BACKGROUND
bacteria belonging to the pseudomonas fluorescens group  <cit>  provide a compelling example of ecological and bacterial lifestyle diversity reflected by the vast range of environmental habitats they occupy.
this group encloses plant-beneficial symbionts, environmental saprophytes and clinical strains of opportunistic human pathogens .
within the p. fluorescens group, root-colonizing pseudomonads are well known for their ability to promote plant growth and to protect plants against soilborne pathogens through a set of diverse and functionally complementary mechanisms.
the capacity to suppress fungal diseases has largely been attributed to the production of secondary metabolites with cytotoxic and antimicrobial activity, in particular  <dig> -diacetylphloroglucinol , phenazines, pyoluteorin, pyrrolnitrin, hydrogen cyanide, and lipopeptides  <cit> .
extensive knowledge has been gathered over the last years on plant disease suppression and plant growth promotion.
surprisingly, it has become only recently apparent that specific strains of plant-associated pseudomonads are able to infect and kill insects .
these observations invoke that particular strains may function as insect pathogens and switch between insect hosts and the plant environment.
insecticidal activity in environmental pseudomonads was, with the exception of pseudomonas entomophila, a pathogen of drosophila , so far only rarely demonstrated.
initially, an insect toxin was discovered in silico when the genome of pseudomonas protegens pf- <dig>  became available  <cit> .
subsequent molecular and mutational characterization revealed that oral and injectable insecticidal activity is linked to the fit  gene, which was described and characterized for the first time in p. protegens strains cha <dig> and pf- <dig>  <cit> .
injection of fit expressing e. coli is sufficient to induce strong melanization and rapid death of the tobacco hornworm manduca sexta and larvae of the greater wax moth galleria mellonella  <cit> .
fit toxin knock-out mutants of cha <dig> have attenuated virulence, both when injected into g. mellonella or fed to african cotton leafworm spodoptera littoralis  <cit> .
the fit gene cluster consists of eight genes  with functions in toxin export, insect toxicity and regulation .
the fit insect toxin gene fitd is flanked upstream by fitabc and downstream by fite encoding components of a type i secretion system.
the products of the fitfgh genes regulate toxin production  <cit> .
fitf is a sensor histidine kinase – response regulator hybrid, detecting the insect environment and activating insecticidal toxin expression via fith and fitg  <cit> .fig.
<dig> organization of the fit gene cluster and their homologues .
in p. protegens and p. chlororaphis the fit gene cluster  is composed of eight orfs encoding the insecticidal toxin , predicted components of a toxin secretion system  including an outer membrane protein , and regulators of toxin production .
the orientation of the toxin gene within the genome is indicated by + for the leading strand and by – for lagging the strand.
transporter homologues  are only found in p. luminescens adjacent to mcf <dig>  a truncated variant of mcf <dig>  x. bovienii ss <dig> and p. asymbiotica attc  <dig> harbor only one mcf variant.
blue arrows indicate flanking genes, which share no homology among the strains represented.
the phylogenetic relationship of p. protegens, p. chlororaphis, photorhabdus spp.
and xenorhabdus spp.
with representative orders/families and groups of γ-proteobacteria is based on concatenated reca, rpob and rpod protein sequences
genome sequencing revealed that certain strains of pseudomonas chlororaphis also harbour the complete fit gene cluster   <cit> .
contribution of the fit toxin to the oral insecticidal activity has been demonstrated for p. chlororaphis pcl <dig> against s. littoralis  <cit> .
the fit insect toxin shares 73 % identity with the makes caterpillars floppy insecticidal toxin mcf <dig> and 67 % with mcf <dig>  both produced by photorhabdus luminescens, a bacterial symbiont of entomopathogenic nematodes  <cit> .
mcf-like toxins are also found in photorhabdus asymbiotica, xenorhabdus nemtophila and xenorhabdus bovienii  .
the mcf <dig> toxin causes rapid disruption of the insect midgut epithelium and hemocytes triggered by a bh-3-like apoptosis control domain  <cit> .
injection of purified mcf <dig> in drosophila embryos leads to a freezing phenotype of hemocytes, due to a rearrangement of the actin cytoskeleton  <cit> .
while mcf toxins are essentially studied in the photorhabdus lineage the evolutionary basis for the homology between fit and mcf toxins has remained unclear.
here, we conducted comparative sequence analysis in combination with virulence assays to yield a better understanding of insect pathogenicity in plant-associated pseudomonads.
in order to study fit/mcf toxin evolution we have sequenced seven pseudomonas genomes to retrieve the entire gene cluster.
we analyzed evolutionary footprints of the pseudomonas fit gene cluster and the related mcf genes of the insect pathogenic photorhabdus and xenorhabdus bacteria and found patterns of recent horizontal transfer.
this study suggests that the fit toxin is restricted to a particular group of plant-colonizing pseudomonads consisting of p. protegens and p. chlororaphis.
we show that the presence of the fit toxin gene strongly correlates with high insect toxicity and thus is a suitable molecular marker for potent insecticidal activity in fluorescent pseudomonads.
absence of the fit toxin gene in closely related pseudomonads and the genomic context suggest that fit genes have evolved in part via exchange of genetic material from phylogenetically distantly related bacteria.
the acquisition of the fit toxin within pseudomonads may represent an ancient event in the evolution towards a distinct ecotype of insect-associated pseudomonads.
our analysis further indicates substantial rearrangements within photorhabdus/xenorhabdus lineage of these insecticidal toxins thereby extending and diversifying the existing toxin repertoire of these entomopathogens.
RESULTS
survey of diverse pseudomonads predicts p. protegens and p. chlororaphis as exclusive fit producers within plant-colonizing pseudomonads
we investigated the occurrence of fit toxin production in plant-associated pseudomonads and tested whether insect toxin production is linked to specific ecological and molecular characteristics.
we screened a large worldwide collection of pseudomonas isolates  from soil and roots of different plant species using generic primers directed against the fit toxin gene fitd .
in addition to the root-associated isolates, we tested  <dig> strains representing the major phylogenetic groups within the genus pseudomonas and strains isolated from different environments including invertebrates such as cyclops, earthworms or isopods for the presence of the fitd gene.
the phylogenetic relationship of the investigated strains based on concatenated sequences of the three housekeeping genes reca, rpob and rpod is shown in fig.
2a.fig.
<dig> insecticidal activity correlates with presence of the fitd gene.
the phylogenetic relationship amongst  <dig> closely related plant-associated fit+ and  <dig> fit− pseudomonads is based on the three concatenated housekeeping genes reca, rpob and rpod consisting of a total of  <dig> nucleotide sites with pseudomonas syringae as outgroup.
strains/subgroups harboring the fit toxin gene and major antifungal compounds produced by the pseudomonads are indicated: dapg, 2-4-diacetylphloroglucinol; phz, phenazines; and plt, pyoluteorin.
the evolutionary history was inferred using phyml  <dig> .
bootstrap values are based on  <dig> replicates and indicated for major nodes indicated by circles.
only the p. protegens and p. chlororaphis subgroups harbor the fit toxin gene.
the p. corrugata subgroup containing dapg producers is indicated as defined by mulet  <cit> .
insect mortality of g. mellonella larvae four days after injection of 4 ×  <dig> cells of  <dig> fit producing and  <dig> non-producing pseudomonas strains.
tested strains are shown in bold in .
each strain was tested on a total of  <dig> larvae .
bars show average of insect mortality for each strain.
error bars show standard error of the mean.
the experiment was repeated with similar results .
fit+ strains were significantly different from fit− based on wilcoxon rank sum test grouped by fit+ and fit− strains
pcr amplification and sequencing showed the presence of the toxin in  <dig> strains .
in addition, pcr results were verified using southern blotting on a subset of isolates .
we detected the fit toxin gene only in two phylogenetic subgroups within the p. fluorescens group .
the first subgroup comprises fluorescent pseudomonads that produce both the antifungal metabolites dapg and plt  <cit>  including our model strain p. protegens cha <dig>  <cit> .
as the second group of fit toxin gene carriers, we were able to identify members of the p. chlororaphis subgroup .
we included in addition to p. protegens cha <dig> and pf- <dig> the sequenced strains p. chlororaphis gp <dig>  <cit> , pseudomonas aureofaciens 30- <dig>  <cit>  and pseudomonas cmr12a  all harboring the fit gene cluster.
cmr12a is placed next to the group of dapg and plt producers, although cmr12a is phylogenetically clearly distinct from these strains  and does not produce the two antifungal compounds  <cit> .
insecticidal activity strongly correlates with the presence of the fit toxin gene
we aimed to test if insecticidal activity is restricted to a particular group of pseudomonads and whether presence of the fit toxin is predictive of insecticidal activity.
to this end, the insecticidal activity of selected strains from phylogenetically and functionally diverse subgroups within the p. fluorescens group  was tested using a previously established assay with larvae of the greater wax moth galleria mellonella  <cit> .
tested strains included representatives of different phylogenetic subgroups of dapg-producers and some dapg non-producing strains .
injection assays with galleria larvae demonstrated that pseudomonas strains harboring the fitd toxin gene display potent insecticidal activity while the naturally fitd-negative sister group fails to induce significant mortality .
since insecticidal activity is restricted to a particular group and substantial toxicity is retained in fitd knock-out mutants  <cit> , insecticidal activity is likely driven by other shared traits.
we have previously shown that the fit toxin gene significantly contributes to the insecticidal activity, but requires additional factors for full activity that are regulated by the gacs/gaca system  <cit> .
nevertheless, the fit toxin gene seems to be predictive of insecticidal activity in root-colonizing fluorescent pseudomonads.
the fit gene cluster is located in a dynamic genomic region
to characterize the fit gene cluster among pseudomonas spp.
we generated high quality assemblies of seven toxin-producing strains  covering the entire fit toxin cluster and flanking regions.
the complete genome sequence of cha <dig> has recently been published  <cit> .
sequences containing the fit genes were aligned to the reference sequence of strains pf- <dig> and 30- <dig>  <cit> .
all seven strains contain the complete fit toxin cluster .
the fit gene cluster in the genome of pf- <dig> is embedded in a large genomic region, with features indicative of horizontal acquisition, such as phage-related proteins and unusual nucleotide composition   <cit> .
the fit cluster in p. chlororaphis strains 30- <dig> and o <dig> is located in a different part of the genome within a 24- <dig> gene insertion   <cit> .
we were able to define a 165-kb region of the pf- <dig> genome flanked by steep residual g + c content clines .
abrupt changes in the residual cumulative g + c content curve may point to foreign genetic elements, such as horizontally acquired genes, phage-derived elements or other mobile elements  <cit> .
the corresponding genomic region in strain pf is highly similar to that of pf- <dig> and in both strains the region is delimited at the 5’-end by a type i restriction modification system and by phage-related proteins at the 3’-end.
interestingly, comparisons of the respective regions among the six p. protegens strains show a high level of insertion-deletion polymorphism.
in particular, the type i restriction-modification system is present in strains pf- <dig>  pf, k <dig>  and brip, but absent in pgnr <dig> and cha <dig> .
the gene cluster encoding the rhizoxin biosynthesis is unique to pf- <dig> and pf as well as the phage-related proteins  <cit> .
a polysaccharide synthesis gene cluster  is common to all p. protegens strains and located upstream of the fit cluster.
in pseudomonas aeruginosa, pel is one of at least three secreted extracellular polysaccharides implicated in biofilm formation  <cit> .fig.
<dig> comparison of the fit cluster harbouring region.
within p. protegens  and p. chlororaphis  subgroups indicates a high degree of absence/presence polymorphism.
the genomic region was defined based on residual cumulative g + c content analysis of p. protegens strain pf- <dig> and p. chlororaphis strain 30- <dig>  where steep slopes indicate local variations in g + c content indicative of foreign elements.
compared with pf- <dig> and pf, the genomic region of strains k <dig> , brip, pgnr <dig> and cha <dig> is notably downsized
the genomic context of the fit genes in p. protegens differs from that in p. chlororaphis .
using the residual cumulative g + c content approach a region of 45 kb composed of  <dig> orfs was defined in p. chlororaphis strain 30- <dig>  similar to p. protegens, there is substantial presence/absence polymorphism.
in strains cd and pcl <dig>  adjacent to a gene encoding a putative membrane-associated transporter upstream of fita, a nine-orf gene cluster involved in ribose uptake and utilization is located, which was assumed to be unique to the p. chlororaphis subgroup of the p. fluorescens group  <cit> .
fit gene cluster: a phylogenetic patchwork
since only strains of p. protegens and p. chlororaphis were identified as carriers of the fit toxin, we were interested in the evolutionary origin of this gene cluster.
high similarities between protein sequences in distantly related species, patchy distribution or phylogenetic incongruence indicate potential horizontal transmission of a gene  <cit> .
blastp searches, revealed a very distinct phylogenetic distribution of fit components.
consistent with horizontal transmission, most of the fit components  have significant best hits outside the pseudomonas .
initial sequence analysis of the fit toxin from p. protegens strain cha <dig> revealed 73 % sequence identity over the entire protein to the insecticidal protein mcf <dig> of p. luminescens strain tt <dig>  <cit> .
mcf <dig> of tt <dig> shares 67 % identity with fitd .
mcf <dig> is also present in p. asymbiotica atcc  <dig>  but this bacterium appears to have lost mcf <dig>  <cit> .
similarly, x. bovienii ss- <dig> and x. nematophila atcc  <dig>  both sister species of photorhabdus, carry an mcf variant, which we call here mcf <dig> with 69 %, respectively, 65 % overall identity to fitd and highest identity  in a  <dig> amino acid n-terminal overlap.
mcf <dig> is also present in photorhabdus temperata  <cit> .
within the genus xenorhabdus mcf <dig> is only found in x. nematophila atcc  <dig> with an overall identity of 64 % to fitd.
mcf-like proteins are also found in other γ-proteobacteria including vibrio and providencia spp.
distantly related fit-like genes   <cit> ,  with a predicted tcda/tcdb pore-forming domain can also be found in p. brassicacearum and diverse fit-negative p. fluorescens strains, but none of the representative strains of this group tested in our virulence tests  caused significant insect mortality .
interestingly, fita, fitb and fitc with predicted function in toxin secretion  <cit>  revealed highest similarities  with the rtx toxin transporter encoded by the genomic region adjacent to the mcf <dig> insect toxin gene of p. luminescens .
similar genes, however, are absent in proximity of mcf <dig> and mcf <dig> as well as of mcf <dig> of xenorhabdus .
the only fit components showing closest identity to the genus pseudomonas are fite  and the regulatory protein fitg .
the other proteins involved in regulation of toxin expression  show closest identity to dechloromonas aromatica  and vibrio sp.
, respectively.
the three regulatory proteins fitf, fitg and fith are absent in photorhabdus/xenorhabdus, suggesting a pseudomonas-specific regulation of fit.
however, we cannot rule out an earlier existence of the whole cluster also in photorhabdus/xenorhabdus but that transport and regulatory genes were subsequently lost.
mcf <dig> in photorhabudus luminescens, for example is still flanked by the three transport genes.fig.
<dig> similarity plot fit and mcf toxin encoding genomic regions in pseudomonas, photorhabdus and xenorhabdus species.
the alignment was conducted using lagan as implemented in mvista  <cit>  with the respective reference sequence of  p. protegens cha <dig> fitd,  p. luminescens tt <dig> mcf <dig>  photorhabdus luminescens tt <dig> mcf <dig> and  x. nematophila atcc  <dig> mcf.
the peaks and valleys graphs represent percent conservation between aligned sequences at a given coordinate on the reference sequence.
regions of high conservation  are colored according to the coding region of the reference sequences in dark red for toxin genes, in orange for transport genes and in green for regulation genes.
regions of high conservation not related to the cluster sequence are colored in pink.
regions colored in blue have high similarity to transposable elements and are only present adjunct to mcf genes in xenorhabdus and photorhabdus.
dotted lines mark the region encoding the tcda/tcdb pore forming domain.
nucleotide key positions discussed in the text are indicated with numbers 1- <dig> in fig.
1a for fitd: 1 = site 1; 2 = site 2707; 3 = site 4849; 4 = site 6831; 5 = site  <dig>  the plot shown of each alignment ranges between 50 % and 100 % identity calculated on a 100 bp window
the patchy phylogenetic distribution of fit/mcf toxins and the absence of the toxin in closely related pseudomonas species e. g members of the p. corrugata subgroup strongly suggest that the fit cluster evolved in part via horizontal acquisition, followed by functional integration of vertically transmitted genes, making up a unique virulence cluster within the subclade of p. chlororaphis and p. protegens.
mosaic composition of fit/mcf toxin variants
aligning the genomic regions of fitd and mcf variants encoding sequences reveals a mosaic like structure of toxin-encoding parts and associated components.
the fitd-encoding region of p. protegens and p. chlororaphis shows extraordinarily high similarity over the entire gene  to mcf <dig> of p. luminescens and p. asymbiotica.
however, immediately adjacent flanking regions drop below the threshold of alignable sequences set at 50 % similarity in a 100 bp window .
short stretches sharing over 70 % nucleotide similarity outside the designated toxin encoding regions indicated by sharp peaks in the similarity plots among p. luminescens, p. asymbiotica as well as between p. luminescens genes mcf <dig> and mcf <dig> include fragments with predicted association to transposable elements .
within the photorhabdus/xenorhabdus lineage three variants of mcf toxins can be determined based on their sequential make up: mcf <dig>  mcf <dig> and mcf <dig>  while insect toxicity has been demonstrated at several levels for mcf <dig> and mcf <dig> from photorhabdus  <cit> , and fitd from pseudomonas  <dig>  <cit> , the functionality of xenorhabdus mcf toxins remains to be tested, in particular of mcf <dig> the most distinct toxin variant discussed in this paper.
the vista alignment presented in fig.
<dig> shows the mosaic-like structure of mcf variants.
while fitd and mcf <dig> share high homology over the entire nucleotide sequence, mcf <dig> in the xenorhabdus lineage and also present in p. temperata shares only the 5′-end  with fitd/mcf <dig> .
including mcf <dig> in the alignment, it becomes apparent, that it is exactly this stretch at the mcf <dig> 5’ -end with high homology to fitd/mcf <dig>  which is entirely missing in mcf <dig> .
blast searches revealed that the sequence towards the 3′-end  of the xenorhabdus mcf <dig> shows for most of this stretch  no similarity to the other mcf/fitd variants and did not allow to identify a putative origin.
the rapid drop from >70 % similarity to 40 % between position  <dig> and  <dig>  coincide with a breakpoint indicated by sbp  analysis.
in the middle of this stretch there is a part  which again shows a low similarity  to the tcda/tcdb pore forming domain of fit, mcf <dig> and mcf <dig>  thus, all investigated toxin variants encode a tcda/tcdb pore-forming domain in this region, however, based on amino acid and nucleotide comparison, it seems that these pore-forming domains originate from at least two different ancestors .
a phylogenetic tree conducted on the tcda/tcdb pore forming domain indeed suggests a monophyletic origin for the pore-forming domain of fitd/mcf <dig> and mcf <dig>  but a different origin for that of mcf <dig> found in xenorhabdus and p. temperata .fig.
<dig> representation of fit and mcf toxin variants.
segments of same color represent highly identical regions  between the different variants.
strains representing the toxin variants are indicated in bold in fig.
1b.
c <dig> peptidase is only present in mcf <dig>  tcda/tcdb indicates the pore-forming domain present in all variants, but most likely of different origin .
the bh <dig> domain present in fitd, mcf <dig> and mcf <dig> has been attributed a functional role in cell death induction  <cit> .
phylogenetic relationship of the bacterial tcda/tcdb pore-forming domain present in all fit and mcf protein variants.
the tcda/tcdb domain from rhodobacteraceae was designated as outgroup.
the tree is based on  <dig> amino acids after removal of segments by gblocks  <cit>  and inferred using phyml  <dig>  with the hky <dig> substituion model and default settings  <cit> .
bootstrap values are based on  <dig> replicates and given for relevant nodes indicated by circles.
the range of amino acid identity of the tcda/tcdb domain compared to the fitd reference sequence of cha <dig> is given next to the colored boxes delimiting the different mcf variants
the mosaic composition of the different fit/mcf variants, the presence of transposable elements and the patchy phylogenetic distribution of the toxin could indicate a highly mobile nature of this gene.
an unusual gc content of a gene/segment, is a further indication of horizontal acquisition, assuming that donor and recipient have a sufficiently high degree of base composition differences  <cit> .
while the average gc content of p. luminescens tt <dig>  and x. nematophila atcc  <dig>  genomes is low, pseudomonas spp.
have high overall gc content .
comparing the gc content of fitd and mcf <dig> to the respective average of all genes within the same strain, the mcf <dig> gene in p. luminescens displays an unusual high gc content , whereas the fitd gene in pseudomonas is within the range of the average gc content of the genome  .
in addition, only  <dig>  % of p. luminescens genes display an equal or higher gc content than mcf <dig> and rtxd and rtxb  .
interestingly, all investigated mcf variants of xenorhabdus spp.
do not show such obvious abnormality  .table  <dig> gc content of fitd and mcf genes
pseudomons protegens
fitd
fitd
fita
fitb
fitc
pseudomonas chlororaphis
fitd
photorhabdus luminescens
mcf1
mcf2
plu3125a
plu3126
a
plu3127
a
photorhabdus asymbiotica
mcf1
xenorhabdus nematophila
mcf
mcf2
xenorhabdus bovienii
mcf
aencode predicted transporters and are homologous to fita, fitb and fitc respectively
the unusual nucleotide composition of mcf <dig> and mcf <dig> in photorhabdus contradicts the hypothesis that pseudomonads acquired the insect toxin from entomopathogenic photorhabdus.
however, horizontal acquisition of mcf in photorhabdus from an unknown ancestral vector is very likely.
in p. chlororaphis and p. protegens the nucleotide composition of the fit genes does not differ from the average of the whole genome .
therefore, one might speculate that pseudomonads have acquired the toxin earlier than photorhabdus and the fit codon usage has already adapted to the pseudomonas background or that pseudomonads have acquired the toxin from a bacterium displaying a similar nucleotide composition.
mcf toxin genes are not only shuffled around in bacteria.
a recent study by ambrose  <cit>  indicates that a mcf-like gene of the fungal grass endosymbiont epichloë poae, which is sufficient to confer a lethal phenotype when expressed in e. coli cells and injected into the black cutworms agrotis ipsilon, has derived from a single lineage-specific horizontal transfer of bacterial origin  <cit> .
CONCLUSIONS
the plant environment was assumed to be the dominant niche of p. fluorescens group bacteria, but it becomes apparent that some members, notably p. protegens and p. chlororaphis, which harbor the fit insect toxin, are capable of colonizing and killing insects .
the present comparative analysis study provides a better understanding of the processes driving the evolution of insect pathogenicity in environmental pseudomonads.
the fit virulence cassette seems to be ubiquitous for p. protegens and p. chlororaphis and is encoded in dynamic portions of the p. protegens and p. chlororaphis genomes with substantial absence/presence polymorphism, phage-related genes and an unusual base composition, while in the photorhabuds/xenorhabdus lineage transposable elements are located in proximity of the mcf gene.
it would therefore appear that evolutionary processes including the acquisition of insecticidal elements, sequence rearrangements  and protein adaptation through domain shuffling  allowed plant-associated pseudomonads to adapt to a new ecological niche.
in line with the genomic arguments of horizontal acquisition are the patchy distributed fit components that share highest homology with bacteria outside the pseudomonadaceae family.
our data show that a specific group of plant-colonizing pseudomonads have evolved a unique virulence gene cluster through diverse evolutionary processes, which contributed to extend their existing repertoire of antifungal and antipredator activities with insecticidal activity.
frequent mobilization and recombination is possibly favored by the common niche of insect hosts shared between these particular pseudomonas, photorhabdus and xenorhabdus bacteria, and may provide a selective advantage by the diversification of the toxin gene repertoire.

 bacteria belonging to the pseudomonas fluorescens group provide a compelling example of ecological and bacterial lifestyle diversity reflected by the vast range of environmental habitats they occupy. this group encloses plant-beneficial symbionts, environmental saprophytes and clinical strains of opportunistic human pathogens. within the p. fluorescens group, root-colonizing pseudomonads are well known for their ability to promote plant growth and to protect plants against soilborne pathogens through a set of diverse and functionally complementary mechanisms. the capacity to suppress fungal diseases has largely been attributed to the production of secondary metabolites with cytotoxic and antimicrobial activity, in particular -diacetylphloroglucinol, phenazines, pyoluteorin, pyrrolnitrin, hydrogen cyanide, and lipopeptides. extensive knowledge has been gathered over the last years on plant disease suppression and plant growth promotion. surprisingly, it has become only recently apparent that specific strains of plant-associated pseudomonads are able to infect and kill insects. these observations invoke that particular strains may function as insect pathogens and switch between insect hosts and the plant environment. insecticidal activity in environmental pseudomonads was, with the exception of pseudomonas entomophila, a pathogen of drosophila, so far only rarely demonstrated. initially, an insect toxin was discovered in silico when the genome of pseudomonas protegens pf- became available. subsequent molecular and mutational characterization revealed that oral and injectable insecticidal activity is linked to the fit gene, which was described and characterized for the first time in p. protegens strains cha and pf-. injection of fit expressing e. coli is sufficient to induce strong melanization and rapid death of the tobacco hornworm manduca sexta and larvae of the greater wax moth galleria mellonella. fit toxin knock-out mutants of cha have attenuated virulence, both when injected into g. mellonella or fed to african cotton leafworm spodoptera littoralis. the fit gene cluster consists of eight genes with functions in toxin export, insect toxicity and regulation. the fit insect toxin gene fitd is flanked upstream by fitabc and downstream by fite encoding components of a type i secretion system. the products of the fitfgh genes regulate toxin production. fitf is a sensor histidine kinase – response regulator hybrid, detecting the insect environment and activating insecticidal toxin expression via fith and fitg.fig. organization of the fit gene cluster and their homologues. in p. protegens and p. chlororaphis the fit gene cluster is composed of eight orfs encoding the insecticidal toxin, predicted components of a toxin secretion system including an outer membrane protein, and regulators of toxin production. the orientation of the toxin gene within the genome is indicated by + for the leading strand and by – for lagging the strand. transporter homologues are only found in p. luminescens adjacent to mcf a truncated variant of mcf x. bovienii ss and p. asymbiotica attc harbor only one mcf variant. blue arrows indicate flanking genes, which share no homology among the strains represented. the phylogenetic relationship of p. protegens, p. chlororaphis, photorhabdus spp. and xenorhabdus spp. with representative orders/families and groups of γ-proteobacteria is based on concatenated reca, rpob and rpod protein sequences genome sequencing revealed that certain strains of pseudomonas chlororaphis also harbour the complete fit gene cluster. contribution of the fit toxin to the oral insecticidal activity has been demonstrated for p. chlororaphis pcl against s. littoralis. the fit insect toxin shares 73 % identity with the makes caterpillars floppy insecticidal toxin mcf and 67 % with mcf both produced by photorhabdus luminescens, a bacterial symbiont of entomopathogenic nematodes. mcf-like toxins are also found in photorhabdus asymbiotica, xenorhabdus nemtophila and xenorhabdus bovienii. the mcf toxin causes rapid disruption of the insect midgut epithelium and hemocytes triggered by a bh-3-like apoptosis control domain. injection of purified mcf in drosophila embryos leads to a freezing phenotype of hemocytes, due to a rearrangement of the actin cytoskeleton. while mcf toxins are essentially studied in the photorhabdus lineage the evolutionary basis for the homology between fit and mcf toxins has remained unclear. here, we conducted comparative sequence analysis in combination with virulence assays to yield a better understanding of insect pathogenicity in plant-associated pseudomonads. in order to study fit/mcf toxin evolution we have sequenced seven pseudomonas genomes to retrieve the entire gene cluster. we analyzed evolutionary footprints of the pseudomonas fit gene cluster and the related mcf genes of the insect pathogenic photorhabdus and xenorhabdus bacteria and found patterns of recent horizontal transfer. this study suggests that the fit toxin is restricted to a particular group of plant-colonizing pseudomonads consisting of p. protegens and p. chlororaphis. we show that the presence of the fit toxin gene strongly correlates with high insect toxicity and thus is a suitable molecular marker for potent insecticidal activity in fluorescent pseudomonads. absence of the fit toxin gene in closely related pseudomonads and the genomic context suggest that fit genes have evolved in part via exchange of genetic material from phylogenetically distantly related bacteria. the acquisition of the fit toxin within pseudomonads may represent an ancient event in the evolution towards a distinct ecotype of insect-associated pseudomonads. our analysis further indicates substantial rearrangements within photorhabdus/xenorhabdus lineage of these insecticidal toxins thereby extending and diversifying the existing toxin repertoire of these entomopathogens.  survey of diverse pseudomonads predicts p. protegens and p. chlororaphis as exclusive fit producers within plant-colonizing pseudomonads we investigated the occurrence of fit toxin production in plant-associated pseudomonads and tested whether insect toxin production is linked to specific ecological and molecular characteristics. we screened a large worldwide collection of pseudomonas isolates from soil and roots of different plant species using generic primers directed against the fit toxin gene fitd. in addition to the root-associated isolates, we tested strains representing the major phylogenetic groups within the genus pseudomonas and strains isolated from different environments including invertebrates such as cyclops, earthworms or isopods for the presence of the fitd gene. the phylogenetic relationship of the investigated strains based on concatenated sequences of the three housekeeping genes reca, rpob and rpod is shown in fig. 2a.fig. insecticidal activity correlates with presence of the fitd gene. the phylogenetic relationship amongst closely related plant-associated fit+ and fit− pseudomonads is based on the three concatenated housekeeping genes reca, rpob and rpod consisting of a total of nucleotide sites with pseudomonas syringae as outgroup. strains/subgroups harboring the fit toxin gene and major antifungal compounds produced by the pseudomonads are indicated: dapg, 2-4-diacetylphloroglucinol; phz, phenazines; and plt, pyoluteorin. the evolutionary history was inferred using phyml. bootstrap values are based on replicates and indicated for major nodes indicated by circles. only the p. protegens and p. chlororaphis subgroups harbor the fit toxin gene. the p. corrugata subgroup containing dapg producers is indicated as defined by mulet. insect mortality of g. mellonella larvae four days after injection of 4 ×  cells of fit producing and non-producing pseudomonas strains. tested strains are shown in bold in. each strain was tested on a total of larvae. bars show average of insect mortality for each strain. error bars show standard error of the mean. the experiment was repeated with similar results. fit+ strains were significantly different from fit− based on wilcoxon rank sum test grouped by fit+ and fit− strains pcr amplification and sequencing showed the presence of the toxin in strains. in addition, pcr results were verified using southern blotting on a subset of isolates. we detected the fit toxin gene only in two phylogenetic subgroups within the p. fluorescens group. the first subgroup comprises fluorescent pseudomonads that produce both the antifungal metabolites dapg and plt including our model strain p. protegens cha. as the second group of fit toxin gene carriers, we were able to identify members of the p. chlororaphis subgroup. we included in addition to p. protegens cha and pf- the sequenced strains p. chlororaphis gp, pseudomonas aureofaciens 30- and pseudomonas cmr12a all harboring the fit gene cluster. cmr12a is placed next to the group of dapg and plt producers, although cmr12a is phylogenetically clearly distinct from these strains and does not produce the two antifungal compounds. insecticidal activity strongly correlates with the presence of the fit toxin gene we aimed to test if insecticidal activity is restricted to a particular group of pseudomonads and whether presence of the fit toxin is predictive of insecticidal activity. to this end, the insecticidal activity of selected strains from phylogenetically and functionally diverse subgroups within the p. fluorescens group was tested using a previously established assay with larvae of the greater wax moth galleria mellonella. tested strains included representatives of different phylogenetic subgroups of dapg-producers and some dapg non-producing strains. injection assays with galleria larvae demonstrated that pseudomonas strains harboring the fitd toxin gene display potent insecticidal activity while the naturally fitd-negative sister group fails to induce significant mortality. since insecticidal activity is restricted to a particular group and substantial toxicity is retained in fitd knock-out mutants, insecticidal activity is likely driven by other shared traits. we have previously shown that the fit toxin gene significantly contributes to the insecticidal activity, but requires additional factors for full activity that are regulated by the gacs/gaca system. nevertheless, the fit toxin gene seems to be predictive of insecticidal activity in root-colonizing fluorescent pseudomonads. the fit gene cluster is located in a dynamic genomic region to characterize the fit gene cluster among pseudomonas spp. we generated high quality assemblies of seven toxin-producing strains covering the entire fit toxin cluster and flanking regions. the complete genome sequence of cha has recently been published. sequences containing the fit genes were aligned to the reference sequence of strains pf- and 30-. all seven strains contain the complete fit toxin cluster. the fit gene cluster in the genome of pf- is embedded in a large genomic region, with features indicative of horizontal acquisition, such as phage-related proteins and unusual nucleotide composition. the fit cluster in p. chlororaphis strains 30- and o is located in a different part of the genome within a 24- gene insertion. we were able to define a 165-kb region of the pf- genome flanked by steep residual g + c content clines. abrupt changes in the residual cumulative g + c content curve may point to foreign genetic elements, such as horizontally acquired genes, phage-derived elements or other mobile elements. the corresponding genomic region in strain pf is highly similar to that of pf- and in both strains the region is delimited at the 5’-end by a type i restriction modification system and by phage-related proteins at the 3’-end. interestingly, comparisons of the respective regions among the six p. protegens strains show a high level of insertion-deletion polymorphism. in particular, the type i restriction-modification system is present in strains pf- pf, k and brip, but absent in pgnr and cha. the gene cluster encoding the rhizoxin biosynthesis is unique to pf- and pf as well as the phage-related proteins. a polysaccharide synthesis gene cluster is common to all p. protegens strains and located upstream of the fit cluster. in pseudomonas aeruginosa, pel is one of at least three secreted extracellular polysaccharides implicated in biofilm formation.fig. comparison of the fit cluster harbouring region. within p. protegens and p. chlororaphis subgroups indicates a high degree of absence/presence polymorphism. the genomic region was defined based on residual cumulative g + c content analysis of p. protegens strain pf- and p. chlororaphis strain 30- where steep slopes indicate local variations in g + c content indicative of foreign elements. compared with pf- and pf, the genomic region of strains k, brip, pgnr and cha is notably downsized the genomic context of the fit genes in p. protegens differs from that in p. chlororaphis. using the residual cumulative g + c content approach a region of 45 kb composed of orfs was defined in p. chlororaphis strain 30- similar to p. protegens, there is substantial presence/absence polymorphism. in strains cd and pcl adjacent to a gene encoding a putative membrane-associated transporter upstream of fita, a nine-orf gene cluster involved in ribose uptake and utilization is located, which was assumed to be unique to the p. chlororaphis subgroup of the p. fluorescens group. fit gene cluster: a phylogenetic patchwork since only strains of p. protegens and p. chlororaphis were identified as carriers of the fit toxin, we were interested in the evolutionary origin of this gene cluster. high similarities between protein sequences in distantly related species, patchy distribution or phylogenetic incongruence indicate potential horizontal transmission of a gene. blastp searches, revealed a very distinct phylogenetic distribution of fit components. consistent with horizontal transmission, most of the fit components have significant best hits outside the pseudomonas. initial sequence analysis of the fit toxin from p. protegens strain cha revealed 73 % sequence identity over the entire protein to the insecticidal protein mcf of p. luminescens strain tt. mcf of tt shares 67 % identity with fitd. mcf is also present in p. asymbiotica atcc but this bacterium appears to have lost mcf. similarly, x. bovienii ss- and x. nematophila atcc both sister species of photorhabdus, carry an mcf variant, which we call here mcf with 69 %, respectively, 65 % overall identity to fitd and highest identity in a amino acid n-terminal overlap. mcf is also present in photorhabdus temperata. within the genus xenorhabdus mcf is only found in x. nematophila atcc with an overall identity of 64 % to fitd. mcf-like proteins are also found in other γ-proteobacteria including vibrio and providencia spp. distantly related fit-like genes, with a predicted tcda/tcdb pore-forming domain can also be found in p. brassicacearum and diverse fit-negative p. fluorescens strains, but none of the representative strains of this group tested in our virulence tests caused significant insect mortality. interestingly, fita, fitb and fitc with predicted function in toxin secretion revealed highest similarities with the rtx toxin transporter encoded by the genomic region adjacent to the mcf insect toxin gene of p. luminescens. similar genes, however, are absent in proximity of mcf and mcf as well as of mcf of xenorhabdus. the only fit components showing closest identity to the genus pseudomonas are fite and the regulatory protein fitg. the other proteins involved in regulation of toxin expression show closest identity to dechloromonas aromatica and vibrio sp., respectively. the three regulatory proteins fitf, fitg and fith are absent in photorhabdus/xenorhabdus, suggesting a pseudomonas-specific regulation of fit. however, we cannot rule out an earlier existence of the whole cluster also in photorhabdus/xenorhabdus but that transport and regulatory genes were subsequently lost. mcf in photorhabudus luminescens, for example is still flanked by the three transport genes.fig. similarity plot fit and mcf toxin encoding genomic regions in pseudomonas, photorhabdus and xenorhabdus species. the alignment was conducted using lagan as implemented in mvista with the respective reference sequence of p. protegens cha fitd, p. luminescens tt mcf photorhabdus luminescens tt mcf and x. nematophila atcc mcf. the peaks and valleys graphs represent percent conservation between aligned sequences at a given coordinate on the reference sequence. regions of high conservation are colored according to the coding region of the reference sequences in dark red for toxin genes, in orange for transport genes and in green for regulation genes. regions of high conservation not related to the cluster sequence are colored in pink. regions colored in blue have high similarity to transposable elements and are only present adjunct to mcf genes in xenorhabdus and photorhabdus. dotted lines mark the region encoding the tcda/tcdb pore forming domain. nucleotide key positions discussed in the text are indicated with numbers 1- in fig. 1a for fitd: 1 = site 1; 2 = site 2707; 3 = site 4849; 4 = site 6831; 5 = site the plot shown of each alignment ranges between 50 % and 100 % identity calculated on a 100 bp window the patchy phylogenetic distribution of fit/mcf toxins and the absence of the toxin in closely related pseudomonas species e. g members of the p. corrugata subgroup strongly suggest that the fit cluster evolved in part via horizontal acquisition, followed by functional integration of vertically transmitted genes, making up a unique virulence cluster within the subclade of p. chlororaphis and p. protegens. mosaic composition of fit/mcf toxin variants aligning the genomic regions of fitd and mcf variants encoding sequences reveals a mosaic like structure of toxin-encoding parts and associated components. the fitd-encoding region of p. protegens and p. chlororaphis shows extraordinarily high similarity over the entire gene to mcf of p. luminescens and p. asymbiotica. however, immediately adjacent flanking regions drop below the threshold of alignable sequences set at 50 % similarity in a 100 bp window. short stretches sharing over 70 % nucleotide similarity outside the designated toxin encoding regions indicated by sharp peaks in the similarity plots among p. luminescens, p. asymbiotica as well as between p. luminescens genes mcf and mcf include fragments with predicted association to transposable elements. within the photorhabdus/xenorhabdus lineage three variants of mcf toxins can be determined based on their sequential make up: mcf mcf and mcf while insect toxicity has been demonstrated at several levels for mcf and mcf from photorhabdus, and fitd from pseudomonas, the functionality of xenorhabdus mcf toxins remains to be tested, in particular of mcf the most distinct toxin variant discussed in this paper. the vista alignment presented in fig.  shows the mosaic-like structure of mcf variants. while fitd and mcf share high homology over the entire nucleotide sequence, mcf in the xenorhabdus lineage and also present in p. temperata shares only the 5′-end with fitd/mcf. including mcf in the alignment, it becomes apparent, that it is exactly this stretch at the mcf 5’ -end with high homology to fitd/mcf which is entirely missing in mcf. blast searches revealed that the sequence towards the 3′-end of the xenorhabdus mcf shows for most of this stretch no similarity to the other mcf/fitd variants and did not allow to identify a putative origin. the rapid drop from >70 % similarity to 40 % between position and coincide with a breakpoint indicated by sbp analysis. in the middle of this stretch there is a part which again shows a low similarity to the tcda/tcdb pore forming domain of fit, mcf and mcf thus, all investigated toxin variants encode a tcda/tcdb pore-forming domain in this region, however, based on amino acid and nucleotide comparison, it seems that these pore-forming domains originate from at least two different ancestors. a phylogenetic tree conducted on the tcda/tcdb pore forming domain indeed suggests a monophyletic origin for the pore-forming domain of fitd/mcf and mcf but a different origin for that of mcf found in xenorhabdus and p. temperata.fig. representation of fit and mcf toxin variants. segments of same color represent highly identical regions between the different variants. strains representing the toxin variants are indicated in bold in fig. 1b. c peptidase is only present in mcf tcda/tcdb indicates the pore-forming domain present in all variants, but most likely of different origin. the bh domain present in fitd, mcf and mcf has been attributed a functional role in cell death induction. phylogenetic relationship of the bacterial tcda/tcdb pore-forming domain present in all fit and mcf protein variants. the tcda/tcdb domain from rhodobacteraceae was designated as outgroup. the tree is based on amino acids after removal of segments by gblocks and inferred using phyml with the hky substituion model and default settings. bootstrap values are based on replicates and given for relevant nodes indicated by circles. the range of amino acid identity of the tcda/tcdb domain compared to the fitd reference sequence of cha is given next to the colored boxes delimiting the different mcf variants the mosaic composition of the different fit/mcf variants, the presence of transposable elements and the patchy phylogenetic distribution of the toxin could indicate a highly mobile nature of this gene. an unusual gc content of a gene/segment, is a further indication of horizontal acquisition, assuming that donor and recipient have a sufficiently high degree of base composition differences. while the average gc content of p. luminescens tt and x. nematophila atcc genomes is low, pseudomonas spp. have high overall gc content. comparing the gc content of fitd and mcf to the respective average of all genes within the same strain, the mcf gene in p. luminescens displays an unusual high gc content, whereas the fitd gene in pseudomonas is within the range of the average gc content of the genome. in addition, only  % of p. luminescens genes display an equal or higher gc content than mcf and rtxd and rtxb. interestingly, all investigated mcf variants of xenorhabdus spp. do not show such obvious abnormality.table gc content of fitd and mcf genes pseudomons protegens fitd fitd fita fitb fitc pseudomonas chlororaphis fitd photorhabdus luminescens mcf1 mcf2 plu3125a plu3126 a plu3127 a photorhabdus asymbiotica mcf1 xenorhabdus nematophila mcf mcf2 xenorhabdus bovienii mcf aencode predicted transporters and are homologous to fita, fitb and fitc respectively the unusual nucleotide composition of mcf and mcf in photorhabdus contradicts the hypothesis that pseudomonads acquired the insect toxin from entomopathogenic photorhabdus. however, horizontal acquisition of mcf in photorhabdus from an unknown ancestral vector is very likely. in p. chlororaphis and p. protegens the nucleotide composition of the fit genes does not differ from the average of the whole genome. therefore, one might speculate that pseudomonads have acquired the toxin earlier than photorhabdus and the fit codon usage has already adapted to the pseudomonas background or that pseudomonads have acquired the toxin from a bacterium displaying a similar nucleotide composition. mcf toxin genes are not only shuffled around in bacteria. a recent study by ambrose indicates that a mcf-like gene of the fungal grass endosymbiont epichloë poae, which is sufficient to confer a lethal phenotype when expressed in e. coli cells and injected into the black cutworms agrotis ipsilon, has derived from a single lineage-specific horizontal transfer of bacterial origin.  the plant environment was assumed to be the dominant niche of p. fluorescens group bacteria, but it becomes apparent that some members, notably p. protegens and p. chlororaphis, which harbor the fit insect toxin, are capable of colonizing and killing insects. the present comparative analysis study provides a better understanding of the processes driving the evolution of insect pathogenicity in environmental pseudomonads. the fit virulence cassette seems to be ubiquitous for p. protegens and p. chlororaphis and is encoded in dynamic portions of the p. protegens and p. chlororaphis genomes with substantial absence/presence polymorphism, phage-related genes and an unusual base composition, while in the photorhabuds/xenorhabdus lineage transposable elements are located in proximity of the mcf gene. it would therefore appear that evolutionary processes including the acquisition of insecticidal elements, sequence rearrangements and protein adaptation through domain shuffling allowed plant-associated pseudomonads to adapt to a new ecological niche. in line with the genomic arguments of horizontal acquisition are the patchy distributed fit components that share highest homology with bacteria outside the pseudomonadaceae family. our data show that a specific group of plant-colonizing pseudomonads have evolved a unique virulence gene cluster through diverse evolutionary processes, which contributed to extend their existing repertoire of antifungal and antipredator activities with insecticidal activity. frequent mobilization and recombination is possibly favored by the common niche of insect hosts shared between these particular pseudomonas, photorhabdus and xenorhabdus bacteria, and may provide a selective advantage by the diversification of the toxin gene repertoire. 
a comparative evolutionary analysis of fit toxin-producing pseudomonas including the insect-pathogenic bacteria photorhabdus and xenorhadus, which produce the fit related mcf toxin, showed that fit genes are part of a dynamic genomic region with substantial presence/absence polymorphism and local variation in gc base composition. screening a large collection of plant-associated pseudomonads for insecticidal activity and presence of the fit toxin revealed that fit is highly indicative of insecticidal activity and predicts that pseudomonas protegens and p. chlororaphis are exclusive fit producers. pseudomonasphotorhabdus and xenorhabdusinsecticidal activitytoxin evolutionissue-copyright-statement© the author 2015 however, the ecological relevance of insect toxicity and the mechanisms driving the evolution of toxin production remain puzzling. some of these bacteria produce an insecticidal toxin suggesting that they may exploit insect hosts as a secondary niche. root-colonizing fluorescent pseudomonads are known for their excellent abilities to protect plants against soil-borne fungal pathogens. the patchy distribution and phylogenetic incongruence of fit genes indicate that the fit cluster evolved via horizontal transfer, followed by functional integration of vertically transmitted genes, generating a unique pseudomonas-specific insect toxin cluster.
 root-colonizing fluorescent pseudomonads are known for their excellent abilities to protect plants against soil-borne fungal pathogens. some of these bacteria produce an insecticidal toxin suggesting that they may exploit insect hosts as a secondary niche. however, the ecological relevance of insect toxicity and the mechanisms driving the evolution of toxin production remain puzzling.  screening a large collection of plant-associated pseudomonads for insecticidal activity and presence of the fit toxin revealed that fit is highly indicative of insecticidal activity and predicts that pseudomonas protegens and p. chlororaphis are exclusive fit producers. a comparative evolutionary analysis of fit toxin-producing pseudomonas including the insect-pathogenic bacteria photorhabdus and xenorhadus, which produce the fit related mcf toxin, showed that fit genes are part of a dynamic genomic region with substantial presence/absence polymorphism and local variation in gc base composition. the patchy distribution and phylogenetic incongruence of fit genes indicate that the fit cluster evolved via horizontal transfer, followed by functional integration of vertically transmitted genes, generating a unique pseudomonas-specific insect toxin cluster.  our findings suggest that multiple independent evolutionary events led to formation of at least three versions of the mcf/fit toxin highlighting the dynamic nature of insect toxin evolution. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords pseudomonasphotorhabdus and xenorhabdusinsecticidal activitytoxin evolutionissue-copyright-statement© the author 2015 