BACKGROUND
infections with the human gastric pathogen h. pylori are paradigmatic examples of chronic, or persistent, bacterial infections in the face of a constant immune response
<cit> .
h. pylori infections are usually contracted during early childhood and persist for the lifetime of the host, but most infected individuals develop only mild gastric inflammation without overt symptoms.
nevertheless, a substantial fraction of infected persons develops more severe consequences, making h. pylori the principal cause of  chronic active gastritis and peptic ulcer disease, and a major risk factor for development of gastric adenocarcinoma and mucosa-associated lymphoid tissue  lymphoma
<cit> .
for survival and persistent growth in the presence of a constant immune response and in an environment which is changing considerably over decades of infection, permanent adaptation of the bacteria is thought to be required
<cit> .
such adaptive processes may include regulatory mechanisms acting on gene expression, but also reversible or irreversible genome changes.
for instance, it has been shown that strains isolated from patients with atrophic gastritis
<cit>  or marginal zone b-cell malt lymphoma
<cit>  have reduced genomes in comparison to gastritis or ulcer strains, and a strain isolated from a gastric cancer patient had lost further genes in comparison to a strain isolated previously from the same patient during atrophic gastritis
<cit> .
that genome plasticity plays a role in bacterial persistence is further supported by the observation that natural transformation competence, which is upregulated upon dna stress
<cit> , promotes persistent colonization in mice
<cit> .
allelic diversity caused by high mutation rates and frequent recombination events is a striking property of h. pylori strains.
genetic fingerprints of individual strains obtained by multilocus sequence typing of housekeeping genes have indicated that clonal transmission is likely to occur, but is followed by a rapid adaptation to the new host, so that h. pylori isolates from different subjects are almost always unique
<cit> .
on the other hand, while recombination events generating allelic diversity are frequent, genome changes involving gain or loss of genes seem to be rare
<cit> .
nevertheless, on the level of gene content, evidence has been presented that h. pylori is a species with an open pan-genome, in which each individual isolate contains a distinct set of non-core, or strain-specific, genes
<cit> .
comparative analysis of the first sequenced h. pylori genomes suggested that these strain-specific genes are often located in genomic regions that had previously been termed plasticity zones or plasticity regions, a designation originally used to describe a particular genetic locus with high variation between the first two h. pylori genome sequences
<cit> .
however, with the availability of more sequencing data and more complete h. pylori genome sequences, it became clear that parts of the plasticity regions are usually organized as genomic islands that may be integrated in one of several different genetic loci.
furthermore, they generally contain complete sets of genes required to produce type iv secretion machineries, as well as genes encoding different dna-processing proteins
<cit> , suggesting that they are actually mobile genetic elements capable of horizontal gene transfer between bacterial cells, and that they might be best described as conjugative transposons or integrating conjugative elements .
the actual plasticity of these islands partly derives from the fact that gene rearrangements, insertions or deletions may have occurred within them, but it is not clear whether they also carry variable passenger genes.
interestingly, intrahost variation among genes of the plasticity zones, including deletions in a type iv secretion system gene, has been found for sequential isolates obtained from a duodenal ulcer patient over a course of 10 years
<cit> .
although several candidate genes of these plasticity regions have been suggested as disease markers, e.g.
dupa for duodenal ulcer
<cit> , or jhp <dig> for marginal zone b-cell malt lymphoma
<cit> , the functions of the plasticity zones are currently not well-understood.
to address the question of plasticity zone prevalence, and of their genetic diversity, we have performed a comparative analysis of these genome islands from a larger number of h. pylori genome sequences, including newly determined genome sequences of nine additional strains from different backgrounds.
we show that these elements have a high prevalence throughout all populations, and that gene evolution within the elements is not congruent with the rest of the genomes.
the wide variety of integration loci together with a conserved sequence motif at each integration site suggests an integration mechanism that depends on a short recognition motif in the dna sequence only.
RESULTS
prevalence of plasticity regions in the h. pylori population
we have reported previously that h. pylori strain p <dig> contains three genome regions with similarity to the prototypical plasticity zones, but only one of them  corresponds to the originally described locus, whereas the other two regions  have a genetic organization typical for genome islands and contain genes for type iv secretion systems that might make them capable of self-transfer
<cit> .
in comparison, the original two genome sequences  contain only truncated and highly rearranged portions of these genome islands .
as reported previously, the most conserved type iv secretion system genes fall into one of two distinct groups, which have been termed either tfs <dig> and tfs3a/b <cit> , or tfs <dig> and tfs <dig> <cit> .
in accordance with ref.
<cit> , where conserved tfs <dig> genes have been shown not to be more closely related to tfs <dig> genes than to the respective comb genes encoding the type iv secretion system used for natural transformation, we consider tfs <dig> and tfs <dig> here as independent systems.
moreover, since there is evidence for horizontal gene transfer of the corresponding islands
<cit> , but not for transposition within a strain, we propose to use the term integrating conjugative elements  and refer to individual islands as icehptfs <dig> or icehptfs <dig>  respectively.
a comparison of different designations of the islands and associated type iv secretion systems is given in table
<dig>  to determine the occurrence of icehptfs <dig> and icehptfs <dig> elements in the h. pylori population and the degree of variation among them, we performed a comparative sequence analysis of these elements from  <dig> completely sequenced h. pylori genomes available in public databases .
n.a., not applicable.
14
<dig>   <dig>  2018
1
1strains  <dig>   <dig> and  <dig> are sequential isolates from a single patient
<cit>  and do not show major differences in their icehptfs <dig> sequences.
however, note that genbank entries ef <dig> , ef <dig>  and ef <dig>  describe icehptfs <dig> clusters in these strains
<cit>  that are not present in the genome sequences.
2helpy <dig> is possibly a vestige of hpp12_1321/pz <dig>
3resulting from insertions of 2- <dig> genomic islands and subsequent rearrangements.
4containing icehptfs4a-type genes close to the left junction, and icehptfs4b-type genes close to the right junction.
5associated with genome rearrangement in comparison to strain p <dig>
6associated with deletion of hpp12_ <dig> to hpp12_ <dig>  including one copy of 5s-23s-rrna.
7associated with a recombination between the two 5s-23s-rrna loci .
8partial duplication of both genes; icehptfs <dig> inserted into truncated icehptfs4b.
9within a restriction-modification system inserted into this region.
10integrated together with a  <dig>  kb fragment of icehptfs <dig> and a putative toxin-antitoxin system.
11integration of icehptfs4a into remnant of icehptfs4b, which is in turn integrated into truncated icehptfs <dig>
12irregular integration, using internal aagaatg motif.
13left and right junctions coincide due to irregular integration.
14numbers in parentheses indicate incomplete ice elements.
15disrupted by a chromosomal inversion from hpp12_ <dig> to hpp12_ <dig>
16size of ice increased by is element insertion.
17interrupted by a chromosomal rearrangement between hpp12_ <dig> and hpp12_ <dig> .
18original integration probably in hpp12_994-5s-rrna locus; from there, relocation of  <dig>  kb fragment via internal aagaatg motifs into hpp12_1510;  <dig>  kb duplication  in both loci.
we found that only  <dig> out of these  <dig> strains do not contain icehptfs <dig> or icehptfs <dig> islands or fragments thereof .
among the remaining  <dig> strains,  <dig> harbour icehptfs <dig> islands,  <dig> of which seem to have complete gene sets, and  <dig> harbour icehptfs <dig> islands,  <dig> of which are complete.
there are  <dig> strains with two different icehptfs <dig> elements, and  <dig> strains which have at least parts of both icehptfs <dig> and icehptfs <dig>  three strains  contain hybrid arrangements of icehptfs <dig> and icehptfs <dig> islands, but these seem to result from dna rearrangements after integration of two independent genome islands .
thus, each complete or truncated island can be assigned to either the icehptfs <dig> or the icehptfs <dig> type.
within the icehptfs <dig> group, two distinct variants can be discriminated, which differ by the presence  or absence  of the pz21-pz <dig> genes .
in contrast, three variants of icehptfs <dig>  defined by orthologous, but variant sets of genes at both ends of the genome islands, or in their central regions, can be distinguished and are termed here icehptfs4a, icehptfs4b and icehptfs4c, respectively .
the third subtype, icehptfs4c, was only found in strain southafrica <dig>  which belongs to the hpafrica <dig> population , and as a plasmid-borne fragment in strain lithuania <dig>  both types of genome island seem to vary considerably in size between strains , but this is often due to small deletions within the islands or to insertion of is elements; therefore, complete icehptfs <dig> islands have “standard” sizes of about  <dig> , or 46 kb, depending on the presence of pz21- <dig> orthologs, while complete icehptfs4a, icehptfs4b and icehptfs4c usually comprise about  <dig>   <dig> , and  <dig>  kb, respectively .
geographic distribution of icehptfs <dig> and icehptfs <dig> islands
it is well-established that h. pylori strains cluster into distinct populations according to their geographic origin when multilocus sequence typing using partial sequences of seven housekeeping genes is employed
<cit> .
in contrast to this allelic variability, which suggests a common evolution of h. pylori and humans, consistent gene content profiles of individual populations could not be found, with the exception of one hypothetical gene  present only in strains from the hpafrica <dig> population
<cit> .
interestingly, comparison of gene content microarray data
<cit>  with icehptfs <dig> composition suggests that most hpafrica <dig> strains contain icehptfs4a genes close to the left junctions and in the mid region , but icehptfs4b genes close to the right junctions , while hpeurope strains variably contain these genes.
since there are only three hpafrica <dig> strains among the  <dig> complete genome sequences analysed , we decided to determine draft genome sequences of three further strains originating from western africa, as well as of six strains isolated in europe, five of which had been tested positive for the presence of an icehptfs4a-type or an icehptfs4b-type virb <dig> gene .
sequence analysis revealed that all strains except one  contain at least 37 kb of icehptfs <dig> and/or icehptfs <dig> sequences .
1
4
5
1inferred from the neighbor-joining tree shown in figure
<dig>
2resulting from insertion of two genome islands and rearrangements associated with is element insertion and two copies of pz21/hpp12_447-like genes.
3associated with a genome rearrangement between hpp12_ <dig> and hpp12_ <dig>
4genomic position of aagaatg motif in strain p <dig>
5numbers in parentheses indicate incomplete ice elements.
6contains 28 kb of prophage-related sequences.
to examine possible variations in plasticity zone distribution among phylogeographic groups, we first constructed a phylogenetic tree based on mlst gene sequences, using all  <dig> fully sequenced strains, the nine strains sequenced in this study, and  <dig> reference strains from the mlst database .
no correlation between phylogeographic groups and the presence or absence of either icehptfs <dig> or icehptfs <dig> could be found.
however, all hpafrica <dig> strains contain truncated versions of icehptfs4b or of an icehptfs4a/b variant similar to the hpafrica <dig> strains mentioned above .
we then calculated neighbor-joining phylogenetic trees using conserved icehptfs <dig> or icehptfs <dig> gene sequences  and compared them with an mlst-derived tree .
interestingly, icehptfs4ab genes clustered in a similar way as housekeeping gene sequences did, except for a much closer relationship of these genes than of housekeeping genes between hpafrica <dig> strain southafrica <dig> and other populations .
in contrast, icehptfs <dig> sequences formed at least three strongly divergent clades that were not congruent with the mlst population structure.
these clades seem to correspond to  the hspamerind population;  a mixture of hspeasia and hpasia <dig> populations; and  a mixture of hpeurope and hpafrica <dig> populations .
however, the number of icehptfs3-positive strains analysed may be too low to definitely draw conclusions from this observation.
<dig>  note that unequivocal classification of strains pecan <dig> and pecan <dig> was not possible.
phylogenetic tree calculated from concatenated virb <dig>  virb <dig> and vird <dig> ortholog sequences of all icehptfs <dig> and icehptfs <dig> islands.
neighbor-joining tree calculated from dna sequences of methylase/helicase  orthologs.
orthologs associated with icehptfs <dig> elements are marked by blue branch lines, and orthologs associated with icehptfs <dig> elements by red branch lines.
black lines indicate hybrid elements or the presence of two different elements in the same strain.
colouring of individual strains by phylogeographic origin is shown according to the tree in figure
<dig>
identification of conserved and ice type-specific genes
since both icehptfs <dig> and icehptfs <dig> islands contain genes for complete type iv secretion systems and may coexist in a single strain, an open question is whether individual genes or groups of genes from one type of island have the capacity to complement deficiencies in the other.
sequence comparisons showed that each of the type iv secretion apparatus components is clearly distinguishable between the different types  of islands, with amino acid sequence similarities ranging from 40% to 80% .
this is also true for putative dna processing or segregation proteins such as xert, para, topa or vird <dig> /hpp12_447; see below), suggesting that the individual secretion systems might be sufficiently divergent to be incompatible.
hptfs4a
-encoded proteins and proteins from ice
hptfs <dig>
and ice
hptfs4b/c
islands
hptfs4b
1
hptfs4c
1
hptfs3
hptfs3
1
2
2
4
6
1numbers printed in normal face correspond to >90% identity , and numbers in bold face to 40-85% similarity.
2genes hpb8_ <dig> and pz <dig>  as well as hpb8_ <dig> and pz <dig> share only 61/73% and 54/70% identity/similarity, respectively, to each other, but are equally similar to hpp12_ <dig> and hpp12_ <dig>  respectively.
3some icehptfs4c islands contain the icehptfs4b versions with lower similarities in these sites.
4similarities confined to short regions only.
5no significant similarity detectable, but gene with similar size and orientation present.
6icehptfs4c and icehptfs <dig> islands contain fusions of hpp12_ <dig> and hpp12_ <dig>
to define further common ice gene products and to identify ice-type-specific genes, we performed similarity searches with all other amino acid sequences as well.
the results show that nine further, hypothetical icehptfs4a genes have similar counterparts in icehptfs3-type islands .
interestingly, orthologs of the conserved hypothetical genes hpb8_ <dig> or hpp12_ <dig> are present in icehptfs <dig>  icehptfs4a and icehptfs4c islands, but absent from icehptfs4b islands.
because of their sequence similarities, we speculate that these hypothetical genes have additional conserved functions for genome island maintenance and/or transfer.
in contrast, genes that are specific for either type of genome island might be cargo proteins of the respective mobile genetic elements, fulfilling more specific roles.
such specific genes for icehptfs <dig> islands are hpp12_ <dig> , hpp12_450/hpg27_ <dig> , hpp12_ <dig>  hpp12_ <dig>  hpp12_ <dig>  hpp12_459- <dig>  and hpp12_ <dig> .
specific genes of icehptfs <dig> islands include hpb8_ <dig>  hpb8_ <dig>  hpb8_ <dig>  hpb8_ <dig>  hpb8_ <dig>  hpb8_ <dig>  hpb8_ <dig>  hpb8_ <dig>  hpb8_ <dig>  hpb8_ <dig>  hpb8_ <dig>  pz <dig> and pz <dig>  interestingly, icehptfs <dig> islands in some strains have insertions of specific genes encoding fic domain-containing or jhp940-like proteins .
the putative dna methylase/helicase gene pz21/hpp12_ <dig> may be found associated with either icehptfs <dig> or icehptfs <dig> islands.
in striking contrast to the above-mentioned divergence between orthologous icehptfs <dig> and icehptfs <dig> genes, the methylase/helicase orthologs present on icehptfs <dig>  and on icehptfs4a/b/c islands  are highly conserved , indicating an evolutionary pressure for this gene which is distinct from other genes on the genome islands.
a neighbor-joining tree of pz21/hpp12_ <dig> orthologs shows a certain clustering according to geographic origin, but this clustering is clearly independent of gene association with either icehptfs <dig> or icehptfs <dig> .
indeed, in cases where both icehptfs <dig> and icehptfs <dig> methylase/helicase orthologs are present in a single strain , these orthologs are always more similar to each other than to icehptfs <dig> or icehptfs <dig> orthologs of geographically related strains, and even more similar than two icehptfs <dig> methylase/helicase orthologs present in a single strain  are to each other .
because of these high sequence similarities, homologous recombination between icehptfs <dig> and icehptfs <dig> methylase/helicase orthologs is possible.
by analysing the gene arrangements of the hybrid icehptfs3-icehptfs <dig> elements mentioned above, we could identify situations where such recombination events seem to have occurred indeed after integration of one ice element into another .
analysis of ice integration sites
originally, the plasticity zone was found located at a distinct position within h. pylori genomes  and one copy of the 5s-23s rrna genes)
<cit> .
however, analysis of strain p <dig>  shi <dig> and g <dig> genome sequences showed that icehptfs <dig> and icehptfs <dig> elements are able to integrate as well into different genomic locations, in a manner similar to conjugative transposons or genome islands
<cit> .
to examine further variations in integration sites, we compared the sequences of ice integration sites and duplicated junction motifs in all genome sequences with recognizable left and/or right icehptfs <dig> and icehptfs <dig> junctions.
in addition to  <dig> different sites described previously
<cit> , we identified further  <dig> chromosomal sites and one plasmid site where complete or partial icehptfs <dig> or icehptfs <dig> elements can be integrated .
although these integration sites cluster in certain genome regions, such as the originally identified ice integration locus , the left border region of icehptfs4a, or a locus containing several restriction-modification system genes , there is no obvious general preference for ice integration.
we also did not observe different patterns of icehptfs <dig> versus icehptfs <dig> integration sites; in fact, some integration sites are used by either icehptfs <dig> or icehptfs <dig> .
all islands with detectable junctions contained the conserved sequence motif aagaatg
<cit> , and this motif is always present in the corresponding empty sites of pz-free strains , suggesting that it represents a minimal requirement for integration of icehptfs <dig> and icehptfs <dig> elements.
to determine whether additional sequences are required to form an integration site, we compared the sequences of the flanking regions of icehptfs <dig> and icehptfs <dig> separately .
there is a certain preference for a or t close to the left junctions of both icehptfs <dig> and icehptfs <dig> islands , but the alignment revealed no significant consensus sequences otherwise.
however, there seems to be a stronger preference of a at the - <dig> position  in icehptfs <dig> than in icehptfs <dig> islands.
furthermore, the low prevalence of the last g at the right junctions of icehptfs <dig> islands may even suggest that only six bases  are used by icehptfs <dig> islands.
<cit> .
the level of sequence conservation is indicated by the height of the letters .
identification of a unique icehptfs <dig> variant in the hpafrica <dig> population
since deletions of single genes or different sets of genes are frequent for both icehptfs <dig> and icehptfs <dig> islands , we checked whether these occur randomly or at conserved sites.
deletions found within icehptfs <dig> variants range from small deletions  to loss of major parts of the island , and mostly seem to occur at random positions and without conserved sequence motifs .
however, we also identified several cases where icehptfs <dig> truncation sites are flanked by aagaatg motifs, suggesting that recombination events similar to ice integration resulted in some deletions .
for icehptfs <dig> islands, we found certain deletions that are more frequent.
for example, four hspeasia strains  have identical truncations of their icehptfs4a islands .
these elements also have identical integration sites  and are accompanied by a common genome rearrangement
<cit> , suggesting that the observed truncations reflect the situation in a common ancestor of all four strains.
in fact, these truncated versions are the only icehptfs4a remnants that we found in hspeasia or hspamerind strains; all other complete or truncated variants in these populations are of the icehptfs4b type.
a second common truncation was found in all hspwafrica strains  and involved a loss of several genes close to the right junctions of their icehptfs4b or icehptfs4a/b islands, including the 5’ regions of the respective virb <dig> genes .
the same deletion occurs in hspwafrica strain j <dig>  where the corresponding virb <dig> gene  is also known as dupa <cit> .
all these icehptfs4b islands have their right junctions deleted and are furthermore inserted at the same genome position , flanked on the truncation site by jhp <dig>  jhp <dig> and jhp <dig> orthologs .
a closer inspection of the right border revealed that truncations have occurred at a cattctt  motif which is conserved in the virb <dig> genes of icehptfs4b  islands.
interestingly, those icehptfs4b variants which contain icehptfs4a genes close to their left borders, all have another small truncation of about 300 bp at their left junctions, which also has occurred at a conserved cattctt motif upstream of the xert gene , indicating that these islands have integrated in an irregular fashion, producing irregular left junctions  and irregular right junctions .
since the nearby jhp <dig> gene has previously been reported to be specifically present in the hpafrica <dig> population
<cit> , we asked whether this truncated right border might be a general signature of hpafrica <dig> strains.
to test this hypothesis, we performed a blast search of draft genome sequences with a 260 bp query sequence spanning the right border of j <dig> .
of  <dig> retrieved draft genome sequences having the same irj,  <dig> also contained the jhp <dig> gene .
furthermore, we checked a panel of h. pylori strains isolated in nigeria for the presence of the irregular icehptfs4b right border .
pcr analysis with primers specific to virb <dig> and jhp <dig>  respectively , confirmed that  <dig> out of  <dig> strains from this population were positive for a similar gene arrangement in this locus and thus for an irj .
<dig>  and asterisks denote frameshift or nonsense mutations  pcr analysis of the icehptfs4b right junction in h. pylori strains from nigeria.
pcr was performed from chromosomal dna of the indicated strains with primers ws <dig> and ws <dig> .
discussion
the unusual genetic heterogeneity of h. pylori has been well-documented in terms of allelic diversity, establishing it as a species with a very high population recombination rate, and allowing for different populations from different geographic regions to be identified
<cit> .
mlst analysis of these populations has revealed important insights into the coevolution of h. pylori and humans, and into migration events of human populations, but relatively little is known about bacterial population-specific properties on a genomic level.
striking differences in the presence or absence of putative host interaction genes have been reported for east asian h. pylori strains in comparison to european strains
<cit> , and many divergent genes were found to evolve under positive selection between east asian and non-asian strains
<cit> .
previous comparative analysis of a small number of h. pylori genome sequences indicated that many strain-specific genes are located either at potential genome rearrangement sites or within the plasticity zones
<cit> .
however, for those plasticity zone regions that are organized in icehptfs <dig> or icehptfs <dig> islands as described here, identification of further novel genes seems unlikely.
instead, the gene content of a given type of icehptfs <dig> or icehptfs <dig> island is, apart from the variable presence of jhp940- or fic domain protein-encoding genes, highly conserved, strongly suggesting that these elements are autonomous elements with fixed contents rather than true regions of genome plasticity.
nevertheless, partial truncations, insertions of restriction-modification systems, is elements or even distinct genome islands, and associated rearrangements
<cit>  are frequent within both types of ice and result in a considerable amount of variation.
rearrangements between icehptfs <dig> and icehptfs <dig> elements may be facilitated by recombination events within pz21/hpp12_ <dig>  orthologs present on both types of islands.
apart from that, icehptfs <dig> and icehptfs <dig> islands are clearly distinct and do not seem to exchange individual genes.
the fact that pz21/hpp12_ <dig> orthologs are the only genes with high similarity between icehptfs <dig> and icehptfs <dig> elements, indicates that these orthologs are either frequently exchanged between both types of island, or that they are subject to strong selective pressures.
interestingly, certain regions of both icehptfs <dig> and icehptfs <dig> islands are much more variable than others.
for instance, we were able to identify  <dig>   <dig>  and  <dig> distinct clades, respectively, for the pz <dig>  pz <dig> and pz <dig> orthologs on icehptfs <dig> elements , whereas all other icehptfs <dig> genes are more conserved.
however, similar to the variability of hpp12_444/ <dig> and hpp12_ <dig> orthologs among icehptfs <dig> islands, where two clades each can be distinguished , no clear correlation of these different clades with individual geographic groups could be found.
likewise, the three different subtypes of icehptfs <dig> islands which are characterized by orthologous, but distinct sets of genes, do not seem to be restricted to certain geographic groups.
we also performed a preliminary analysis of two further hpafrica <dig> strain genome sequences
<cit>  and one hpsahul strain genome sequence
<cit>  that were published after completion of our comparative analysis.
both hpafrica <dig> strains contain one full-length icehptfs4b element, and the hpsahul strain harbours a full-length icehptfs4b and a partial icehptfs <dig> element , which further supports the notion that these elements are present in all phylogeographic groups.
the modular structure of icehptfs <dig> islands indicates that parts of these elements can easily be exchanged, and that all variants may coexist in a given h. pylori population.
indeed, icehptfs4a, b and c islands all have some common genes which may be used for exchange of modules.
however, it is striking that all members of icehptfs4b subtypes consistently lack hpp12_ <dig> orthologs and that hybrid elements between different icehptfs <dig> subtypes do not occur.
an exception is the combination of icehptfs4a  with icehptfs4b , which seems to occur in hpafrica <dig> strains only, and always in a truncated version.
these restrictions on modular exchange suggest that there is a selective pressure on maintenance of cognate left and right icehptfs <dig> ends, for example by an inability of hybrid elements to be excised and/or transferred.
the presence of icehptfs3-like islands in other helicobacter species, such as h. cetorum <cit>  and h. suis <cit> , indicates that these elements were acquired a long time ago .
whereas microdiversity within cag pathogenicity island genes correlates with microdiversity in housekeeping genes, this is not the case for icehptfs <dig> or icehptfs <dig> genes, which shows again that these islands are subject to more frequent horizontal gene transfer.
horizontal gene transfer of typical ices involves several steps
<cit> : first, the element is usually excised from the chromosome by a recombinase to generate a circular intermediate; second, this circular form is transferred from the donor to a recipient cell by conjugation; and third, the ice integrates into the recipient cell chromosome via site-specific or unspecific recombination.
in the case of icehptfs <dig>  the first step is dependent on the xert recombinase
<cit> , and the second on the vird <dig> relaxase
<cit> , both of which are encoded on the ice.
it is likely, but has not been shown yet, that the ice-encoded type iv secretion system is responsible for the conjugative transfer process.
it is also currently unclear whether the xert recombinase catalyzes integration of the ice into the recipient cell chromosome as well.
an interesting finding of this study was the presumptive minimal requirement for integration of both icehptfs <dig> and icehptfs <dig> islands, the sequence motif aagaatg , as suspected previously
<cit> .
thus, the total number of possible insertion sites might be limited only by the number of these motifs in intergenic regions or in non-essential genes.
in total, we identified more than  <dig> different integration sites, but the total number of possible integration sites might be significantly higher, given that aagaatg sequences are found approximately  <dig> times within individual h. pylori genomes .
many well-characterized ices integrate into a unique position in the host cell genome , often in the 3’ regions of trna loci
<cit> .
in the absence of primary attachment sites, these elements are sometimes capable of integrating into secondary sites with much less specificity, but this may result in ice immobility or even toxicity for the host cell
<cit> .
in contrast, other ice-like elements, which are often termed conjugative transposons, have very low integration site specificities, with as many as  <dig>  possible integration sites in a given host strain
<cit> .
in this regard, icehptfs <dig> and icehptfs <dig> seem to integrate with an intermediate specificity, but still with the potential to insert into coding regions and thereby to disrupt essential genes.
possible integration sites are also located on the ice elements themselves, and we found several cases where one ice is integrated into another.
we could also identify situations where these internal sites were used for irregular ice integration, associated with truncation of the left and/or right ice ends, and possibly an incapability of these elements to excise.
finally, despite the presence of genes encoding host interaction factors such as jhp940
<cit> , or correlated with disease outcome, such as dupa <cit> , the  functions of icehptfs <dig> and icehptfs <dig> islands are currently unclear.
in our analysis, a total of  <dig> strains were positive for dupa , and  <dig> additional strains were found positive for icehptfs4a or icehptfs4c virb <dig> genes, which are likely to have the same functions.
because of this, and since not all of these strains have complete ices or even complete type iv secretion systems, testing for the presence of the dupa gene alone, and correlations of dupa with pathology is probably not useful.
it has been shown that a more complete analysis of type iv secretion system genes is more significant as a virulence marker
<cit> .
therefore, future correlation studies should determine the presence of the complete set of genes.
CONCLUSIONS
taken together, our comparative analysis reinforces the notion that major parts of the h. pylori plasticity zones described earlier should in fact be considered as mobile genetic elements with conserved gene content, rather than regions of genome plasticity.
although horizontal gene transfer of complete icehptfs <dig> or icehptfs <dig> elements remains to be demonstrated experimentally, the number of different integration sites indicates a considerable mobility, possibly also within individual h. pylori genomes.
in this regard, these elements differ from the cag pathogenicity island, for which only one integration site is known .
the high prevalence and wide distribution of these ices throughout all h. pylori populations suggest that they might provide an as yet unknown fitness benefit to their hosts.

 infections with the human gastric pathogen h. pylori are paradigmatic examples of chronic, or persistent, bacterial infections in the face of a constant immune response. h. pylori infections are usually contracted during early childhood and persist for the lifetime of the host, but most infected individuals develop only mild gastric inflammation without overt symptoms. nevertheless, a substantial fraction of infected persons develops more severe consequences, making h. pylori the principal cause of chronic active gastritis and peptic ulcer disease, and a major risk factor for development of gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma. for survival and persistent growth in the presence of a constant immune response and in an environment which is changing considerably over decades of infection, permanent adaptation of the bacteria is thought to be required. such adaptive processes may include regulatory mechanisms acting on gene expression, but also reversible or irreversible genome changes. for instance, it has been shown that strains isolated from patients with atrophic gastritis or marginal zone b-cell malt lymphoma have reduced genomes in comparison to gastritis or ulcer strains, and a strain isolated from a gastric cancer patient had lost further genes in comparison to a strain isolated previously from the same patient during atrophic gastritis. that genome plasticity plays a role in bacterial persistence is further supported by the observation that natural transformation competence, which is upregulated upon dna stress, promotes persistent colonization in mice. allelic diversity caused by high mutation rates and frequent recombination events is a striking property of h. pylori strains. genetic fingerprints of individual strains obtained by multilocus sequence typing of housekeeping genes have indicated that clonal transmission is likely to occur, but is followed by a rapid adaptation to the new host, so that h. pylori isolates from different subjects are almost always unique. on the other hand, while recombination events generating allelic diversity are frequent, genome changes involving gain or loss of genes seem to be rare. nevertheless, on the level of gene content, evidence has been presented that h. pylori is a species with an open pan-genome, in which each individual isolate contains a distinct set of non-core, or strain-specific, genes. comparative analysis of the first sequenced h. pylori genomes suggested that these strain-specific genes are often located in genomic regions that had previously been termed plasticity zones or plasticity regions, a designation originally used to describe a particular genetic locus with high variation between the first two h. pylori genome sequences. however, with the availability of more sequencing data and more complete h. pylori genome sequences, it became clear that parts of the plasticity regions are usually organized as genomic islands that may be integrated in one of several different genetic loci. furthermore, they generally contain complete sets of genes required to produce type iv secretion machineries, as well as genes encoding different dna-processing proteins, suggesting that they are actually mobile genetic elements capable of horizontal gene transfer between bacterial cells, and that they might be best described as conjugative transposons or integrating conjugative elements. the actual plasticity of these islands partly derives from the fact that gene rearrangements, insertions or deletions may have occurred within them, but it is not clear whether they also carry variable passenger genes. interestingly, intrahost variation among genes of the plasticity zones, including deletions in a type iv secretion system gene, has been found for sequential isolates obtained from a duodenal ulcer patient over a course of 10 years. although several candidate genes of these plasticity regions have been suggested as disease markers, e.g. dupa for duodenal ulcer, or jhp for marginal zone b-cell malt lymphoma, the functions of the plasticity zones are currently not well-understood. to address the question of plasticity zone prevalence, and of their genetic diversity, we have performed a comparative analysis of these genome islands from a larger number of h. pylori genome sequences, including newly determined genome sequences of nine additional strains from different backgrounds. we show that these elements have a high prevalence throughout all populations, and that gene evolution within the elements is not congruent with the rest of the genomes. the wide variety of integration loci together with a conserved sequence motif at each integration site suggests an integration mechanism that depends on a short recognition motif in the dna sequence only.  prevalence of plasticity regions in the h. pylori population we have reported previously that h. pylori strain p contains three genome regions with similarity to the prototypical plasticity zones, but only one of them corresponds to the originally described locus, whereas the other two regions have a genetic organization typical for genome islands and contain genes for type iv secretion systems that might make them capable of self-transfer. in comparison, the original two genome sequences contain only truncated and highly rearranged portions of these genome islands. as reported previously, the most conserved type iv secretion system genes fall into one of two distinct groups, which have been termed either tfs and tfs3a/b, or tfs and tfs. in accordance with ref., where conserved tfs genes have been shown not to be more closely related to tfs genes than to the respective comb genes encoding the type iv secretion system used for natural transformation, we consider tfs and tfs here as independent systems. moreover, since there is evidence for horizontal gene transfer of the corresponding islands, but not for transposition within a strain, we propose to use the term integrating conjugative elements and refer to individual islands as icehptfs or icehptfs respectively. a comparison of different designations of the islands and associated type iv secretion systems is given in table  to determine the occurrence of icehptfs and icehptfs elements in the h. pylori population and the degree of variation among them, we performed a comparative sequence analysis of these elements from completely sequenced h. pylori genomes available in public databases. n.a., not applicable. 14 2018 1 1strains and are sequential isolates from a single patient and do not show major differences in their icehptfs sequences. however, note that genbank entries ef, ef and ef describe icehptfs clusters in these strains that are not present in the genome sequences. 2helpy is possibly a vestige of hpp12_1321/pz 3resulting from insertions of 2- genomic islands and subsequent rearrangements. 4containing icehptfs4a-type genes close to the left junction, and icehptfs4b-type genes close to the right junction. 5associated with genome rearrangement in comparison to strain p 6associated with deletion of hpp12_ to hpp12_ including one copy of 5s-23s-rrna. 7associated with a recombination between the two 5s-23s-rrna loci. 8partial duplication of both genes; icehptfs inserted into truncated icehptfs4b. 9within a restriction-modification system inserted into this region. 10integrated together with a  kb fragment of icehptfs and a putative toxin-antitoxin system. 11integration of icehptfs4a into remnant of icehptfs4b, which is in turn integrated into truncated icehptfs 12irregular integration, using internal aagaatg motif. 13left and right junctions coincide due to irregular integration. 14numbers in parentheses indicate incomplete ice elements. 15disrupted by a chromosomal inversion from hpp12_ to hpp12_ 16size of ice increased by is element insertion. 17interrupted by a chromosomal rearrangement between hpp12_ and hpp12_. 18original integration probably in hpp12_994-5s-rrna locus; from there, relocation of  kb fragment via internal aagaatg motifs into hpp12_1510;  kb duplication in both loci. we found that only out of these strains do not contain icehptfs or icehptfs islands or fragments thereof. among the remaining strains, harbour icehptfs islands, of which seem to have complete gene sets, and harbour icehptfs islands, of which are complete. there are strains with two different icehptfs elements, and strains which have at least parts of both icehptfs and icehptfs three strains contain hybrid arrangements of icehptfs and icehptfs islands, but these seem to result from dna rearrangements after integration of two independent genome islands. thus, each complete or truncated island can be assigned to either the icehptfs or the icehptfs type. within the icehptfs group, two distinct variants can be discriminated, which differ by the presence or absence of the pz21-pz genes. in contrast, three variants of icehptfs defined by orthologous, but variant sets of genes at both ends of the genome islands, or in their central regions, can be distinguished and are termed here icehptfs4a, icehptfs4b and icehptfs4c, respectively. the third subtype, icehptfs4c, was only found in strain southafrica which belongs to the hpafrica population, and as a plasmid-borne fragment in strain lithuania both types of genome island seem to vary considerably in size between strains, but this is often due to small deletions within the islands or to insertion of is elements; therefore, complete icehptfs islands have “standard” sizes of about, or 46 kb, depending on the presence of pz21- orthologs, while complete icehptfs4a, icehptfs4b and icehptfs4c usually comprise about, and  kb, respectively. geographic distribution of icehptfs and icehptfs islands it is well-established that h. pylori strains cluster into distinct populations according to their geographic origin when multilocus sequence typing using partial sequences of seven housekeeping genes is employed. in contrast to this allelic variability, which suggests a common evolution of h. pylori and humans, consistent gene content profiles of individual populations could not be found, with the exception of one hypothetical gene present only in strains from the hpafrica population. interestingly, comparison of gene content microarray data with icehptfs composition suggests that most hpafrica strains contain icehptfs4a genes close to the left junctions and in the mid region, but icehptfs4b genes close to the right junctions, while hpeurope strains variably contain these genes. since there are only three hpafrica strains among the complete genome sequences analysed, we decided to determine draft genome sequences of three further strains originating from western africa, as well as of six strains isolated in europe, five of which had been tested positive for the presence of an icehptfs4a-type or an icehptfs4b-type virb gene. sequence analysis revealed that all strains except one contain at least 37 kb of icehptfs and/or icehptfs sequences. 1 4 5 1inferred from the neighbor-joining tree shown in figure  2resulting from insertion of two genome islands and rearrangements associated with is element insertion and two copies of pz21/hpp12_447-like genes. 3associated with a genome rearrangement between hpp12_ and hpp12_ 4genomic position of aagaatg motif in strain p 5numbers in parentheses indicate incomplete ice elements. 6contains 28 kb of prophage-related sequences. to examine possible variations in plasticity zone distribution among phylogeographic groups, we first constructed a phylogenetic tree based on mlst gene sequences, using all fully sequenced strains, the nine strains sequenced in this study, and reference strains from the mlst database. no correlation between phylogeographic groups and the presence or absence of either icehptfs or icehptfs could be found. however, all hpafrica strains contain truncated versions of icehptfs4b or of an icehptfs4a/b variant similar to the hpafrica strains mentioned above. we then calculated neighbor-joining phylogenetic trees using conserved icehptfs or icehptfs gene sequences and compared them with an mlst-derived tree. interestingly, icehptfs4ab genes clustered in a similar way as housekeeping gene sequences did, except for a much closer relationship of these genes than of housekeeping genes between hpafrica strain southafrica and other populations. in contrast, icehptfs sequences formed at least three strongly divergent clades that were not congruent with the mlst population structure. these clades seem to correspond to the hspamerind population; a mixture of hspeasia and hpasia populations; and a mixture of hpeurope and hpafrica populations. however, the number of icehptfs3-positive strains analysed may be too low to definitely draw conclusions from this observation. note that unequivocal classification of strains pecan and pecan was not possible. phylogenetic tree calculated from concatenated virb virb and vird ortholog sequences of all icehptfs and icehptfs islands. neighbor-joining tree calculated from dna sequences of methylase/helicase orthologs. orthologs associated with icehptfs elements are marked by blue branch lines, and orthologs associated with icehptfs elements by red branch lines. black lines indicate hybrid elements or the presence of two different elements in the same strain. colouring of individual strains by phylogeographic origin is shown according to the tree in figure  identification of conserved and ice type-specific genes since both icehptfs and icehptfs islands contain genes for complete type iv secretion systems and may coexist in a single strain, an open question is whether individual genes or groups of genes from one type of island have the capacity to complement deficiencies in the other. sequence comparisons showed that each of the type iv secretion apparatus components is clearly distinguishable between the different types of islands, with amino acid sequence similarities ranging from 40% to 80%. this is also true for putative dna processing or segregation proteins such as xert, para, topa or vird /hpp12_447; see below), suggesting that the individual secretion systems might be sufficiently divergent to be incompatible. hptfs4a -encoded proteins and proteins from ice hptfs and ice hptfs4b/c islands hptfs4b 1 hptfs4c 1 hptfs3 hptfs3 1 2 2 4 6 1numbers printed in normal face correspond to >90% identity, and numbers in bold face to 40-85% similarity. 2genes hpb8_ and pz as well as hpb8_ and pz share only 61/73% and 54/70% identity/similarity, respectively, to each other, but are equally similar to hpp12_ and hpp12_ respectively. 3some icehptfs4c islands contain the icehptfs4b versions with lower similarities in these sites. 4similarities confined to short regions only. 5no significant similarity detectable, but gene with similar size and orientation present. 6icehptfs4c and icehptfs islands contain fusions of hpp12_ and hpp12_ to define further common ice gene products and to identify ice-type-specific genes, we performed similarity searches with all other amino acid sequences as well. the results show that nine further, hypothetical icehptfs4a genes have similar counterparts in icehptfs3-type islands. interestingly, orthologs of the conserved hypothetical genes hpb8_ or hpp12_ are present in icehptfs icehptfs4a and icehptfs4c islands, but absent from icehptfs4b islands. because of their sequence similarities, we speculate that these hypothetical genes have additional conserved functions for genome island maintenance and/or transfer. in contrast, genes that are specific for either type of genome island might be cargo proteins of the respective mobile genetic elements, fulfilling more specific roles. such specific genes for icehptfs islands are hpp12_, hpp12_450/hpg27_, hpp12_ hpp12_ hpp12_ hpp12_459- and hpp12_. specific genes of icehptfs islands include hpb8_ hpb8_ hpb8_ hpb8_ hpb8_ hpb8_ hpb8_ hpb8_ hpb8_ hpb8_ hpb8_ pz and pz interestingly, icehptfs islands in some strains have insertions of specific genes encoding fic domain-containing or jhp940-like proteins. the putative dna methylase/helicase gene pz21/hpp12_ may be found associated with either icehptfs or icehptfs islands. in striking contrast to the above-mentioned divergence between orthologous icehptfs and icehptfs genes, the methylase/helicase orthologs present on icehptfs and on icehptfs4a/b/c islands are highly conserved, indicating an evolutionary pressure for this gene which is distinct from other genes on the genome islands. a neighbor-joining tree of pz21/hpp12_ orthologs shows a certain clustering according to geographic origin, but this clustering is clearly independent of gene association with either icehptfs or icehptfs. indeed, in cases where both icehptfs and icehptfs methylase/helicase orthologs are present in a single strain, these orthologs are always more similar to each other than to icehptfs or icehptfs orthologs of geographically related strains, and even more similar than two icehptfs methylase/helicase orthologs present in a single strain are to each other. because of these high sequence similarities, homologous recombination between icehptfs and icehptfs methylase/helicase orthologs is possible. by analysing the gene arrangements of the hybrid icehptfs3-icehptfs elements mentioned above, we could identify situations where such recombination events seem to have occurred indeed after integration of one ice element into another. analysis of ice integration sites originally, the plasticity zone was found located at a distinct position within h. pylori genomes and one copy of the 5s-23s rrna genes). however, analysis of strain p shi and g genome sequences showed that icehptfs and icehptfs elements are able to integrate as well into different genomic locations, in a manner similar to conjugative transposons or genome islands. to examine further variations in integration sites, we compared the sequences of ice integration sites and duplicated junction motifs in all genome sequences with recognizable left and/or right icehptfs and icehptfs junctions. in addition to different sites described previously, we identified further chromosomal sites and one plasmid site where complete or partial icehptfs or icehptfs elements can be integrated. although these integration sites cluster in certain genome regions, such as the originally identified ice integration locus, the left border region of icehptfs4a, or a locus containing several restriction-modification system genes, there is no obvious general preference for ice integration. we also did not observe different patterns of icehptfs versus icehptfs integration sites; in fact, some integration sites are used by either icehptfs or icehptfs. all islands with detectable junctions contained the conserved sequence motif aagaatg, and this motif is always present in the corresponding empty sites of pz-free strains, suggesting that it represents a minimal requirement for integration of icehptfs and icehptfs elements. to determine whether additional sequences are required to form an integration site, we compared the sequences of the flanking regions of icehptfs and icehptfs separately. there is a certain preference for a or t close to the left junctions of both icehptfs and icehptfs islands, but the alignment revealed no significant consensus sequences otherwise. however, there seems to be a stronger preference of a at the - position in icehptfs than in icehptfs islands. furthermore, the low prevalence of the last g at the right junctions of icehptfs islands may even suggest that only six bases are used by icehptfs islands.. the level of sequence conservation is indicated by the height of the letters. identification of a unique icehptfs variant in the hpafrica population since deletions of single genes or different sets of genes are frequent for both icehptfs and icehptfs islands, we checked whether these occur randomly or at conserved sites. deletions found within icehptfs variants range from small deletions to loss of major parts of the island, and mostly seem to occur at random positions and without conserved sequence motifs. however, we also identified several cases where icehptfs truncation sites are flanked by aagaatg motifs, suggesting that recombination events similar to ice integration resulted in some deletions. for icehptfs islands, we found certain deletions that are more frequent. for example, four hspeasia strains have identical truncations of their icehptfs4a islands. these elements also have identical integration sites and are accompanied by a common genome rearrangement, suggesting that the observed truncations reflect the situation in a common ancestor of all four strains. in fact, these truncated versions are the only icehptfs4a remnants that we found in hspeasia or hspamerind strains; all other complete or truncated variants in these populations are of the icehptfs4b type. a second common truncation was found in all hspwafrica strains and involved a loss of several genes close to the right junctions of their icehptfs4b or icehptfs4a/b islands, including the 5’ regions of the respective virb genes. the same deletion occurs in hspwafrica strain j where the corresponding virb gene is also known as dupa. all these icehptfs4b islands have their right junctions deleted and are furthermore inserted at the same genome position, flanked on the truncation site by jhp jhp and jhp orthologs. a closer inspection of the right border revealed that truncations have occurred at a cattctt motif which is conserved in the virb genes of icehptfs4b islands. interestingly, those icehptfs4b variants which contain icehptfs4a genes close to their left borders, all have another small truncation of about 300 bp at their left junctions, which also has occurred at a conserved cattctt motif upstream of the xert gene, indicating that these islands have integrated in an irregular fashion, producing irregular left junctions and irregular right junctions. since the nearby jhp gene has previously been reported to be specifically present in the hpafrica population, we asked whether this truncated right border might be a general signature of hpafrica strains. to test this hypothesis, we performed a blast search of draft genome sequences with a 260 bp query sequence spanning the right border of j. of retrieved draft genome sequences having the same irj, also contained the jhp gene. furthermore, we checked a panel of h. pylori strains isolated in nigeria for the presence of the irregular icehptfs4b right border. pcr analysis with primers specific to virb and jhp respectively, confirmed that out of strains from this population were positive for a similar gene arrangement in this locus and thus for an irj. and asterisks denote frameshift or nonsense mutations pcr analysis of the icehptfs4b right junction in h. pylori strains from nigeria. pcr was performed from chromosomal dna of the indicated strains with primers ws and ws. discussion the unusual genetic heterogeneity of h. pylori has been well-documented in terms of allelic diversity, establishing it as a species with a very high population recombination rate, and allowing for different populations from different geographic regions to be identified. mlst analysis of these populations has revealed important insights into the coevolution of h. pylori and humans, and into migration events of human populations, but relatively little is known about bacterial population-specific properties on a genomic level. striking differences in the presence or absence of putative host interaction genes have been reported for east asian h. pylori strains in comparison to european strains, and many divergent genes were found to evolve under positive selection between east asian and non-asian strains. previous comparative analysis of a small number of h. pylori genome sequences indicated that many strain-specific genes are located either at potential genome rearrangement sites or within the plasticity zones. however, for those plasticity zone regions that are organized in icehptfs or icehptfs islands as described here, identification of further novel genes seems unlikely. instead, the gene content of a given type of icehptfs or icehptfs island is, apart from the variable presence of jhp940- or fic domain protein-encoding genes, highly conserved, strongly suggesting that these elements are autonomous elements with fixed contents rather than true regions of genome plasticity. nevertheless, partial truncations, insertions of restriction-modification systems, is elements or even distinct genome islands, and associated rearrangements are frequent within both types of ice and result in a considerable amount of variation. rearrangements between icehptfs and icehptfs elements may be facilitated by recombination events within pz21/hpp12_ orthologs present on both types of islands. apart from that, icehptfs and icehptfs islands are clearly distinct and do not seem to exchange individual genes. the fact that pz21/hpp12_ orthologs are the only genes with high similarity between icehptfs and icehptfs elements, indicates that these orthologs are either frequently exchanged between both types of island, or that they are subject to strong selective pressures. interestingly, certain regions of both icehptfs and icehptfs islands are much more variable than others. for instance, we were able to identify and distinct clades, respectively, for the pz pz and pz orthologs on icehptfs elements, whereas all other icehptfs genes are more conserved. however, similar to the variability of hpp12_444/ and hpp12_ orthologs among icehptfs islands, where two clades each can be distinguished, no clear correlation of these different clades with individual geographic groups could be found. likewise, the three different subtypes of icehptfs islands which are characterized by orthologous, but distinct sets of genes, do not seem to be restricted to certain geographic groups. we also performed a preliminary analysis of two further hpafrica strain genome sequences and one hpsahul strain genome sequence that were published after completion of our comparative analysis. both hpafrica strains contain one full-length icehptfs4b element, and the hpsahul strain harbours a full-length icehptfs4b and a partial icehptfs element, which further supports the notion that these elements are present in all phylogeographic groups. the modular structure of icehptfs islands indicates that parts of these elements can easily be exchanged, and that all variants may coexist in a given h. pylori population. indeed, icehptfs4a, b and c islands all have some common genes which may be used for exchange of modules. however, it is striking that all members of icehptfs4b subtypes consistently lack hpp12_ orthologs and that hybrid elements between different icehptfs subtypes do not occur. an exception is the combination of icehptfs4a with icehptfs4b, which seems to occur in hpafrica strains only, and always in a truncated version. these restrictions on modular exchange suggest that there is a selective pressure on maintenance of cognate left and right icehptfs ends, for example by an inability of hybrid elements to be excised and/or transferred. the presence of icehptfs3-like islands in other helicobacter species, such as h. cetorum and h. suis, indicates that these elements were acquired a long time ago. whereas microdiversity within cag pathogenicity island genes correlates with microdiversity in housekeeping genes, this is not the case for icehptfs or icehptfs genes, which shows again that these islands are subject to more frequent horizontal gene transfer. horizontal gene transfer of typical ices involves several steps: first, the element is usually excised from the chromosome by a recombinase to generate a circular intermediate; second, this circular form is transferred from the donor to a recipient cell by conjugation; and third, the ice integrates into the recipient cell chromosome via site-specific or unspecific recombination. in the case of icehptfs the first step is dependent on the xert recombinase, and the second on the vird relaxase, both of which are encoded on the ice. it is likely, but has not been shown yet, that the ice-encoded type iv secretion system is responsible for the conjugative transfer process. it is also currently unclear whether the xert recombinase catalyzes integration of the ice into the recipient cell chromosome as well. an interesting finding of this study was the presumptive minimal requirement for integration of both icehptfs and icehptfs islands, the sequence motif aagaatg, as suspected previously. thus, the total number of possible insertion sites might be limited only by the number of these motifs in intergenic regions or in non-essential genes. in total, we identified more than different integration sites, but the total number of possible integration sites might be significantly higher, given that aagaatg sequences are found approximately times within individual h. pylori genomes. many well-characterized ices integrate into a unique position in the host cell genome, often in the 3’ regions of trna loci. in the absence of primary attachment sites, these elements are sometimes capable of integrating into secondary sites with much less specificity, but this may result in ice immobility or even toxicity for the host cell. in contrast, other ice-like elements, which are often termed conjugative transposons, have very low integration site specificities, with as many as possible integration sites in a given host strain. in this regard, icehptfs and icehptfs seem to integrate with an intermediate specificity, but still with the potential to insert into coding regions and thereby to disrupt essential genes. possible integration sites are also located on the ice elements themselves, and we found several cases where one ice is integrated into another. we could also identify situations where these internal sites were used for irregular ice integration, associated with truncation of the left and/or right ice ends, and possibly an incapability of these elements to excise. finally, despite the presence of genes encoding host interaction factors such as jhp940, or correlated with disease outcome, such as dupa, the functions of icehptfs and icehptfs islands are currently unclear. in our analysis, a total of strains were positive for dupa, and additional strains were found positive for icehptfs4a or icehptfs4c virb genes, which are likely to have the same functions. because of this, and since not all of these strains have complete ices or even complete type iv secretion systems, testing for the presence of the dupa gene alone, and correlations of dupa with pathology is probably not useful. it has been shown that a more complete analysis of type iv secretion system genes is more significant as a virulence marker. therefore, future correlation studies should determine the presence of the complete set of genes.  taken together, our comparative analysis reinforces the notion that major parts of the h. pylori plasticity zones described earlier should in fact be considered as mobile genetic elements with conserved gene content, rather than regions of genome plasticity. although horizontal gene transfer of complete icehptfs or icehptfs elements remains to be demonstrated experimentally, the number of different integration sites indicates a considerable mobility, possibly also within individual h. pylori genomes. in this regard, these elements differ from the cag pathogenicity island, for which only one integration site is known. the high prevalence and wide distribution of these ices throughout all h. pylori populations suggest that they might provide an as yet unknown fitness benefit to their hosts. 
their genetic diversity is incongruent with variations in the rest of the genome, suggesting that they are subject to horizontal gene transfer within h. pylori populations. plasticity zonehelicobacter pyloriintegrating conjugative elementtype iv secretion systemhorizontal gene transfer we show that these regions contain fixed gene sets, rather than being true regions of genome plasticity, but two different types and several subtypes with partly diverging gene content can be distinguished. we have comparatively analysed such clusters, which are commonly termed plasticity zones, in a high number of h. pylori strains of varying geographical origin. the significant number of possible integration sites, together with the requirement for a short conserved integration motif and the high level of gene conservation, indicates that these elements are best described as integrating conjugative elements with an intermediate integration site specificity. strain-specific genes are dispersed throughout the genome, but clusters of genes organized as genomic islands may also collectively be present or absent. we identified distinct integration sites in genome sequences, with a conserved heptanucleotide motif that seems to be the minimal requirement for integration.
 the human gastric pathogen helicobacter pylori is a paradigm for chronic bacterial infections. its persistence in the stomach mucosa is facilitated by several mechanisms of immune evasion and immune modulation, but also by an unusual genetic variability which might account for the capability to adapt to changing environmental conditions during long-term colonization. this variability is reflected by the fact that almost each infected individual is colonized by a genetically unique strain. strain-specific genes are dispersed throughout the genome, but clusters of genes organized as genomic islands may also collectively be present or absent.  we have comparatively analysed such clusters, which are commonly termed plasticity zones, in a high number of h. pylori strains of varying geographical origin. we show that these regions contain fixed gene sets, rather than being true regions of genome plasticity, but two different types and several subtypes with partly diverging gene content can be distinguished. their genetic diversity is incongruent with variations in the rest of the genome, suggesting that they are subject to horizontal gene transfer within h. pylori populations. we identified distinct integration sites in genome sequences, with a conserved heptanucleotide motif that seems to be the minimal requirement for integration.  the significant number of possible integration sites, together with the requirement for a short conserved integration motif and the high level of gene conservation, indicates that these elements are best described as integrating conjugative elements with an intermediate integration site specificity. plasticity zonehelicobacter pyloriintegrating conjugative elementtype iv secretion systemhorizontal gene transfer 