- Research article
- Open Access
Insecticidal genes of Yersinia spp.: taxonomical distribution, contribution to toxicity towards Manduca sexta and Galleria mellonella, and evolution
https://doi.org/10.1186/1471-2180-8-214
© Fuchs et al; licensee BioMed Central Ltd. 2008
- Received: 22 July 2008
- Accepted: 08 December 2008
- Published: 08 December 2008
Abstract
Background
Toxin complex (Tc) proteins termed TcaABC, TcdAB, and TccABC with insecticidal activity are present in a variety of bacteria including the yersiniae.
Results
The tc gene sequences of thirteen Yersinia strains were compared, revealing a high degree of gene order conservation, but also remarkable differences with respect to pseudogenes, sequence variability and gene duplications. Outside the tc pathogenicity island (tc-PAI Ye ) of Y. enterocolitica strain W22703, a pseudogene (tccC2'/3') encoding proteins with homology to TccC and similarity to tyrosine phosphatases at its C-terminus was identified. PCR analysis revealed the presence of the tc-PAI Ye and of tccC2'/3'-homologues in all biotype 2–5 strains tested, and their absence in most representatives of biotypes 1A and 1B. Phylogenetic analysis of 39 TccC sequences indicates the presence of the tc-PAI Ye in an ancestor of Yersinia. Oral uptake experiments with Manduca sexta revealed a higher larvae lethality of Yersinia strains harbouring the tc-PAI Ye in comparison to strains lacking this island. Following subcutaneous infection of Galleria mellonella larvae with five non-human pathogenic Yersinia spp. and four Y. enterocolitica strains, we observed a remarkable variability of their insecticidal activity ranging from 20% (Y. kristensenii) to 90% (Y. enterocolitica strain 2594) dead larvae after five days. Strain W22703 and its tcaA deletion mutant did not exhibit a significantly different toxicity towards G. mellonella. These data confirm a role of TcaA upon oral uptake only, and suggest the presence of further insecticidal determinants in Yersinia strains formerly unknown to kill insects.
Conclusion
This study investigated the tc gene distribution among yersiniae and the phylogenetic relationship between TccC proteins, thus contributing novel aspects to the current discussion about the evolution of insecticidal toxins in the genus Yersinia. The toxic potential of several Yersinia spp. towards M. sexta and G. mellonella demonstrated here for the first time points to insects as a natural reservoir for yersiniae.
Keywords
- Insecticidal Activity
- Inverse Polymerase Chain Reaction
- Insecticidal Toxin
- Enterocolitica Strain
- Pestis Strain
Background
The toxin complex (Tc) proteins whose insecticidal potential resembles that of the Bacillus thuringiensis Bt-toxin were first purified from Photorhabdus luminescens which lives in symbiosis with nematodes [1]. They have also been identified in other insect-parasitizing bacteria such as Serratia entomophila, Xenorhabdus nematophilus, or Pseudomonas entomophila [2, 3]. Homologous insecticidal toxin genes are present in most genomes of Yersinia strains sequenced so far, including Y. mollaretii, seven Yersinia pestis strains and three Y. pseudotuberculosis strains. They have also been found in Y. frederiksenii and in two Y. enterocolitica strains, T83 and W22703, for which a genome sequence is not yet available [4–6]. However, tc genes are absent in Y. bercovieri and in Y. enterocolitica strain 8081 [7]. Interestingly, Tc proteins of three Serratia species and of Y. frederiksenii are plasmid-encoded, indicating that these sepABC-like genes are part of a horizontally mobile region [4].
Little is known about the biological role of the tc genes in Yersinia spp. The genes of the tc operons have been classified into three types according to their homology, namely tcdA/tcaAB/tccAB (type [A]), tcdB/tcaC (type [B]), and tccC (type [C]) [8]. Tc proteins have recently been shown to be secreted in a type III-dependent manner in Y. pestis [9]. Type [A] and [B] Tc proteins are presumably toxins directed against invertebrate and mammalian gut cells, and the variability in terms of Tc composition and Tc sequences may be due to insect- and tissue-specific activity [8, 10]. A role of the Tc proteins from Y. enterooclitica strain T83, Y. pseudotuberculosis strain IP32953 and Y. pestis KIM in mice gut colonization and in the actin cytoskeleton rearrangement of human gut cells and mouse fibroblast cells, respectively, has been reported [5, 8, 11]. The function of TccC remains unknown, but it has been suggested that TccC homologs could contribute to stable biofilm formation in fleas or combatting yet unknown antibacterial effectors in fleas [12], or that they act as universal activator of, or chaperons for, different toxin proteins [13].
Y. enterocolitica was the first member of the Yersinia genus for which insecticidal activity has been experimentally demonstrated, and tcaA encoding a subunit of the toxin complex was identified to be necessary for this activity [6]. The transcription of tcaA in Y. enterocolitica is completely repressed at 37°C, but strongly induced at lower temperatures with a maximum at approximately 10°C to 15°C. In contrast to Y. enterocolitica W22703, tcaABC expression in Y. pseudotuberculosis strain IP32953 was observed at 15°C and at 37°C [14]. Upregulation of tcaA and tcaB, but not tccC, upon temperature shift from 37°C to 26°C have been shown in two Y. pestis strains [15, 16]. The IP32953 Tc proteins are toxic against M. sexta larvae when expressed heterologously in E. coli [14]. Temperature-independent, but weak oral toxicity of several Y. pseudotuberculosis to this tobacco hornworm has been reported. Y. pseudotuberculosis, unlike Y. pestis, causes acute oral toxicity to fleas [12]. However, when the tcaAB gene pair from Y. pseudotuberculosis was heterologously expressed in E. coli, the lysates did not cause excess mortality in fleas, and a Y. pseudotuberculosis mutant deleted of the tc genes remained toxic toward the arthropod [8]. This is in line with the finding that two Y. enterocolitica strains containing a tcdB-tccC gene pair (strain CS080) or lacking any tc-like genes (strain 8081) were equally toxic to fleas [12].
The insecticidal potential of a variety of Yersinia spp. has not been tested in an insect infection assay, and the correlation of virulence to the presence or absence of tc operons in yersiniae is unknown. The phylogenetic relationship of the insecticidal toxins is not well understood. Here, we report a genome-based comparison of the tc genes in Y. enterocolitica strain W22703, the phylogenetic analysis of tccC genes in Yersinia species, the tc-PAI Ye distribution among six biotypes, and the insecticidal activity of Yersinia spp. towards two model organisms, the greater wax moth G. mellonella and the tobacco hornworm M. sexta.
Results
The tc-PAI Ye in Yersinia spp
Comparison of the tc -PAI Ye in Yersiniae. Three homology groups are depicted, namely tcaAB/tcdA, tcaC/tcdB, and tccC. tcdA/tcdB homologous are present only in Y. enterocolitica strain T83. tcaR1 (left) encoding a regulator and tldD encoding a putative DNA gyrase modulator (right, checkered) mark the island insertion site common for all Yersinia strains that harbour tc homologues. Identically coloured arrows mark homologous genes. A transposase-like gene (tnp) is present in the genome of Y. pestis Angola (black arrow). The overall gene organisation is similar for all strains harbouring insecticidal determinants, but differences with respect to gene homology, hypothetical ORFs, the presence of transposase-like elements and the number of tccC genes are also visible. Gene lengths and intergenic regions are in scale. Asterisks mark frameshifts. With the exception of tcaC, all frameshifts result in two ORFs. Y. ptb., Y. pseudotuberculosis.
Homologues of tccC located outside tc-PAI Ye
By screening a Tn5 luxCDABE reporter library of strain W22703 for genes induced upon low-temperature [17], we identified a transposon insertion located outside the tc-PAI Ye . A 4,595 bp sequence encompassing the transposon insertion site was derived, revealing two strain-specific ORFs termed tccC2' (1083 bp) and tccC3' (1680 bp) due to homologies to other Yersinia tccC loci. Obviously, a frameshift had splitted a tccC homologue into two ORFs. Exploring the available genome sequences of Yersinia strains, two additional tccC genes located outside the tc-PAI Ye were identified in Y. pestis strains Antiqua, CO92, Nepal516, Orientalis IP275 and 91001, and in Y. pseudotuberculosis IP32953, and one further tccC gene in Y. pestis strains KIM and Angola, and in Y. pseudotuberculosis IP31758. A truncated tccC2' gene with a 1953 bp deletion in comparison to tccC2 of strain IP32953 is present in the genome of Y. pseudotuberculosis YPIII. tccC2' and tccC3' of strain W22703 are located between two genes encoding a lipid A biosynthesis lauroyl acyltransferase, HtrB (YE1612), and a putative membrane protein (YE1611) of strain 8081. In contrast, the non-clustered tccC loci of all other strains are inserted into two equivalent locations on the common Yersinia backbone. These locations are exemplified as between the Y. pestis CO92 genes YPO2379/YPO2381 encoding an N-ethylmaleimide reductase and a lactoylglutathione lyase, and between YPO2311/YPO2313 coding for the insertion element IS1541 and a hypothetical protein. In addition, domain structure of TccC with similarity to a protein tyrosine phosphatase of undefined specificity was identified in the sequence of Y. pestis CO92 TccC2 and, albeit with lower probability, of W22703.
Cladogram based on 39 TccC amino acid sequence data. Four TccC sequences of P. luminescens subspecies laumondii strain TT01 served as outgroup. The phylogenetic analysis was performed with the neighbour-joining method and calculated using the two parameter model of Kimura [31]. Values on each branch indicate the occurrence (%) of the branching order in 500 bootstrapped trees. *insertion site between genes coding for the insertion element IS1541 and a hypothetical protein, **insertion site between genes encoding a N-ethylmaleimide reductase and a lactoylglutathione lyase. The frameshift of tccC in strain W22703 was not considered here; hence, one coherent amino acid sequence was used for the alignment. Bar represents 0.1% sequence divergence. Y. ptb., Y. pseudotuberculosis.
Presence of tc genes in Y. enterocolitica strains
Distribution of the tc -PAI Ye in Y. enterocolitica strains. Strains investigated are described in Table 3. The lines below the tc-PAI Ye are in scale and mark the fragments amplified by PCR. The PCR numbers correspond to those indicated in Additional file 1. Asterisks mark frameshifts in tcaB and tcaC. Dark grey: fragment amplification, light grey: biased results, white: no amplification; nd, not defined, e.g. no PCR was performed. See text for further details.
Oral infection of M. sexta
Oral infection of M. sexta
strain | tc-PAI Ye | total number | dead | alive | dead [%] ± sd |
---|---|---|---|---|---|
Y. enterocolitica 2594 | present2) | 18 | 15 | 3 | 83 ± 3 |
Y. mollaretii | present1) | 25 | 12 | 13 | 48 ± 9 |
Y. enterocolitica 4466 | absent2) | 36 | 15 | 21 | 42 ± 15 |
W22703 | present2) | 33 | 10 | 23 | 30 ± 14 |
Y. ruckeri | absent2) | 19 | 4 | 15 | 21 ± 26 |
W22703-tcaA::Tn5 lux | present, but tcaA knockout | 21 | 2 | 19 | 10 ± 11 |
Y. aldovae | absent2) | 23 | 2 | 21 | 9 ± 2 |
control | |||||
DH5α | absent1) | 21 | 1 | 20 | 5 ± 7 |
Subcutaneous infection of G. mellonella
Subcutaneous infection of G. mellonella
1:10 | 1:100 | total | |||||||
---|---|---|---|---|---|---|---|---|---|
strain | tc -PAI Ye | total number | dead | alive | dead | alive | dead | alive | dead [%] ± sd |
Y. enterocolitica 2594 | present2) | 79 | 36 | 4 | 34 | 5 | 70 | 9 | 90 ± 9 |
Y. enterocolitica 4466 | absent2) | 96 | 47 | 1 | 34 | 14 | 81 | 15 | 88 ± 11 |
Y. mollaretii | present1) | 64 | 30 | 2 | 24 | 8 | 54 | 10 | 84 ± 5 |
Y. bercovieri | absent1) | 64 | 28 | 8 | 23 | 5 | 51 | 13 | 81 ± 11 |
Y. ruckeri | absent2) | 85 | 33 | 11 | 20 | 21 | 53 | 32 | 53 ± 21 |
W22703- tcaA::Tn 5 lux | present, but tcaA knockout | 93 | 30 | 25 | 18 | 20 | 48 | 45 | 51 ± 13 |
W22703 | present 2) | 114 | 32 | 25 | 20 | 37 | 52 | 62 | 41 ± 17 |
Y. aldovae | absent2) | 68 | 23 | 13 | 4 | 28 | 27 | 41 | 41 ± 6 |
Y. kristensenii | absent2) | 88 | 19 | 27 | 1 | 41 | 20 | 68 | 20 ± 12 |
controls | |||||||||
S. typhimurium | absent1) | 88 | 10 | 33 | 10 | 35 | 20 | 68 | 18 ± 9 |
DH5α | absent1) | 63 | 5 | 34 | 2 | 22 | 7 | 56 | 13 ± 6 |
LB | 64 | 3 | 61 | 5 ± 0 |
Discussion
Two basic methods have been used here to determine the insecticidal potential of Yersinia spp., namely the oral application of viable cells and the subcutaneous injection of protein extracts or living bacterial cells. Upon oral application of W22703 and W22703-tcaA::Tn5lux protein extract to M. sexta larvae, we could recently demonstrate the role of TcaA in Y. enterocolitica toxicity towards insects [6]. Further five Yersinia strains were tested here for the first time with respect to their oral toxicity in the M. sexta model (Table 1). The presence of the tc-PAI Ye correlates with a higher toxicity of yersiniae towards larvae of the tobacco hornworm, while strains such as Y. ruckeri or Y. aldovae lacking the tc genes are less insecticidal in this assay. The variable insecticidal activity of strains with tc genes might be the result of sequence variations, or the presence of further insecticidal components. Due to a higher toxin concentration, the feeding of protein extracts led to higher lethality rates using strain W22703 [8]. In contrast, subcutaneous infection of G. mellonella does not result in a significantly different toxicity of these strains (Table 2). In comparison to P. luminescens that causes death of G. mellonella larvae within 24 hours following injection of several thousand cells [18], approximately 5 × 105 Y. enterocolitica strain W22730 cells are required to kill G. mellonella within five days. The most surprising result of the injection study performed here was the high variability of the insecticidal potential among Yersinia strains that is probably caused by tc-independent determinants. Examples for factors required for full virulence towards insect larvae are the hemolysin XhlA of X. nematophila or the gene mcf of P. luminescens. [13, 19]. In Y. enterocolitica, XaxAB, an apoptotic AB toxin, and the putative macrophage toxin MT have been identified as candidates with potential insecticidal activity, but their biological role still remains to be uncovered [20]. The overall results of the Galleria bioassay correlate with the finding that among 147 Yersinia isolates from the environment, 15.6% were Y. enterocolitica, but only 0.7% belonged to Y. kristensenii [21].
Although the biological role of Tc proteins has still to be experimentally defined, sequence analysis already revealed several interesting aspects. Regions of significant sequence similarities have been identified in all TcdA-like elements characterized so far [14]. Especially, TcaC is well conserved within the Yersinia genus, but TcaB and TcaA show significant sequence variability [8]. When the TccC sequences derived from the tc-PAI Ye of yersiniae were aligned, a high degree of sequence conservation was obtained at amino acids 1–680, followed by a remarkably high sequence diversity [14] as is confirmed by the TccC cladogram (Fig. 2). Some Tc sequences show evidence of undergoing degradation with frameshifts that often result in the splitting of tc genes into two separate ORFs (Fig. 1). Frameshifts in Y. pestis, especially in tcaB of CO92, are discussed as a critical step in the recent evolution of flea-borne transmission in the genus Yersinia due to the loss of one or more of those insect gut toxins [12, 14]. This data indicates that the tc genes of yersiniae may be under diversifying selection [8] which might result in insecticidal proteins with host-specific activity and with varying insecticidal potential.
It has been suggested that the genomes of different strains have taken up different tc genes after strain separation [22]. However, the data presented here point to a common Yersinia ancestor that has aquired the tc-PAI Ye . The plasmid-encoded Tc proteins in Y. frederiksenii and a transposon-like element downstream of Y. pestis Angola tcaC hint to putative mechanisms that might have played a role during horizontal transfer of insecticidal toxin genes (Fig. 1). This hypothesis is strongly supported by the common insertion site of the tc-PAI Ye that indicates one horizontal gene transfer (HGT) event, by the highly conserved phage-related genes within the tc-PAI Ye , and by a similar gene order including tccC1 and tccC2 in all islands investigated. Moreover, the cladogram derived from a comprehensive alignment of TccC protein sequences (Fig. 2) essentially reflects the phylogeny of Yersinia based on 16S rDNA sequences, including the clonal diversity among Y. enterocolitica strains [23]. As an additional insecticidal determinant, tccC genes located outside the tc-PAI Ye might have been acquired by a further HGT event following the separation of Y. pseudotuberculosis and Y. enterocolitica, because all available genomes of the Y. pseudotuberculosis and Y. pestis subline share two tccC insertion sites. Thus, reductive evolution by genetic drift might explain the lack of tc-PAI Ye in several Yersinia species and strains (Table 2) as examplified by the identification of rudimentary tc genes in biotypes 1A and 1B (Fig. 3).
Conclusion
The prevalence of the tc-PAI Ye in many genomes, its proven functionality in Y. enterocolitica and Y. pseudotuberculosis, as well as the common insecticidal potential of Yersinia spp. towards M. sexta and G. mellonella, hints to insects as yet unknown host organisms of yersiniae. This is in line with the hypothesis that environmental predators such as nematodes or insect larvae play a role in the evolution of pathogens [22, 24]. The tc-PAI Ye has probably been acquired by an ancestral Yersinia strain before the separation of Y. pestis, Y. pseudotuberculosis, Y. enterocolitica, and others. This ancestor strain could then have evolved the ability to exploit invertebrates by the acquisition of further genetic determinants required for the interaction of yersiniae with those hosts [20]. Distinct sequence variation, and reductive evolution especially within the genomes of Y. pestis serovars, might have allowed yersiniae to occupy specific ecological niches [22]. The role of the tc genes and other insecticidal determinants in proliferation and transmission of the three human pathogenic Yersinia species remains to be elucidated in more detail.
Methods
Bacterial strains and growth conditions
Y. enterocolitica strains used in this study
WS no. | Biotype | Serotype | Strain | Geographic origin | Biological origin |
---|---|---|---|---|---|
1968 | 1A | n. d. | MZ0124a) | n. d. | Concentrate of whey |
4346 | 1A | O:5 | Y755c) | France | Pony |
2602 | 1A | O:5 | H79/83b) | Germany | Man |
2603 | 1A | O:5 | H1527/93b) | Germany | Man |
4259 | 1A | O:41,43 | SZ593/04b) | Germany | Baby food |
4260 | 1A | O:41,43 | SZ554/04b) | Germany | Food |
4266 | 1A | O:4,33 | SZ1167/04b) | Germany | Man |
4267 | 1A | O:10 | SZ671/04b) | Germany | Man |
4268 | 1A | O:41,43 | SZ634/04b) | Germany | Man |
4313 | 1A | O:5 | NFOc) | New Foundland | Man |
4346 | 1A | O:5 | Y755c) | France | Pony |
3760 | 1B | O:8 | 8081g) | USA | Man |
4263 | 1B | O:8 | SZ506/04b) | Germany | Man |
4265 | 1B | O:8 | SZ375/04b) | Germany | Man |
4314 | 1B | O:8 | WA-314c) | USA | Man |
4348 | 1B | O:8 | Y286d) | USA | n. d. |
4349 | 1B | O:13 | Y293d) | n. d. | n. d. |
4466 | 1B | O:21 | 209–36/84b) | Germany | Man |
2594 | 2 | O:9 | H692/94b) | Germany | n. d. |
2595 | 2 | O:9 | H621/87b) | Germany | Man |
2599 | 2 | O:5,27 | H280/83b) | Germany | n. d. |
3372 | 2 | O:9 | W22703 h) | n. d. | n. d. |
4264 | 2 | O:5,27 | SZ1249/0b)4 | Germany | Man |
4317 | 2 | O:9 | Y738c) | France | Man |
4347 | 2 | O:9 | Y127d) | n. d. | n. d. |
2596 | 3 | O:9 | H324/78b) | n. d. | Pig |
2597 | 3 | O:9 | H7580/93b) | n. d. | n. d. |
2598 | 3 | O:9 | H7692/93b) | n. d. | n. d. |
2600 | 3 | O:5,27 | H230/89b) | Germany | Man |
2601 | 3 | O:5,27 | H582/87b) | n. d. | Man |
3371 | 3 | O:1 | NCTC 10460f) | Denmark | Chinchilla |
4322 | 3 | O:3 | Y745 c) | Japan | Man |
4323 | 3 | O:3 | Y746c) | Japan | Man |
2589 | 4 | O:3 | H270/78b) | n. d. | Dog feces |
2590 | 4 | O:3 | H31/80b) | n. d. | Pig |
2591 | 4 | O:3 | H608/87b) | n. d. | Man |
2592 | 4 | O:3 | H450/87b) | n. d. | Man |
2593 | 4 | O:3 | H469/87b) | n. d. | Pig |
4261 | 4 | O:3 | SZ425/04 b) | Germany | Pig tongue |
4262 | 4 | O:3 | SZ687/04b) | Germany | Dog feces |
4315 | 4 | O:3 | Y-108c) | Germany | Man |
4324 | 4 | O:3 | Y747c) | Sweden | Man |
4325 | 4 | O:3 | Y750c) | China | Man |
4326 | 4 | O:3 | Y751c) | Great Britain | Man |
4327 | 4 | O:3 | Y752c) | Brazil | Man |
4328 | 4 | O:3 | Y753c) | New Caledonia | Man |
4329 | 4 | O:3 | Y754c) | New Caledonia | Man |
4330 | 4 | O:3 | Y755c) | South Africa | Man |
4331 | 4 | O:3 | Y756c) | South Africa | Man |
4332 | 4 | O:3 | Y757c) | Hungary | Man |
4333 | 4 | O:3 | Y758c) | Hungary | Man |
4334 | 4 | O:3 | Y759c) | Canada | Man |
4335 | 4 | O:3 | Y763c) | Canada | Man |
4336 | 4 | O:3 | Y764c) | Canada | Man |
4337 | 4 | O:3 | Y765c) | Australia | Man |
4338 | 4 | O:3 | Y766c) | Australia | Man |
4339 | 4 | O:3 | Y767c) | Australia | Man |
4340 | 4 | O:3 | Y768c) | Australia | Man |
4341 | 4 | O:3 | Y769c) | New Zealand | Man |
4342 | 4 | O:3 | Y770c) | New Zealand | Man |
4343 | 4 | O:3 | Y771c) | New Zealand | Man |
4316 | 4 | O:3 | Y11 d) | n. d. | n. d. |
4344 | 5 | O:2a,2b,3 | Y772c) | France | Hare |
4345 | 5 | O:2a,2b,3 | Y773 c) | France | Hare |
4318 | 2/3 | O:5,27 | 237 c) | USA | n. d. |
4319 | 2/3 | O:5,27 | 238 c) | Great Britain | n. d. |
General molecular techniques
DNA and RNA manipulation was performed according to standard procedures [25]. To isolate chromosomal DNA, 1.5 ml of a bacterial culture was centrifuged, and the sediment was resuspended in 400 μl of lysis buffer (100 mM Tris pH 8.0, 5 mM EDTA, 200 mM NaCl). After incubation for 15 min on ice, 10 μl of 10% SDS and 5 μl of proteinase K (10 mg/ml) were added, and the sample was incubated overnight at 55°C. The chromosomal DNA was then precipitated with 500 μl of isopropanol, washed in ethanol, dried, and dissolved in 500 μl of TE buffer (10 mM Tris-HCl, 1 mM Na2 EDTA, pH 7.4) containing 1 μl of RNase (10 mg/ml). Polymerase chain reactions (PCR) were carried out with Taq polymerase (Fermentas, Vilnius, Lithunia) and the following programme: one cycle at 95°C for 2 min; 30 cycles at 95°C for 10 sec, at the appropriate annealing temperature for 30 sec, at 72°C for 45 sec to 180 sec depending on the expected fragment length; one cycle at 72°C for 10 min. All primers used are listed in Additional file 1. 4 μl of chromosomal DNA (100 ng ml-1) was used as template for PCR amplification, and the GeneRuler DNA mix (Fermentas) served as DNA ladder.
Inverse PCR and DNA sequencing
Identification of the transposon insertion site in mutant W22703-tccC(405)::Tn5lux was performed as described previously [17]. Briefly, 400 ng chromosomal DNA of the transposon mutant was completely digested with Cla I, Hind III or Ssp I (Fermentas), enzymes were heat-inactivated, and fragments were treated with T4 DNA ligase (Invitrogen, Carlsbad, USA) to allow self-ligation resulting in circular molecules. Inverse PCR [26] was then performed using transposon-specific primers [17], and the resulting fragments were sequenced with primers hybridizing to transposon regions near the O-end and the I-end. Sequencing of the strain-specific DNA was performed following inverse PCR using the restriction enzymes Hae III (USB, Cleveland, USA), Hha I, Hind III, Hpa I, Msp I, Mun I, Rsa I, Ssp I and Vsp I (Fermentas), and primers derived from the sequence already obtained. Sequencing was done by 4 base lab (Reutlingen, Germany) and by MWG-Biotech (Ebersberg, Germany).
Bioinformatics
Mapping of the mini-Tn5 luxCDABE insertion was performed using the Y. enterocolitica Blast Server from the Sanger Institute http://www.sanger.ac.uk/cgi-bin/blast/submitblast/y_enterocolitica. The reference genome sequence was that of Y. enterocolitica 8081 (accession numbers AM286415 and AM286416). Sequence assembly was done with Vector NTI Advance™ (Invitrogen, Carlsbad, USA). The resulting sequence was annotated using the NCBI ORF-Finder http://www.ncbi.nlm.nih.gov/gorf/gorf.html. Homology searches of predicted proteins were performed by BLAST analysis http://www.ncbi.nlm.nih.gov/BLAST/. Genome sequences of Yersinia strains were obtained from the NCBI database and compared using the homepage http://www.microbesonline.org/. Protein sequence alignment was done with the ClustalW program [27], and cladogram was constructed with TREECON [28]. Promoter sequences located upstream of the identified genes were deduced with BPROM http://www.softberry.com/. The accession number of the W22703 tccC2' and tccC3' sequence is AM941739.
Bioassays
M. sexta were reared as described recently [29]. For oral bioassays, bacteria were grown at 15°C (Yersinia strains) or 37°C (DH5α) until stationary phase. 50 μl of a culture was applied to 4-mm3 disks of an agar-based artificial diet [30]. The liquid was allowed to soak into the agar block which was then dried under a laminar flow. First-instar M. sexta neonate larvae were then placed on the disk and incubated at 22°C. The application of bacterial culture aliquots was repeated after three days, and the larvae mortality was recorded after 5 days.
Larvae of the greater wax moth, G. mellonella, were obtained from the Zoo-Fachmarkt (München, Germany), and stored for less than one week at room temperature. Bacterial strains were grown to stationary phase at 15°C (Yersinia spp.) or 37°C (S. enterica serovar Typhimurium and DH5α) and then diluted 1:10 and 1:100. 5–7.5 μl of each dilution corresponding to approximately 5–7.5 × 105 and 5–7.5 × 104 viable cells were subcutaneously injected into larvae of 2–3 cm length and of 90–140 mg weight using a sterilized micro syringe (Hamilton 1702 RN, 25 μl). Infected larvae were then incubated for five days at 15°C, and the numbers of killed and living larvae were enumerated.
Declarations
Acknowledgements
We thank Peter Roggentin, Alexander Rakin, Jürgen Heesemann, Henry Derschum and Herbert Nattermann for providing Y. enterocolitica strains, and Patrick Schiwek for technical assistance. This work was supported by a grant of the Hochschul- und Wissenschaftsprogramm (HWPII): Fachprogramm "Chancengleichheit für Frauen in Forschung und Lehre" to G. B.
Authors’ Affiliations
References
- Bowen DJ, Ensign JC: 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.PubMed CentralPubMedGoogle Scholar
- Waterfield NR, Bowen DJ, Fetherston JD, Perry RD, ffrench-Constant RH: The tc genes of Photorhabdus: a growing family. Trends Microbiol. 2001, 9: 185-191.View ArticlePubMedGoogle Scholar
- Vodovar N, Vallenet D, Cruveiller S, Rouy Z, Barbe V, Acosta C, Cattolico L, Jubin C, Lajus A, Segurens B, Vacherie B, Wincker P, Weissenbach J, Lemaitre B, Medigue C, Boccard F: Complete genome sequence of the entomopathogenic and metabolically versatile soil bacterium Pseudomonas entomophila. Nat Biotechnol. 2006, 24: 673-679.View ArticlePubMedGoogle Scholar
- Dodd SJ, Hurst MR, Glare TR, O'Callaghan M, Ronson CW: Occurrence of sep insecticidal toxin complex genes in Serratia spp. and Yersinia frederiksenii. Appl Environ Microbiol. 2006, 72: 6584-6592.PubMed CentralView ArticlePubMedGoogle Scholar
- Tennant SM, Skinner NA, Joe A, Robins-Browne RM: Homologues of insecticidal toxin complex genes in Yersinia enterocolitica biotype 1A and their contribution to virulence. Infect Immun. 2005, 73: 6860-6867.PubMed CentralView ArticlePubMedGoogle Scholar
- Bresolin G, Morgan JA, Ilgen D, Scherer S, Fuchs TM: Low temperature-induced insecticidal activity of Yersinia enterocolitica. Mol Microbiol. 2006, 59: 503-512.View ArticlePubMedGoogle Scholar
- Thomson NR, Howard S, Wren BW, Holden MT, Crossman L, Challis GL, Churcher C, Mungall K, Brooks K, Chillingworth T, Feltwell T, Abdellah Z, Hauser H, Jagels K, Maddison M, Moule S, Sanders M, Whitehead S, Quail MA, Dougan G, Parkhill J, Prentice MB: The complete genome sequence and comparative genome analysis of the high pathogenicity Yersinia enterocolitica strain 8081. PLoS Genet. 2006, 2: e206-PubMed CentralView ArticlePubMedGoogle Scholar
- Waterfield N, Hares M, Hinchliffe S, Wren B, ffrench-Constant R: The insect toxin complex of Yersinia. Adv Exp Med Biol. 2007, 603: 247-257.View ArticlePubMedGoogle Scholar
- Gendlina I, Held KG, Bartra SS, Gallis BM, Doneanu CE, Goodlett DR, Plano GV, Collins CM: Identification and type III-dependent secretion of the Yersinia pestis insecticidal-like proteins. Mol Microbiol. 2007, 64: 1214-1227.View ArticlePubMedGoogle Scholar
- Silva CP, Waterfield NR, Daborn PJ, Dean P, Chilver T, Au CP, Sharma S, Potter U, Reynolds SE, ffrench-Constant RH: Bacterial infection of a model insect: Photorhabdus luminescens and Manduca sexta. Cell Microbiol. 2002, 4: 329-339.View ArticlePubMedGoogle Scholar
- Hares MC, Hinchliffe SJ, Strong PC, Eleftherianos I, Dowling AJ, ffrench-Constant RH, Waterfield N: The Yersinia pseudotuberculosis and Yersinia pestis toxin complex is active against cultured mammalian cells. Microbiology. 2008, 154: 3503-3517.View ArticlePubMedGoogle Scholar
- Erickson DL, Waterfield NR, Vadyvaloo V, Long D, Fischer ER, ffrench-Constant R, Hinnebusch BJ: Acute oral toxicity of Yersinia pseudotuberculosis to fleas: implications for the evolution of vector-borne transmission of plague. Cell Microbiol. 2007, 9: 2658-2666.View ArticlePubMedGoogle Scholar
- ffrench-Constant R, Waterfield N, Daborn P, Joyce S, Bennett H, Au C, Dowling A, Boundy S, Reynolds S, Clarke D: Photorhabdus: towards a functional genomic analysis of a symbiont and pathogen. FEMS Microbiol Rev. 2003, 26: 433-456.View ArticlePubMedGoogle Scholar
- Pinheiro VB, Ellar DJ: Expression and insecticidal activity of Yersinia pseudotuberculosis and Photorhabdus luminescens toxin complex proteins. Cell Microbiol. 2007, 9: 2372-2380.View ArticlePubMedGoogle Scholar
- Motin VL, Georgescu AM, Fitch JP, Gu PP, Nelson DO, Mabery SL, Garnham JB, Sokhansanj BA, Ott LL, Coleman MA, Elliott JM, Kegelmeyer LM, Wyrobek AJ, Slezak TR, Brubaker RR, Garcia E: Temporal global changes in gene expression during temperature transition in Yersinia pestis. J Bacteriol. 2004, 186: 6298-6305.PubMed CentralView ArticlePubMedGoogle Scholar
- Han Y, Zhou D, Pang X, Song Y, Zhang L, Bao J, Tong Z, Wang J, Guo Z, Zhai J, Du Z, Wang X, Zhang X, Wang J, Huang P, Yang R: Microarray analysis of temperature-induced transcriptome of Yersinia pestis. Microbiol Immunol. 2004, 48: 791-805.View ArticlePubMedGoogle Scholar
- Bresolin G, Neuhaus K, Scherer S, Fuchs TM: Transcriptional analysis of long-term adaptation of Yersinia enterocolitica to low-temperature growth. J Bacteriol. 2006, 188: 2945-2958.PubMed CentralView ArticlePubMedGoogle Scholar
- Bowen DJ, Ensign JC: Isolation and characterization of intracellular protein inclusions produced by the entomopathogenic bacterium Photorhabdus luminescens. Appl Environ Microbiol. 2001, 67: 4834-4841.PubMed CentralView ArticlePubMedGoogle Scholar
- Herbert EE, Goodrich-Blair H: Friend and foe: the two faces of Xenorhabdus nematophila. Nat Rev Microbiol. 2007, 5: 634-646.View ArticlePubMedGoogle Scholar
- Heermann R, Fuchs TM: Comparative analysis of the Photorhabdus luminescens and the Yersinia enterocolitica genomes: uncovering candidate genes involved in insect pathogenicity. BMC Genomics. 2008, 9: 40-PubMed CentralView ArticlePubMedGoogle Scholar
- Shayegani M, DeForge I, McGlynn DM, Root T: Characteristics of Yersinia enterocolitica and related species isolated from human, animal, and environmental sources. J Clin Microbiol. 1981, 14: 304-312.PubMed CentralPubMedGoogle Scholar
- Waterfield NR, Wren BW, ffrench-Constant RH: Invertebrates as a source of emerging human pathogens. Nat Rev Microbiol. 2004, 2: 833-841.View ArticlePubMedGoogle Scholar
- Ibrahim A, Goebel BM, Liesack W, Griffiths M, Stackebrandt E: The phylogeny of the genus Yersinia based on 16S rDNA sequences. FEMS Microbiol Lett. 1993, 114: 173-177.View ArticlePubMedGoogle Scholar
- Hilbi H, Weber SS, Ragaz C, Nyfeler Y, Urwyler S: Environmental predators as models for bacterial pathogenesis. Environ Microbiol. 2007, 9: 563-575.View ArticlePubMedGoogle Scholar
- Sambrook J, Russell DW: Molecular cloning: a laboratory manual. 2001, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y, 3Google Scholar
- Ochman H, Ajioka JW, Garza D, Hartl DL: Inverse polymerase chain reaction. Biotechnology (NY). 1990, 8: 759-760.View ArticleGoogle Scholar
- Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994, 22: 4673-4680.PubMed CentralView ArticlePubMedGoogle Scholar
- Peer Van de Y, De Wachter R: TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Comput Appl Biosci. 1994, 10: 569-570.PubMedGoogle Scholar
- Schachtner J, Huetteroth W, Nighorn A, Honegger HW: Copper/zinc superoxide dismutase-like immunoreactivity in the metamorphosing brain of the sphinx moth Manduca sexta. J Comp Neurol. 2004, 469: 141-152.View ArticlePubMedGoogle Scholar
- David WAL, Gardiner BOC: Rearing Pieris brassicae larvae on a semi-synthetic diet. Nature. 1965, 207: 882-883.View ArticleGoogle Scholar
- Kimura M: A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol. 1980, 16: 111-120.View ArticlePubMedGoogle Scholar
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