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

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. AQ989457–AQ991805.

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.

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. luminescens^a

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

^aSee Fig. 2 for genomic locations. 

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

^cUnp., unpublished data. 

^dClone 00357 represents a sequence containing two different ORFs. 

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

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

^bUnp., 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

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

^bUnp., 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 proteins^a

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

^aSee Fig. 3 for putative genomic organizations. 

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

^cThe numbers in parentheses are percentages. 

^dClone 01175 represents a sequence containing two different ORFs. 

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.

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

^bThe numbers in parentheses are percent amino acid identities. 

^cUnp., 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

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

^bThe numbers in parentheses are percent amino acid identities. 

^cUnp., 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.

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

^bThe numbers in parentheses are percent amino acid identities. 

^cUnp., 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

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

^bUnp., 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.

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

^bUnp., 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 elements^a

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

^aData for phage and phage-related proteins are not included. 

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

^cThe numbers in parentheses are percent amino acid identities. 

^dUnp., 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.