BACKGROUND
mobile genetic elements such as phages, plasmids and transposable elements play a vital role in horizontal gene transfer and genome rearrangement in bacteria and archaea  <cit> .
among transposable elements, insertion sequence  elements are particularly widespread within bacterial and archaeal genomes, and are considered the most abundant and ubiquitous genes in nature  <cit> .
is elements can have profound effects on chromosome structure and evolution.
due to their ability to disrupt genes and to induce rearrangements such as inversions, duplications and deletions they are key mediators of genome plasticity  <cit> .
although is elements are perceived primarily as genomic parasites, their activity can also be beneficial.
as composite transposons is elements are able to mobilize adjacent genes, thereby mediating the spread of antibiotic resistance genes and genes involved in the catabolism of complex xenobiotics  <cit> .
is elements may also promote adaptation of their host genomes as demonstrated in experimental evolution experiments  <cit> .
in addition, is elements can influence or activate the expression of adjacent genes, e.g.
by forming hybrid or fusion promoters or by containing outward-directed promoters  <cit> .
is elements are usually less than  <dig>  kbp in length and have a relatively simple genetic organization.
most is elements are flanked both by inverted and direct repeats and generally encode no function other than those involved in mobility, which is mediated by transposases  <cit> .
is elements have been classified into several families based on the degree of sequence conservation of their transposases and its catalytic site, similar genetic organization such as size, number of open reading frames  and potential coding sequences , inverted repeats, and genome target sites  <cit> .
the majority of is elements encode transposases containing the so-called dde-motif consisting of the three amino acids aspartic acid, aspartic acid, and glutamic acid.
these residues form the catalytic triad necessary for transposition.
they are found in three regions  of the transposase amino acid sequence separated by spacers of various lengths  <cit> .
although is elements are found in the majority of sequenced bacterial and archaeal genomes  <cit> , their distribution is patchy, and their occurrence within single genomes is usually below 3%  <cit> .
is elements are very rare in the genomes of most ancient host-restricted symbionts or pathogens such as mutualistic insect and clam symbionts or chlamydiae  <cit> .
on the other hand, elevated numbers of is elements has been observed in the genomes of bacteria which adapted only recently to an intracellular or pathogenic lifestyle  <cit> .
however, this view has been challenged by the recent detection of is element-rich genomes in ancient symbionts such as wolbachia spp.
or orientia tsutsugamushi  <cit> .
interestingly, the genomes containing the highest percentages of is elements are from obligate intracellular bacteria: orientia tsutsugamushi  <cit> , the γ <dig> symbiont of the marine oligochaete olavius algarvensis  <cit> , the symbionts of grain weevils  <cit> , and the amoeba symbiont amoebophilus asiaticus 5a <dig>  <cit> .
amoebophilus asiaticus is a gram-negative, obligate intracellular symbiont, which has been discovered within an amoeba isolated from alkaline lake sediment  <cit> .
highly similar a. asiaticus strains have been recovered from various sources worldwide  <cit> .
a. asiaticus shows highest 16s rrna similarity to 'candidatus cardinium hertigii', an obligate intracellular parasite of arthropods able to manipulate the reproduction of its hosts  <cit> .
both organisms belong to the phylum bacteroidetes and form a monophyletic lineage in 16s rrna-based phylogenetic trees  <cit> , consisting only of symbionts and sequences retrieved from coral samples  <cit> .
the a. asiaticus genome is only moderately reduced in size compared to many other obligate intracellular bacteria  <cit>  but nevertheless, its biosynthetic capabilities are extremely limited  <cit> .
the a. asiaticus genome encodes a hitherto unparalleled high number of proteins with eukaryotic domains such as ankyrin repeats, tpr/sel <dig> repeats, leucine-rich repeats and domains from the eukaryotic ubiquitin system, and it contains an unusually large number of transposase genes  corresponding to 23% of all genes  <cit> .
here, we report on the in-depth analysis of the is elements in the a. asiaticus genome.
we classified them and describe their main characteristics.
we demonstrated that other symbionts closely related to a. asiaticus contain highly similar is elements, and we could show that although they are transcribed, they exhibited no transpositional acitivity on a population level during a time period of almost  <dig>  days.
taking into account evidence that no major rearrangements have occurred recently in the a. asiaticus genome, this suggests that the is elements are evolutionary older components of the a. asiaticus genome, which likely played an important role during genome reduction and adaptation to an obligate intracellular life style.
RESULTS
diversity of is elements in the a. asiaticus 5a <dig> genome
is elements make up  <dig> kbp  of the a. asiaticus genome.
in total,  <dig> transposase genes  were identified in the detailed and manually curated analysis performed here .
compared to other sequenced prokaryotic genomes, the percentage of is elements as well as the number of is elements per megabase genome is among the highest in a. asiaticus .
we were able to assign the vast majority of these transposase genes  to  <dig> different is elements , which belong to eight different is element families, with is <dig> family is elements being most abundant in the a. asiaticus genome .
each of the  <dig> is elements is present in  <dig> to  <dig> full-length copies in the a. asiaticus genome, the only exception being iscaa <dig>  which was identified earlier by the isfinder website  <cit>  and is only present as a single full-length copy .
this results in a total copy number of  <dig> full-length is elements that are evenly spread across the a. asiaticus genome  <cit> .
a high number of is elements in a. asiaticus is truncated , and in some cases  there are more truncated than full-length copies present .
truncation sites were generally not conserved, i.e.
truncations occurred in different regions, and truncated is elements show varying lengths .
for most of the full-length is element copies  we could not identify direct repeats .
in the following sections we shortly describe few selected is elements of a. asiaticus in more detail.
* note that is elements can consist of more than one transposase gene
is1031
a number of base pairs conserved between left and right end repeats/length of the repeat
b length of direct repeat/number of isoforms with direct repeat
c iscaa <dig> was identified by the isfinder website http://www-is.biotoul.fr/})
iscaa4
iscaa <dig> is the most abundant is element in a. asiaticus.
it is present in  <dig> full-length copies,  <dig> of these copies should be able to produce an intact, functional transposase.
iscaa <dig> belongs to the is <dig> family and shows a typical is <dig> family dde-motif  <cit> .
similar to other is <dig> family members, the iscaa <dig> transposase is encoded by two overlapping orfs, which are probably translated into a  <dig> amino acid transposase by - <dig> ribosomal frameshifting .
translational frameshifting is often found in is elements and represents an important mechanism regulating the expression of the transposases at a translational level  <cit> .
translation starts at the first orf  and shifts to the - <dig> reading frame at the so-called slippery site and continues in a second overlapping orf  resulting in a transframe orfab protein.
the predicted frameshift site in iscaa <dig>  is highly shift-prone in bacteria such as a. asiaticus that have only a single trnalys  and lack the trnalys with the anticodon uuc  <cit> .
in iscaa <dig> five nucleotides downstream of the putative slippery site a stem-loop structure is predicted  .
such stem-loop structures have been shown to be stimulatory for - <dig> ribosomal frameshifting  <cit> .
interestingly, iscaa <dig> shows highest amino acid sequence identity  to uncharacterized is elements from methanogenic archaea of the family methanosarcinaceae; the similarity to other transposases is lower than 40%.
in phylogenetic trees, iscaa <dig> forms a stable monophyletic group with these archaeal transposases, indicating interdomain horizontal gene transfer between methanogenic archaea and a. asiaticus .
iscaa3
iscaa <dig> is present in ten full-length copies in a. asiaticus and belongs to isl <dig> group within the is <dig> family  whose transposases typically consist of a single orf  <cit> .
the transposase of iscaa <dig> however, is encoded by two overlapping orfs most likely translated into a  <dig> amino acid protein by - <dig> ribosomal frameshifting.
in contrast to other is elements with canonical slippery sites like iscaa <dig> and iscaa <dig>  no stimulatory stem-loop structure possibly enhancing ribosomal frameshifting is predicted downstream of the slippery site in iscaa <dig> .
iscaa <dig> shows highest amino acid sequence identity  to iscaa <dig> and is elements found in the intracellular bacteria orientia tsutsugamushi, legionella drancourtii, regiella insecticola, and parachlamydia acanthamoebae.
in phylogenetic analyses iscaa <dig>  iscaa <dig> and related is elements from intracellular bacteria consistently group together in all treeing methods applied, suggesting horizontal transfer of is elements between these intracellular bacteria .
interestingly, a number of cyanobacterial is elements form a sister group with the iscaa3-related is elements.
iscaa9
iscaa <dig> is an  <dig> bp is element which is present in  <dig> almost identical copies .
iscaa <dig> belongs to the is <dig> family and shows highest amino acid sequence identity  to ismac <dig> from methanosarcina acetivorans c2a, and 40% amino acid identity to iswpi <dig>  an is element found in many wolbachia strains  <cit> .
the iscaa <dig> transposase is encoded by three consecutive and overlapping orfs which are translated into a  <dig> amino acid protein .
we propose a stop codon read-through to occur at the stop codon  at nucleotide positions  <dig> to  <dig>  which is supported by the presence of the stop codon in all  <dig> iscaa <dig> copies in a. asiaticus, the absence of a stem-loop structure indicative of a terminator downstream of the stop codon, and the observation that ugaa is a weak stop codon quartet  <cit> .
we predict that the stop codon is recoded into tryptophane , a common feature of ugaa stop codon quartets  <cit> .
in addition, the majority of iscaa9-related transposases encodes a tryptophane at the position of the stop codon read-through in iscaa <dig> .
we predict a translational - <dig> frameshifting at a slippery site  between orfb and orfc .
five nucleotides downstream of this putative slippery site, a stem-loop structure  is predicted in iscaa <dig> .
the iscaa <dig> transposase contains a dde-motif, which is most similar to the is <dig> group within the is <dig> family, the transposases of this group however, are usually encoded by a single orf  <cit> .
iscaa10
iscaa <dig> is with a length of  <dig> bp a very short is element that contains a single orf encoding a  <dig> amino acid transposase.
it belongs to the is200/is <dig> family and is <dig> group of is elements comprising the shortest known transposases  <cit> .
members of the is <dig> group are unusual is elements because their transposases do not contain the dde-motif found in most transposases.
instead they belong to the y <dig> transposases with a catalytic tyrosine residue and a conserved huh motif   <cit> .
interestingly, this motif is present only in two of seven iscaa <dig> copies; in the others, the second histidine is replaced by tyrosine, which might render these copies nonfunctional.
other unusual features of is <dig> is elements, that are also found in iscaa <dig>  are the absence of both direct and terminal inverted repeats and the presence of secondary structures leading to low transcriptional and transpositional activity  <cit> .
for example, is <dig> from salmonella typhimurium lt <dig> forms two stem-loop structures: the first is a transcriptional repressor terminating impinging transcripts, the second acts at the translational level and occludes the ribosome binding site  <cit> .
similarly, a stem-loop structure is predicted ten nucleotides upstream of the start codon of the iscaa <dig> transposase and close to the 3' end of iscaa <dig> .
iscaa <dig> shows highest amino acid sequence identity  to  is <dig> family transposases from xenorhabdus nematophila .
unclassified is elements
twenty-five transposase genes could not be assigned to either of the  <dig> a. asiaticus is elements under the criteria applied here.
among these unclassified full-length transposases two transposases belong to the is <dig> family ; and to the tn <dig> family ; one belongs to the is <dig> family ; and two belong to the yhga-like family of putative transposases .
conservation of is elements among different a. asiaticus strains
in order to analyze whether the is elements found in the genome of a. asiaticus 5a <dig> are also present in closely related a. asiaticus strains, we performed pcr using primers targeting the  <dig> most abundant is elements  with genomic dna from a. asiaticus strain eids <dig>  <cit>  as well as from two novel a. asiaticus isolates, a. asiaticus us <dig> and a. asiaticus wr.
these strains show  <dig> %,  <dig> %, and  <dig> % 16s rrna sequence similarity to a. asiaticus 5a <dig>  respectively, corresponding to strain and species level diversity, respectively.
six out of the  <dig> is elements analyzed here were detected in all four a. asiaticus isolates.
cloning and sequencing of pcr products obtained from a. asiaticus eids <dig> revealed nucleic and amino acid sequence identities to consensus sequences of the a. asiaticus 5a <dig> is elements of 87% to 98% .
the lack of pcr products for some is elements indicates either the absence of these is elements in the investigated a. asiaticus strains or a low degree of conservation and hence the absence of or mismatches with the primer target sites.
transcription but lack of transpositional activity of the a. asiaticus is elements
the large copy number and the high degree of conservation of some is elements identified in the a. asiaticus 5a <dig> genome might indicate that they are transpositionally active.
to investigate this, we first asked whether the is elements are transcribed during intracellular replication of a. asiaticus in its amoeba host.
using reverse transcriptase -pcr, we analyzed the transcription of those  <dig> is elements that are present in at least three copies in the genome .
the detection of transcripts of all  <dig> is elements demonstrates that at least one copy each is actively transcribed .
next, we used southern hybridizations to check for chromosomal rearrangements resulting from transposition events  <cit> .
we analyzed the same  <dig> is elements for which we could show transcription and compared dna from the same a. asiaticus culture isolated in november  <dig> and in july  <dig>  respectively, a period of  <dig> days.
we could not detect differences in the banding pattern indicative for chromosomal rearrangements in southern hybridizations for any of the is elements tested .
contiguous transcription of is elements and their downstream genes
some of the a. asiaticus is elements are in close proximity to their downstream genes .
as previous reports have shown that is elements can influence the transcription of neighboring genes  <cit> , we investigated whether contiguous transcripts between a. asiaticus is elements and downstream genes occur.
we analyzed ten selected loci where is elements and their downstream genes are encoded on the same strand and have the same orientation .
using rt-pcr we could show contiguous transcripts of the investigated is elements with their downstream genes at  <dig> out of  <dig> analyzed loci .
we performed two control experiments in order to exclude that the observed transcripts from rt-pcr derive from unspecific background noise transcriptional read-through.
one control targeted an unlikely contiguous transcript between two genes located on different strands and oriented in opposite directions .
we could not detect transcripts in this control reaction , indicating that the observed transcripts from the nine loci of is elements and their downstream genes are above unspecific read-through transcription.
this is further supported by a second, semi-quantitative control experiment in which we compared rt-pcr products  from contiguous transcripts between is elements and their downstream genes with the products from rt-pcr reactions targeting only the downstream genes .
in all cases the obtained bands were of similar intensity, providing further evidence that the observed contiguous transcripts are above unspecific transcriptional read-through.
discussion
mobile genetic elements such as is elements move within and between genomes.
owing to its intracellular lifestyle in free-living amoebae a. asiaticus is, however, largely shielded from other bacteria.
although horizontal gene transfer seems unlikely to occur under these circumstances, previous studies proposed that amoebae may serve as hot spots for horizontal gene transfer among intracellular bacteria  <cit> , and according to the 'intracellular arena' hypothesis genetic material may move in and out of communities of obligate intracellular bacteria that co-infect the same intracellular host environment  <cit> .
we identified four is elements in a. asiaticus that were likely involved in horizontal gene transfer although the direction of the transfer cannot be inferred .
three of these is elements group with is elements from several other intracellular bacteria related to rickettsiae, legionellae and chlamydiae, and consistent with previous findings it is conceivable that amoebae or other protozoa served as a common habitat for these microbes.
one is element of a. asiaticus is most closely related to is elements found in free-living methanogenic archaea .
anoxic aquatic sediments, where free-living amoebae and methanogenic archaea can be found, might represent a possible shared habitat facilitating horizontal gene transfer  <cit> .
horizontal gene transfer of is elements between distantly related organisms is rather rare  <cit> .
hence the discovery of related is elements in three different bacterial phyla  and the archaea might be surprising.
however, a recent study based on the analysis of  <dig> bacterial and archaeal genomes showed that although the majority of horizontal gene transfer events occur between closely related organisms there is a considerable number of large-distance horizontal gene transfer events  <cit> .
our observations expand our view on the extent of horizontal gene transfer of is elements among distantly related microbes, and they provide a glimpse into past interactions of a. asiaticus with other microbes during its evolutionary history.
several lines of evidence point to an ancient origin of many is elements in a. asiaticus.
first, iscaa <dig>  iscaa <dig>  iscaa <dig>  and iscaa <dig>  which together make up 46% of all full-length a. asiaticus is elements, show a remarkably low degree of sequence conservation among their different copies .
this is in contrast to high sequence similarities expected if is elements have entered a genome and spread only recently  <cit> .
second, the high number  of truncated is element copies suggests that these is elements have been present in the a. asiaticus genome for extended time periods during which they disintegrated slowly.
third, the gc-content of the a. asiaticus is elements  is similar to the overall gc content of the a. asiaticus genome .
this suggests that considerable time has elapsed to allow the base composition of the is elements to adapt towards the general base composition of the a. asiaticus genome  <cit> .
finally, at least six is elements are conserved among four different a. asiaticus strains, some of which show a relatively high divergence .
taking into account that our pcr based screening likely underestimates the actual number of shared is elements , this indicates that many - if not most - a. asiaticus is elements were already present in the last common ancestor of the a. asiaticus strains investigated here.
taken together, there is compelling evidence that the is elements have been residing in the a. asiaticus genome for considerable evolutionary time periods.
we noted previously that the a. asiaticus genome shows a gc skew pattern typical for most bacterial genomes with two major shifts at the origin and terminus of replication and only few local deviations, which are indicative of recent genome rearrangements  <cit> .
this is remarkable because with the exception of lactobacillus helveticus dpc  <dig> and shigella sonnei ss <dig>  <cit> , all other bacteria with high numbers of is elements do not show such a regular genomic gc skew pattern .
thus, despite of the high number of is elements, the a. asiaticus genome has not been reshuffled extensively recently, which indicates that most is elements are transpositionally inactive and also that recombination events between highly similar is element copies have not occurred.
a mechanism by which apparently inactive, non-functional is elements can be maintained in bacterial genomes is gene conversion, which was described recently for the genome of wolbachia wbm, a mutualistic symbiont of the nematode brugia malayi whose genome contains a number of highly similar is element copies rendered non-functional by multiple stop codons and frame shifts  <cit> .
in contrast to wolbachia wbm, a. asiaticus still encodes intact copies of each is element, and many is elements show relatively high sequence divergence .
in addition, the transposase genes of different non-functional is element copies show variable pseudogenization states.
this largely rules out gene conversion as the main mechanism for maintenance of is elements in a. asiaticus.
rather unexpectedly, we detected transcription of  <dig> a. asiaticus 5a <dig> is elements during intracellular growth in amoebae .
generally, is elements are among the lowest expressed genes due to their potentially detrimental effects on the host genome  <cit> .
for several a. asiaticus is elements  stable hairpin structures within the first  <dig> bp of the is elements are predicted, which might interfere with expression both at the transcriptional and the translational level, thus controlling the activity of these is elements.
in addition, evidence for programmed translational frameshifting, another regulatory mechanism, can be found in six a. asiaticus is elements .
translational frameshifting acts at the level of translation elongation between two consecutive  open reading frames where the ribosome slides one basepair up- or downstream at a so-called slippery site  <cit> .
for several is elements of a. asiaticus, the occurrence of frameshifting is supported by the presence of a canonical slippery site, of stimulatory secondary structures downstream of the slippery site and, most importantly, the merged amino acid sequences of the is elements transposase orfs show more significant blast hits than the single orfs alone .
in summary, transcription of several is elements occurs in a. asiaticus, but there is evidence that many is elements are tightly regulated both at the transcriptional and the translational level.
southern hybridizations demonstrated the absence of major transposition events and genome rearrangements for a. asiaticus during a time period of  <dig> days .
with an estimated generation time of acanthamoeba sp.
5a <dig> infected with a. asiaticus of  <dig> h , this time period corresponds to approximately  <dig> generations of the acanthamoeba host.
although the generation time of a. asiaticus is unknown, it must be shorter than that of its amoeba host .
the analyzed time period thus corresponds to considerably more than  <dig> a. asiaticus generations.
for e. coli and lactococcus lactis, the first is element-mediated genomic changes  occurred already after  <dig> to  <dig> generations  <cit> .
this indicates that the time period monitored in our study should be sufficient to detect is element-mediated genomic rearrangements.
however, in contrast to our experiment, in these studies bacterial cultures were exposed to environmental stress conditions with respect to nutrient availability, temperature, or oxygen, facilitating adaptive changes.
although no genome rearrangements were observed for a. asiaticus, we cannot exclude the possibility that transposition events occurred in individual a. asiaticus cells which subsequently became not fixed at the population level and would thus be undetectable by our experimental approach.
however, southern blot is a highly sensitive method  <cit> , and we have estimated that we should be able to monitor changes in southern blot patterns in subpopulations consisting of only a few to a few hundred of amoeba host cells .
taking into account typical densities of acanthamoeba sp.
5a <dig> infected with a. asiaticus during in vitro cultivation of  <dig> up to  <dig> cells/ml, the sensitivity of our assay should thus be sufficient to detect variations even in very small subpopulations.
the is elements in a. asiaticus are therefore most likely transpositional inactive.
their abundance is explained by transpositional activity in the evolutionary past of a. asiaticus, and while still being transcriptionally active, most is elements are transpositionally inactive in extant a. asiaticus.
in addition to a tight transcriptional and translational control there are several other conceivable explanations for this observation.
for example, a. asiaticus might lack host factors required for transposition activity of is elements although most of those are specific for certain is elements; they act at different steps and cellular processes and their exact role in transposition is still largely unclear  <cit> .
alternatively, the small, reduced genome of a. asiaticus, which is highly adapted to the intracellular life style and optimized for host cell interactions, might not allow for major rearrangements as most transposition events would be deleterious rendering the cell nonviable.
one reason why some is elements were retained in the a. asiaticus genome despite of the apparent lack of transpositional activity might be their influence on the transcription of downstream genes.
indeed, we could show contiguous transcripts of is elements with their downstream genes at  <dig> out of  <dig> tested loci .
in some cases, the distance between the is element and the start codon of the downstream gene is too short to include known bacteroidetes shine-dalgarno sequences, which are located at - <dig> and - <dig> bp relative to the transcription initiation site  <cit> .
expression of the respective downstream genes might thus depend on promoter sequences located within the upstream is element , a feature often found in is elements  <cit> , or on the endogenous promoter of the is element.
in other cases the distance between the analyzed is elements and their downstream genes was larger .
similar polycistronic mrnas starting from is elements including downstream genes have been described recently for two is elements in francisella tularensis  <cit>  and in mycobacterium tuberculosis is <dig>  <cit> .
it is striking that many of the genes whose transcription is affected by the presence of is elements in a. asiaticus likely play an important role .
for example, aasi_ <dig> is an uncharacterized membrane protein conserved among most bacteroidetes and chlorobi; aasi_ <dig> contains six tpr/sel <dig> repeats, eukaryotic domains that can be involved in host cell interaction  <cit> , and aasi_ <dig> is a ferritin homolog involved in iron storage.
furthermore, a genomic organization of is elements and downstream genes similar to the loci analyzed in this study was found in  <dig> other regions on the a. asiaticus genome , suggesting that contiguous transcripts between is elements and downstream genes are even more widespread and represent a more general feature of a. asiaticus.
genome reduction is an important process during the adaptation of bacteria to an obligate intracellular life style, and is elements are considered to be important in this process  <cit> .
the genome of a. asiaticus is only moderately reduced compared to other obligate intracellular bacteria  <cit> .
its genome size is with  <dig>  mbp notably larger than that of other related symbionts in the bacteroidetes , but smaller than those of free-living relatives .
the genome of a. asiaticus thus represents a transitional stage in genome reduction.
we argue that the is elements in the a. asiaticus genome are evolutionary remnants.
they have been present in the a. asiaticus genome for extended time periods and reflect the organism's evolutionary history.
the is elements proliferated and were important during the adaptation of a. asiaticus to the intracellular life style, but they became increasingly redundant.
the a. asiaticus genome thus represents a snapshot of a bacterial genome which was shaped by the activity of is elements but whose is elements are largely inactive and in the process of further degradation at the present stage.
CONCLUSIONS
analysis and characterization of the a. asiaticus is elements provides evidence for an extremely is element-rich genome, which seems to be evolutionary surprisingly stable - a feature not found in other is element-rich genomes.
the presence of contiguous transcripts between is elements and their downstream genes indicates that these is elements influence the transcription of their downstream genes, most of which likely play an important role for a. asiaticus.
proliferation of is elements in the evolutionary past of a. asiaticus might thus have been an important process during the adaptation of a. asiaticus to an intracellular life style in which its genome was shaped by their activity.

 mobile genetic elements such as phages, plasmids and transposable elements play a vital role in horizontal gene transfer and genome rearrangement in bacteria and archaea. among transposable elements, insertion sequence elements are particularly widespread within bacterial and archaeal genomes, and are considered the most abundant and ubiquitous genes in nature. is elements can have profound effects on chromosome structure and evolution. due to their ability to disrupt genes and to induce rearrangements such as inversions, duplications and deletions they are key mediators of genome plasticity. although is elements are perceived primarily as genomic parasites, their activity can also be beneficial. as composite transposons is elements are able to mobilize adjacent genes, thereby mediating the spread of antibiotic resistance genes and genes involved in the catabolism of complex xenobiotics. is elements may also promote adaptation of their host genomes as demonstrated in experimental evolution experiments. in addition, is elements can influence or activate the expression of adjacent genes, e.g. by forming hybrid or fusion promoters or by containing outward-directed promoters. is elements are usually less than kbp in length and have a relatively simple genetic organization. most is elements are flanked both by inverted and direct repeats and generally encode no function other than those involved in mobility, which is mediated by transposases. is elements have been classified into several families based on the degree of sequence conservation of their transposases and its catalytic site, similar genetic organization such as size, number of open reading frames and potential coding sequences, inverted repeats, and genome target sites. the majority of is elements encode transposases containing the so-called dde-motif consisting of the three amino acids aspartic acid, aspartic acid, and glutamic acid. these residues form the catalytic triad necessary for transposition. they are found in three regions of the transposase amino acid sequence separated by spacers of various lengths. although is elements are found in the majority of sequenced bacterial and archaeal genomes, their distribution is patchy, and their occurrence within single genomes is usually below 3%. is elements are very rare in the genomes of most ancient host-restricted symbionts or pathogens such as mutualistic insect and clam symbionts or chlamydiae. on the other hand, elevated numbers of is elements has been observed in the genomes of bacteria which adapted only recently to an intracellular or pathogenic lifestyle. however, this view has been challenged by the recent detection of is element-rich genomes in ancient symbionts such as wolbachia spp. or orientia tsutsugamushi. interestingly, the genomes containing the highest percentages of is elements are from obligate intracellular bacteria: orientia tsutsugamushi, the γ symbiont of the marine oligochaete olavius algarvensis, the symbionts of grain weevils, and the amoeba symbiont amoebophilus asiaticus 5a. amoebophilus asiaticus is a gram-negative, obligate intracellular symbiont, which has been discovered within an amoeba isolated from alkaline lake sediment. highly similar a. asiaticus strains have been recovered from various sources worldwide. a. asiaticus shows highest 16s rrna similarity to'candidatus cardinium hertigii', an obligate intracellular parasite of arthropods able to manipulate the reproduction of its hosts. both organisms belong to the phylum bacteroidetes and form a monophyletic lineage in 16s rrna-based phylogenetic trees, consisting only of symbionts and sequences retrieved from coral samples. the a. asiaticus genome is only moderately reduced in size compared to many other obligate intracellular bacteria but nevertheless, its biosynthetic capabilities are extremely limited. the a. asiaticus genome encodes a hitherto unparalleled high number of proteins with eukaryotic domains such as ankyrin repeats, tpr/sel repeats, leucine-rich repeats and domains from the eukaryotic ubiquitin system, and it contains an unusually large number of transposase genes corresponding to 23% of all genes. here, we report on the in-depth analysis of the is elements in the a. asiaticus genome. we classified them and describe their main characteristics. we demonstrated that other symbionts closely related to a. asiaticus contain highly similar is elements, and we could show that although they are transcribed, they exhibited no transpositional acitivity on a population level during a time period of almost days. taking into account evidence that no major rearrangements have occurred recently in the a. asiaticus genome, this suggests that the is elements are evolutionary older components of the a. asiaticus genome, which likely played an important role during genome reduction and adaptation to an obligate intracellular life style.  diversity of is elements in the a. asiaticus 5a genome is elements make up kbp of the a. asiaticus genome. in total, transposase genes were identified in the detailed and manually curated analysis performed here. compared to other sequenced prokaryotic genomes, the percentage of is elements as well as the number of is elements per megabase genome is among the highest in a. asiaticus. we were able to assign the vast majority of these transposase genes to different is elements, which belong to eight different is element families, with is family is elements being most abundant in the a. asiaticus genome. each of the is elements is present in to full-length copies in the a. asiaticus genome, the only exception being iscaa which was identified earlier by the isfinder website and is only present as a single full-length copy. this results in a total copy number of full-length is elements that are evenly spread across the a. asiaticus genome. a high number of is elements in a. asiaticus is truncated, and in some cases there are more truncated than full-length copies present. truncation sites were generally not conserved, i.e. truncations occurred in different regions, and truncated is elements show varying lengths. for most of the full-length is element copies we could not identify direct repeats. in the following sections we shortly describe few selected is elements of a. asiaticus in more detail. * note that is elements can consist of more than one transposase gene is1031 a number of base pairs conserved between left and right end repeats/length of the repeat b length of direct repeat/number of isoforms with direct repeat c iscaa was identified by the isfinder website http://www-is.biotoul.fr/}) iscaa4 iscaa is the most abundant is element in a. asiaticus. it is present in full-length copies, of these copies should be able to produce an intact, functional transposase. iscaa belongs to the is family and shows a typical is family dde-motif. similar to other is family members, the iscaa transposase is encoded by two overlapping orfs, which are probably translated into a amino acid transposase by - ribosomal frameshifting. translational frameshifting is often found in is elements and represents an important mechanism regulating the expression of the transposases at a translational level. translation starts at the first orf and shifts to the - reading frame at the so-called slippery site and continues in a second overlapping orf resulting in a transframe orfab protein. the predicted frameshift site in iscaa is highly shift-prone in bacteria such as a. asiaticus that have only a single trnalys and lack the trnalys with the anticodon uuc. in iscaa five nucleotides downstream of the putative slippery site a stem-loop structure is predicted. such stem-loop structures have been shown to be stimulatory for - ribosomal frameshifting. interestingly, iscaa shows highest amino acid sequence identity to uncharacterized is elements from methanogenic archaea of the family methanosarcinaceae; the similarity to other transposases is lower than 40%. in phylogenetic trees, iscaa forms a stable monophyletic group with these archaeal transposases, indicating interdomain horizontal gene transfer between methanogenic archaea and a. asiaticus. iscaa3 iscaa is present in ten full-length copies in a. asiaticus and belongs to isl group within the is family whose transposases typically consist of a single orf. the transposase of iscaa however, is encoded by two overlapping orfs most likely translated into a amino acid protein by - ribosomal frameshifting. in contrast to other is elements with canonical slippery sites like iscaa and iscaa no stimulatory stem-loop structure possibly enhancing ribosomal frameshifting is predicted downstream of the slippery site in iscaa. iscaa shows highest amino acid sequence identity to iscaa and is elements found in the intracellular bacteria orientia tsutsugamushi, legionella drancourtii, regiella insecticola, and parachlamydia acanthamoebae. in phylogenetic analyses iscaa iscaa and related is elements from intracellular bacteria consistently group together in all treeing methods applied, suggesting horizontal transfer of is elements between these intracellular bacteria. interestingly, a number of cyanobacterial is elements form a sister group with the iscaa3-related is elements. iscaa9 iscaa is an bp is element which is present in almost identical copies. iscaa belongs to the is family and shows highest amino acid sequence identity to ismac from methanosarcina acetivorans c2a, and 40% amino acid identity to iswpi an is element found in many wolbachia strains. the iscaa transposase is encoded by three consecutive and overlapping orfs which are translated into a amino acid protein. we propose a stop codon read-through to occur at the stop codon at nucleotide positions to which is supported by the presence of the stop codon in all iscaa copies in a. asiaticus, the absence of a stem-loop structure indicative of a terminator downstream of the stop codon, and the observation that ugaa is a weak stop codon quartet. we predict that the stop codon is recoded into tryptophane, a common feature of ugaa stop codon quartets. in addition, the majority of iscaa9-related transposases encodes a tryptophane at the position of the stop codon read-through in iscaa. we predict a translational - frameshifting at a slippery site between orfb and orfc. five nucleotides downstream of this putative slippery site, a stem-loop structure is predicted in iscaa. the iscaa transposase contains a dde-motif, which is most similar to the is group within the is family, the transposases of this group however, are usually encoded by a single orf. iscaa10 iscaa is with a length of bp a very short is element that contains a single orf encoding a amino acid transposase. it belongs to the is200/is family and is group of is elements comprising the shortest known transposases. members of the is group are unusual is elements because their transposases do not contain the dde-motif found in most transposases. instead they belong to the y transposases with a catalytic tyrosine residue and a conserved huh motif. interestingly, this motif is present only in two of seven iscaa copies; in the others, the second histidine is replaced by tyrosine, which might render these copies nonfunctional. other unusual features of is is elements, that are also found in iscaa are the absence of both direct and terminal inverted repeats and the presence of secondary structures leading to low transcriptional and transpositional activity. for example, is from salmonella typhimurium lt forms two stem-loop structures: the first is a transcriptional repressor terminating impinging transcripts, the second acts at the translational level and occludes the ribosome binding site. similarly, a stem-loop structure is predicted ten nucleotides upstream of the start codon of the iscaa transposase and close to the 3' end of iscaa. iscaa shows highest amino acid sequence identity to is family transposases from xenorhabdus nematophila. unclassified is elements twenty-five transposase genes could not be assigned to either of the a. asiaticus is elements under the criteria applied here. among these unclassified full-length transposases two transposases belong to the is family; and to the tn family; one belongs to the is family; and two belong to the yhga-like family of putative transposases. conservation of is elements among different a. asiaticus strains in order to analyze whether the is elements found in the genome of a. asiaticus 5a are also present in closely related a. asiaticus strains, we performed pcr using primers targeting the most abundant is elements with genomic dna from a. asiaticus strain eids as well as from two novel a. asiaticus isolates, a. asiaticus us and a. asiaticus wr. these strains show %, %, and % 16s rrna sequence similarity to a. asiaticus 5a respectively, corresponding to strain and species level diversity, respectively. six out of the is elements analyzed here were detected in all four a. asiaticus isolates. cloning and sequencing of pcr products obtained from a. asiaticus eids revealed nucleic and amino acid sequence identities to consensus sequences of the a. asiaticus 5a is elements of 87% to 98%. the lack of pcr products for some is elements indicates either the absence of these is elements in the investigated a. asiaticus strains or a low degree of conservation and hence the absence of or mismatches with the primer target sites. transcription but lack of transpositional activity of the a. asiaticus is elements the large copy number and the high degree of conservation of some is elements identified in the a. asiaticus 5a genome might indicate that they are transpositionally active. to investigate this, we first asked whether the is elements are transcribed during intracellular replication of a. asiaticus in its amoeba host. using reverse transcriptase -pcr, we analyzed the transcription of those is elements that are present in at least three copies in the genome. the detection of transcripts of all is elements demonstrates that at least one copy each is actively transcribed. next, we used southern hybridizations to check for chromosomal rearrangements resulting from transposition events. we analyzed the same is elements for which we could show transcription and compared dna from the same a. asiaticus culture isolated in november and in july respectively, a period of days. we could not detect differences in the banding pattern indicative for chromosomal rearrangements in southern hybridizations for any of the is elements tested. contiguous transcription of is elements and their downstream genes some of the a. asiaticus is elements are in close proximity to their downstream genes. as previous reports have shown that is elements can influence the transcription of neighboring genes, we investigated whether contiguous transcripts between a. asiaticus is elements and downstream genes occur. we analyzed ten selected loci where is elements and their downstream genes are encoded on the same strand and have the same orientation. using rt-pcr we could show contiguous transcripts of the investigated is elements with their downstream genes at out of analyzed loci. we performed two control experiments in order to exclude that the observed transcripts from rt-pcr derive from unspecific background noise transcriptional read-through. one control targeted an unlikely contiguous transcript between two genes located on different strands and oriented in opposite directions. we could not detect transcripts in this control reaction, indicating that the observed transcripts from the nine loci of is elements and their downstream genes are above unspecific read-through transcription. this is further supported by a second, semi-quantitative control experiment in which we compared rt-pcr products from contiguous transcripts between is elements and their downstream genes with the products from rt-pcr reactions targeting only the downstream genes. in all cases the obtained bands were of similar intensity, providing further evidence that the observed contiguous transcripts are above unspecific transcriptional read-through. discussion mobile genetic elements such as is elements move within and between genomes. owing to its intracellular lifestyle in free-living amoebae a. asiaticus is, however, largely shielded from other bacteria. although horizontal gene transfer seems unlikely to occur under these circumstances, previous studies proposed that amoebae may serve as hot spots for horizontal gene transfer among intracellular bacteria, and according to the'intracellular arena' hypothesis genetic material may move in and out of communities of obligate intracellular bacteria that co-infect the same intracellular host environment. we identified four is elements in a. asiaticus that were likely involved in horizontal gene transfer although the direction of the transfer cannot be inferred. three of these is elements group with is elements from several other intracellular bacteria related to rickettsiae, legionellae and chlamydiae, and consistent with previous findings it is conceivable that amoebae or other protozoa served as a common habitat for these microbes. one is element of a. asiaticus is most closely related to is elements found in free-living methanogenic archaea. anoxic aquatic sediments, where free-living amoebae and methanogenic archaea can be found, might represent a possible shared habitat facilitating horizontal gene transfer. horizontal gene transfer of is elements between distantly related organisms is rather rare. hence the discovery of related is elements in three different bacterial phyla and the archaea might be surprising. however, a recent study based on the analysis of bacterial and archaeal genomes showed that although the majority of horizontal gene transfer events occur between closely related organisms there is a considerable number of large-distance horizontal gene transfer events. our observations expand our view on the extent of horizontal gene transfer of is elements among distantly related microbes, and they provide a glimpse into past interactions of a. asiaticus with other microbes during its evolutionary history. several lines of evidence point to an ancient origin of many is elements in a. asiaticus. first, iscaa iscaa iscaa and iscaa which together make up 46% of all full-length a. asiaticus is elements, show a remarkably low degree of sequence conservation among their different copies. this is in contrast to high sequence similarities expected if is elements have entered a genome and spread only recently. second, the high number of truncated is element copies suggests that these is elements have been present in the a. asiaticus genome for extended time periods during which they disintegrated slowly. third, the gc-content of the a. asiaticus is elements is similar to the overall gc content of the a. asiaticus genome. this suggests that considerable time has elapsed to allow the base composition of the is elements to adapt towards the general base composition of the a. asiaticus genome. finally, at least six is elements are conserved among four different a. asiaticus strains, some of which show a relatively high divergence. taking into account that our pcr based screening likely underestimates the actual number of shared is elements, this indicates that many - if not most - a. asiaticus is elements were already present in the last common ancestor of the a. asiaticus strains investigated here. taken together, there is compelling evidence that the is elements have been residing in the a. asiaticus genome for considerable evolutionary time periods. we noted previously that the a. asiaticus genome shows a gc skew pattern typical for most bacterial genomes with two major shifts at the origin and terminus of replication and only few local deviations, which are indicative of recent genome rearrangements. this is remarkable because with the exception of lactobacillus helveticus dpc and shigella sonnei ss, all other bacteria with high numbers of is elements do not show such a regular genomic gc skew pattern. thus, despite of the high number of is elements, the a. asiaticus genome has not been reshuffled extensively recently, which indicates that most is elements are transpositionally inactive and also that recombination events between highly similar is element copies have not occurred. a mechanism by which apparently inactive, non-functional is elements can be maintained in bacterial genomes is gene conversion, which was described recently for the genome of wolbachia wbm, a mutualistic symbiont of the nematode brugia malayi whose genome contains a number of highly similar is element copies rendered non-functional by multiple stop codons and frame shifts. in contrast to wolbachia wbm, a. asiaticus still encodes intact copies of each is element, and many is elements show relatively high sequence divergence. in addition, the transposase genes of different non-functional is element copies show variable pseudogenization states. this largely rules out gene conversion as the main mechanism for maintenance of is elements in a. asiaticus. rather unexpectedly, we detected transcription of a. asiaticus 5a is elements during intracellular growth in amoebae. generally, is elements are among the lowest expressed genes due to their potentially detrimental effects on the host genome. for several a. asiaticus is elements stable hairpin structures within the first bp of the is elements are predicted, which might interfere with expression both at the transcriptional and the translational level, thus controlling the activity of these is elements. in addition, evidence for programmed translational frameshifting, another regulatory mechanism, can be found in six a. asiaticus is elements. translational frameshifting acts at the level of translation elongation between two consecutive open reading frames where the ribosome slides one basepair up- or downstream at a so-called slippery site. for several is elements of a. asiaticus, the occurrence of frameshifting is supported by the presence of a canonical slippery site, of stimulatory secondary structures downstream of the slippery site and, most importantly, the merged amino acid sequences of the is elements transposase orfs show more significant blast hits than the single orfs alone. in summary, transcription of several is elements occurs in a. asiaticus, but there is evidence that many is elements are tightly regulated both at the transcriptional and the translational level. southern hybridizations demonstrated the absence of major transposition events and genome rearrangements for a. asiaticus during a time period of days. with an estimated generation time of acanthamoeba sp. 5a infected with a. asiaticus of h, this time period corresponds to approximately generations of the acanthamoeba host. although the generation time of a. asiaticus is unknown, it must be shorter than that of its amoeba host. the analyzed time period thus corresponds to considerably more than a. asiaticus generations. for e. coli and lactococcus lactis, the first is element-mediated genomic changes occurred already after to generations. this indicates that the time period monitored in our study should be sufficient to detect is element-mediated genomic rearrangements. however, in contrast to our experiment, in these studies bacterial cultures were exposed to environmental stress conditions with respect to nutrient availability, temperature, or oxygen, facilitating adaptive changes. although no genome rearrangements were observed for a. asiaticus, we cannot exclude the possibility that transposition events occurred in individual a. asiaticus cells which subsequently became not fixed at the population level and would thus be undetectable by our experimental approach. however, southern blot is a highly sensitive method, and we have estimated that we should be able to monitor changes in southern blot patterns in subpopulations consisting of only a few to a few hundred of amoeba host cells. taking into account typical densities of acanthamoeba sp. 5a infected with a. asiaticus during in vitro cultivation of up to cells/ml, the sensitivity of our assay should thus be sufficient to detect variations even in very small subpopulations. the is elements in a. asiaticus are therefore most likely transpositional inactive. their abundance is explained by transpositional activity in the evolutionary past of a. asiaticus, and while still being transcriptionally active, most is elements are transpositionally inactive in extant a. asiaticus. in addition to a tight transcriptional and translational control there are several other conceivable explanations for this observation. for example, a. asiaticus might lack host factors required for transposition activity of is elements although most of those are specific for certain is elements; they act at different steps and cellular processes and their exact role in transposition is still largely unclear. alternatively, the small, reduced genome of a. asiaticus, which is highly adapted to the intracellular life style and optimized for host cell interactions, might not allow for major rearrangements as most transposition events would be deleterious rendering the cell nonviable. one reason why some is elements were retained in the a. asiaticus genome despite of the apparent lack of transpositional activity might be their influence on the transcription of downstream genes. indeed, we could show contiguous transcripts of is elements with their downstream genes at out of tested loci. in some cases, the distance between the is element and the start codon of the downstream gene is too short to include known bacteroidetes shine-dalgarno sequences, which are located at - and - bp relative to the transcription initiation site. expression of the respective downstream genes might thus depend on promoter sequences located within the upstream is element, a feature often found in is elements, or on the endogenous promoter of the is element. in other cases the distance between the analyzed is elements and their downstream genes was larger. similar polycistronic mrnas starting from is elements including downstream genes have been described recently for two is elements in francisella tularensis and in mycobacterium tuberculosis is. it is striking that many of the genes whose transcription is affected by the presence of is elements in a. asiaticus likely play an important role. for example, aasi_ is an uncharacterized membrane protein conserved among most bacteroidetes and chlorobi; aasi_ contains six tpr/sel repeats, eukaryotic domains that can be involved in host cell interaction, and aasi_ is a ferritin homolog involved in iron storage. furthermore, a genomic organization of is elements and downstream genes similar to the loci analyzed in this study was found in other regions on the a. asiaticus genome, suggesting that contiguous transcripts between is elements and downstream genes are even more widespread and represent a more general feature of a. asiaticus. genome reduction is an important process during the adaptation of bacteria to an obligate intracellular life style, and is elements are considered to be important in this process. the genome of a. asiaticus is only moderately reduced compared to other obligate intracellular bacteria. its genome size is with mbp notably larger than that of other related symbionts in the bacteroidetes, but smaller than those of free-living relatives. the genome of a. asiaticus thus represents a transitional stage in genome reduction. we argue that the is elements in the a. asiaticus genome are evolutionary remnants. they have been present in the a. asiaticus genome for extended time periods and reflect the organism's evolutionary history. the is elements proliferated and were important during the adaptation of a. asiaticus to the intracellular life style, but they became increasingly redundant. the a. asiaticus genome thus represents a snapshot of a bacterial genome which was shaped by the activity of is elements but whose is elements are largely inactive and in the process of further degradation at the present stage.  analysis and characterization of the a. asiaticus is elements provides evidence for an extremely is element-rich genome, which seems to be evolutionary surprisingly stable - a feature not found in other is element-rich genomes. the presence of contiguous transcripts between is elements and their downstream genes indicates that these is elements influence the transcription of their downstream genes, most of which likely play an important role for a. asiaticus. proliferation of is elements in the evolutionary past of a. asiaticus might thus have been an important process during the adaptation of a. asiaticus to an intracellular life style in which its genome was shaped by their activity. 
despite this high is element load, the a. asiaticus genome displays a gc skew pattern typical for most bacterial genomes, indicating that no major rearrangements have occurred recently. however, we detected contiguous transcripts between is elements and their downstream genes at nine loci in the a. asiaticus genome, indicating that some is elements influence the transcription of downstream genes, some of which might be important for host cell interaction. additionally, the high sequence divergence of some is elements, the high number of truncated is element copies, as well as the absence of direct repeats in most is elements suggest that the is elements of a. asiaticus are transpositionally inactive. although we could show transcription of is elements, we did not find experimental evidence for transpositional activity, corroborating our results from sequence analyses. the mbp genome of the obligate intracellular amoeba symbiont amoebophilus asiaticus contains an unusually large number of transposase genes. the transposase genes in the a. asiaticus genome can be assigned to different is elements termed iscaa to iscaa which are represented by to full-length copies, respectively. taken together, the is elements in the a. asiaticus genome are currently in the process of degradation and largely represent reflections of the evolutionary past of a. asiaticus in which its genome was shaped by their activity. insertion sequence elements are important mediators of genome plasticity and are widespread among bacterial and archaeal genomes.
 insertion sequence elements are important mediators of genome plasticity and are widespread among bacterial and archaeal genomes. the mbp genome of the obligate intracellular amoeba symbiont amoebophilus asiaticus contains an unusually large number of transposase genes.  the transposase genes in the a. asiaticus genome can be assigned to different is elements termed iscaa to iscaa which are represented by to full-length copies, respectively. despite this high is element load, the a. asiaticus genome displays a gc skew pattern typical for most bacterial genomes, indicating that no major rearrangements have occurred recently. additionally, the high sequence divergence of some is elements, the high number of truncated is element copies, as well as the absence of direct repeats in most is elements suggest that the is elements of a. asiaticus are transpositionally inactive. although we could show transcription of is elements, we did not find experimental evidence for transpositional activity, corroborating our results from sequence analyses. however, we detected contiguous transcripts between is elements and their downstream genes at nine loci in the a. asiaticus genome, indicating that some is elements influence the transcription of downstream genes, some of which might be important for host cell interaction.  taken together, the is elements in the a. asiaticus genome are currently in the process of degradation and largely represent reflections of the evolutionary past of a. asiaticus in which its genome was shaped by their activity. insertion sequence elementendosymbiontbacteroidetesgenome evolution 