequence comparisons) and instead describe three different levels of stringency below. This form of presentation provides not only a sense of the absolute number of sequences that are similar but also a sense of the strength of the similarities. It should be noted that although the hits are distributed, albeit unevenly, around the K-12 map, this does not necessarily indicate that there is colinearity, and indeed the sizes of the two genomes are probably different, which could account for some of the gaps and sparse regions in Fig. 1. In all, 1,133 W14 clones exhibited no significant matches in even the lowest-stringency analysis (E < E−05), and from this we inferred that approximately 53% (1,133 of the 2,122 clones examined) of the P. luminescens genome is clearly distinct from the genome of E. coli K-12.

FIG. 1.

FIG. 1

Graphic display of sample sequence similarities to E. coli K-12 nucleotide and protein sequences, generated from a BLAST search of P. luminescens sequences performed with K-12. The three concentric sets of data show nucleotide (outer ring) and protein (inner ring) hits plotted at the coordinates of the K-12 target. From the outside, the three data sets show hits with BLAST expected value (E) limits of <10e−05 (2,765 protein and 729 nucleotide hits), <10e−20 (1,227 protein and 376 nucleotide hits), and <10e−40 (664 protein and 234 nucleotide hits), respectively. The positions of the genetic markers ori, ter, and rrnA through rrnH are shown as landmarks to orient the circle. The figure was generated by using the program Genescene (DNASTAR).

Old and new toxin complex (tc) loci.

We previously cloned and sequenced four Tc-encoding loci, tca, tcb, tcc, and tcd, from P. luminescens W14 (22); each of these loci encodes a different high-molecular-weight insecticidal Tc (Tca, Tcb, Tcc, and Tcd, respectively). The Tc proteins are secreted into the supernatant by P. luminescens grown in liquid culture (23). Despite the fact that the Tc toxins exhibit both oral and injectable activity against a range of insects (22, 23), their precise role as potential virulence factors in the infection process remains to be determined. However, one of the complexes, Tca, has highly specific histopathological effects on the lepidopteran midgut (18), suggesting that Tca proteins may be used by the bacterium to destroy the insect midgut and effectively stop feeding. In the sample sequence analysis, BLASTX searches gave 19 hits for the four known tc loci (22), but 27 additional sequences (Table 1) were also identified that could not be ascribed to the previously identified tc loci after careful examination of the sequence chromatographs (Fig. 2A). This suggests that there are other tc-like loci in the P. luminescens W14 genome in addition to those already reported. The matches with new tc-like loci were classified as tca-like (3 hits), tcc-like (13 hits), or tcb/tcd-like (11 hits; tcb and tcd are close homologs of one another).

TABLE 1.

Hits to predicted products of known tc loci and putative new tc-like loci from P. luminescensa

Gene function Accession no. No. of Hitsb BLASTX E value(s) Clone(s) Reference
Known tc loci
 TcaABCZ AF046867 3 (1) 3e-11 to 1e-93 01421, 01421f, 02319 22
 TcbA AF04757 5 (0) 2e-25 to 1e-102 00513, 00753, 00753f, 00949, 01893 22
 TccABCZ AF47028 9 (3) 1e-53 to 8e-91 00707, 00707f, 01179, 01478, 01478f, 02015, 02197, 002197f, 02327 22
 TcdA AF188483 2 (0) 3e-34 to 6e-84 00617, 01839, 02280 Unp.c
Putative new tc-like loci
 TcaA-like AF046867 1 (0) 2.6 01461 22
 TcaC-like AF046867 2 (0) 6e-14, 1e-14 00357, 01661d 22
 TcbA- and TcdA-like AF04757 11 (5) 7e-04 to 2e-51 00508, 00508f, 00598, 00598f, 00878, 01303, 01303f, 01508, 01744, 01939, 02105, 02189, 02507, 02507f, 02474, 02474f 22
 TccA-like AF047028 5 (1) 0.01 to 2e-37 00093, 00093f, 01817, 01483, 02281, 02349 22
 TccB-like AF047028 3 (0) 9e-16 to 4e-34 01932, 01515, 01932 22
 TccC-like AF047028 5 (1) 6e-07 to 3e-79 00357f, 00763, 00869, 01403, 01498, 02049 22
a

See Fig. 2 for genomic locations. 

b

Number of sequence hits. The numbers in parentheses are the numbers of flipped sequences (indicated by the suffix f in the clone designations). 

c

Unp., unpublished data. 

d

Clone 00357 represents a sequence containing two different ORFs. 

FIG. 2.

FIG. 2

Diagrams showing the relative locations of hits to known tc loci and new tc-like loci. (A) Locations of individual sequencing reads (arrows above the diagrams) and their associated contigs and BLASTX matches (boxes below the diagrams). Note that the predicted amino acid sequences for tcb and tcd are sufficiently similar that we could not distinguish matches with either locus. (B) One example of a difference in genomic organization of a new tc-like locus inferred from a small contig and adjacent flipped sequence. Note that two TccC-like BLASTX matches are located next to a TcaC-like ORF with a phage remnant in between (see text). aa, amino acid.

The hypothesis that there are more than four tc loci in the W14 genome was confirmed by several other lines of evidence. First, extended sequencing of DNA surrounding the tcdA locus revealed not only the presence of a second open reading frame (ORF) immediately downstream of tcdA (designated tcdB) but also the presence of a second tccC-like locus further downstream from the tcdAB locus (unpublished results). As suggested by the sample sequence, this proves that there are at least two copies of tccC in the W14 genome. Second, sequencing of the opposite ends of flipped tc-containing clones showed that some of the new tc-like loci occupy novel genomic positions beyond the positions established for the four known loci. For example, clone 02349 is a tccA-like sequence whose flip (clone 02349f) is a lon protease, and clone 01515 is a tccB-like sequence whose flip is an exochitinase. Clone 00763 contains a tccC-like sequence which forms a contig with three other clones (00763f, 00339, and 02380), which also contain a yfiP-encoded lipase. Finally and perhaps most interestingly, one sequence (00357) contains both tccC-like and tcaC-like sequences but has phage sequences inserted between them (Fig. 2B shows the implied genomic organization of this contig). The abundance and potential implications of phagelike sequences in the P. luminescens W14 genome are discussed below. However, together, the sequence and inferred position data provide firm evidence that additional tc loci are present in the W14 genome. The implications for the potentially increased variety of encoded insecticidal Tc toxins remain unclear.

Antibiotics and antibiotic resistance.

Having destroyed the insect gut, presumably by using the tc-encoded Tc (18), P. luminescens must then defend the insect cadaver from a wide range of other colonizing organisms, such as bacteria (including other strains of P. luminescens), fungi, and/or nematodes. It seems reasonable to assume that during this process P. luminescens W14 deploys a range of antimicrobial agents, such as antibiotics and antifungal agents, as documented for other Photorhabdus strains and also Xenorhabdus strains (52), in order to maintain a bacterial monoculture in the insect cadaver. Thus, in the sample sequence one of the largest classes of hits was hits for polyketide synthetase-like genes. This class of genes is responsible for nonribosomal synthesis of a diverse array of compounds involved in processes ranging from fatty acid synthesis to antibiotic production (including production of inhibitors of eukaryotic protein phosphatases [41]). Even if we took into account the effects of the large sizes of some of the polyketide synthetase loci (up to 28 kb of repeated subunits), these classes of hits were still some of the predominant classes of hits in the sample sequence, accounting for 3.7% (80 hits) of the total sequences. Of the matches with polyketide synthetase-like sequences, 31 were with a syringomycin synthetase from Pseudomonas syringae pv. syringae (Table 2). Interestingly, the syringomycin synthetase gene cluster is thought to provide a link between prokaryotic and eukaryotic peptide synthetases (62), while syringomycin itself has a wide range of antibacterial and antifungal properties. Deployment of similar antibiotics by P. luminescens W14 may, therefore, help maintain a bacterial monoculture in an insect cadaver. Furthermore, it is interesting to note that P. luminescens also contains a sequence that is similar to tolaasin (another lipodepsipeptide), which is used for self-protection in Pseudomonas tolaasii, which implies that W14 may employ this peptidoglycan-associated lipoprotein in self-protection against its own antibiotics. In addition to potentially deploying broad-spectrum antibiotics to repel other organisms that might colonize the insect cadaver, strain W14 also contains sequences similar to colicin activity proteins (CeaAB), colicin transport proteins (BtuB), and pyocin immunity proteins (S3). A sequence similar to colicin lysis protein was not found, although there was a match with a similar VlyS lysis protein S from lambda phage (BLASTX E value, 2e-16). Although the role of the colicin- or pyocin-like sequences in P. luminescens remains to be determined, they may be used to produce toxins and antitoxins designed to kill non-self bacteria.

TABLE 2.

Hits to polyketide synthetase-like proteins, colicin, and pyocins

Gene function Organism Accession no. No. of hitsa BLASTX E value(s) Clone(s) Reference
Polyketide synthetases: syringomycin synthetase-like
 Syringomycin synthetase (Pseudomonas) P. syringae pv. syringae AF047828 31 (22) 2e-09 to 3e-54 00006, 00006f, 00033, 00033f2, 00104, 00134, 00134f, 00033, 00033f, 00104, 00134, 00134f, 00554, 00554f, 00564, 00564f, 00967, 00976, 00967f, 01218, 01247, 01458, 01458f, 01986, 00015, 00037, 00037f, 00380, 00380f, 00665, 00665f, 01057, 01190, 01190f, 01258, 01385, 01385f, 01513, 01519, 01519f, 01762, 01901, 01901f, 02274, 01973f, 00649, 00502f, 00060, 00466f, 00498f, 00533f, 00379, 00772f, 01029, 01170, 01258f, 01633, 01946 62
Other polyketide synthetase-like proteins
 BacA, bacitracin synthetase 1 Bacillus licheniformis AF007865 2 (0) 1e-06 to 3e-29 01477, 00946 97
 BacC, bacitracin synthetase 3 B. licheniformis AF007865 7 (2) 4e-06 to 3e-29 00058, 00058f, 00996f, 01242, 01614, 01782, 01783 97
 Saframycin Mx1 synthetase A Myxococcus xanthus U24657 1 (0) 1e-04 02574 143
 Saframycin Mx1 synthetase B M. xanthus U24657 5 (2) 1e-11 to 4e-29 00060f, 004466, 01119, 01119f, 02368 143
 Pristinamycin I synthetase Streptomyces pristinaespiralis X98690 3 (1) 4e-04 to 2e-24 00502, 00649f, 00772 39
 LicA, lichenysin synthetase A B. licheniformis U95370 3  4e-09 to 1e-26 00750, 01729, 01171 96
 LicB, lichenysin synthetase B B. licheniformis U95370 2 (2) 4e-09, 2e-14 00750f, 00946f 96
 Tyrocidine synthetases 1, 2, and 3 Bacillus brevis AF004835 4 (2) 5e-13 to 3e-16 00641, 00738, 01242f, 02574f 126
 PksK, polyketide synthetase Bacillus subtilis P40803 2 (1) 0.4, 7e-35 00269f, 01717 3
 PksL polyketide synthetase B. subtilis P40803 1  0.04 02466 3
 PksR, polyketide synthetase B. subtilis P40803 1  4e-04 01729f 3
 Surfactin synthetase subunit 2 B. subtilis Q04747 3 (1) 4e-10 to 7e-36 00104f, 00738f, 02068 20
 Peptide synthetase-like B. subtilis AF087452 3 (0) 3e-07 to 7e-08 00153, 00136, 01474 Unp.b
 Lysobactin synthetase Lysobacter sp. X96558 3 (1) 6e-17 to 6e-23 00533, 00641f, 00996 17
 Daptomycin synthetase-like Streptomyces roseosporus AF021263 2 (0) 1.1, 4e-34 02364, 01805 121
 Polyketide synthetase 6-like Mycobacterium tuberculosis Z84725 2 (0) 1e-12 to 3e-14 01953, 02416 34
 Danorubicin-like Streptomyces peucetius L35560 1 (0) 8e-09 02548 170
 Pyoverdine synthetase D-like P. aeruginosa S53999 2 (0) 9e-07, 6e-39 00812, 01517 122
 Gramicidin S synthetase 2 B. brevis JX0340 1 (0) 9e-31 00097 152
 Microcystin synthetase B Microcystin aeruginosa U97078 1 (0) 2e-25 01315 41
 Saccharopolyspora PKS Saccharopolyspora hirsuta S35197 1 (0) 2e-14 01493 104
Self-protection to lipodepsipeptides
 Pal, peptidoglycan-associated lipoprotein E. coli P07176 1 (0) 3e-38 01801 32
 Tolassin self-protection P. tolaasii U16024 1e-09 (Second match) Unp.
Colicins and pycocins
 CeaA, colicin A Citrobacter freundii plasmid P04480 1 (0) 6e-24 01036 127
 CeaB, colicin activity protein E. coli pColE2 P04419 1 (0) 4e-34 00284 116
 Pyocin S3 immunity protein P. aeruginosa P12 B56394 3 (1) 4e-06 to 2e-15 01787, 02024, 02024f 44
 BtuB, transport of E colicins C. freundii Y09059 1 (0) 1e-59 00358 Unp.
 YebA, hypothetical lysostaphin E. coli P24204 1 (0) 2e-60 07734 76
a

Number of sequence hits. The numbers in parentheses are the numbers of flipped sequences (indicated by the suffix f in the clone designations). 

b

Unp., unpublished data. 

In addition to genes for specific mechanisms for antibiotic production and self-protection, the W14 genome contains numerous sequences that exhibit homology to genes for other antibiotic resistance mechanisms. These sequences include genes involved in resistance to penicillin (penicillinase and penicillin-binding protein), bicyclomycin, and a range of other antibiotics (tetracycline, rifampin, and kasugamycin) via a variety of different mechanisms (Table 3). Most notable in this respect are the large number of sequences that exhibit homology to genes for different multiple-drug-like export systems, including Emr-like and Mdl-like systems that export drugs ranging from chloramphenicol to acriflavin. These multiple-drug export systems are also very similar to the hemolysin B export systems, as discussed below, and begin to describe a large family of exportlike genes in the P. luminescens genome. Also present are sequences similar to cation resistance genes in other enteropathogenic bacteria, notably sequences that encode resistance to tellurite (TelA) in E. coli plasmid RK2 (Table 3).

TABLE 3.

Hits to antibiotic and drug resistance-associated proteins

Gene function Organism Accession no. No. of hitsa BLASTX E value(s) Clone(s) Reference
Penicillinase and penicillin-binding proteins
 BlaC, penicillinase Y. enterocolitica Q01166 2 (0) 3e-11, 5e-59 02059, 02237 157
 PbpB, penicillin-binding protein 1B E. coli AE000124 1 (0) 3e-11 01997 56
 PbpE, penicillin-binding protein 4 Bacillus subtilis P32959 2 (0) 2e-07, 2e-10 00417, 02075 142
Bicyclomycin resistance proteins
 YnfM, bicyclomycin resistance E. coli D90801 2 (0) 3e-51, 2e-52 00114, 02573 Unp.b
 Bcr, bicyclomycin resistance E. coli AE000308 1 (0) 3e-18 00511 19
 Bcr, bicyclomycin resistance H. influenzae P45123 1 (0) 3e-13 02347 51
Other antibiotic resistance proteins
 TypA, GTPase E. coli AJ224871 1 (0) 1e-76 01716 47
 LpxD, glucosamine N-acyltransferase (rifampin) Y. enterocolitica P32203 1 (0) 4e-29 00423 180
 KsgA, dimethyladenosine transferase (kasugamycin) E. coli P06992 1 (0) 2e-74 00122 175
Multiple-drug-like efflux systems
Streptomyces chloramphenicol resistance-like B. subtilis AB001488 2 (0) 3e-09, 3e-13 01467, 01540 Unp.
 EnvD, protein D (acriflavin) E. coli D90846 3 (0) 3e-26 to 6e-71 01292, 01981, 02167 91
 AcrE, acriflavin resistance-like Aquifex aeolicus AE000702 1 (0) 5e-06 01439 Unp.
Emr multiple-drug-resistance proteins
 EmrD, protein D E. coli P31442 4 (0) 3e-10 to 3e-56 00349, 01526, 01647, 01875 129
 EmrY, protein Y E. coli P52600 1 (0) 3e-53 01983 19
Mdl multiple-drug protein
 Mdl, ATP binding E. coli U82664 2 (0) 4e-53, 5e-76 01456, 01960 Unp.
Other putative resistance-associated translocases
 YqjV, resistance protein-like B. subtilis P54559 2 (0) 1e-07, 1e-09 00530, 01766 Unp.
 YfkF, resistance protein-like B. subtilis D83967 2 (0) 0.002 to 6e-04 01266, 02238 99
 YgeD, resistance protein-like E. coli P39196 1 (0) 2e-50 00624 77
 YieO, resistance protein-like E. coli P31474 1 (0) 4e-18 02452 29
 YbhF, ATP binding E. coli P75776 2 (1) 3e-40, 3e-43 02378, 02377f 19
Ethidium bromide resistance
 E1 protein (putative chaperone) E. coli D90802 4 (0) 1e-08 to 3e-27 00009, 00030, 00077, 00666 1
Cation and solvent resistance-like
 YaaN, toxic cation resistance B. subtilis P37535 1 (0) 6e-41 02039 133
 TelA, tellurite resistance E. coli pRK2 Q52328 2e-29 60
 OstA, organic solvent tolerance E. coli P31554 2 (0) 3e-68, 5e-70 00135, 00828 6
 Ttg2F, toluene tolerance Pseudomonas putida AF106002 1 (0) 2e-09 01702 90
a

Number of sequence hits. The numbers in parentheses are the numbers of flipped sequences (indicated by the suffix f in the clone designations). 

b

Unp., unpublished data. 

Rtx-like homologs.

Another large class of database matches comprises sequences similar to both Rtx-like and hemolysin A-like toxins and their associated export systems (Table 4). The RTX (repeats in toxin) toxins are cytolytic toxins that are virulence factors in many pathogenic gram-negative bacteria (182). The RTX elements of other gram-negative bacteria share certain aspects of genomic organization, including the presence of three elements: an exported protein (RtxA-like), an ATP-binding cassette ABC protein (RtxB-like), and a membrane fusion protein (RtxD-like). Figure 3 shows the sequences similar to each of these elements alongside the loci to which they are most similar as determined by BLASTX searches. This figure shows that the Photorhabdus sample sequence contains sequences similar to the sequences of RtxA and RtxB of Vibrio cholerae, ShlA and ShlB of Serratia marcescens, EthA and EthB of Erwinia tarda, and HecA and HecB of Erwinia chrysanthemi. We also discerned sequences similar to both HlyB and CvaA/CvaB of E. coli, which are involved in hemolysin secretion and colicin V secretion, respectively. Notably, even if we took into account the large predicted ORF size (size of rtxA, ∼12 kb), there were still 24 hits with RtxA-like sequences alone, suggesting that more than one locus may be present. Furthermore, BLASTX E values were highly significant (e-25 to e-78), suggesting that there is a high level of amino acid conservation. We also observed that there is a sequence similar to TolC which is unlinked but is required both for hemolysin export and for colicin V export (58). We can only speculate as to the number of loci that these sequences correspond to and to the likely role of the encoded toxins in P. luminescens infection. However, given the propensity of Rtx-like toxins to attack host phagocytes (182), we postulate that the sequences may be important in attacking the insect cellular immune system, the hemocytes. This hypothesis could be tested by deleting the toxin loci or their export machinery and examining the infection process in their presence and absence.

TABLE 4.

Rtx-like operon homologs, including proteins exported by these operons and their accompanying export machinery and activating and modulating proteinsa

Gene function Organism Accession no. No. of hitsb BLASTX E value(s)c Clone(s) Reference
Exported proteins
 RtxA-like V. cholerae AF119150 17 (2) 2e-25 to 8e-78 00028, 00397, 00486, 00727, 00987, 00987f, 01071, 01071f, 01700, 01342, 01389, 01452, 01551, 02082, 02096, 02396, 02287, 02505, 02505f, 00727f, 02082f, 02333 112
 EthA, hemolysin E. tarda D89876 4 (1) 0.003 to 4e-29 01557f, 01727, 01896, 02259, 01618, 00774 67
 ShlA, hemolysin A S. marcescens P15320 2 (0) 1.8 (50), 1e-42 00369, 02511f, 00489f, 00369f 141
 HmpA, hemolysin A P. mirabilis P16466 1 (1) 0.12 (30) 01539 174
 PrtA, metalloproteinase A E. chrysanthemi JN0891 1 (1) 2e-47 01175f 24
 Prt1, metalloproteinase A Erwinia carotovora Q99132 1 (1) 3e-18 00148 100
Activator or modulating proteins
 ShlB-like, hemolysin secretion S. marcescens P15321 6 (0) 3e-15 to 4e-54 00489, 01578, 02066, 02427, 02511, 02512 141
 HecB-like, hemolysin secretion E. chrysanthemi L39897 5 (0) 1e-07 to 5e-13 00075, 00149, 00200, 01845, 02556, 00904f, 00149f, 01657 12
 Protease inhibitor E. chrysanthemi AF071511 2 (0) 5.2 (47), 1e-15 00886, 01175d 106
Secretion functions: ATP-binding cassette proteins
 RtxB-like V. cholerae AF119150 2 (0) 5e-56, 2e-77 00848, 02333, 00848f 112
 HlyB-like, hemolysin secretion E. coli P08716 2 (0) 2e-33, 4e-57 00909, 01791, 00909f 48
 LipB, protease transporter S. marcescens D49826 7 (1) 6e-10 to 4e-65 00179, 00264, 00337, 01175*, 01512, 01512f, 02149 2
 CvaB, colicin V secretion E. coli P22520 2 (1) 1e-09, 5e-13 00916, 00949f 58
Membrane fusion proteins
 CvaA, colicin V secretion E. coli P22519 2 (0) 1e-15, 2e-13 01283, 01780 58
V. cholerae AF119150 1e-15, 2e-08 112
 LipC, protease transporter S. marcescens D49826 2 (1) 5e-37, 2e-50 00179f, 02010 2
Outer membrane proteins
 TolC, outer membrane protein Salmonella enteritidis Q54001 1 (0) 3e-61 00783 156
 TolA, outer membrane protein E. coli P19934 1 (0) 3e-15 01818 108
 LipD-like (PrtF, TolC) E. chrysanthemi P23598 1 (0) 0.8 (27) 00181 107
Hemolysin coregulated protein
 Hcp, 28-kDa secreted protein V. cholerae S911006 1 (0) 2e-09 01146 184
a

See Fig. 3 for putative genomic organizations. 

b

Number of sequence hits. The numbers in parentheses are the numbers of flipped sequences (indicated by the suffix f in the clone designations). 

c

The numbers in parentheses are percentages. 

d

Clone 01175 represents a sequence containing two different ORFs. 

FIG. 3.

FIG. 3

Inferred genomic organization of different putative Rtx-like operons (rtx, shl, eth, hec, hly, and cva) from the sample sequence. The relative predicted positions of sequencing hits are shown below each predicted locus, and the range of percent identity values is shown. The putative operons are shaded in order to indicate their potential functions as either an exported protein, a activity regulator, an ATP-binding protein, or a membrane fusion protein.

In addition to an RtxA-like export system, we also found evidence of other Rtx-like export systems, including an Rtx-like metalloprotease and its accompanying export machinery. The Rtx-like metalloprotease itself is similar to PrtA-encoded metalloproteinase A of E. chrysanthemi, while its associated export machinery is similar to the LipBCD-like ABC transporter of S. marcescens, which also exports a protease. Between the protease and its associated ABC transporter there is a small protease inhibitor (as confirmed by our extended sequencing of the operon). This genomic organization of an Rtx-like metalloprotease and its associated LipBCD-like transporter shows that P. luminescens uses different combinations of Rtx-like genes to export virulence factors and stresses the potential importance of these systems for anti-insect virulence.

Other putative virulence factors.

In addition to the specific Tc-like and Rtx-like toxins discussed above, we also identified a wide range of other sequences related to a diverse array of genes that are potentially involved in infection and virulence. These genes include genes that encode factors involved in bioluminescence, other proteases, lipases, hemmaglutinins, chitinases, and other toxins, such as non-Rtx hemolysins and ADP-ribosyltransferases (Table 5). They also include genes involved in two-component sensor systems that have previously been implicated in regulation of virulence both in Photorhabdus strains and in other bacteria.

TABLE 5.

Putative virulence factors, genes expressed in infection, and two-component sensors

Gene function Organism Accession no. No. of hitsa BLASTX E value(s)b Clone(s) Reference
Bioluminescence
 Luciferase, beta subunit P. luminescens C35411 1 (0) 3e-66 00415 c79
 UbiB, NAD(P) H-flavin reductase P. luminescens P43129 3 (0) 1e-39 to 1e-71 00817, 02182, 02377 193
 LumQ transcriptional regulator (linked to lux operon) Photobacterium leiognathi Q5187 1 (0) 2e-04 00131 111
Proteases, peptidases, and lipases
 Triaglycerol lipase 1 P. luminescens P40601 4 (2) 4e-13 to 9e-86 00639, 00639f, 01676, 01676f 181
 YfiP, lipase Bacillus subtilis D78508 2 (0) 2e-04, 2e-05 00339, 02380 188
 Lipase (cold adapted) Pseudomonas sp. AF034088 1 (0) 3e-17 00082 33
 PldB, lysophospholipase L2 E. coli P07000 2 (0) 2e-20, 3e-49 02035, 02458 95
 Lys-X cysteine protease P. gingivalis U83995 2 (0) 0.9 (66), 6e-04 01699, 02180 109
 YegQ, putative collagenase E. coli P76403 2 (0) 4e-17, 7e-31 01523, 01829 19
 OpdA, oligopeptidase A E. coli A43329 1 (0) 4e-50 01206 35
 PrtB, oligopeptidase B E. coli P24555 3 (0) 3e-22 to 4e-47 00189, 01995, 01877 81
 Protease IV E. coli P08395 2 (0) 1e-07, 1e-32 01602, 02400 74
 Peptidase B E. coli P37095 1 (0) 4e-34 00116 189
 Putative peptidase E. coli AE000321 1 (0) 4e-58 00083 19
 Yae1, hypothetical E. coli P37764 1 (0) 2e-48 01938 19
 Metalloprotease Chlamydia pneumoniae AE001618 6e-11 Unp.c
 Matrix metalloprotease type 1 Gallus gallus AF062392 1 (0) 7e-07 01471 Unp.
 Major seed albumin Pea P08688 1 (0) 5e-04 01183 Unp.
 Matrix metalloprotease type 2 Limulus A40774 0.001 (second score) 113
Chitinases (N-acetyl-beta-glucosamidase)
 ChB, chitobiase S. marcescens Q54468 3 (0) 7e-34 to 1e-81 00174, 00222, 01723 171
 Exochitinase chitinase C-like S endosymbiont Y11391 4 (2) 0.07 (61) to 9e-27 00596, 01515f, 02526, 02526f Unp.
Hemagglutinins
 Putative secreted protein Neisseria meningitidis AF030941 3 (0) 2e-09 to 2e-34 00904, 01541, 01709 Unp.
 FhaB, filamentous hemmaglutinin B B. pertussis P12255 0.04 (35) to 7e-05 148
 Hemagglutinin neuramidase Newcastle disease virus M22110 1 (0) 4.1 (40) 01642 120
 PalL, PA-I galactophilic lectin (galactophilic hemagglutinin) P. aeruginosa Q05097 1 (0) 3e-06 01333 8
Hemolysins (non-RTX)
 Ybex, hemolysin E. coli P77392 2 (0) 1e-48, 2e-78 00477, 01423 135
 Hemolysin erythrocyte lysis protein 2 Prevotella intermedia AF052516 1 (0) 1e-24 01236 14
ADP-ribosyltransferases and B. thuringiensis
 Cytotoxic necrotizing factor type 2 E. coli 711 A55260 1 (0) 3e-13 01649 137
 ToxA, exotoxin A P. aeruginosa P11439 1 (0) 0.8 (44) 00003 110
 ToxA, exotoxin A C. difficile A37052 1 (0) 0.05 (32) 02134 154
 Halovibrin V. fisheri U38815 1 (0) 6e-19 00730 147
 ExsA, exoenzyme S synthesis regulatory protein P. aeruginosa P26993 1 (0) 6e-50 01619 53
 VirF, virulence regulon transcriptional regulator Y. enterocolitica P13225 2e-44 (Second match) 53
B. thuringiensis delta-endotoxin B. thuringiensis L07025 2 (0) 8.5 (26), 0.8 (30) 01891, 01973 101
Other virulence-associated factors
 VacB, RNase II H. influenzae P44907 1 (0) 5e-33 01226 51
 VacB, RNase II E. coli P21499 2 (1) 1e-28, 9e-70 01226f, 02032 172
 VapD, virulence-associated protein D H. influenzae C64069 1 (0) 2e-13 00582 51
 VapZ, virulence-associated protein A′ D. nodosus Q46561 2 (0) 0.2 (32), 8e-12 01633, 01947 86
 KicA, killing factor E. coli S43912 1 (0) 2e-60 01813 49
 MviM, virulence factor E. coli D90805 1 (0) 2e-45 02187 1
Two-component sensors
 EnvZ, osmolarity sensor Y. enterocolitica Y08950 1 (0) 2e-71 00849 43
 CheA, chemotaxis protein S. typhimurium P09384 1 (0) 5e-37 01857 164
 ExpA E. carotovora X95564 2 (0) 0.1 (25), 1e-39 00055, 02245 45
 TctE S. typhi AF029846 1 (0) 2e-61 01665 Unp.
 BaeS, sensory kinase E. coli P30847 1 (0) 3e-34 01887 128
Outer membrane proteins
 OmpF, porin S. marcescens 033980 1 (0) 4e-43 01121 73
 OmpW, outer membrane protein W E. coli P21364 1 (0) 4e-20 01233 125
 OprF, outer membrane porin F Pseudomonas fluorescens AF117969 1 (0) 5e-06 02506 Unp.
a

Number of sequence hits. The numbers in parentheses are the numbers of flipped sequences (indicated by the suffix f in the clone designations). 

b

The numbers in parentheses are percent amino acid identities. 

c

Unp., unpublished data. 

Bioluminescence (from which P. luminescens obtained its specific epithet) occurs shortly after bacteria invade an insect, but its biological role is unclear. The genes that encode the luciferase beta subunit and NAD(P)H-flavin reductase, which reduces flavin mononucleotide for bioluminescence, have previously been cloned, and hits to these genes were highly significant (BLASTX E values, 1e-39 to 3e-66), which again confirmed the quality and coverage of the sample sequence. Compared to other classes of potential virulence factors, we found several sequences similar to sequences of non-RTX-like proteases and lipases, which have previously been implicated in virulence. One of these, triaglycerol lipase 1, has been cloned previously, and hits to this sequence were highly significant (BLASTX E values, 4e-13 to 9e-86). Other proteins, like the Lys-X cysteine protease of Porphyromonas gingivalis, have been implicated in virulence during soft-tissue infections (109). Another subclass of hits in this category are hits to matrix metalloprotease-like sequences. These are interesting because one of the proteins, limunectin (from the horseshoe crab, Limulus sp.), binds bacterial cells, fixed amebocytes, and extracellular matrix molecules (113). If Photorhabdus cells do indeed make a similar protein, the protein may play some role in bacterial aggregation, previously termed nodulation (52), or in attachment to host cells. Other hits to proteins potentially involved in host cell binding included hits to several hemagglutinins. For example, deletion of the filamentous hemagglutinin locus in Bordetella pertussis results in loss of binding to ciliated eukaryotic host cells (148). As well as degrading host cells via proteolytic activity, much of the insect exoskeleton is composed of chitin. Therefore, hits to chitinases (N-acetyl-beta-glucosaminidase) probably indicate that there are several chitin-degrading enzymes, notably an enzyme similar to the chB-encoded chitinase of S. marcescens and a chitinase C-like product of an insect (Glossina morsitans) S endosymbiont (Table 5).

In addition to the Tc and Rtx-like toxins discussed above, W14 also appears to contain several non-Rtx hemolysins and other classes of toxins. One of the non-Rtx hemolysins, hemocyte erythrocyte lysis protein 2 from Prevotella intermedia, is notable in that searches of DNA and protein databases have not previously revealed any significant homologies (14); the Photorhabdus homology reported here is, therefore, possibly such a match. Other classes of toxins include two ADP-ribosyltransferases and cytotoxic necrotizing factor 2, all of which are cytotoxins. The ADP-ribosyltransferases are like exotoxin A from Clostridium difficile and Pseudomonas aeruginosa. Although the BLASTX E values for these hits are of low significance (0.05 and 0.8), the narrow ranges of homology values indicate that the levels of predicted amino acid identity are high (44% [17 of 38 residues] and 32% [24 of 73 residues], respectively). Cytotoxic necrotizing factor 2 from E. coli acts on the small GTP-binding protein Rho involved in actin cytoskeleton assembly and causes stress fiber formation in target cells (137). It is also interesting that there was a hit to halovibrin from Vibrio fisheri, which is a member of a novel class of ADP ribosyltransferases with no significant sequence homology to other ADP ribosyltransferases (147). In relation to potential ADP ribosyltransferase regulation, the sample sequence had a highly significant (BLASTX E value, 6e-50) hit to ExsA, the exoenzyme S synthesis regulatory protein (53). Exoenzyme S is another ADP ribosyltransferase that is distinct from exotoxin A and is secreted by P. aeruginosa, and ExsA is an AraC-like transcriptional regulator of its production. This sequence is also similar (BLASTX E value, 2e-44) to the VirF virulence regulon transcriptional regulator which controls the yop regulon (see below). Finally, with regard to other non-Tc toxins, there were two low-scoring hits to the delta-endotoxins from Bacillus thuringiensis, whose significance is uncertain.

Hits on other potential virulence factors included matches to Vac, Vap, and Kic-like proteins. There were hits on VacB from both E. coli and Haemophilus influenzae. Disruption of this gene in enteroinvasive E. coli results in reduced expression of virulence phenotypes, suggesting that it is necessary for full expression of virulence (172). We also found sequences similar to both VapD and VapZ from Dichelobacter nodosus. These are virulence-associated proteins homologous to ORFs found on the F plasmid of E. coli (86). Furthermore, we found a KicA-like sequence; this protein is thought to suppress the killing function of the kicB gene product (49). The putative KicA-like protein in P. luminescens may, therefore, function as a toxin-antitoxin system for killing non-self bacteria, like the colicins and pycocins discussed above.

The sample sequence revealed five sequences that exhibit similarity to known two-component sensors: EnvZ, CheA, ExpA, BaeS, and TctE. Of these, only EnvZ and CheA have been characterized in detail. The ompR-envZ regulatory system has been shown to contribute to virulence in a number of enteric bacterial pathogens. For example, an isogenic ompR mutant of Yersinia enterocolitica exhibited increased sensitivity to high osmolarity, high temperature, and low pH and also offered partial protection against wild-type challenge in a murine yersiniosis model (43). The ompR and envZ signal transduction genes have also been cloned from another entomopathogenic nematode-associated bacterium, Xenorhabdus nematophilus (168). Deletion of envZ in a Xenorhabdus strain suggests that the gene regulates some outer membrane proteins during the stationary growth phase, implying that it has a potential role in virulence (see below). The CheA protein is required to initiate the response of the flagellar motor to the binding of stimulatory ligands to chemoreceptors during bacterial chemotaxis. The hit to ExpA is of great interest as this protein and its relatives appear to play a key role in regulating expression of a range of secreted virulence factors in different gram-negative bacteria. The relatives include SirA in Salmonella typhimurium, ExpA in Erwinia spp., and GacA in Pseudomonas spp. For example, in S. typhimurium SirA is needed for expression of the type III secretion apparatus. Furthermore, upon sensing of a mammalian microenvironment, SirA phosphorylation initiates a cascade of transcription factor synthesis that leads not only to invasion gene transcription but also to Ssp secretion and bacterial epithelial invasion (78). Deletion of such a locus from a Photorhabdus strain would, therefore, allow us to test the hypothesis that a similar system is effective for sensing the insect hemocoel and subsequently initiating virulence-associated transcription.

Locomotion, attachment, and invasion.

During its complex life cycle, a Photorhabdus strain not only needs to detect which environment it is in (e.g., nematode gut versus insect hemocoel) but presumably also needs to recognize specific surfaces for attachment and potentially invasion. Although we have little understanding of when and where Photorhabdus strains go during the insect infection process, we do know that their titers in the hemolymph change rapidly (52) and that during the infection process the insect midgut is specifically destroyed (18). Thus, we do not know if the bacteria replicate in the insect hemocytes or if they invade the gut directly. However, even in the absence of substantial information concerning the basic biology of these organisms, we can make inferences about the likely infection process based on the array of genes that they carry which are putatively involved in locomotion and tissue-specific attachment and/or invasion. Most notable in the latter case are two hits to the attachment invasion locus (ail) found in Yersinia spp. (Table 6). In Y. enterocolitica this locus is responsible for the ability of the pathogen to cross the epithelium of the gut on its way to replicate in the reticuloendothelial system. Again, although we have no direct evidence that a putative homolog plays a similar role in Photorhabdus strains, we can test whether P. luminescens W14 can invade the gut (presumably from the hemocoel, not the lumen) and, if it can, whether deletion of the ail-like locus interferes with this ability. With respect to attachment, we also found a sequence similar to intimins, which are proteins homologous to the invasins of Yersinia spp. and which play a role in attachment and effacing of the brush border membrane (54).

TABLE 6.

Genes encoding proteins important in attachment and locomotion including fimbriae, pili, and adhesins

Gene function Organism Accession no. No. of hitsa BLASTX E valueb Clone(s) Reference
Virulence-associated attachment
 Ail, attachment invasion Y. enterocolitica P16454 1 (0) 2e-15 00876 124
 Ail, attachment invasion Y. pseudotuberculosis L49439 1 (0) 5e-13 02118 190
 Int, intimin (invasin) E. coli AF043226 1 (0) 3.2 (40) 01861 54
 TcpJ, toxin coregulated pilus leader peptidase V. cholerae P27717 1 (0) 1.3 (28) 01360 87
Fimbriae, chaperones, ushers, and adhesins
 AtfA, subunit of type 1 fimbria P. mirabilis Z78535 1 (0) 1.5 (27) 00198 117
 YehA, type 1 fimbrial protein E. coli P33340 1 (0) 0.03 (30) 01345 19
 FimA, type 1 fimbrial subunit S. marcescens P22595 1 (0) 0.2 (38) 00054 130
 FimB, recombinase E. coli S533063 1 (0) 3e-15 01186 Unp.c
 LpfB, chaperone (FimC-like) S. typhimurium P43661 1 (1) 1e-23 01029f 13
 Putative chaperone (FimC-like) E. coli P77249 1 (0) 2e-11 01718 Unp.
 Fimbrial chaperone (type 1) E. coli L77091 1 (0) 3e-10 00929 Unp.
 YraI, fimbrial chaperone E. coli P42914 1 (0) 1.9 (31) 01426 Unp.
 MrpD, fimbrial chaperone P. mirabilis Z32686 1 (0) 4e-77 02555 10
 FimD, outer membrane usher S. typhimurium P37924 1 (1) 2e-16 00377f Unp.
 FimD, outer membrane usher E. coli P30130 2 (0) 3e-17, 4e-22 01205, 01626 92
 HtrA, FimD-like usher E. coli P33129 1 (0) 1e-29 00506 145
 MrpC, outer membrane usher P. mirabilis Z32686 2 (0) 9e-45, 9e-51 00479, 00155 10
 Caf1A, F1 capsule anchoring (adhesin-like) Y. pestis P26949 1 (0) 7e-12 00377 83
 S fimbrial adhesin E. coli 1713397E 1 (0) 8e-24 01362 Unp.
 Putative adhesin H. influenzae AF053125 1 (0) 2e-31 01905 114
 B precursor (Zn binding)
 Fibronectin-binding protein B E. coli D90745 1 (0) 3e-13 01325 135
Prepilin peptidase and dependent protein
 TapD, prepilin peptidase type IV Aerumonas salmonicida AF059249 1 (0) 5e-27 02076 Unp.
 Prepilin peptidase type IV Pseudomonas stutzeri AJ132364 1 (0) 0.004 01150 Unp.
 PpdD, prepilin peptidase-dependent protein E. coli P36647 1 (0) 3e-09 01704 183
Flh and Fli
 Flagellar hook-associated protein 2 (FliD-like) X. nematophilus X91047 2 (0) 9e-55, 4e-63 01895, 01903 59
 FliF, flagellar M-ring protein E. coli P25798 1 (0) 4e-68 00243 150
 FliL, flagellar protein S. typhimurium P26417 1 (0) 2e-33 02318 89
 FliQ, flagellar protein S. typhimurium P54701 1 (0) 2e-22 01686 Unp.
 FliS, flagellar protein S. typhimurium P26609 1 (0) 1e-30 00187 88
 FliZ, flagellar protein S. typhimurium AB010947 1 (0) 2e-28 02310 75
 FlhA, flagellar biosynthesis P. mirabilis Q51910 1 (0) 3e-57 00007 Unp.
 FlhE, flagellar protein E. coli P76297 1 (0) 0.19 (33) 01798 76
 FlgA, flagellar basal body P. mirabilis U82214 1 (0) 3e-26 00676 64
 P-ring formation protein
 FlgI, flagellar P-ring protein Agrobacterium Q44340 1 (0) 0.9 (37) 02471 38
 FlgL, flagellar hook-associated protein (HAP3) S. typhimurium P13326 1 (0) 2e-08 01534 69
 FlgN, flagellar synthesis protein P. mirabilis U82214 1 (0) 2e-32 00234 64
 Flagellum-specific ATP synthase E. coli P52612 1 (0) 2e-68 01021 76
a

Number of sequence hits. The numbers in parentheses are the numbers of flipped sequences (indicated by the suffix f in the clone designations). 

b

The numbers in parentheses are percent amino acid identities. 

c

Unp., unpublished data. 

Another class of proteins involved in recognition of specific tissues is the class that includes the fimbriae and the associated adhesins. It has been hypothesized that in X. nematophilus fimbriae are involved in establishment of the specific association between the bacterium and the nematode gut (52). In the Photorhabdus sample sequence, we detected numerous matches with sequences encoding fimbrial type 1 subunits, fimbrial chaperones, and the outer membrane ushers associated with fimbrial export and assembly (Table 6). Although it is difficult to predict from these sequence matches the likely fimbrial composition of P. luminescens W14, we found both mrpC-like and mrpD-like loci, which encode the outer membrane usher and fimbrial chaperone from the Mrp (mannose-resistant, Proteus-like) fimbriae of Proteus mirabilis, respectively, and also FimD-like ushers and FimC-like chaperones from E. coli. The FimD sequence also exhibits similarity to S fimbrial adhesins, filamentous hemaglutinin A, and bovine colonization factor, implying that it may also play a role in virulence-associated adhesion. A second indication that there is another group of genes involved in a diverse array of functions that include fimbrial biogenesis, protein secretion, and DNA uptake (68) is the presence of sequences similar to those encoding a prepilin type of leader peptidase. Again, the significance of the presence of these sequences in Photorhabdus sp. is not clear, but this topic warrants further investigation.

Flagella are important in bacterial locomotion, and phase I Xenorhabdus cells exhibit swarming motility when they are grown on suitable solid media (52). Correspondingly, extracts from phase I variants appear to contain flagellar filaments (flagellin), whereas phase II cells do not (52). Although the molecular mechanism of this defect in flagellin synthesis is unclear, we found several ORFs in both fli-like and flh-like operons in P. luminescens W14 (Table 6). These ORFs include the FliD-like hook-associated protein 2 previously cloned from X. nematophilus (59), which is differentially transcribed in the two phase variants. Previous experiments have shown that insertion of a transposon into the flgN gene of P. mirabilis resulted in a mutant which was still motile but had lost the ability to swarm (64). This suggests that specific flagella are independently responsible for the swarming and motility phenotypes. Identification of the genes encoding these two classes of flagella in P. luminescens may, therefore, enable us to elucidate not only what types of flagella are produced by the bacterium but in which phase variants they are expressed and what function they perform.

Finally, we found three different sequences that potentially encode outer membrane proteins (Omp). The outer membrane protein composition of X. nematophilus changes as the organism enters the stationary phase of growth, and the outer membrane proteins, which are thought to form pores, may be responsible for functions that are necessary for survival under stress conditions (52). For example, expression of cloned ompF of S. marcescens is increased in E. coli under high-osmolarity conditions (73). It has also been hypothesized that the X. nematophilus outer membrane proteins play a role in specific interactions with the nematode host (52). Production of these proteins is regulated by EnvZ, as discussed above.

Secretion and transport.

One of the most striking features of P. luminescens grown in a liquid culture is the large number of proteins that are secreted into the supernatant. Some of these proteins have been well characterized, including the Tc toxins, proteases, and lipases discussed above. However, most of the secreted proteins are poorly characterized, and, perhaps equally importantly, their mechanisms of export are not known. Thus, for example, the mechanism and timing of secretion of the Tc toxins in the insect host remain obscure. Below we discuss sequences similar to different types of secretion machinery, notably type III-like secretion systems and ABC transporters. We observed a series of hits to the Yop type III secretion system of Yersinia species, including sequences similar to both Yop proteins, Ysc secretion proteins, and Syc Yop-specific chaperones (Table 7). The yop virulon enables Yersinia cells to survive and multiply in the lymphoid tissues of their hosts (36). The Yop proteins are encoded on the pYV plasmid at the low-calcium-response locus, and virulent Yersinia cells secrete these virulence determinants when they are incubated at 37°C in the absence of Ca2+ ions. The Yop proteins themselves are involved in contact-dependent delivery of toxins and effector molecules. Thus, in P. luminescens they could potentially be responsible for delivering toxins to either the gut or the insect hemocytes. As discussed above, the virF virulence regulon transcriptional regulator (BLASTX E value, 2e-44) (Table 5) regulates production of Yop proteins. This gene is, therefore, a very interesting candidate for knockout in P. luminescens, as its loss may alter the pathogenesis of Photorhabdus cells with different insect tissues and potentially ascribe a function to the presence of the Yop-like sequences in strain W14.

TABLE 7.

Secretion and transport, including Yop and low-calcium response-like sequences and ABC transporters

Gene function Organism Accession no. No. of hitsa BLASTX E value(s)b Clone(s) Reference
Yops and low-calcium response-like stimulon (type III secretion)
 Yop37, outer membrane protein Y. enterocolitica plasmid pYV 153665 1 (0) 5e-46 01174 11
 YopT, Yop effector Y. enterocolitica AF12990 1 (0) 0.015 (31) 02522 Unp.
 Invasin precursor/Yop1 adhesin Y. pseudotuberculosis P10858 1 (0) 4.1 (31) 02074 151
 YscC, Yop secretion protein C Y. enterocolitica plasmid pYV Q01244 1 (0) 3e-23 02011 123
 YscO, Yop secretion protein O Y. pestis AF020214 1 (0) 7e-13 01758 Unp.c
 YscP, Yop secretion protein P Y. pestis P40295 1 (1) 1e-11 01758f 50
 YscQ, Yop secretion protein Q Y. pseudotuberculosis P40296 1 (0) 1e-14 02311 16
 SycN, YopN chaperone Y. enterocolitica pYV03 M32097 1 (0) 4e-38 00164 178
General secretory pathway proteins
 General pathway protein F (cholera toxin secretion) V. cholerae P45780 1 (0) 2e-10 01002 Unp.
 General pathway protein F Burkholderia pseudomallei AF110185 1 (0) 2.0 (27) 02509 40
ABC transporters: peptide and amino acid
 OppA, oligopeptide binding S. typhimurium P06202 2 (0) 7e-44, 6e-48 01442, 01703 66
 OppB, oligopeptide transport E. coli P31132 1 (0) 1e-61 01507 84
 DppA, dipeptide transport E. coli P23847 1 (0) 2e-19 00410 134
 SapC, peptide transport E. coli Q47624 1 (0) 1e-68 01143 Unp.
 ArtP, arginine transport E. coli P30858 1 (0) 1e-74 00618 19
 ProU, glycine betaine transport E. coli P14175 1 (0) 2e-32 01868 163
 GltL, amino acid transport H. influenzae P45022 1 (0) 3e-46 01437 51
 TauB, taurine transport E. coli Q47538 1 (0) 8e-23 01789 176
 Thiamine ABC transporter H. influenzae U32782 1 (0) 2e-32 00615 51
 YvrO, amino acid transport Bacillus subtilis AJ223978 1 (0) 3e-08 00910 185
 LivM, branched amino acids S. typhimurium P30296 1 (0) 3e-40 00622 118
 CelB, cellobiose permease Borrelia burgdorferi AE000792 1 (0) 1e-25 02398 55
Sugar transport (Pts and Rbs)
 PtsG, glucose specific B. burgdorferi AE001166 1 (0) 1e-17 01900 Unp.
 PTS, mannitol specific E. coli P00550 1 (0) 1e-35 01409 177
 PTS, mannose subunit V. furnissii U65015 1 (0) 1e-35 01307 21
 YidK, glucose transport-like E. coli P31448 3 (0) 4e-10 to 2e-38 01297, 01302, 01310 29
 RbsA, ribose transport E. coli P04983 1 (0) 4e-89 01654 26
 RbsC, ribose transport E. coli P04984 2 (0) 8e-11, 3e-28 00853, 00957 15
 MalF, maltose transport Enterobacter aerogenes P18812 1 (0) 4e-38 01788 37
 YbbA, heterocyst maturation H. influenzae P45247 1 (0) 4e-23 01616 51
 YbbI, transcriptional regulator E. coli P77565 1 (0) 8e-27 02591 Unp.
a

Number of sequence hits. The numbers in parentheses are the numbers of flipped sequences (indicated by the suffix f in the clone designations). 

b

The numbers in parentheses are percent amino acid identities. 

c

Unp., unpublished data. 

In addition to contact-dependent secretion, the ABC transporters represent a large family of transporter systems with a diverse array of functions, including transport of peptides, amino acids, sugars, and metal ions. We, therefore, catalogued some of the sequences similar to ABC-like transporters (Table 7), and below we discuss some of their potential functions in P. luminescens. There were several sequences similar to peptide and amino acid transporters. Two potential homologs, OppA and ProU, are of special interest. OppA is located in the periplasm and is required for uptake of peptide antibiotics in E. coli and S. typhimurium (66). ProU, the product of the proU locus (also found in both E. coli and S. typhimurium), is a high-affinity glycine betaine transport system which plays an important role in survival under osmotic stress conditions (163). There were also several sequences similar to various sugar transporters and their transcriptional regulators. Central among these was the bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS), which catalyzes cellular uptake and subsequent phosphorylation of carbohydrates and also plays a crucial role in the global regulation of various metabolic pathways (177). The presence of PTS-like sequences in Photorhabdus cells is potentially important because chitin-degrading bacteria, such as Vibrio furnissii, rely on PTSs in the chitin catabolic cascade (21), and P. luminescens may therefore utilize a similar system for degrading insect chitin.

Polysaccharide biosynthesis.

Another striking feature of the P. luminescens culture supernatant is the large amount of LPS present. LPS production has been directly implicated in virulence in P. luminescens, as it has been in a wide range of other bacteria. For example, in B. pertussis the LPS is biologically active and is both toxic and immunogenic (5). LPS can also act as a recognition or binding site for extracellular agents. Thus, core LPS can act as a binding site for bacteriocins (alongside the outer membrane proteins OmpA and OmpF, as discussed above), while the trsG operon (Table 8) is required for biosynthesis of the bacteriophage Phi R1-37 receptor structures (158). The lipid A-core component of LPS is synthesized by sequential addition of sugars and fatty acids, and several sequences similar those involved in LPS biosynthesis were found in the sample sequence. These include envA (lpxC), which encodes an enzyme necessary for synthesis of the lipid A moiety (94), and rfaC, which is required for LPS inner-core synthesis (31). We also found genes likely to encode polysaccharide export functions, such as rcsF, which confers a mucoid phenotype (57), and wza, which encodes an outer membrane lipoprotein probably responsible for colanic acid (extracellular polysaccharide) export (161). Finally, genes encoding pullulanaselike proteins (starch-debranching enzymes) are also present; these proteins may play a role in recycling of the cell wall.

TABLE 8.

Polysaccharide biosynthesis, secretion, and recycling

Gene function Organism Accession no. No. of hitsa BLASTX E value(s) Clone(s) Reference
Core LPS biosynthesis
 Putative glycosyltransferase S. marcescens U52844 2 (0) 1e-29, 7e-34 00798, 00944 61
 Glycosyltransferase homolog B. pertussis S70676 1 (0) 1e-19 02392 5
 TrsG, mannosyltransferase Y. enterocolitica S51266 1 (0) 3e-23, 3e-43 02267, 02222 158
 RfaC, LPS heptosyltransferase 1 E. coli P24173 1 (0) 8e-50 00086 31
 RfaD, ADP-l-glycero-d-manno-heptose-6-epimerase H. influenzae P45048 1 (0) 2e-53 00230 51
 RfbU-like, LPS biosynthesis Methanobacterium thermoautotrophicum AE000829 1 (0) 1e-16 00285 Unp.b
 YfbE, spore coat polysaccharide E. coli P77690 2 (0) 7e-44, 2e-61 00452, 01453 Unp.
 MulI, murein-lipoprotein Erwinia amylovora P02939 2 (0) 1e-30, 1e-30 01917, 02090 187
 LpxA, UDP-N-acetylglucosamine acyltransferase P. mirabilis P72215 1 (0) 5e-8501015 Unp.
 EnvA (LpxC), N-acetylglucosamine deacetylase E. coli P07652 1 (0) 3e-68 01963 94
 RcsF, exopolysaccharide synthesis regulator E. coli P28633 1 (0) 2e-17 00997 57
 Wza, polysaccharide export E. coli P76388 1 (0) 5e-81 00362 161
 PulA, pullulanase Klebsiella aerogenes M16187 2 (0) 2e-18, 1e-71 00894, 00974 85
 PulA, pullulanase Klebsiella pneumoniae P07206 1 (0) 5e-36 02084 98
 GalE, UDP-glucose 4-epimerase Bacillus subtilis P55180 1 (0) 3e-05 02567 191
 GalR, galactose operon repressor E. coli P03024 2 (0) 1e-09, 4e-23 01736, 01949 179
 GalT, galactose-1-P uridyltransferase E. coli X06226 1 (0) 4e-28 02336 105
a

Number of sequence hits. The numbers in parentheses are the numbers of flipped sequences (indicated by the suffix f in the clone designations). 

b

Unp., unpublished data. 

Iron acquisition and transport.

As iron is often a rate-limiting growth factor in the host, many pathogenic bacteria have high-affinity iron-binding systems which can capture iron from host iron chelators. Thus, P. luminescens W14 has sequences which predict proteins similar to those involved in biosynthesis, transport, and reception of the siderophore yersiniabactin. Yersiniabactin (Ybt) has a high affinity for ferric iron, and similar siderophore-dependent iron transport systems are found in Yersinia pestis, Yersinia pseudotuberculosis, and Y. enterocolitica (140). A similar system may, therefore, also be used by P. luminescens. The irp1 and irp2 genes are required for yersiniabactin synthesis, as is ybtE, which encodes yersiniabactin dihydroxybenzoate ligase (Table 9). Transport of the iron-yersiniabactin complex back into the cell requires the TonB-dependent surface receptor FyuA, which may also be present in P. luminescens W14. This receptor is highly conserved and is found in all pesticin-sensitive bacteria, including E. coli (146). The sample sequence also contained hits to an R4-like ferric siderophore receptor from E. coli, which may perform a similar function in P. luminescens, and a putative operon (pvcABCD) involved in synthesis of the chromophore moiety of the P. aeruginosa siderophore pyoverdine (162).

TABLE 9.

Iron assimilation: ferric siderophore biosynthesis and transport and regulation of iron and other metals

Gene function Organism Accession no. No. of hitsa BLASTX E value(s) Clone(s) Reference
Biosynthesis and reception of yersiniabactin-like siderophore
 Irp1, HMWP1-like Y. enterocolitica Y12527 12 (3) 4e-07 to 4e-35 00066, 00066f, 00245, 00269, 00498, 00647, 01200, 01491, 01491f, 01492, 01510, 01614f 139
 Irp2, HMWP2-like Y. enterocolitica P48633 4 (2) 1e-15 to 5e-62 00062, 00062f, 00758, 00758f 139
 Irp5, YbtE-like Y. pestis U50364 1 (0) 3e-59 01573 139
Ferric siderophore receptor-like
 FyuA, yersiniabactin receptor Y. enterocolitica P46360 1 (0) 2e-25 02508 146
 R4, ferric siderophore receptor E. coli P27772 2 (0) 8e-06, 5e-30 00567, 01023 63
Pyoverdin siderophore synthesis
 PvcA, pyoverdin chromophore P. aeruginosa AF002222 1 (0) 1e-48 00220 162
 PvcC, pyoverdin chromophore P. aeruginosa AF002222 1 (0) 1e-10 02521 162
Other iron and hemin transport systems
 YfeE, YfeABCD regulator Y. pestis Q56956 1 (0) 3e-32 01897 Unp.b
 HmuR, outer membrane receptor Y. pestis Q56989 1 (0) 2e-62 002244 71
 HmuS, transport protein Y. pestis Q56990 1 (0) 6e-57 00590 71
 HemT, hemin binding periplasmic Y. enterocolitica X77867 1 (0) 8e-64 00516 165
 HemT, hemin binding periplasmic Y. pestis Q56991 3e-45 71
 FecA, outer membrane E. coli P13036 1 (0) e-119 01194 159
 FecC, cytosolic E. coli P15030 1 (0) 8e-38 02411 159
 FecE, ATP binding Synechocystis sp. D90899 1 (0) 1e-12 00752 82
 FeoB, ferrous iron transport E. coli P336650 2 (0) 5e-49, 3e-72 01462, 02346 80
Iron regulation and regulated proteins
 Fur, ferric uptake regulation E. coli P06975 1 (1) 2e-52 00174f 155
 DtxR, diphtheria toxin repressor (iron dependent) Corynebacterium diphtheriae U20617 1 (0) 8e-18 02031 156
Hem biosynthesis
 Hem2, porphobilinogen synthase P. aeruginosa Q59643 1 (0) 7e-48 02146 Unp.
 Hem6, coproporphyrinogen III oxidase, aerobic S. typhimurium P33771 2 (0) 8e-31, 2e-87 00810, 00883 186
 HemE-like (DcuP) uroporphyrinogen decarboxylase E. coli P29680 1 (0) 2e-59 00202 131
 HemN, coproporphyrinogen III oxidase, oxygen independent S. typhimurium P37129 1 (0) 8e-33 01830 186
 HemN, coproporphyrinogonen III oxidase, oxygen independent Bacillus subtilis P54304 1 (0) 0.001 02315 70
 HemY, protohem IX synthesis E. coli P09128 1 (0) 4e-14 00743 4
 HemZ, ferrocheletase Y. enterocolitica P43413 1 (0) 8e-37 00814 165
 NirJ-2, heme biosynthesis Archaeoglobus fulgidus AE000964 1 (0) 1e-05 00348 93
 CysG, uroporphyrinogen III methylase, CG site 893 E. coli P11098 2 (0) 1e-11, 5e-18 00955, 01812 138
 CysI, sulfite reductase hemoprotein component E. coli M23008 1 (0) 3e-20 01919 136
Ferredoxin-like proteins
 Fer, ferredoxin E. coli P25528 1 (0) 2e-40 01022 167
 YfhL, ferredoxin-like E. coli P52102 2 (0) 3e-33, 1e-38 01294, 01535 Unp.
 HcaD-like, ferredoxin reductase Sphingomonas aromaticivorans AF079317 1 (0) 3e-08 01745 Unp.
a

Number of sequence hits. The numbers in parentheses are the numbers of flipped sequences (indicated by the suffix f in the clone designations). 

b

Unp., unpublished data. 

P. luminescens, like Yersinia spp., also appears to contain alternative iron and hemin transport systems, as indicated by hits to genes similar to yfeE, the yfeABCD ferric iron uptake operon regulator, and members of the hmu hemin utilization system. The latter system is essential in Y. pestis for utilization of free hemin and heme-protein complexes, which are the bacterium's sole sources of iron (71). P. luminescens also contains sequences similar to members of both the feo iron(II) (80) and fec iron(III) (159) transport systems. Finally, like numerous other gram-negative bacteria, P. luminescens also has a sequence similar to the fur gene sequence. This gene is involved in iron regulation, and in the presence of excess iron, the fur gene product generally represses expression of iron-regulated genes (140). Together, these sequences suggest that scavenging and transporting iron are important in P. luminescens, as they are in many pathogenic bacteria.

Extrachromosomal elements.

Like the genomes of other bacteria, the P. luminescens sample sequence contains many sequences similar to sequences found in a wide range of phage and insertion sequence elements. These sequences are important because they may begin to explain how P. luminescens, an insect pathogen, acquired virulence factors previously associated only with vertebrate pathogenicity, such as sequences similar to the low-calcium-response stimulon from Yersinia discussed above. Numerous hits to tail proteins from P2-like bacteriophages (46) (P2, P4, 186, and HP1) and a range of other phage-related proteins were observed (n = 51). There were also 10 hits (BLASTX P values, 0.01 to 2e-86) to products of the integrase (int) gene, which controls phage site-specific integration. Notably, in the range of phage homologies there were hits (although with relatively low significance [BLASTX E values, 0.3 to 5e-15]) to three different ORFs (ORFs 16, 20, and 25) of the P. aeruginosa cytotoxin converting phage Phi CTX (cholera toxin). We note that the rtx gene cluster is physically linked to the CTX toxin element in the V. cholerae genome (112). Therefore, it will be interesting to investigate whether this element is linked with the rtxA-like sequences found in P. luminescens W14, suggesting that it could have been responsible for horizontal transfer of the toxin-encoding genes. Numerous transposon-like sequences were also found (n = 33), including 10 hits to a transposase from plasmid ColIb-P9 (BLASTX E values, 1e-04 to 4e-86). Again, although these sequences indicate that transfer events occurred, it is not known how long these transposons have been present and if any of them have retained functionality. Finally, the P. luminescens W14 genome contains numerous sequences related to sequences involved in plasmid maintenance and stability (Table 10). However, we cannot at this stage distinguish which of these sequences are plasmid encoded (plasmids have been found previously in Xenorhabdus spp. [103]) and which are chromosomal. The presence of these sequences, therefore, raises the possibility of plasmid maintenance in P. luminescens W14 but is not strictly indicative.

TABLE 10.

Extrachromosomal elements or inserted elements, including transposons and insertion sequence elementsa

Gene function Organism Accession no. No. of hitsb BLASTX E value(s)c Clone(s) Reference
Plasmid associated and plasmid stability
 Orf4, hypothetical Enterobacteriaceae plasmid R100 S28661 1 (0) 4.4 (33) 01224 7
 YY08, predicted Orf Methanococcus jannaschii plasmid pURB801 Q60307 1 (0) 2.0 (26) 00214 27
 ParD, stabilization factor E. coli pRP4 P22995 1 (0) 2e-32 00808 149
 StbB, stability protein P. syringae Q52562 1 (0) 1e-17 01196 Unp.d
 StbD, stability protein Morganella morganii AF072126 1 (0) 7e-12 01412 65
 Plasmid stability-like Thiobacillus ferrooxidans U73041 1 (0) 2.0 (50) 01275 42
 Dma7, DNA adenine methylase E. coli retron EC67 P21311 1 (0) 0.013, 8e-23 00173 72
 Orf1, hypothetical E. coli retron EC67 P21323 1 (0) 8e-23 01168 72
Replication, repair, transformation, and conjugation
 PriA, replication factor N′ E. coli A35505 2 (0) 5e-69, 1e-87 01203, 01446 132
 PriC, replication factor N" E. coli P23862 1 (0) 3e-07 01634 192
 UvrA, ABC excinuclease A S. typhimurium P37434 1 (0) 4e-58 01967 Unp.
 UvrB, ABC exinuclease B E. coli P07025 1 (0) 1e-95 02230 9
 UvrC, ABC exinuclease C E. coli P07028 1 (0) 4e-28 01063 153
 TfoX, DNA transformation H. influenzae P43779 1 (0) 3e-05 00715 194
 ComE, DNA transformation H. influenzae P31772 1 (0) 2e-24 00510 102
 DNA transformation-like H. influenzae JH0436 1 (0) 3e-26 01234 173
 Ex5A, exodeoxyribonuclease E. coli P04993 3 (0) 7e-32 to 2e-70 01529, 01652, 01842 19
Restriction enzymes and their control
 Tlr1, type I restriction enzyme EcoR124II E. coli P10486 1 (0) 2e-84 02345 144
 Tls1, type 1 restriction enzyme EcoRI24II specificity E. coli P10485 1 (0) 6e-30 00989 144
NgoMI, restriction enzyme type II Neisseria gonorrhoeae P31032 1 (1) 3e-07 00484f 160
NaeI modification methylase cytosine specific Nocardia aerocolonigenes P50188 1 (1) 3e-20 01699f 169
BamHI control element Bacillus amyloliquefaciens X55285 1 (0) 4e-06 00175 25
a

Data for phage and phage-related proteins are not included. 

b

Number of sequence hits. The numbers in parentheses are the numbers of flipped sequences (indicated by the suffix f in the clone designations). 

c

The numbers in parentheses are percent amino acid identities. 

d

Unp., unpublished data. 

Conclusions.

P. luminescens has a life cycle which introduces it into a diverse array of environments, and in only one of these environments, the insect environment, is the bacterium pathogenic. The sample sequence of strain W14 revealed sequences similar to the sequences of a diverse array of potential virulence factor-encoding genes, including the genes for several classes of toxins, proteases, lipases, and LPS. It also gave us some indication of the diversity of the transport and metabolic systems present. Furthermore, Photorhabdus spp. also seem to share potential virulence factors (Yops, a yersiniabactin-like siderophore, and the low-calcium-response stimulon) with distantly related vertebrate pathogens, such as members of the genus Yersinia. This hypothesis is supported by the presence of numerous phagelike and transposon-like sequences in the P. luminescens genome. The potential for horizontal transfer raises the intriguing possibility that the virulence factors present in invertebrate pathogens may also be present in vertebrate pathogens. Given the far greater diversity of invertebrates and, potentially, their associated pathogens, this raises interesting questions about the diversity and origins of potential vertebrate virulence factors. In relation to P. luminescens itself, complete elucidation of the genome sequence of strain W14 and other strains should allow us to begin to understand the roles of individual genes via targeted disruption and to begin to compare the diversity of virulence factors found in different invertebrate pathogens. Our findings are consistent with the hypothesis of Burland et al. (30), who hypothesized that all of the pathogenic genes shared by enteric bacteria form a pool or “pathosphere”; however, here we emphasize that the pool must be extended to include both invertebrate and vertebrate pathogens. Furthermore, as invertebrates evolved before vertebrates, this also raises the interesting possibility that pathogens such as P. luminescens include the progenitors of virulence factors in vertebrate pathogens.

ACKNOWLEDGMENTS

This work was supported by grants to R.F.C from the BBSRC, the Wellcome Trust (JIF), and DowAgroSciences, which we thank for their interest and support.

We thank all of the technical staff of the Wisconsin Genome Project for their help with sequencing and assembly. We thank David Clarke for comments on the manuscript.

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