BACKGROUND
the toxin complex  proteins whose insecticidal potential resembles that of the bacillus thuringiensis bt-toxin were first purified from photorhabdus luminescens which lives in symbiosis with nematodes  <cit> .
they have also been identified in other insect-parasitizing bacteria such as serratia entomophila, xenorhabdus nematophilus, or pseudomonas entomophila  <cit> .
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, t <dig> and w <dig>  for which a genome sequence is not yet available  <cit> .
however, tc genes are absent in y. bercovieri and in y. enterocolitica strain  <dig>  <cit> .
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  <cit> .
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 , tcdb/tcac , and tccc   <cit> .
tc proteins have recently been shown to be secreted in a type iii-dependent manner in y. pestis  <cit> .
type  and  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  <cit> .
a role of the tc proteins from y. enterooclitica strain t <dig>  y. pseudotuberculosis strain ip <dig> 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  <cit> .
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  <cit> , or that they act as universal activator of, or chaperons for, different toxin proteins  <cit> .
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  <cit> .
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 w <dig>  tcaabc expression in y. pseudotuberculosis strain ip <dig> was observed at 15°c and at 37°c  <cit> .
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  <cit> .
the ip <dig> tc proteins are toxic against m. sexta larvae when expressed heterologously in e. coli  <cit> .
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  <cit> .
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  <cit> .
this is in line with the finding that two y. enterocolitica strains containing a tcdb-tccc gene pair  or lacking any tc-like genes  were equally toxic to fleas  <cit> .
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 w <dig>  the phylogenetic analysis of tccc genes in yersinia species, the tc-paiye 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-paiye in yersinia spp
chromosomal loci encoding tc proteins have first been sequenced from p. luminescens, s. entomophila and x. nematophilus.
in the past few years, the genome sequences of several yersinia strains became available.
most of them, namely seven y. pestis strains, three y. pseudotuberculosis strains and y. mollaretii, carry a common tc gene cluster termed the tc pathogenicity island of yersiniae .
further dna fragments encoding insecticidal toxins were detected in the genomes of two y. enterocolitica strains w <dig> and t <dig>  <cit> , and on a plasmid of y. frederiksenii strain  <dig>  <cit> .
a comparison of the chromosomal loci of yersiniae containing tc homologues is shown in fig.
<dig>  the length of the sequences ranges from ~ <dig> kb to ~ <dig> kb.
this variation is mainly due to the presence of one to four tccc  homologues.
in all cases, tc-paisye are inserted into an equivalent location with respect to the common yersinia genome backbone  <cit> , namely between the genes encoding a putative dna-binding transcriptional regulator and a putative dna gyrase modulator.
these genes are ye <dig>  and ye <dig>  with respect to the chromosomal sequence of y. enterocolitica strain  <dig>  the overall organisation is similar for all strains, including a second gene, tcar <dig> that encodes a homolog of lysr-like regulators, followed by genes belonging to the homology types tcaab/tcda, tcac/tcdb, and tccc.
tccc is separated from tcac/tcdb by two small phage-related genes and two orfs of unknown function.
according to the classification of waterfield et al.
<cit>  described above, all tc genes belong to tcd operons.
beyond these similarities, several differences were revealed by homology analysis and re-annotation.
y. enterocolitica strain w <dig> is characterised by a  <dig> bp sequence located between tccc and ye <dig>  harbouring two orfs of unknown function, one of which is also present in the y. pestis genomes.
a transposon-related sequence  was identified in front of the first phage-related gene of y. pestis angola.
frameshifts  are present in tcac of strain w <dig>  in tcaa of strain antiqua and of y. mollaretii, and in tcab of strains w <dig>  orientalis ip <dig> and co <dig>
homologues of tccc located outside tc-paiye
by screening a tn <dig> luxcdabe reporter library of strain w <dig> for genes induced upon low-temperature  <cit> , we identified a transposon insertion located outside the tc-paiye.
a  <dig>  bp sequence encompassing the transposon insertion site was derived, revealing two strain-specific orfs termed tccc2'  and tccc3'  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-paiye were identified in y. pestis strains antiqua, co <dig>  nepal <dig>  orientalis ip <dig> and  <dig>  and in y. pseudotuberculosis ip <dig>  and one further tccc gene in y. pestis strains kim and angola, and in y. pseudotuberculosis ip <dig>  a truncated tccc2' gene with a  <dig> bp deletion in comparison to tccc <dig> of strain ip <dig> is present in the genome of y. pseudotuberculosis ypiii.
tccc2' and tccc3' of strain w <dig> are located between two genes encoding a lipid a biosynthesis lauroyl acyltransferase, htrb , and a putative membrane protein  of strain  <dig>  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 co <dig> genes ypo2379/ypo <dig> encoding an n-ethylmaleimide reductase and a lactoylglutathione lyase, and between ypo2311/ypo <dig> coding for the insertion element is <dig> 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 co <dig> tccc <dig> and, albeit with lower probability, of w <dig>
a total of  <dig> tccc amino acid sequences, derived from yersiniae genes either located within or outside the tc-paiye, was compared here by a clustlx alignment.
the cladogram of the yersiniae tccc proteins tc-paiye exhibits a significant sequence variability between island- and non island-encoded tcccs .
the tccc proteins expressed from loci outside the tc-paiye compose two groups that are characterised by the two common insertion sites as described above.
the only exception is tccc2'/3' of strain w <dig> that is more closely related to the tc-paiye-encoded tccc proteins.
tccc sequences derived from genes located within the tc-paiye show a more complex relationship.
one group of tcccs represents tccc <dig> proteins, a second group tccc <dig> proteins, indicating a highly conserved linear order of tccc <dig> and tccc <dig> genes in the yersiniae genomes.
interestingly, tccc <dig> of y. pestis angola encoded by a tccc gene located nearby a transposase-like gene  appears to be more closely related to tccc <dig> proteins.
two further sublines that show a higher sequence variability are represented by tccc2- <dig> of y. pseudotuberculosis ip <dig>  and by tccc <dig> of y. mollaretii and y. enterocolitica strains w <dig> and t <dig>
presence of tc genes in y. enterocolitica strains
in a previous study, we had studied a restricted number of y. enterocolitica strains by pcr and southern blot for the presence of tcaa, tcab, and tcac in representatives of five biotypes  <cit> .
here, we performed a more thorough investigation for the presence, absence, variability and genetic organisation of tc genes in a total of  <dig> y. enterocolitica strains belonging to six biotypes.
chromosomal dna of all strains was successfully subjected to pcr with oligonucleotides specific for 16s rdna as control.
a series of  <dig> pcrs designed to amplify intragenic and gene-overlapping fragments was performed .
dna of strains w <dig> and  <dig> served as a positive and a negative control.
first, tcar <dig> was confirmed as part of the common yersiniae genomic backbone.
the second regulatory gene, tcar <dig>  is present in all strains of biotypes 2– <dig>  but absent in biotypes 1a and 1b.
two different primer combinations failed to amplify tcaa-specific fragments from dna of several strains.
sequencing of four fragments revealed mismatches, and conserved tcaa regions were therefore used to design more appropriate oligonucleotides that showed the presence of tcaa in biotypes 2– <dig>  but not 1a and 1b .
similar patterns were obtained following amplification of intragenic tcab and tcac fragments , with the exception that tcac revealed also to be present in one biotype 1a and one biotype 1b strain.
using oligonucleotides homologous to the 3'-end of tcaa and the 5'-end of tcab, the operon organisation was confirmed for most, but not all, biotype 2– <dig> strains .
negative results in case of pcr  <dig> predominantly with dna of biotype  <dig> strains might be due to a frameshift in tcab similar to that in strain w <dig>  resulting in an unfunctional and possibly degenerated 5'-end of tcab.
remnants of the phage-related gene cassette which are present in all biotype 2– <dig> strains could also be amplified from dna of biotype 1a and 1b strains .
the results of pcrs 18– <dig> indicate a highly conserved 5'-region and an otherwise variable tccc sequence.
we therefore performed a pcr with primers specific for tccc of strain t <dig> and found the respective fragment also in three biotype  <dig> strains and in one biotype  <dig> strain.
to investigate whether tc genes homologous to that of biotype 1a strain t <dig> are present in other y. enterocolitica strains, pcrs  <dig> and  <dig>  with oligonucleotides specific for tcda of strain t <dig> were performed.
fragments of the expected length were obtained using dna of four biotype 1a strains, a finding that correlates well with the negative result of pcr  <dig> for fragments overlapping the insertion site of the tc-paiye.
finally, orf <dig> is present in all but one biotype 2– <dig> strains, but absent in most other yersinia spp.
.
taken together, the tc operons of strain w <dig> are present in all biotype 2– <dig> representatives tested here.
a tc gene cluster homologous to that of strain t <dig> is probably present in biotype 1a strains  <dig>   <dig> and  <dig>  in comparison to all other tc genes, tccc exhibits a higher sequence variability .
oral infection of m. sexta
some yersinia spp.
besides the three human pathogenic species have not yet been investigated for their insecticidal activity.
oral infection of first-instar m. sexta neonates was performed by soaking small blocks of an artificial diet with  <dig> μl aliquots of overnight cultures grown at 15°c  and 37°c .
yersinia strains applied were y. mollaretii, y. aldovae, y. ruckeri, and four y. enterocolitica strains .
their toxicity towards m. sexta as percentage of dead larvae five days after infection is shown in table  <dig>  the experimental setting hampered the reproducibility of the assay, resulting in larger standard deviations in comparison to the g. mellonella infection model .
the strains y. enterocolitica  <dig> and y. mollaretii possessing the tc-paiye showed the highest killing rates.
strains without tc genes such as y. ruckeri and y. aldovae were less toxic towards m. sexta.
the only exception was the tc-negative y. enterocolitica strain  <dig> with a lethality rate of 42%, suggesting the presence of further gut-active insecticidal determinants in its genome.
the toxicity of strain w <dig>  in comparison to that of the tcaa knockout mutant w22703-tcaa::tn5lux  confirms our recent experiments using concentrated protein extracts instead of viable cells.
taken together, the average killing rates of table  <dig> show that the tc genes play an important role in the insecticidal activity of yersinia spp.
upon oral uptake.
yersinia strains were orally fed to first-instar m. sexta neonates.
each experiment was independently performed at least three times with a minimum of six larvae, and the average dead rates are shown.
y. enterocolitica w22703: biotype  <dig>  serotype o:9; y. enterocolitica 2594: biotype  <dig>  serotype o:9; y. enterocolitica 4466: biotype 1b, serotype o: <dig>  1) according to the genome sequence, 2) according to pcr analysis performed in this study.
subcutaneous infection of g. mellonella
for an additional bioassay, we chose the model organism g. mellonella that is also used to determine the toxicity of p. luminescens and x. nematophilus.
we infected larvae of this greater wax moth subcutaneously with 1: <dig> and 1: <dig> diluted aliquots of y. mollaretii, y. kristensenii, y. bercovieri, y. aldovae, y. ruckeri, and four y. enterocolitica strains  incubated at 15°c until they reached stationary phase.
after five days, only 10% of the larvae had survived an infection of strains  <dig> or  <dig>  in total, four strains showed high killing rates of 81–90% .
to determine whether the toxicity towards the insect larvae depends on the presence of tc proteins, we analysed chromosomal dna of y. ruckeri, y. aldovae, y. kristensenii, and y. enterocolitica strain  <dig> with primers described in additional file  <dig> and shown in fig.
<dig>  no fragments were obtained with primers specific to tcar <dig> , tcaa , tcab , tcab-tcac , tcac , orf11-ye <dig> , and tccc of y. enterocolitica strain t <dig> .
as a control, amplification of a tcar <dig> fragment which is part of the common yersinia backbone sequence  succeeded with dna of y. aldovae and y. enterocolitica strain  <dig>  therefore, we conclude that the tc-paiye is absent in these strains .
thus, only two of the strains exhibiting a killing rate of at least 81%, namely y. mollaretii, and the biotype  <dig> strain  <dig>  harbour the tc-paiye, while it is absent in y. enterocolitica strain  <dig> and y. bercovieri.
on the other hand, y. ruckeri and y. aldovae killed g. mellonella larvae in approximately 40–50% of all infection experiments, and the insecticidal activity of y. kristensenii is only slightly higher than that of the control strain s. enterica serovar typhimurium.
these three strains do not carry tc genes, too.
no significant difference  in the insecticidal activity between w <dig> and its tcaa knockout mutant could be observed.
plasmid-encoded sep-like genes of y. frederiksenii isolate  <dig> could also not be detected using primers specific for tcyf <dig>  tcyf <dig> or tcyf <dig>  in bacterial lysates of y. enterocolitica strain  <dig>  y. ruckeri, y. aldovae, or y. kristensenii.
according to blast analysis, homologous of type iii secretion system  genes are present in the genomes of w <dig> and the other strains from table  <dig> sequenced so far, but the role of t3ss and insect virulence as shown for y. pestis remains to be determined for these strains.
taken together, this data strongly suggests that insecticidal activity of yersinia spp.
towards g. mellonella upon subcutaneous infection is not caused by tc proteins, and that yet unknown determinants contribute to the insecticidal activity of yersinia strains towards the insect larvae.
the similar lethality of strain w <dig> and its tcaa-negative mutant not only confirms the assumption of tc-independent killing, but also of a gut-related tcaa activity following oral infection as demonstrated above and recently suggested  <cit> .
g. mellonella larvae were subcutaneously infected with  <dig> μl of diluted overnight cultures.
strain w <dig> and its tcaa knockout mutant confer similar toxicity towards the larvae .
each experiment was independently performed at least three times, and the average dead rates are shown.
y. enterocolitica w22703: biotype  <dig>  serotype o:9; y. enterocolitica 2594: biotype  <dig>  serotype o:9; y. enterocolitica 4466: biotype 1b, serotype o: <dig>  1) according to the genome sequence, 2) according to pcr analysis performed in this study.
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 w <dig> and w22703-tcaa::tn5lux protein extract to m. sexta larvae, we could recently demonstrate the role of tcaa in y. enterocolitica toxicity towards insects  <cit> .
further five yersinia strains were tested here for the first time with respect to their oral toxicity in the m. sexta model .
the presence of the tc-paiye 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 w <dig>  <cit> .
in contrast, subcutaneous infection of g. mellonella does not result in a significantly different toxicity of these strains .
in comparison to p. luminescens that causes death of g. mellonella larvae within  <dig> hours following injection of several thousand cells  <cit> , approximately  <dig> ×  <dig> y. enterocolitica strain w <dig> 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.
<cit> .
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  <cit> .
the overall results of the galleria bioassay correlate with the finding that among  <dig> yersinia isolates from the environment,  <dig> % were y. enterocolitica, but only  <dig> % belonged to y. kristensenii  <cit> .
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  <cit> .
especially, tcac is well conserved within the yersinia genus, but tcab and tcaa show significant sequence variability  <cit> .
when the tccc sequences derived from the tc-paiye of yersiniae were aligned, a high degree of sequence conservation was obtained at amino acids 1– <dig>  followed by a remarkably high sequence diversity  <cit>  as is confirmed by the tccc cladogram .
some tc sequences show evidence of undergoing degradation with frameshifts that often result in the splitting of tc genes into two separate orfs .
frameshifts in y. pestis, especially in tcab of co <dig>  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  <cit> .
this data indicates that the tc genes of yersiniae may be under diversifying selection  <cit>  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  <cit> .
however, the data presented here point to a common yersinia ancestor that has aquired the tc-paiye.
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 .
this hypothesis is strongly supported by the common insertion site of the tc-paiye that indicates one horizontal gene transfer  event, by the highly conserved phage-related genes within the tc-paiye, and by a similar gene order including tccc <dig> and tccc <dig> in all islands investigated.
moreover, the cladogram derived from a comprehensive alignment of tccc protein sequences  essentially reflects the phylogeny of yersinia based on 16s rdna sequences, including the clonal diversity among y. enterocolitica strains  <cit> .
as an additional insecticidal determinant, tccc genes located outside the tc-paiye 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-paiye in several yersinia species and strains  as examplified by the identification of rudimentary tc genes in biotypes 1a and 1b .
CONCLUSIONS
the prevalence of the tc-paiye 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  <cit> .
the tc-paiye 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  <cit> .
distinct sequence variation, and reductive evolution especially within the genomes of y. pestis serovars, might have allowed yersiniae to occupy specific ecological niches  <cit> .
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.

 the toxin complex proteins whose insecticidal potential resembles that of the bacillus thuringiensis bt-toxin were first purified from photorhabdus luminescens which lives in symbiosis with nematodes. they have also been identified in other insect-parasitizing bacteria such as serratia entomophila, xenorhabdus nematophilus, or pseudomonas entomophila. 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, t and w for which a genome sequence is not yet available. however, tc genes are absent in y. bercovieri and in y. enterocolitica strain. 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. 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, tcdb/tcac, and tccc. tc proteins have recently been shown to be secreted in a type iii-dependent manner in y. pestis. type and 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. a role of the tc proteins from y. enterooclitica strain t y. pseudotuberculosis strain ip 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. 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, or that they act as universal activator of, or chaperons for, different toxin proteins. 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. 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 w tcaabc expression in y. pseudotuberculosis strain ip was observed at 15°c and at 37°c. 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. the ip tc proteins are toxic against m. sexta larvae when expressed heterologously in e. coli. 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. 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. this is in line with the finding that two y. enterocolitica strains containing a tcdb-tccc gene pair or lacking any tc-like genes were equally toxic to fleas. 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 w the phylogenetic analysis of tccc genes in yersinia species, the tc-paiye 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.  the tc-paiye in yersinia spp chromosomal loci encoding tc proteins have first been sequenced from p. luminescens, s. entomophila and x. nematophilus. in the past few years, the genome sequences of several yersinia strains became available. most of them, namely seven y. pestis strains, three y. pseudotuberculosis strains and y. mollaretii, carry a common tc gene cluster termed the tc pathogenicity island of yersiniae. further dna fragments encoding insecticidal toxins were detected in the genomes of two y. enterocolitica strains w and t, and on a plasmid of y. frederiksenii strain. a comparison of the chromosomal loci of yersiniae containing tc homologues is shown in fig. the length of the sequences ranges from ~ kb to ~ kb. this variation is mainly due to the presence of one to four tccc homologues. in all cases, tc-paisye are inserted into an equivalent location with respect to the common yersinia genome backbone, namely between the genes encoding a putative dna-binding transcriptional regulator and a putative dna gyrase modulator. these genes are ye and ye with respect to the chromosomal sequence of y. enterocolitica strain the overall organisation is similar for all strains, including a second gene, tcar that encodes a homolog of lysr-like regulators, followed by genes belonging to the homology types tcaab/tcda, tcac/tcdb, and tccc. tccc is separated from tcac/tcdb by two small phage-related genes and two orfs of unknown function. according to the classification of waterfield et al. described above, all tc genes belong to tcd operons. beyond these similarities, several differences were revealed by homology analysis and re-annotation. y. enterocolitica strain w is characterised by a bp sequence located between tccc and ye harbouring two orfs of unknown function, one of which is also present in the y. pestis genomes. a transposon-related sequence was identified in front of the first phage-related gene of y. pestis angola. frameshifts are present in tcac of strain w in tcaa of strain antiqua and of y. mollaretii, and in tcab of strains w orientalis ip and co homologues of tccc located outside tc-paiye by screening a tn luxcdabe reporter library of strain w for genes induced upon low-temperature, we identified a transposon insertion located outside the tc-paiye. a bp sequence encompassing the transposon insertion site was derived, revealing two strain-specific orfs termed tccc2' and tccc3' 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-paiye were identified in y. pestis strains antiqua, co nepal orientalis ip and and in y. pseudotuberculosis ip and one further tccc gene in y. pestis strains kim and angola, and in y. pseudotuberculosis ip a truncated tccc2' gene with a bp deletion in comparison to tccc of strain ip is present in the genome of y. pseudotuberculosis ypiii. tccc2' and tccc3' of strain w are located between two genes encoding a lipid a biosynthesis lauroyl acyltransferase, htrb, and a putative membrane protein of strain 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 co genes ypo2379/ypo encoding an n-ethylmaleimide reductase and a lactoylglutathione lyase, and between ypo2311/ypo coding for the insertion element is 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 co tccc and, albeit with lower probability, of w a total of tccc amino acid sequences, derived from yersiniae genes either located within or outside the tc-paiye, was compared here by a clustlx alignment. the cladogram of the yersiniae tccc proteins tc-paiye exhibits a significant sequence variability between island- and non island-encoded tcccs. the tccc proteins expressed from loci outside the tc-paiye compose two groups that are characterised by the two common insertion sites as described above. the only exception is tccc2'/3' of strain w that is more closely related to the tc-paiye-encoded tccc proteins. tccc sequences derived from genes located within the tc-paiye show a more complex relationship. one group of tcccs represents tccc proteins, a second group tccc proteins, indicating a highly conserved linear order of tccc and tccc genes in the yersiniae genomes. interestingly, tccc of y. pestis angola encoded by a tccc gene located nearby a transposase-like gene appears to be more closely related to tccc proteins. two further sublines that show a higher sequence variability are represented by tccc2- of y. pseudotuberculosis ip and by tccc of y. mollaretii and y. enterocolitica strains w and t presence of tc genes in y. enterocolitica strains in a previous study, we had studied a restricted number of y. enterocolitica strains by pcr and southern blot for the presence of tcaa, tcab, and tcac in representatives of five biotypes. here, we performed a more thorough investigation for the presence, absence, variability and genetic organisation of tc genes in a total of y. enterocolitica strains belonging to six biotypes. chromosomal dna of all strains was successfully subjected to pcr with oligonucleotides specific for 16s rdna as control. a series of pcrs designed to amplify intragenic and gene-overlapping fragments was performed. dna of strains w and served as a positive and a negative control. first, tcar was confirmed as part of the common yersiniae genomic backbone. the second regulatory gene, tcar is present in all strains of biotypes 2– but absent in biotypes 1a and 1b. two different primer combinations failed to amplify tcaa-specific fragments from dna of several strains. sequencing of four fragments revealed mismatches, and conserved tcaa regions were therefore used to design more appropriate oligonucleotides that showed the presence of tcaa in biotypes 2– but not 1a and 1b. similar patterns were obtained following amplification of intragenic tcab and tcac fragments, with the exception that tcac revealed also to be present in one biotype 1a and one biotype 1b strain. using oligonucleotides homologous to the 3'-end of tcaa and the 5'-end of tcab, the operon organisation was confirmed for most, but not all, biotype 2– strains. negative results in case of pcr predominantly with dna of biotype strains might be due to a frameshift in tcab similar to that in strain w resulting in an unfunctional and possibly degenerated 5'-end of tcab. remnants of the phage-related gene cassette which are present in all biotype 2– strains could also be amplified from dna of biotype 1a and 1b strains. the results of pcrs 18– indicate a highly conserved 5'-region and an otherwise variable tccc sequence. we therefore performed a pcr with primers specific for tccc of strain t and found the respective fragment also in three biotype strains and in one biotype strain. to investigate whether tc genes homologous to that of biotype 1a strain t are present in other y. enterocolitica strains, pcrs and with oligonucleotides specific for tcda of strain t were performed. fragments of the expected length were obtained using dna of four biotype 1a strains, a finding that correlates well with the negative result of pcr for fragments overlapping the insertion site of the tc-paiye. finally, orf is present in all but one biotype 2– strains, but absent in most other yersinia spp.. taken together, the tc operons of strain w are present in all biotype 2– representatives tested here. a tc gene cluster homologous to that of strain t is probably present in biotype 1a strains and in comparison to all other tc genes, tccc exhibits a higher sequence variability. oral infection of m. sexta some yersinia spp. besides the three human pathogenic species have not yet been investigated for their insecticidal activity. oral infection of first-instar m. sexta neonates was performed by soaking small blocks of an artificial diet with μl aliquots of overnight cultures grown at 15°c and 37°c. yersinia strains applied were y. mollaretii, y. aldovae, y. ruckeri, and four y. enterocolitica strains. their toxicity towards m. sexta as percentage of dead larvae five days after infection is shown in table the experimental setting hampered the reproducibility of the assay, resulting in larger standard deviations in comparison to the g. mellonella infection model. the strains y. enterocolitica and y. mollaretii possessing the tc-paiye showed the highest killing rates. strains without tc genes such as y. ruckeri and y. aldovae were less toxic towards m. sexta. the only exception was the tc-negative y. enterocolitica strain with a lethality rate of 42%, suggesting the presence of further gut-active insecticidal determinants in its genome. the toxicity of strain w in comparison to that of the tcaa knockout mutant w22703-tcaa::tn5lux confirms our recent experiments using concentrated protein extracts instead of viable cells. taken together, the average killing rates of table show that the tc genes play an important role in the insecticidal activity of yersinia spp. upon oral uptake. yersinia strains were orally fed to first-instar m. sexta neonates. each experiment was independently performed at least three times with a minimum of six larvae, and the average dead rates are shown. y. enterocolitica w22703: biotype serotype o:9; y. enterocolitica 2594: biotype serotype o:9; y. enterocolitica 4466: biotype 1b, serotype o: 1) according to the genome sequence, 2) according to pcr analysis performed in this study. subcutaneous infection of g. mellonella for an additional bioassay, we chose the model organism g. mellonella that is also used to determine the toxicity of p. luminescens and x. nematophilus. we infected larvae of this greater wax moth subcutaneously with 1: and 1: diluted aliquots of y. mollaretii, y. kristensenii, y. bercovieri, y. aldovae, y. ruckeri, and four y. enterocolitica strains incubated at 15°c until they reached stationary phase. after five days, only 10% of the larvae had survived an infection of strains or in total, four strains showed high killing rates of 81–90%. to determine whether the toxicity towards the insect larvae depends on the presence of tc proteins, we analysed chromosomal dna of y. ruckeri, y. aldovae, y. kristensenii, and y. enterocolitica strain with primers described in additional file and shown in fig. no fragments were obtained with primers specific to tcar, tcaa, tcab, tcab-tcac, tcac, orf11-ye, and tccc of y. enterocolitica strain t. as a control, amplification of a tcar fragment which is part of the common yersinia backbone sequence succeeded with dna of y. aldovae and y. enterocolitica strain therefore, we conclude that the tc-paiye is absent in these strains. thus, only two of the strains exhibiting a killing rate of at least 81%, namely y. mollaretii, and the biotype strain harbour the tc-paiye, while it is absent in y. enterocolitica strain and y. bercovieri. on the other hand, y. ruckeri and y. aldovae killed g. mellonella larvae in approximately 40–50% of all infection experiments, and the insecticidal activity of y. kristensenii is only slightly higher than that of the control strain s. enterica serovar typhimurium. these three strains do not carry tc genes, too. no significant difference in the insecticidal activity between w and its tcaa knockout mutant could be observed. plasmid-encoded sep-like genes of y. frederiksenii isolate could also not be detected using primers specific for tcyf tcyf or tcyf in bacterial lysates of y. enterocolitica strain y. ruckeri, y. aldovae, or y. kristensenii. according to blast analysis, homologous of type iii secretion system genes are present in the genomes of w and the other strains from table sequenced so far, but the role of t3ss and insect virulence as shown for y. pestis remains to be determined for these strains. taken together, this data strongly suggests that insecticidal activity of yersinia spp. towards g. mellonella upon subcutaneous infection is not caused by tc proteins, and that yet unknown determinants contribute to the insecticidal activity of yersinia strains towards the insect larvae. the similar lethality of strain w and its tcaa-negative mutant not only confirms the assumption of tc-independent killing, but also of a gut-related tcaa activity following oral infection as demonstrated above and recently suggested. g. mellonella larvae were subcutaneously infected with μl of diluted overnight cultures. strain w and its tcaa knockout mutant confer similar toxicity towards the larvae. each experiment was independently performed at least three times, and the average dead rates are shown. y. enterocolitica w22703: biotype serotype o:9; y. enterocolitica 2594: biotype serotype o:9; y. enterocolitica 4466: biotype 1b, serotype o: 1) according to the genome sequence, 2) according to pcr analysis performed in this study. 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 w and w22703-tcaa::tn5lux protein extract to m. sexta larvae, we could recently demonstrate the role of tcaa in y. enterocolitica toxicity towards insects. further five yersinia strains were tested here for the first time with respect to their oral toxicity in the m. sexta model. the presence of the tc-paiye 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 w. in contrast, subcutaneous infection of g. mellonella does not result in a significantly different toxicity of these strains. in comparison to p. luminescens that causes death of g. mellonella larvae within hours following injection of several thousand cells, approximately × y. enterocolitica strain w 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.. 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. the overall results of the galleria bioassay correlate with the finding that among yersinia isolates from the environment, % were y. enterocolitica, but only % belonged to y. kristensenii. 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. especially, tcac is well conserved within the yersinia genus, but tcab and tcaa show significant sequence variability. when the tccc sequences derived from the tc-paiye of yersiniae were aligned, a high degree of sequence conservation was obtained at amino acids 1– followed by a remarkably high sequence diversity as is confirmed by the tccc cladogram. some tc sequences show evidence of undergoing degradation with frameshifts that often result in the splitting of tc genes into two separate orfs. frameshifts in y. pestis, especially in tcab of co 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. this data indicates that the tc genes of yersiniae may be under diversifying selection 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. however, the data presented here point to a common yersinia ancestor that has aquired the tc-paiye. 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. this hypothesis is strongly supported by the common insertion site of the tc-paiye that indicates one horizontal gene transfer event, by the highly conserved phage-related genes within the tc-paiye, and by a similar gene order including tccc and tccc in all islands investigated. moreover, the cladogram derived from a comprehensive alignment of tccc protein sequences essentially reflects the phylogeny of yersinia based on 16s rdna sequences, including the clonal diversity among y. enterocolitica strains. as an additional insecticidal determinant, tccc genes located outside the tc-paiye 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-paiye in several yersinia species and strains as examplified by the identification of rudimentary tc genes in biotypes 1a and 1b.  the prevalence of the tc-paiye 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. the tc-paiye 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. distinct sequence variation, and reductive evolution especially within the genomes of y. pestis serovars, might have allowed yersiniae to occupy specific ecological niches. 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. 
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. oral uptake experiments with manduca sexta revealed a higher larvae lethality of yersinia strains harbouring the tc-paiye in comparison to strains lacking this island. 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. towards m. sexta and g. mellonella demonstrated here for the first time points to insects as a natural reservoir for yersiniae. 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. strain w and its tcaa deletion mutant did not exhibit a significantly different toxicity towards g. mellonella. following subcutaneous infection of galleria mellonella larvae with five non-human pathogenic yersinia spp. the toxic potential of several yersinia spp. pcr analysis revealed the presence of the tc-paiye and of tccc2'/3'-homologues in all biotype 2– strains tested, and their absence in most representatives of biotypes 1a and 1b. and four y. enterocolitica strains, we observed a remarkable variability of their insecticidal activity ranging from 20% to 90% dead larvae after five days. phylogenetic analysis of tccc sequences indicates the presence of the tc-paiye in an ancestor of yersinia. toxin complex proteins termed tcaabc, tcdab, and tccabc with insecticidal activity are present in a variety of bacteria including the yersiniae. outside the tc pathogenicity island of y. enterocolitica strain w a pseudogene encoding proteins with homology to tccc and similarity to tyrosine phosphatases at its c-terminus was identified.
 toxin complex proteins termed tcaabc, tcdab, and tccabc with insecticidal activity are present in a variety of bacteria including the yersiniae.  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 of y. enterocolitica strain w a pseudogene 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-paiye and of tccc2'/3'-homologues in all biotype 2– strains tested, and their absence in most representatives of biotypes 1a and 1b. phylogenetic analysis of tccc sequences indicates the presence of the tc-paiye in an ancestor of yersinia. oral uptake experiments with manduca sexta revealed a higher larvae lethality of yersinia strains harbouring the tc-paiye 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% to 90% dead larvae after five days. strain w 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.  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. 