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. 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 |
See Fig. 2 for genomic locations.
Number of sequence hits. The numbers in parentheses are the numbers of flipped sequences (indicated by the suffix f in the clone designations).
Unp., unpublished data.
Clone 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 |
Number of sequence hits. The numbers in parentheses are the numbers of flipped sequences (indicated by the suffix f in the clone designations).
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 |
Number of sequence hits. The numbers in parentheses are the numbers of flipped sequences (indicated by the suffix f in the clone designations).
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 |
See Fig. 3 for putative genomic organizations.
Number of sequence hits. The numbers in parentheses are the numbers of flipped sequences (indicated by the suffix f in the clone designations).
The numbers in parentheses are percentages.
Clone 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. |
Number of sequence hits. The numbers in parentheses are the numbers of flipped sequences (indicated by the suffix f in the clone designations).
The numbers in parentheses are percent amino acid identities.
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 |
Number of sequence hits. The numbers in parentheses are the numbers of flipped sequences (indicated by the suffix f in the clone designations).
The numbers in parentheses are percent amino acid identities.
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. |
Number of sequence hits. The numbers in parentheses are the numbers of flipped sequences (indicated by the suffix f in the clone designations).
The numbers in parentheses are percent amino acid identities.
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 |
Number of sequence hits. The numbers in parentheses are the numbers of flipped sequences (indicated by the suffix f in the clone designations).
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. |
Number of sequence hits. The numbers in parentheses are the numbers of flipped sequences (indicated by the suffix f in the clone designations).
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 |
Data for phage and phage-related proteins are not included.
Number of sequence hits. The numbers in parentheses are the numbers of flipped sequences (indicated by the suffix f in the clone designations).
The numbers in parentheses are percent amino acid identities.
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.
REFERENCES
- 1.Aiba H, Baba T, Hayashi K, Inada T, Isono K, Itoh T, Kasai H, Kashimoto K, Kimura S, Kitakawa M, Kitagawa M, Makino K, Miki T, Mizobuchi K, Mori H, Mori T, Motomura K, Nakade S, Nakamura Y, Nashimoto H, Nishio Y, Oshima T, Saito N, Sampei G, Horiuchi T, et al. A 570-kb DNA sequence of the Escherichia coli K-12 genome corresponding to the 28.0-40.1 min region on the linkage map. DNA Res. 1996;3:363–377. doi: 10.1093/dnares/3.6.363. [DOI] [PubMed] [Google Scholar]
- 2.Akatsuka H, Kawai E, Omori K, Shibatani T. The three genes lipB, lipC, and lipD involved in the extracellular secretion of the Serratia marcescens lipase which lacks an N-terminal signal peptide. J Bacteriol. 1995;177:6381–6389. doi: 10.1128/jb.177.22.6381-6389.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Albertini A M, Caramori T, Scoffone F, Scotti C, Galizzi A. Sequence around the 159 degree region of the Bacillus subtilis genome: the pksX locus spans 33.6 kb. Microbiology. 1995;141:299–309. doi: 10.1099/13500872-141-2-299. [DOI] [PubMed] [Google Scholar]
- 4.Alefounder P R, Abell C, Battersby A R. The sequence of hemC, hemD and two additional E. coli genes. Nucleic Acids Res. 1988;16:9871. doi: 10.1093/nar/16.20.9871. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Allen A, Maskell D. The identification, cloning and mutagenesis of a genetic locus required for lipopolysaccharide biosynthesis in Bordetella pertussis. Mol Microbiol. 1996;19:37–52. doi: 10.1046/j.1365-2958.1996.354877.x. [DOI] [PubMed] [Google Scholar]
- 6.Aono R, Negishi T, Nakajima H. Cloning of organic solvent tolerance gene ostA that determines n-hexane tolerance level in Escherichia coli. Appl Environ Microbiol. 1994;60:4624–4626. doi: 10.1128/aem.60.12.4624-4626.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Armstrong K A, Ohtsubo H, Bauer W R, Yoshioka Y, Miyazaki C, Maeda Y, Ohtsubo E. Characterization of the gene products produced in minicells by pSM1, a derivative of R100. Mol Gen Genet. 1986;205:56–65. doi: 10.1007/BF02428032. [DOI] [PubMed] [Google Scholar]
- 8.Avichezer D, Gilboa-Garber N, Garber N C, Katcoff D J. Pseudomonas aeruginosa PA-I lectin gene molecular analysis and expression in Escherichia coli. Biochim Biophys Acta. 1994;1218:11–20. doi: 10.1016/0167-4781(94)90095-7. [DOI] [PubMed] [Google Scholar]
- 9.Backendorf C, Spaink H, Barbeiro A P, van de Putte P. Structure of the uvrB gene of Escherichia coli. Homology with other DNA repair enzymes and characterization of the uvrB5 mutation. Nucleic Acids Res. 1986;14:2877–2890. doi: 10.1093/nar/14.7.2877. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Bahrani F K, Mobley H L. Proteus mirabilis MR/P fimbrial operon: genetic organization, nucleotide sequence, and conditions for expression. J Bacteriol. 1994;176:3412–3419. doi: 10.1128/jb.176.11.3412-3419.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Barteneva N S, Evstafieva A G, Gorelov V N, Wenzel B E. Identification and sequencing of a plasmid (pYV96)-encoded gene product of Yersinia enterocolitica recognized by antibodies in sera of patients with autoimmune thyroid disease. Ann N Y Acad Sci. 1994;730:345–347. doi: 10.1111/j.1749-6632.1994.tb44287.x. [DOI] [PubMed] [Google Scholar]
- 12.Bauer D W, Wei Z M, Beer S V, Collmer A. Erwinia chrysanthemi harpinEch: an elicitor of the hypersensitive response that contributes to soft-rot pathogenesis. Mol Plant-Microbe Interact. 1995;8:484–491. doi: 10.1094/mpmi-8-0484. [DOI] [PubMed] [Google Scholar]
- 13.Baumler A J, Heffron F. Identification and sequence analysis of lpfABCDE, a putative fimbrial operon of Salmonella typhimurium. J Bacteriol. 1995;177:2087–2097. doi: 10.1128/jb.177.8.2087-2097.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Beem J E, Nesbitt W E, Leung K P. Cloning of Prevotella intermedia loci demonstrating multiple hemolytic domains. Oral Microbiol Immunol. 1999;14:143–152. doi: 10.1034/j.1399-302x.1999.140302.x. [DOI] [PubMed] [Google Scholar]
- 15.Bell A W, Buckel S D, Groarke J M, Hope J N, Kingsley D H, Hermodson M A. The nucleotide sequences of the rbsD, rbsA, and rbsC genes of Escherichia coli K12. J Biol Chem. 1986;261:7652–7658. [PubMed] [Google Scholar]
- 16.Bergman T, Erickson K, Galyov E, Persson C, Wolf-Watz H. The lcrB (yscN/U) gene cluster of Yersinia pseudotuberculosis is involved in Yop secretion and shows high homology to the spa gene clusters of Shigella flexneri and Salmonella typhimurium. J Bacteriol. 1994;176:2619–2626. doi: 10.1128/jb.176.9.2619-2626.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Bernhard F, Demel G, Soltani K, Dohren H V, Blinov V. Identification of genes encoding for peptide synthetases in the gram-negative bacterium Lysobacter sp. ATCC 53042 and the fungus Cylindrotrichum oligospermum. DNA Seq. 1996;6:319–330. doi: 10.3109/10425179609047570. [DOI] [PubMed] [Google Scholar]
- 18.Blackburn M, Golubeva E, Bowen D, ffrench-Constant R H. A novel insecticidal toxin from Photorhabdus luminescens, Toxin complex a (Tca), and its histopathological effects on the midgut of Manduca sexta. Appl Environ Microbiol. 1998;64:3036–3041. doi: 10.1128/aem.64.8.3036-3041.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Blattner F R, Plunkett G, 3rd, Bloch C A, Perna N T, Burland V, Riley M, Collado-Vides J, Glasner J D, Rode C K, Mayhew G F, Gregor J, Davis N W, Kirkpatrick H A, Goeden M A, Rose D J, Mau B, Shao Y. The complete genome sequence of Escherichia coli K-12. Science. 1997;277:1453–1474. doi: 10.1126/science.277.5331.1453. [DOI] [PubMed] [Google Scholar]
- 20.Borchert S, Patil S S, Marahiel M A. Identification of putative multifunctional peptide synthetase genes using highly conserved oligonucleotide sequences derived from known synthetases. FEMS Microbiol Lett. 1992;71:175–180. doi: 10.1016/0378-1097(92)90508-l. [DOI] [PubMed] [Google Scholar]
- 21.Bouma C L, Roseman S. Sugar transport by the marine chitinolytic bacterium Vibrio furnissii. Molecular cloning and analysis of the mannose/glucose permease. J Biol Chem. 1996;271:33468–33475. doi: 10.1074/jbc.271.52.33468. [DOI] [PubMed] [Google Scholar]
- 22.Bowen D, Rocheleau T A, Blackburn M, Andreev O, Golubeva E, Bhartia R, ffrench-Constant R H. Insecticidal toxins from the bacterium Photorhabdus luminescens. Science. 1998;280:2129–2132. doi: 10.1126/science.280.5372.2129. [DOI] [PubMed] [Google Scholar]
- 23.Bowen D J, Ensign J C. Purification and characterization of a high-molecular-weight insecticidal protein complex produced by the entomopathogenic bacterium Photorhabdus luminescens. Appl Environ Microbiol. 1998;64:3029–3035. doi: 10.1128/aem.64.8.3029-3035.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Boyd C, Keen N T. Characterization of the prtA and prtB genes of Erwinia chrysanthemi EC16. Gene. 1993;133:115–118. doi: 10.1016/0378-1119(93)90234-t. [DOI] [PubMed] [Google Scholar]
- 25.Brooks J E, Nathan P D, Landry D, Sznyter L A, Waite-Rees P, Ives C L, Moran L S, Slatko B E, Benner J S. Characterization of the cloned BamHI restriction modification system: its nucleotide sequence, properties of the methylase, and expression in heterologous hosts. Nucleic Acids Res. 1991;19:841–850. doi: 10.1093/nar/19.4.841. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Buckel S D, Bell A W, Rao J K, Hermodson M A. An analysis of the structure of the product of the rbsA gene of Escherichia coli K12. J Biol Chem. 1986;261:7659–7662. [PubMed] [Google Scholar]
- 27.Bult C J, White O, Olsen G J, Zhou L, Fleischmann R D, Sutton G G, Blake J A, FitzGerald L M, Clayton R A, Gocayne J D, Kerlavage A R, Dougherty B A, Tomb J F, Adams M D, Reich C I, Overbeek R, Kirkness E F, Weinstock K G, Merrick J M, Glodek A, Scott J L, Geoghagen N S M, Venter J C. Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii. Science. 1996;273:1058–1073. doi: 10.1126/science.273.5278.1058. [DOI] [PubMed] [Google Scholar]
- 28.Burland V, Daniels D L, d. Plunkett G, Blattner F R. Genome sequencing on both strands: the Janus strategy. Nucleic Acids Res. 1993;21:3385–3390. doi: 10.1093/nar/21.15.3385. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Burland V, d. Plunkett G, Daniels D L, Blattner F R. DNA sequence and analysis of 136 kilobases of the Escherichia coli genome: organizational symmetry around the origin of replication. Genomics. 1993;16:551–561. doi: 10.1006/geno.1993.1230. [DOI] [PubMed] [Google Scholar]
- 30.Burland V, Shao Y, Perna N T, Plunkett G, Sofia H J, Blattner F R. The complete DNA sequence and analysis of the large virulence plasmid of Escherichia coli O157:H7. Nucleic Acids Res. 1998;26:4196–4204. doi: 10.1093/nar/26.18.4196. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Chen L, Coleman W G., Jr Cloning and characterization of the Escherichia coli K-12 rfa-2 (rfaC) gene, a gene required for lipopolysaccharide inner core synthesis. J Bacteriol. 1993;175:2534–2540. doi: 10.1128/jb.175.9.2534-2540.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Chen R, Henning U. Nucleotide sequence of the gene for the peptidoglycan-associated lipoprotein of Escherichia coli K12. Eur J Biochem. 1987;163:73–77. doi: 10.1111/j.1432-1033.1987.tb10738.x. [DOI] [PubMed] [Google Scholar]
- 33.Choo D W, Kurihara T, Suzuki T, Soda K, Esaki N. A cold-adapted lipase of an Alaskan psychrotroph, Pseudomonas sp. strain B11-1: gene cloning and enzyme purification and characterization. Appl Environ Microbiol. 1998;64:486–491. doi: 10.1128/aem.64.2.486-491.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Cole S T, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, Gordon S V, Eiglmeier K, Gas S, Barry C E, 3rd, Tekaia F, Badcock K, Basham D, Brown D, Chillingworth T, Connor R, Davies R, Devlin K, Feltwell T, Gentles S, Hamlin N, Holroyd S, Hornsby T, Jagels K, Barrell B G, et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature. 1998;393:537–544. doi: 10.1038/31159. [DOI] [PubMed] [Google Scholar]
- 35.Conlin C A, Trun N J, Silhavy T J, Miller C G. Escherichia coli prlC encodes an endopeptidase and is homologous to the Salmonella typhimurium opdA gene. J Bacteriol. 1992;174:5881–5887. doi: 10.1128/jb.174.18.5881-5887.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Cornelis G R, Wolf-Watz H. The Yersinia Yop virulon: a bacterial system for subverting eukaryotic cells. Mol Microbiol. 1997;23:861–867. doi: 10.1046/j.1365-2958.1997.2731623.x. [DOI] [PubMed] [Google Scholar]
- 37.Dahl M K, Francoz E, Saurin W, Boos W, Manson M D, Hofnung M. Comparison of sequences from the malB regions of Salmonella typhimurium and Enterobacter aerogenes with Escherichia coli K12: a potential new regulatory site in the interoperonic region. Mol Gen Genet. 1989;218:199–207. doi: 10.1007/BF00331269. [DOI] [PubMed] [Google Scholar]
- 38.Deakin W J, Furniss C S, Parker V E, Shaw C H. Isolation and characterisation of a linked cluster of genes from Agrobacterium tumefaciens encoding proteins involved in flagellar basal-body structure. Gene. 1997;189:135–137. doi: 10.1016/s0378-1119(96)00780-9. [DOI] [PubMed] [Google Scholar]
- 39.de Crecy-Lagard V, Blanc V, Gil P, Naudin L, Lorenzon S, Famechon A, Bamas-Jacques N, Crouzet J, Thibaut D. Pristinamycin I biosynthesis in Streptomyces pristinaespiralis: molecular characterization of the first two structural peptide synthetase genes. J Bacteriol. 1997;179:705–713. doi: 10.1128/jb.179.3.705-713.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.DeShazer D, Brett P J, Burtnick M N, Woods D E. Molecular characterization of genetic loci required for secretion of exoproducts in Burkholderia pseudomallei. J Bacteriol. 1999;181:4661–4664. doi: 10.1128/jb.181.15.4661-4664.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41.Dittmann E, Neilan B A, Erhard M, von Dohren H, Borner T. Insertional mutagenesis of a peptide synthetase gene that is responsible for hepatotoxin production in the cyanobacterium Microcystis aeruginosa PCC 7806. Mol Microbiol. 1997;26:779–787. doi: 10.1046/j.1365-2958.1997.6131982.x. [DOI] [PubMed] [Google Scholar]
- 42.Dominy C N, Deane S M, Rawlings D E. A geographically widespread plasmid from Thiobacillus ferrooxidans has genes for ferredoxin-, FNR-, prismane- and NADH-oxidoreductase-like proteins which are also located on the chromosome. Microbiology. 1997;143:3123–3136. doi: 10.1099/00221287-143-10-3123. [DOI] [PubMed] [Google Scholar]
- 43.Dorrell N, Li S R, Everest P H, Dougan G, Wren B W. Construction and characterization of a Yersinia enterocolitica O:8 ompR mutant. FEMS Microbiol Lett. 1998;165:145–151. doi: 10.1111/j.1574-6968.1998.tb13139.x. [DOI] [PubMed] [Google Scholar]
- 44.Duport C, Baysse C, Michel-Briand Y. Molecular characterization of pyocin S3, a novel S-type pyocin from Pseudomonas aeruginosa. J Biol Chem. 1995;270:8920–8927. doi: 10.1074/jbc.270.15.8920. [DOI] [PubMed] [Google Scholar]
- 45.Eriksson A R, Andersson R A, Pirhonen M, Palva E T. Two-component regulators involved in the global control of virulence in Erwinia carotovora subsp. carotovora. Mol Plant-Microbe Interact. 1998;11:743–752. doi: 10.1094/MPMI.1998.11.8.743. [DOI] [PubMed] [Google Scholar]
- 46.Esposito D, Fitzmaurice W P, Benjamin R C, Goodman S D, Waldman A S, Scocca J J. The complete nucleotide sequence of bacteriophage HP1 DNA. Nucleic Acids Res. 1996;24:2360–2368. doi: 10.1093/nar/24.12.2360. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 47.Farris M, Grant A, Richardson T B, O'Connor C D. BipA: a tyrosine-phosphorylated GTPase that mediates interactions between enteropathogenic Escherichia coli (EPEC) and epithelial cells. Mol Microbiol. 1998;28:265–279. doi: 10.1046/j.1365-2958.1998.00793.x. [DOI] [PubMed] [Google Scholar]
- 48.Felmlee T, Pellett S, Welch R A. Nucleotide sequence of an Escherichia coli chromosomal hemolysin. J Bacteriol. 1985;163:94–105. doi: 10.1128/jb.163.1.94-105.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 49.Feng J, Yamanaka K, Niki H, Ogura T, Hiraga S. New killing system controlled by two genes located immediately upstream of the mukB gene in Escherichia coli. Mol Gen Genet. 1994;243:136–147. doi: 10.1007/BF00280310. [DOI] [PubMed] [Google Scholar]
- 50.Fields K A, Plano G V, Straley S C. A low-Ca2+ response (LCR) secretion (ysc) locus lies within the lcrB region of the LCR plasmid in Yersinia pestis. J Bacteriol. 1994;176:569–579. doi: 10.1128/jb.176.3.569-579.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 51.Fleishmann R D, Adams M D, White O, Clayton R A, Kirkness E F, Kerlavage A R, Bult C J, Tomb J F, Dougherty B A, Merrick J M, et al. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science. 1995;269:496–512. doi: 10.1126/science.7542800. [DOI] [PubMed] [Google Scholar]
- 52.Forst S, Nealson K. Molecular biology of the symbiotic-pathogenic bacteria Xenorhabdus spp. and Photorhabdus spp. Microbiol Rev. 1996;60:21–43. doi: 10.1128/mr.60.1.21-43.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 53.Frank D W, Iglewski B H. Cloning and sequence analysis of a trans-regulatory locus required for exoenzyme S synthesis in Pseudomonas aeruginosa. J Bacteriol. 1991;173:6460–6468. doi: 10.1128/jb.173.20.6460-6468.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 54.Frankel G, Candy D C, Everest P, Dougan G. Characterization of the C-terminal domains of intimin-like proteins of enteropathogenic and enterohemorrhagic Escherichia coli, Citrobacter freundii, and Hafnia alvei. Infect Immun. 1994;62:1835–1842. doi: 10.1128/iai.62.5.1835-1842.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 55.Fraser C M, Casjens S, Huang W M, Sutton G G, Clayton R, Lathigra R, White O, Ketchum K A, Dodson R, Hickey E K, Gwinn M, Dougherty B, Tomb J F, Fleishmann R D, Richardson D, Peterson J, Kerlavage A R, Quackenbush J, Salzberg S, Hanson M, van Vugt R, Palmer N, Adams M D, Gocayne J, Venter J C, et al. Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi. Nature. 1997;390:580–586. doi: 10.1038/37551. [DOI] [PubMed] [Google Scholar]
- 56.Garcia del Portillo F, de Pedro M A, Ayala J A. Identification of a new mutation in Escherichia coli that suppresses a pbpB (Ts) phenotype in the presence of penicillin-binding protein 1B. FEMS Microbiol Lett. 1991;68:7–13. doi: 10.1016/0378-1097(91)90386-o. [DOI] [PubMed] [Google Scholar]
- 57.Gervais F G, Drapeau G R. Identification, cloning, and characterization of rcsF, a new regulator gene for exopolysaccharide synthesis that suppresses the division mutation ftsZ84 in Escherichia coli K-12. J Bacteriol. 1992;174:8016–8022. doi: 10.1128/jb.174.24.8016-8022.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 58.Gilson L, Mahanty H K, Kolter R. Genetic analysis of an MDR-like export system: the secretion of colicin V. EMBO J. 1990;9:3875–3894. doi: 10.1002/j.1460-2075.1990.tb07606.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 59.Givaudan A, Lanois A, Boemare N. Cloning and nucleotide sequence of a flagellin encoding genetic locus from Xenorhabdus nematophilus: phase variation leads to differential transcription of two flagellar genes (fliCD) Gene. 1996;183:243–253. doi: 10.1016/s0378-1119(96)00452-0. [DOI] [PubMed] [Google Scholar]
- 60.Goncharoff P, Saadi S, Chang C H, Saltman L H, Figurski D H. Structural, molecular, and genetic analysis of the kilA operon of broad- host-range plasmid RK2. J Bacteriol. 1991;173:3463–3477. doi: 10.1128/jb.173.11.3463-3477.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 61.Guasch J F, Pique N, Climent N, Ferrer S, Merino S, Rubires X, Tomas J M, Regue M. Cloning and characterization of two Serratia marcescens genes involved in core lipopolysaccharide biosynthesis. J Bacteriol. 1996;178:5741–5747. doi: 10.1128/jb.178.19.5741-5747.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 62.Guenzi E, Galli G, Grgurina I, Gross D C, Grandi G. Characterization of the syringomycin synthetase gene cluster: a link between prokaryotic and eukaryotic peptide synthetases. J Biol Chem. 1998;49:32857–32863. doi: 10.1074/jbc.273.49.32857. [DOI] [PubMed] [Google Scholar]
- 63.Guyer D M, Kao J S, Mobley H L. Genomic analysis of a pathogenicity island in uropathogenic Escherichia coli CFT073: distribution of homologous sequences among isolates from patients with pyelonephritis, cystitis, and catheter-associated bacteriuria and from fecal samples. Infect Immun. 1998;66:4411–4417. doi: 10.1128/iai.66.9.4411-4417.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 64.Gygi D, Fraser G, Dufour A, Hughes C. A motile but non-swarming mutant of Proteus mirabilis lacks FlgN, a facilitator of flagella filament assembly. Mol Microbiol. 1997;25:597–604. doi: 10.1046/j.1365-2958.1997.5021862.x. [DOI] [PubMed] [Google Scholar]
- 65.Hayes F. A family of stability determinants in pathogenic bacteria. J Bacteriol. 1998;180:6415–6418. doi: 10.1128/jb.180.23.6415-6418.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 66.Hiles I D, Higgins C F. Peptide uptake by Salmonella typhimurium. The periplasmic oligopeptide-binding protein. Eur J Biochem. 1986;158:561–567. doi: 10.1111/j.1432-1033.1986.tb09791.x. [DOI] [PubMed] [Google Scholar]
- 67.Hirono I, Tange N, Aoki T. Iron-regulated haemolysin gene from Edwardsiella tarda. Mol Microbiol. 1997;24:851–856. doi: 10.1046/j.1365-2958.1997.3971760.x. [DOI] [PubMed] [Google Scholar]
- 68.Hobbs M, Mattick J S. Common components in the assembly of type 4 fimbriae, DNA transfer systems, filamentous phage and protein-secretion apparatus: a general system for the formation of surface-associated protein complexes. Mol Microbiol. 1993;10:233–243. doi: 10.1111/j.1365-2958.1993.tb01949.x. [DOI] [PubMed] [Google Scholar]
- 69.Homma M, DeRosier D J, Macnab R M. Flagellar hook and hook-associated proteins of Salmonella typhimurium and their relationship to other axial components of the flagellum. J Mol Biol. 1990;213:819–832. doi: 10.1016/S0022-2836(05)80266-9. [DOI] [PubMed] [Google Scholar]
- 70.Homuth G, Heinemann M, Zuber U, Schumann W. The genes of lepA and hemN form a bicistronic operon in Bacillus subtilis. Microbiology. 1996;142:1641–1649. doi: 10.1099/13500872-142-7-1641. [DOI] [PubMed] [Google Scholar]
- 71.Hornung J M, Jones H A, Perry R D. The hmu locus of Yersinia pestis is essential for utilization of free haemin and haem-protein complexes as iron sources. Mol Microbiol. 1996;20:725–739. doi: 10.1111/j.1365-2958.1996.tb02512.x. [DOI] [PubMed] [Google Scholar]
- 72.Hsu M Y, Inouye M, Inouye S. Retron for the 67-base multicopy single-stranded DNA from Escherichia coli: a potential transposable element encoding both reverse transcriptase and Dam methylase functions. Proc Natl Acad Sci USA. 1990;87:9454–9458. doi: 10.1073/pnas.87.23.9454. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 73.Hutsul J A, Worobec E. Molecular characterization of the Serratia marcescens OmpF porin, and analysis of S. marcescens OmpF and OmpC osmoregulation. Microbiology. 1997;143:2797–2806. doi: 10.1099/00221287-143-8-2797. [DOI] [PubMed] [Google Scholar]
- 74.Ichihara S, Suzuki T, Suzuki M, Mizushima S. Molecular cloning and sequencing of the sppA gene and characterization of the encoded protease IV, a signal peptide peptidase, of Escherichia coli. J Biol Chem. 1986;261:9405–9411. [PubMed] [Google Scholar]
- 75.Ikebe T, Iyoda S, Kutsukake K. Structure and expression of the fliA operon of Salmonella typhimurium. Microbiology. 1999;145:1389–1396. doi: 10.1099/13500872-145-6-1389. [DOI] [PubMed] [Google Scholar]
- 76.Itoh T, Aiba H, Baba T, Hayashi K, Inada T, Isono K, Kasai H, Kimura S, Kitakawa M, Kitagawa M, Makino K, Miki T, Mizobuchi K, Mori H, Mori T, Motomura K, Nakade S, Nakamura Y, Nashimoto H, Nishio Y, Oshima T, Saito N, Sampei G, Seki Y, Horiuchi T, et al. A 460-kb DNA sequence of the Escherichia coli K-12 genome corresponding to the 40.1-50.0 min region on the linkage map. DNA Res. 1996;3:379–392. doi: 10.1093/dnares/3.6.379. [DOI] [PubMed] [Google Scholar]
- 77.Jackowski S, Jackson P D, Rock C O. Sequence and function of the aas gene in Escherichia coli. J Biol Chem. 1994;269:2921–2928. [PubMed] [Google Scholar]
- 78.Johnston C, Pegues D A, Hueck C J, Lee A, Miller S I. Transcriptional activation of Salmonella typhimurium invasion genes by a member of the phosphorylated response-regulator superfamily. Mol Microbiol. 1996;22:715–727. doi: 10.1046/j.1365-2958.1996.d01-1719.x. [DOI] [PubMed] [Google Scholar]
- 79.Johnston T C, Rucker E B, Cochrum L, Hruska K S, Vandegrift V. The nucleotide sequence of the luxA and luxB genes of Xenorhabdus luminescens HM and a comparison of the amino acid sequences of luciferases from four species of bioluminescent bacteria. Biochem Biophys Res Commun. 1990;170:407–415. doi: 10.1016/0006-291x(90)92106-a. [DOI] [PubMed] [Google Scholar]
- 80.Kammler M, Schon C, Hantke K. Characterization of the ferrous iron uptake system of Escherichia coli. J Bacteriol. 1993;175:6212–6219. doi: 10.1128/jb.175.19.6212-6219.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 81.Kanatani A, Masuda T, Shimoda T, Misoka F, Lin X S, Yoshimoto T, Tsuru D. Protease II from Escherichia coli: sequencing and expression of the enzyme gene and characterization of the expressed enzyme. J Biochem (Tokyo) 1991;110:315–320. doi: 10.1093/oxfordjournals.jbchem.a123577. [DOI] [PubMed] [Google Scholar]
- 82.Kaneko T, Sato S, Kotani H, Tanaka A, Asamizu E, Nakamura Y, Miyajima N, Hirosawa M, Sugiura M, Sasamoto S, Kimura T, Hosouchi T, Matsuno A, Muraki A, Nakazaki N, Naruo K, Okumura S, Shimpo S, Takeuchi C, Wada T, Watanabe A, Yamada M, Yasuda M, Tabata S. Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Res. 1996;3:109–136. doi: 10.1093/dnares/3.3.109. [DOI] [PubMed] [Google Scholar]
- 83.Karlyshev A V, Galyov E E, Smirnov O, Guzayev A P, Abramov V M, Zav'yalov V P. A new gene of the f1 operon of Y. pestis involved in the capsule biogenesis. FEBS Lett. 1992;297:77–80. doi: 10.1016/0014-5793(92)80331-a. [DOI] [PubMed] [Google Scholar]
- 84.Kashiwagi K, Yamaguchi Y, Sakai Y, Kobayashi H, Igarashi K. Identification of the polyamine-induced protein as a periplasmic oligopeptide binding protein. J Biol Chem. 1990;265:8387–8391. [PubMed] [Google Scholar]
- 85.Katsuragi N, Takizawa N, Murooka Y. Entire nucleotide sequence of the pullulanase gene of Klebsiella aerogenes W70. J Bacteriol. 1987;169:2301–2306. doi: 10.1128/jb.169.5.2301-2306.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 86.Katz M E, Wright C L, Gartside T S, Cheetham B F, Doidge C V, Moses E K, Rood J I. Genetic organization of the duplicated vap region of the Dichelobacter nodosus genome. J Bacteriol. 1994;176:2663–2669. doi: 10.1128/jb.176.9.2663-2669.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 87.Kaufman M R, Seyer J M, Taylor R K. Processing of TCP pilin by TcpJ typifies a common step intrinsic to a newly recognized pathway of extracellular protein secretion by gram-negative bacteria. Genes Dev. 1991;5:1834–1846. doi: 10.1101/gad.5.10.1834. [DOI] [PubMed] [Google Scholar]
- 88.Kawagishi I, Muller V, Williams A W, Irikura V M, Macnab R M. Subdivision of flagellar region III of the Escherichia coli and Salmonella typhimurium chromosomes and identification of two additional flagellar genes. J Gen Microbiol. 1992;138:1051–1065. doi: 10.1099/00221287-138-6-1051. [DOI] [PubMed] [Google Scholar]
- 89.Kihara M, Homma M, Kutsukake K, Macnab R M. Flagellar switch of Salmonella typhimurium: gene sequences and deduced protein sequences. J Bacteriol. 1989;171:3247–3257. doi: 10.1128/jb.171.6.3247-3257.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 90.Kim K, Lee S, Lee K, Lim D. Isolation and characterization of toluene-sensitive mutants from the toluene-resistant bacterium Pseudomonas putida GM73. J Bacteriol. 1998;180:3692–3696. doi: 10.1128/jb.180.14.3692-3696.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 91.Klein J R, Henrich B, Plapp R. Molecular analysis and nucleotide sequence of the envCD operon of Escherichia coli. Mol Gen Genet. 1991;230:230–240. doi: 10.1007/BF00290673. [DOI] [PubMed] [Google Scholar]
- 92.Klemm P, Christiansen G. The fimD gene required for cell surface localization of Escherichia coli type 1 fimbriae. Mol Gen Genet. 1990;220:334–338. doi: 10.1007/BF00260505. [DOI] [PubMed] [Google Scholar]
- 93.Klenk H P, Clayton R A, Tomb J F, White O, Nelson K E, Ketchum K A, Dodson R J, Gwinn M, Hickey E K, Peterson J D, Richardson D L, Kerlavage A R, Graham D E, Kyrpides N C, Fleischmann R D, Quackenbush J, Lee N H, Sutton G G, Gill S, Kirkness E F, Dougherty B A, McKenney K, Adams M D, Loftus B, Venter J C, et al. The complete genome sequence of the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus. Nature. 1997;390:364–370. doi: 10.1038/37052. [DOI] [PubMed] [Google Scholar]
- 94.Kloser A W, Laird M W, Misra R. asmB, a suppressor locus for assembly-defective OmpF mutants of Escherichia coli, is allelic to envA (lpxC) J Bacteriol. 1996;178:5138–5143. doi: 10.1128/jb.178.17.5138-5143.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 95.Kobayashi T, Kudo I, Karasawa K, Mizushima H, Inoue K, Nojima S. Nucleotide sequence of the pldB gene and characteristics of deduced amino acid sequence of lysophospholipase L2 in Escherichia coli. J Biochem (Tokyo) 1985;98:1017–1025. doi: 10.1093/oxfordjournals.jbchem.a135347. [DOI] [PubMed] [Google Scholar]
- 96.Konz D, Doekel S, Marahiel M A. Molecular and biochemical characterization of the protein template controlling biosynthesis of the lipopeptide lichenysin. J Bacteriol. 1999;181:133–140. doi: 10.1128/jb.181.1.133-140.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 97.Konz D, Klens A, Schorgendorfer K, Marahiel M A. The bacitracin biosynthesis operon of Bacillus licheniformis ATCC 10716: molecular characterization of three multi-modular peptide synthetases. Chem Biol. 1997;4:927–937. doi: 10.1016/s1074-5521(97)90301-x. [DOI] [PubMed] [Google Scholar]
- 98.Kornacker M G, Pugsley A P. Molecular characterization of pulA and its product, pullulanase, a secreted enzyme of Klebsiella pneumoniae UNF5023. Mol Microbiol. 1990;4:73–85. doi: 10.1111/j.1365-2958.1990.tb02016.x. [DOI] [PubMed] [Google Scholar]
- 99.Kunst F, Ogasawara N, Moszer I, Albertini A M, Alloni G, Azevedo V, Bertero M G, Bessieres P, Bolotin A, Borchert S, Borriss R, Boursier L, Brans A, Braun M, Brignell S C, Bron S, Brouillet S, Bruschi C V, Caldwell B, Capuano V, Carter N M, Choi S K, Codani J J, Connerton I F, Danchin A, et al. The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature. 1997;390:249–256. doi: 10.1038/36786. [DOI] [PubMed] [Google Scholar]
- 100.Kyostio S R, Cramer C L, Lacy G H. Erwinia carotovora subsp. carotovora extracellular protease: characterization and nucleotide sequence of the gene. J Bacteriol. 1991;173:6537–6546. doi: 10.1128/jb.173.20.6537-6546.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 101.Lambert B, Buysse L, Decock C, Jansens S, Piens C, Saey B, Seurinck J, Van Audenhove K, Van Rie J, Van Vliet A, Peferoen M. A Bacillus thuringiensis insecticidal crystal protein with a high activity against members of the family Noctuidae. Appl Environ Microbiol. 1996;62:80–86. doi: 10.1128/aem.62.1.80-86.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 102.Larson T G, Goodgal S H. Sequence and transcriptional regulation of com101A, a locus required for genetic transformation in Haemophilus influenzae. J Bacteriol. 1991;173:4683–4691. doi: 10.1128/jb.173.15.4683-4691.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 103.Leclerc M C, Boemare N E. Plasmids and phase variation in Xenorhabdus spp. Appl Environ Microbiol. 1991;57:2597–2601. doi: 10.1128/aem.57.9.2597-2601.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 104.Le Gouill C, Desmarais D, Dery C V. Saccharopolyspora hirsuta 367 encodes clustered genes similar to ketoacyl synthase, ketoacyl reductase, acyl carrier protein, and biotin carboxyl carrier protein. Mol Gen Genet. 1993;240:146–150. doi: 10.1007/BF00276894. [DOI] [PubMed] [Google Scholar]
- 105.Lemaire H G, Muller-Hill B. Nucleotide sequences of the galE gene and the galT gene of E. coli. Nucleic Acids Res. 1986;14:7705–7711. doi: 10.1093/nar/14.19.7705. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 106.Letoffe S, Delepelaire P, Wandersman C. Characterization of a protein inhibitor of extracellular proteases produced by Erwinia chrysanthemi. Mol Microbiol. 1989;3:79–86. doi: 10.1111/j.1365-2958.1989.tb00106.x. [DOI] [PubMed] [Google Scholar]
- 107.Letoffe S, Delepelaire P, Wandersman C. Protease secretion by Erwinia chrysanthemi: the specific secretion functions are analogous to those of Escherichia coli alpha-haemolysin. EMBO J. 1990;9:1375–1382. doi: 10.1002/j.1460-2075.1990.tb08252.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 108.Levengood S K, Webster R E. Nucleotide sequences of the tolA and tolB genes and localization of their products, components of a multistep translocation system in Escherichia coli. J Bacteriol. 1989;171:6600–6609. doi: 10.1128/jb.171.12.6600-6609.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 109.Lewis J P, Macrina F L. IS195, an insertion sequence-like element associated with protease genes in Porphyromonas gingivalis. Infect Immun. 1998;66:3035–3042. doi: 10.1128/iai.66.7.3035-3042.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 110.Li M, Dyda F, Benhar I, Pastan I, Davies D R. Crystal structure of the catalytic domain of Pseudomonas exotoxin A complexed with a nicotinamide adenine dinucleotide analog: implications for the activation process and for ADP ribosylation. Proc Natl Acad Sci USA. 1996;93:6902–6906. doi: 10.1073/pnas.93.14.6902. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 111.Lin J W, Yu K Y, Chao Y F, Weng S F. The lumQ gene is linked to the lumP gene and the lux operon in Photobacterium leiognathi. Biochem Biophys Res Commun. 1995;217:684–695. doi: 10.1006/bbrc.1995.2828. [DOI] [PubMed] [Google Scholar]
- 112.Lin W, Fullner K J, Clayton R, Sexton J A, Rogers M B, Calia K E, Calderwood S B, Fraser C, Mekalanos J J. Identification of a Vibrio cholerae RTX toxin gene cluster that is tightly linked to the cholera toxin prophage. Proc Natl Acad Sci USA. 1999;96:1071–1076. doi: 10.1073/pnas.96.3.1071. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 113.Liu T, Lin Y, Cislo T, Minetti C A, Baba J M, Liu T Y. Limunectin. A phosphocholine-binding protein from Limulus amebocytes with adhesion-promoting properties. J Biol Chem. 1991;266:14813–14821. [PubMed] [Google Scholar]
- 114.Lu D, Boyd B, Lingwood C A. Identification of the key protein for zinc uptake in Haemophilus influenzae. J Biol Chem. 1997;272:29033–29038. doi: 10.1074/jbc.272.46.29033. [DOI] [PubMed] [Google Scholar]
- 115.Mahillon J, Kirkpatrick H A, Kijenski H L, Bloch C A, Rode C K, Mayhew G F, Rose D J, Plunkett G, 3rd, Burland V, Blattner F R. Subdivision of the Escherichia coli K-12 genome for sequencing: manipulation and DNA sequence of transposable elements introducing unique restriction sites. Gene. 1998;223:47–54. doi: 10.1016/s0378-1119(98)00365-5. [DOI] [PubMed] [Google Scholar]
- 116.Masaki H, Toba M, Ohta T. Structure and expression of the ColE2-P9 immunity gene. Nucleic Acids Res. 1985;13:1623–1635. doi: 10.1093/nar/13.5.1623. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 117.Massad G, Fulkerson J F, Jr, Watson D C, Mobley H L. Proteus mirabilis ambient-temperature fimbriae: cloning and nucleotide sequence of the aft gene cluster. Infect Immun. 1996;64:4390–4395. doi: 10.1128/iai.64.10.4390-4395.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 118.Matsubara K, Ohnishi K, Kiritani K. Nucleotide sequences and characterization of liv genes encoding components of the high-affinity branched-amino acid transport system in Salmonella typhimurium. J Biochem (Tokyo) 1992;112:93–101. doi: 10.1093/oxfordjournals.jbchem.a123872. [DOI] [PubMed] [Google Scholar]
- 119.McClelland M, Wilson R K. Comparison of sample sequences of the Salmonella typhi genome to the sequence of the complete Escherichia coli K-12 genome. Infect Immun. 1998;66:4305–4312. doi: 10.1128/iai.66.9.4305-4312.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 120.McGinnes L W, Wilde A, Morrison T G. Nucleotide sequence of the gene encoding the Newcastle disease virus hemagglutinin-neuraminidase protein and comparisons of paramyxovirus hemagglutinin-neuraminidase protein sequences. Virus Res. 1987;7:187–202. doi: 10.1016/0168-1702(87)90027-x. [DOI] [PubMed] [Google Scholar]
- 121.McHenney M A, Hosted T J, Dehoff B S, Rosteck P R, Jr, Baltz R H. Molecular cloning and physical mapping of the daptomycin gene cluster from Streptomyces roseosporus. J Bacteriol. 1998;180:143–51. doi: 10.1128/jb.180.1.143-151.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 122.Merriman T R, Merriman M E, Lamont I L. Nucleotide sequence of pvdD, a pyoverdine biosynthetic gene from Pseudomonas aeruginosa: PvdD has similarity to peptide synthetases. J Bacteriol. 1995;177:252–258. doi: 10.1128/jb.177.1.252-258.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 123.Michiels T, Vanooteghem J C, Lambert de Rouvroit C, China B, Gustin A, Boudry P, Cornelis G R. Analysis of virC, an operon involved in the secretion of Yop proteins by Yersinia enterocolitica. J Bacteriol. 1991;173:4994–5009. doi: 10.1128/jb.173.16.4994-5009.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 124.Miller V L, Bliska J B, Falkow S. Nucleotide sequence of the Yersinia enterocolitica ail gene and characterization of the Ail protein product. J Bacteriol. 1990;172:1062–1069. doi: 10.1128/jb.172.2.1062-1069.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 125.Molloy M P, Herbert B R, Walsh B J, Tyler M I, Traini M, Sanchez J C, Hochstrasser D F, Williams K L, Gooley A A. Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis. Electrophoresis. 1998;19:837–844. doi: 10.1002/elps.1150190539. [DOI] [PubMed] [Google Scholar]
- 126.Mootz H D, Marahiel M A. The tyrocidine biosynthesis operon of Bacillus brevis: complete nucleotide sequence and biochemical characterization of functional internal adenylation domains. J Bacteriol. 1997;179:6843–6850. doi: 10.1128/jb.179.21.6843-6850.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 127.Morlon J, Lloubes R, Varenne S, Chartier M, Lazdunski C. Complete nucleotide sequence of the structural gene for colicin A, a gene translated at non-uniform rate. J Mol Biol. 1983;170:271–285. doi: 10.1016/s0022-2836(83)80148-x. [DOI] [PubMed] [Google Scholar]
- 128.Nagasawa S, Ishige K, Mizuno T. Novel members of the two-component signal transduction genes in Escherichia coli. J Biochem (Tokyo) 1993;114:350–357. doi: 10.1093/oxfordjournals.jbchem.a124180. [DOI] [PubMed] [Google Scholar]
- 129.Naroditskaya V, Schlosser M J, Fang N Y, Lewis K. An E. coli gene emrD is involved in adaptation to low energy shock. Biochem Biophys Res Commun. 1993;196:803–809. doi: 10.1006/bbrc.1993.2320. [DOI] [PubMed] [Google Scholar]
- 130.Nichols W A, Clegg S, Brown M R. Characterization of the type 1 fimbrial subunit gene (fimA) of Serratia marcescens. Mol Microbiol. 1990;4:2119–2126. doi: 10.1111/j.1365-2958.1990.tb00573.x. [DOI] [PubMed] [Google Scholar]
- 131.Nishimura K, Nakayashiki T, Inokuchi H. Cloning and sequencing of the hemE gene encoding uroporphyrinogen III decarboxylase (UPD) from Escherichia coli K-12. Gene. 1993;133:109–113. doi: 10.1016/0378-1119(93)90233-s. [DOI] [PubMed] [Google Scholar]
- 132.Nurse P, DiGate R J, Zavitz K H, Marians K J. Molecular cloning and DNA sequence analysis of Escherichia coli priA, the gene encoding the primosomal protein replication factor Y. Proc Natl Acad Sci USA. 1990;87:4615–4619. doi: 10.1073/pnas.87.12.4615. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 133.Ogasawara N, Nakai S, Yoshikawa H. Systematic sequencing of the 180 kilobase region of the Bacillus subtilis chromosome containing the replication origin. DNA Res. 1994;1:1–14. doi: 10.1093/dnares/1.1.1. [DOI] [PubMed] [Google Scholar]
- 134.Olson E R, Dunyak D S, Jurss L M, Poorman R A. Identification and characterization of dppA, an Escherichia coli gene encoding a periplasmic dipeptide transport protein. J Bacteriol. 1991;173:234–244. doi: 10.1128/jb.173.1.234-244.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 135.Oshima T, Aiba H, Baba T, Fujita K, Hayashi K, Honjo A, Ikemoto K, Inada T, Itoh T, Kajihara M, Kanai K, Kashimoto K, Kimura S, Kitagawa M, Makino K, Masuda S, Miki T, Mizobuchi K, Mori H, Motomura K, Nakamura Y, Nashimoto H, Nishio Y, Saito N, Horiuchi T, et al. A 718-kb DNA sequence of the Escherichia coli K-12 genome corresponding to the 12.7-28.0 min region on the linkage map. DNA Res. 1996;3:137–155. doi: 10.1093/dnares/3.3.137. [DOI] [PubMed] [Google Scholar]
- 136.Ostrowski J, Wu J Y, Rueger D C, Miller B E, Siegel L M, Kredich N M. Characterization of the cysJIH regions of Salmonella typhimurium and Escherichia coli B. DNA sequences of cysI and cysH and a model for the siroheme-Fe4S4 active center of sulfite reductase hemoprotein based on amino acid homology with spinach nitrite reductase. J Biol Chem. 1989;264:15726–15737. [PubMed] [Google Scholar]
- 137.Oswald E, Sugai M, Labigne A, Wu H C, Fiorentini C, Boquet P, O'Brien A D. Cytotoxic necrotizing factor type 2 produced by virulent Escherichia coli modifies the small GTP-binding proteins Rho involved in assembly of actin stress fibers. Proc Natl Acad Sci USA. 1994;91:3814–3818. doi: 10.1073/pnas.91.9.3814. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 138.Peakman T, Crouzet J, Mayaux J F, Busby S, Mohan S, Harborne N, Wootton J, Nicolson R, Cole J. Nucleotide sequence, organisation and structural analysis of the products of genes in the nirB-cysG region of the Escherichia coli K-12 chromosome. Eur J Biochem. 1990;191:315–323. doi: 10.1111/j.1432-1033.1990.tb19125.x. [DOI] [PubMed] [Google Scholar]
- 139.Pelludat C, Rakin A, Jacobi C A, Schubert S, Heesemann J. The yersiniabactin biosynthetic gene cluster of Yersinia enterocolitica: organization and siderophore-dependent regulation. J Bacteriol. 1998;180:538–546. doi: 10.1128/jb.180.3.538-546.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 140.Perry R D, Fetherston J D. Yersinia pestis—etiologic agent of plague. Clin Microbiol Rev. 1997;10:35–66. doi: 10.1128/cmr.10.1.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 141.Poole K, Schiebel E, Braun V. Molecular characterization of the hemolysin determinant of Serratia marcescens. J Bacteriol. 1988;170:3177–3188. doi: 10.1128/jb.170.7.3177-3188.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 142.Popham D L, Setlow P. Cloning, nucleotide sequence, and regulation of the Bacillus subtilis pbpE operon, which codes for penicillin-binding protein 4* and an apparent amino acid racemase. J Bacteriol. 1993;175:2917–2925. doi: 10.1128/jb.175.10.2917-2925.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 143.Pospiech A, Cluzel B, Bietenhader J, Schupp T. A new Myxococcus xanthus gene cluster for the biosynthesis of the antibiotic saframycin Mx1 encoding a peptide synthetase. Microbiology. 1995;141:1793–1803. doi: 10.1099/13500872-141-8-1793. [DOI] [PubMed] [Google Scholar]
- 144.Price C, Lingner J, Bickle T A, Firman K, Glover S W. Basis for changes in DNA recognition by the EcoR124 and EcoR124/3 type I DNA restriction and modification enzymes. J Mol Biol. 1989;205:115–125. doi: 10.1016/0022-2836(89)90369-0. [DOI] [PubMed] [Google Scholar]
- 145.Raina S, Missiakas D, Baird L, Kumar S, Georgopoulos C. Identification and transcriptional analysis of the Escherichia coli htrE operon which is homologous to pap and related pilin operons. J Bacteriol. 1993;175:5009–5021. doi: 10.1128/jb.175.16.5009-5021.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 146.Rakin A, Saken E, Harmsen D, Heesemann J. The pesticin receptor of Yersinia enterocolitica: a novel virulence factor with dual function. Mol Microbiol. 1994;13:253–263. doi: 10.1111/j.1365-2958.1994.tb00420.x. [DOI] [PubMed] [Google Scholar]
- 147.Reich K A, Schoolnik G K. Halovibrin, secreted from the light organ symbiont Vibrio fischeri, is a member of a new class of ADP-ribosyltransferases. J Bacteriol. 1996;178:209–215. doi: 10.1128/jb.178.1.209-215.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 148.Relman D A, Domenighini M, Tuomanen E, Rappuoli R, Falkow S. Filamentous hemagglutinin of Bordetella pertussis: nucleotide sequence and crucial role in adherence. Proc Natl Acad Sci USA. 1989;86:2637–2641. doi: 10.1073/pnas.86.8.2637. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 149.Roberts R C, Helinski D R. Definition of a minimal plasmid stabilization system from the broad-host-range plasmid RK2. J Bacteriol. 1992;174:8119–8132. doi: 10.1128/jb.174.24.8119-8132.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 150.Roman S J, Frantz B B, Matsumura P. Gene sequence, overproduction, purification and determination of the wild-type level of the Escherichia coli flagellar switch protein FliG. Gene. 1993;133:103–108. doi: 10.1016/0378-1119(93)90232-r. [DOI] [PubMed] [Google Scholar]
- 151.Rosqvist R, Skurnik M, Wolf-Watz H. Increased virulence of Yersinia pseudotuberculosis by two independent mutations. Nature. 1988;334:522–524. doi: 10.1038/334522a0. [DOI] [PubMed] [Google Scholar]
- 152.Saito F, Hori K, Kanda M, Kurotsu T, Saito Y. Entire nucleotide sequence for Bacillus brevis Nagano Grs2 gene encoding gramicidin S synthetase 2: a multifunctional peptide synthetase. J Biochem (Tokyo) 1994;116:357–367. doi: 10.1093/oxfordjournals.jbchem.a124532. [DOI] [PubMed] [Google Scholar]
- 153.Sancar G B, Sancar A, Rupp W D. Sequence of the E. coli uvrC gene and protein. Nucleic Acids Res. 1984;12:4593–4608. doi: 10.1093/nar/12.11.4593. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 154.Sauerborn M, von Eichel-Streiber C. Nucleotide sequence of Clostridium difficile toxin A. Nucleic Acids Res. 1990;18:1629–1630. doi: 10.1093/nar/18.6.1629. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 155.Schaffer S, Hantke K, Braun V. Nucleotide sequence of the iron regulatory gene fur. Mol Gen Genet. 1985;200:110–113. doi: 10.1007/BF00383321. [DOI] [PubMed] [Google Scholar]
- 156.Schmitt M P, Holmes R K. Cloning, sequence, and footprint analysis of two promoter/operators from Corynebacterium diphtheriae that are regulated by the diphtheria toxin repressor (DtxR) and iron. J Bacteriol. 1994;176:1141–1149. doi: 10.1128/jb.176.4.1141-1149.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 157.Seoane A, Garcia Lobo J M. Nucleotide sequence of a new class A beta-lactamase gene from the chromosome of Yersinia enterocolitica: implications for the evolution of class A beta-lactamases. Mol Gen Genet. 1991;228:215–220. doi: 10.1007/BF00282468. [DOI] [PubMed] [Google Scholar]
- 158.Skurnik M, Venho R, Toivanen P, al-Hendy A. A novel locus of Yersinia enterocolitica serotype O:3 involved in lipopolysaccharide outer core biosynthesis. Mol Microbiol. 1995;17:575–594. doi: 10.1111/j.1365-2958.1995.mmi_17030575.x. [DOI] [PubMed] [Google Scholar]
- 159.Staudenmaier H, Van Hove B, Yaraghi Z, Braun V. Nucleotide sequences of the fecBCDE genes and locations of the proteins suggest a periplasmic-binding-protein-dependent transport mechanism for iron(III) dicitrate in Escherichia coli. J Bacteriol. 1989;171:2626–2633. doi: 10.1128/jb.171.5.2626-2633.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 160.Stein D C, Chien R, Seifert H S. Construction of a Neisseria gonorrhoeae MS11 derivative deficient in NgoMI restriction and modification. J Bacteriol. 1992;174:4899–4906. doi: 10.1128/jb.174.15.4899-4906.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 161.Stevenson G, Andrianopoulos K, Hobbs M, Reeves P R. Organization of the Escherichia coli K-12 gene cluster responsible for production of the extracellular polysaccharide colanic acid. J Bacteriol. 1996;178:4885–4893. doi: 10.1128/jb.178.16.4885-4893.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 162.Stintzi A, Johnson Z, Stonehouse M, Ochsner U, Meyer J M, Vasil M L, Poole K. The pvc gene cluster of Pseudomonas aeruginosa: role in synthesis of the pyoverdine chromophore and regulation by PtxR and PvdS. J Bacteriol. 1999;181:4118–4124. doi: 10.1128/jb.181.13.4118-4124.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 163.Stirling D A, Hulton C S, Waddell L, Park S F, Stewart G S, Booth I R, Higgins C F. Molecular characterization of the proU loci of Salmonella typhimurium and Escherichia coli encoding osmoregulated glycine betaine transport systems. Mol Microbiol. 1989;3:1025–1038. doi: 10.1111/j.1365-2958.1989.tb00253.x. [DOI] [PubMed] [Google Scholar]
- 164.Stock A, Chen T, Welsh D, Stock J. CheA protein, a central regulator of bacterial chemotaxis, belongs to a family of proteins that control gene expression in response to changing environmental conditions. Proc Natl Acad Sci USA. 1988;85:1403–1407. doi: 10.1073/pnas.85.5.1403. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 165.Stojiljkovic I, Hantke K. Transport of haemin across the cytoplasmic membrane through a haemin-specific periplasmic binding-protein-dependent transport system in Yersinia enterocolitica. Mol Microbiol. 1994;13:719–732. doi: 10.1111/j.1365-2958.1994.tb00465.x. [DOI] [PubMed] [Google Scholar]
- 166.Stone B J, Miller V L. Salmonella enteritidis has a homologue of tolC that is required for virulence in BALB/c mice. Mol Microbiol. 1995;17:701–712. doi: 10.1111/j.1365-2958.1995.mmi_17040701.x. [DOI] [PubMed] [Google Scholar]
- 167.Ta D T, Vickery L E. Cloning, sequencing, and overexpression of a [2Fe-2S] ferredoxin gene from Escherichia coli. J Biol Chem. 1992;267:11120–11125. [PubMed] [Google Scholar]
- 168.Tabatabai N, Forst S. Molecular analysis of the two-component genes, ompR and envZ, in the symbiotic bacterium Xenorhabdus nematophilus. Mol Microbiol. 1995;17:643–652. doi: 10.1111/j.1365-2958.1995.mmi_17040643.x. [DOI] [PubMed] [Google Scholar]
- 169.Taron C H, Van Cott E M, Wilson G G, Moran L S, Slatko B E, Hornstra L J, Benner J S, Kucera R B, Guthrie E P. Cloning and expression of the NaeI restriction endonuclease-encoding gene and sequence analysis of the NaeI restriction-modification system. Gene. 1995;155:19–25. doi: 10.1016/0378-1119(94)00806-4. [DOI] [PubMed] [Google Scholar]
- 170.Tercero J A, Espinosa J C, Lacalle R A, Jimenz A. The biosynthetic pathway of the aminonucleoside antibiotic puromycin, as deduced from the molecular analysis of the pur cluster of Streptomyces alboniger. J Biol Chem. 1996;271:1579–1590. doi: 10.1074/jbc.271.3.1579. [DOI] [PubMed] [Google Scholar]
- 171.Tews I, Vincentelli R, Vorgias C E. N-Acetylglucosaminidase (chitobiase) from Serratia marcescens: gene sequence, and protein production and purification in Escherichia coli. Gene. 1996;170:63–67. doi: 10.1016/0378-1119(95)00848-9. [DOI] [PubMed] [Google Scholar]
- 172.Tobe T, Sasakawa C, Okada N, Honma Y, Yoshikawa M. vacB, a novel chromosomal gene required for expression of virulence genes on the large plasmid of Shigella flexneri. J Bacteriol. 1992;174:6359–6367. doi: 10.1128/jb.174.20.6359-6367.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 173.Tomb J F, el-Hajj H, Smith H O. Nucleotide sequence of a cluster of genes involved in the transformation of Haemophilus influenzae Rd. Gene. 1991;104:1–10. doi: 10.1016/0378-1119(91)90457-m. [DOI] [PubMed] [Google Scholar]
- 174.Uphoff T S, Welch R A. Nucleotide sequencing of the Proteus mirabilis calcium-independent hemolysin genes (hpmA and hpmB) reveals sequence similarity with the Serratia marcescens hemolysin genes (shlA and shlB) J Bacteriol. 1990;172:1206–1216. doi: 10.1128/jb.172.3.1206-1216.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 175.van Buul C P, van Knippenberg P H. Nucleotide sequence of the ksgA gene of Escherichia coli: comparison of methyltransferases effecting dimethylation of adenosine in ribosomal RNA. Gene. 1985;38:65–72. doi: 10.1016/0378-1119(85)90204-5. [DOI] [PubMed] [Google Scholar]
- 176.van der Ploeg J R, Weiss M A, Saller E, Nashimoto H, Saito N, Kertesz M A, Leisinger T. Identification of sulfate starvation-regulated genes in Escherichia coli: a gene cluster involved in the utilization of taurine as a sulfur source. J Bacteriol. 1996;178:5438–5446. doi: 10.1128/jb.178.18.5438-5446.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 177.van Montfort R L, Pijning T, Kalk K H, Hangyi I, Kouwijzer M L, Robillard G T, Dijkstra B W. The structure of the Escherichia coli phosphotransferase IIA mannitol reveals a novel fold with two conformations of the active site. Structure. 1998;6:377–388. doi: 10.1016/s0969-2126(98)00039-2. [DOI] [PubMed] [Google Scholar]
- 178.Viitanen A M, Toivanen P, Skurnik M. The lcrE gene is part of an operon in the lcr region of Yersinia enterocolitica O:3. J Bacteriol. 1990;172:3152–3162. doi: 10.1128/jb.172.6.3152-3162.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 179.von Wilcken-Bergmann B, Muller-Hill B. Sequence of galR gene indicates a common evolutionary origin of lac and gal repressor in Escherichia coli. Proc Natl Acad Sci USA. 1982;79:2427–2431. doi: 10.1073/pnas.79.8.2427. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 180.Vuorio R, Harkonen T, Tolvanen M, Vaara M. The novel hexapeptide motif found in the acyltransferases LpxA and LpxD of lipid A biosynthesis is conserved in various bacteria. FEBS Lett. 1994;337:289–292. doi: 10.1016/0014-5793(94)80211-4. [DOI] [PubMed] [Google Scholar]
- 181.Wang H, Dowds B C. Phase variation in Xenorhabdus luminescens: cloning and sequencing of the lipase gene and analysis of its expression in primary and secondary phases of the bacterium. J Bacteriol. 1993;175:1665–1673. doi: 10.1128/jb.175.6.1665-1673.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 182.Welch R A, Bauer M E, Kent A D, Leeds J A, Moayeri M, Regassa L B, Swenson D L. Battling against host phagocytes: the wherefore of the RTX family of toxins? Infect Agents Dis. 1995;4:254–272. [PubMed] [Google Scholar]
- 183.Whitchurch C B, Mattick J S. Escherichia coli contains a set of genes homologous to those involved in protein secretion, DNA uptake and the assembly of type-4 fimbriae in other bacteria. Gene. 1994;150:9–15. doi: 10.1016/0378-1119(94)90851-6. [DOI] [PubMed] [Google Scholar]
- 184.Williams S G, Varcoe L T, Attridge S R, Manning P A. Vibrio cholerae Hcp, a secreted protein coregulated with HlyA. Infect Immun. 1996;64:283–289. doi: 10.1128/iai.64.1.283-289.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 185.Wipat A, Brignell S C, Guy B J, Rose M, Emmerson P T, Harwood C R. The yvsA-yvqA (293 degrees-289 degrees) region of the Bacillus subtilis chromosome containing genes involved in metal ion uptake and a putative sigma factor. Microbiology. 1998;144:1593–1600. doi: 10.1099/00221287-144-6-1593. [DOI] [PubMed] [Google Scholar]
- 186.Xu K, Elliott T. An oxygen-dependent coproporphyrinogen oxidase encoded by the hemF gene of Salmonella typhimurium. J Bacteriol. 1993;175:4990–4999. doi: 10.1128/jb.175.16.4990-4999.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 187.Yamagata H, Nakamura K, Inouye M. Comparison of the lipoprotein gene among the Enterobacteriaceae. DNA sequence of Erwinia amylovora lipoprotein gene. J Biol Chem. 1981;256:2194–2198. [PubMed] [Google Scholar]
- 188.Yamamoto H, Uchiyama S, Sekiguchi J. The Bacillus subtilis chromosome region near 78 degrees contains the genes encoding a new two-component system, three ABC transporters and a lipase. Gene. 1996;181:147–151. doi: 10.1016/s0378-1119(96)00495-7. [DOI] [PubMed] [Google Scholar]
- 189.Yamamoto Y, Aiba H, Baba T, Hayashi K, Inada T, Isono K, Itoh T, Kimura S, Kitagawa M, Makino K, Miki T, Mitsuhashi N, Mizobuchi K, Mori H, Nakade S, Nakamura Y, Nashimoto H, Oshima T, Oyama S, Saito N, Sampei G, Satoh Y, Sivasundaram S, Tagami H, Horiuchi T, et al. Construction of a contiguous 874-kb sequence of the Escherichia coli K12 genome corresponding to 50.0-68.8 min on the linkage map and analysis of its sequence features. DNA Res. 1997;4:91–113. doi: 10.1093/dnares/4.2.91. [DOI] [PubMed] [Google Scholar]
- 190.Yang Y, Merriam J J, Mueller J P, Isberg R R. The psa locus is responsible for thermoinducible binding of Yersinia pseudotuberculosis to cultured cells. Infect Immun. 1996;64:2483–2489. doi: 10.1128/iai.64.7.2483-2489.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 191.Yoshida K, Shindo K, Sano H, Seki S, Fujimura M, Yanai N, Miwa Y, Fujita Y. Sequencing of a 65 kb region of the Bacillus subtilis genome containing the lic and cel loci, and creation of a 177 kb contig covering the gnt-sacXY region. Microbiology. 1996;142:3113–3123. doi: 10.1099/13500872-142-11-3113. [DOI] [PubMed] [Google Scholar]
- 192.Zavitz K H, DiGate R J, Marians K J. The priB and priC replication proteins of Escherichia coli. Genes, DNA sequence, overexpression, and purification. J Biol Chem. 1991;266:13988–13995. [PubMed] [Google Scholar]
- 193.Zenno S, Saigo K. Identification of the genes encoding NAD(P)H-flavin oxidoreductases that are similar in sequence to Escherichia coli Fre in four species of luminous bacteria: Photorhabdus luminescens, Vibrio fischeri, Vibrio harveyi, and Vibrio orientalis. J Bacteriol. 1994;176:3544–3551. doi: 10.1128/jb.176.12.3544-3551.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 194.Zulty J J, Barcak G J. Identification of a DNA transformation gene required for com101A+ expression and supertransformer phenotype in Haemophilus influenzae. Proc Natl Acad Sci USA. 1995;92:3616–3620. doi: 10.1073/pnas.92.8.3616. [DOI] [PMC free article] [PubMed] [Google Scholar]