BACKGROUND
prokaryotic microbes have sex.
this has been known for very long, individuals exchange genetic material to create hybrids that have different properties from the donor cells.
however, contrastingly with eukaryotes genetic exchange is independent from reproduction  and can act on smaller sections of the genome rather than the complete hybrid generated by meiosis in eukaryotes.
as a consequence, there are fewer restrains regarding donors and receptors  <cit> .
one direct consequence of prokaryotic sex is that cells of the same species can have very different gene complements, what has led to a new paradigm, the pangenome  <cit> , a theoretical catalog of the gene complement of the species at large.
the pangenome includes a core of genes that is common to all  of the strains in the taxon considered and a flexible pool that varies from strain to strain.
since the discovery and latter proposal of this term, the numbers of genes in prokaryotic pangenomes have not stopped of amazing microbiologists.
a recent census in escherichia coli reached close to  <dig>  different gene families with  <dig>  strain genomes analyzed and remarkably the discovery of new genes continue at a pace of ca.
<dig> new per strain sequenced  <cit> .
however, in spite of the apparent enormous size of prokaryotic flexible genomes, there has to be rules that are applicable to how these genes circulate through the population.
for some years we have studied the marine bacterium alteromonas with the focus on understanding its genomic make up and dynamics .
this microbe is a typical r-selected specialist in which success is based in high growth rates and exploitation of transient  niches.
its species have large cells and genomes and can grow very fast when nutrients are available.
the analyses of genome sequences of alteromonas species isolated from all around the world revealed remarkable conservation of synteny what has allowed us to study the physical arrangement of the core and flexible regions in the genomes of representatives of this genus  <cit> .
using comparative genomics we proposed a model of short term evolution  <cit>  in which clones diverge forming different clonal lineages, the smallest unit of differentiation of prokaryotes , by acquisition of specific glycosydic receptors that were called glycotypes  <cit> .
it has been already established that the flexible genome is largely collected in genomic islands that appear at equivalent positions in the genomes of the different isolates known as flexible genomic islands .
there are two types of fgis with different mechanisms of variation: i) additive fgis vary by addition or subtraction of gene cassettes, largely by site directed recombination, which movement among lineages appears to be relatively fast, and ii) replacement fgis that code for major exposed structures of the cell  and are exchanged by double crossover homologous recombination, using the neighboring highly conserved genes as substrate  <cit> .
they vary at a much slower pace and remain linked for relatively longer times preventing population-wide gene sweeps  <cit> , making a specific clone, characterized by its surface glycotype and recognized by specific populations of phages, a single selection unit  <cit> .
to get a more complete picture of the mechanistic variation of alteromonas genomes an important element was missing.
what are the mechanisms by which gene pools travel from one clone to another?
alteromonas is not known to have natural transformation and transmission through phages, even specialized gene transfer agents, are unlikely to mediate the transfer of large genomic regions such as replacement fgis.
for these large transfers conjugation mediated by plasmids or integrative and conjugative elements  would be the most likely route in a gram negative bacterium such as alteromonas.
both, conjugative plasmids and ices  have been described before in alteromonas .
plasmids are extra-chromosomal and auto-replicating dna molecules  composed by a segment containing genetic information required for their replication and maintenance, as well as other accessory genes.
plasmid genes are normally dispensable for the essential functions of the cell.
they are transferred using the tra system coded by themselves or by other replicons and can mobilize large segments of the chromosome that can thus be moved to different chromosomal backgrounds.
ices are also transferable by conjugation but, unlike plasmids, they are always integrated into the host chromosome  and have been found in both gram-positive and gram-negative bacteria  <cit> .
integration in the chromosome occurs via site-specific recombination.
although they are not as well-known as plasmids recently guglielmini et al.
<cit>  showed that they are the most abundant conjugative elements in practically all prokaryotic clades.
these elements contain a group of core genes clustered into distinct modules required for integration/excision, conjugative transfer and regulation  <cit> .
in addition they can acquire new dna in specific sites proposed as hotspots by beaber et al.
<cit> .
most of cargo genes  in these modules encode for restriction modification, antibiotic and metal resistance systems  <cit> .
the aim of this study was to make a detailed comparative analysis of ces obtained from different strains of the genus alteromonas to explore the evolutionary relationships not only among the different ces but also with the chromosomes and how they could play a role in the genomic variation  detected at the genomic level among strains and species of the alteromonas genus.
the patterns found might be extrapolated at least to other aquatic gram negative bacteria.
RESULTS
in order to characterize ces we examined all the strains available in databases of the genus alteromonas searching for plasmids or ices.
in addition we are reporting here six new genomes of strains from different locations that have been fully sequenced and assembled and contain ces .
in order to investigate the phylogenomic relationships of the new alteromonas strains within the alteromonas genus, whole-genome phylogeny was inferred from a concatenate of the core genome .
presently, strains of the genus alteromonas are classified into six different species  but using an average nucleotide identity  similarity threshold of 95% the thirty-seven strains in the tree of fig.
<dig> can be classified into  <dig> genospecies.
our data also indicates that the new strains ar <dig>  cp <dig>  cp <dig> and rg <dig> are closely related and clearly belong to a. mediterranea.
however, strains alteromonas sp.
mex <dig> and alteromonas sp.
rw2a <dig> had similarities ca.
75% to the closest genospecies and could be defined as distant and novel genospecies.
the general features of the new strain genomes used in this study are shown in additional file 1: table s <dig> and fig.
<dig> fig.
<dig> phylogenomic tree constructed using a concatenated of the core proteome  in all available alteromonas genomes.
pseudoalteromonas atlantica t6c was used as outgroup.
strains with genomes reported here for the first time are highlighted in red.
strains that belong to the same species are highlighted by the same colour.
presence of either plasmids or ices are indicated with circles or triangles, same colour indicates same version  of the genomic element.
the number in the corner of the rectangles indicates the number of genospecies
plasmids
among the analysed alteromonas strains only  <dig> had plasmids  totalling nine different replicons  that were found in only three different species .
three of them are reported here for the first time .
another plasmid  has been described in the strain a. macleodii ad <dig>  <cit>  but we decided not to include it since it was identified as a putative defective phage.
with the exception of pamde1- <dig> that was found with nearly identical sequence in different strains belonging to different a. mediterranea strains, and the pair pamcp48-600/pamcp49- <dig> that only differ in  <dig> kb, the others were found only in one strain.
plasmid size range was from  <dig> kb  to  <dig> kb  .
the average gc content was always lower than the host chromosome with the highest difference  observed for pamrg65- <dig> .
based on their mobility , plasmids can be classified in three different classes .
all alteromonas largest plasmids > <dig> kb can be classified as conjugative plasmids since they have a mob  and mpf  genes  <cit> , except paste61- <dig>  which has an incomplete t4ss  and thus should be classified as mobilizable .
the other three plasmids < <dig> kb , all of them belonging to a. macleodii strains, lack the conjugative modules and were classified as non-mobilizable.
these small plasmids probably spread by natural transformation or transduction  <cit> .table  <dig> general features of the plasmids and ices found in alteromonas genomes
ce conjugative element
c conjugative
mb mobilizable
nmb non mobilizable
sequence similarity  among alteromonas plasmids within and between all the populations of plasmids described at the ncbi belonging to the order alteromonadales  is displayed in fig.
2a, only  <dig> showed any connection.
we identified two major clusters of plasmids belonging to the genera shewanella and alteromonas.
the shewanella plasmid network  is made up by eight plasmids isolated from seven shewanella baltica strains and a megaplasmid  from shewanella oneidensis mr- <dig>  <cit> .
the t4ss gene cluster was their main connection .
shewanella ana- <dig> plasmid interconnects both networks through similarity with glaciecola sp.
4h-3-7 + ye- <dig> plasmid pglaag <dig> within the alteromonas plasmid network .
apn contains all the alteromonas plasmids except the nearly identical couple pamcp48-600/pamcp49- <dig>  and pamrg65- <dig>  the last only share a few connections with the first two, all hypothetical proteins.fig.
<dig> network representing protein sharing among all the alteromonadales plasmids.
a a network representation of alteromonas plasmids within and between all the available plasmids described at the ncbi belonging to the order alteromonadales was produced using cytoscape at 90% identity.
alteromonas plasmids are in red.
b alignment of the plasmids associated with the alteromonas plasmid network
in order to analyze more in detail the relationships among the members of the apn we performed an alignment of the sequences .
the non-mobilizable pam1a <dig>  <cit>  had a region of high similarity with pglaag <dig> that corresponds nearly exactly with a large gene cassette coding for metal resistance  and a hydrogenase  <cit>  that in a. mediterranea de is located in the chromosome at an additive fgi  <cit> .
several genomes of alteromonas contain different versions of this island at the same location  <cit> .
the presence in the plasmid of a series of gene cassettes nearly identical to some found in the a. mediterranea de chromosome, indicates that plasmids are important vectors for the dispersion of these cassettes among groups of strains even beyond the genus barriers, and also how a specific combination of different operons  can be acquired in a single step.
the other non-mobilizable plasmids pamad6- <dig> and pamad37- <dig> showed high number of small clusters in common.
these plasmids were isolated from the same sample .
they might share a common ancestor that lost the capacity to mobilize and since then each has diverged differentially.
the two strains containing these two plasmids have an ani of ca.
97%.
this is consistent with them belonging to different clonal frames within the same species  <cit> .
interestingly, pamde1- <dig> and paste61- <dig> showed a highly syntenic region  with glaciecola plasmid pglaag <dig> .
this region coincides with the presumed machinery for conjugal transfer and plasmid maintenance and could be designed as the plasmid “core”.
all these plasmids contain also a flexible region that contains a high accumulation of is elements that are hotspots for different gene cluster integrations .
as have been previously described, in this flexible region pamde1- <dig> contained a hybrid nrps-pks cluster  <cit>  and pglaag <dig> genes involved in metal resistance.
however, paste61- <dig> seems that have lost not only most of the gene clusters but also some important tra genes suggesting that this plasmid alone cannot carry out conjugation.
all this evidence illustrates how plasmids are modular, dynamic and flexible genetic scaffolds driving gene flux in their bacterial hosts over a range that might overlap at least a whole genus and beyond.
to try to detect the relationships between plasmids and chromosomes, we have investigated the presence of homologs to alteromonas plasmid proteins in the genome of  <dig> genomes  belonging to the eight families of the alteromonadales order with an identity threshold set to 70% .
the results revealed that out of  <dig>  plasmid proteins only  <dig> appeared in chromosomes at this level of similarity.
most of these homologs  corresponded to the plasmid pam1a <dig> and were heavy metal efflux pumps and components of the copper resistance operon  <cit> , present in the additive fgi mentioned above.
the next plasmid with more homologs  was pamcp48- <dig>  in the latter we found that the homologs were housekeeping proteins, such as reca, the carbon storage regulator  or several subunits of the ribonucleotide-diphosphate reductase.
additional file 2: figure s <dig> shows the abundance of plasmid similar proteins of the alteromonas plasmids in all the genera within the alteromonadales order normalized by the number of genomes within each genus.
in addition, we used a 16s rrna phylogenetic tree with  <dig> sequences belonging to this order to infer phylogenetic relationships .
most of the homologs are found among closely related strains and there seems to be a negative correlation with phylogenetic distance.
these results support that these plasmids have been transferred preferentially among closely related genera.
first alteromonas chromid
the megaplasmids pamcp48- <dig> and pamcp49- <dig>  were found in two strains of a. mediterranea cp <dig> and cp <dig> isolated from the same place in the western mediterranean sea .
although the strain chromosomes are relatively divergent  the plasmids were nearly identical, with the main difference being a segment of  <dig> kb absent in pamcp48- <dig> containing three genes encoding hypothetical proteins .
interestingly, we found another strain  isolated from a slightly different location  with a nearly identical chromosome to cp <dig>  but that did not have the plasmid .
this indicates that this replicon is not essential for cell survival.
the major difference between these strains is that ar <dig> has lost a small genomic island containing two gene clusters, one of them the chromosomal crispr/cas system present in cp <dig> and cp <dig> .
strain cp <dig> although clearly belonging to a different clonal frame from cp <dig> , possesses an identical crispr system  inserted in the same position .
the conservation of an identical set of spacers in both crispr clusters is indicative of very recent common ancestry.
this island  is located next to a single met-trna that probably acted facilitating the integration of the whole island, since we found the duplicated 3’-end of the trna gene at the other end , a hallmark of a single integration event.
in this location strains cp <dig> also have a small deletion of  <dig> kb containing three genes coding for a restriction modification system type i .
the chromosomal crispr system belongs to subtype i-f based on the classification made by  <cit> , sharing identical direct repeats with another strain belonging to a different species within the genus, a. australica de <dig>  isolated from the south adriatic sea  <cit> .
however, no match was obtained in the blast searches of the  <dig> cp <dig> spacers against ncbi nr databases.
strain ar <dig>  with a nearly identical genome to cp <dig> , had no trace of the island or the duplicated trna fragment.fig.
<dig> whole-genome alignment of linearized a. mediterranea ar <dig>  cp <dig> and cp <dig> genomes.
the panel below indicates fragments recruited in err <dig> tara expedition metagenomes and their similarity to the homologous region of the cp <dig> chromosome and chromid.
the two variables regions  are indicated.
crispr clusters are marked in red.
arrows under the genome stretch represented  indicate the 3’ end of the trna gene section that is duplicated and represents a hallmark of an integration event.
annotations of proteins in the lower panel are only shown for the interesting regions
these megaplasmids are by far the largest alteromonas plasmids published and one of the largest of all alteromonadales and contain all the hallmarks of a “chromid”  <cit>  including a plasmid-type replication system and paralog copies of housekeeping genes already present in the main chromosome .
we found  <dig> putative protein-coding sequences  75% of which were of unknown function.
when analyzed in detail, these plasmids had actually two different segments .
one that retains the typical features of a plasmid, containing the tra gene cluster, the plasmid origin of replication and multiple hypothetical proteins we have called this half “plasmid module”.
we have defined the other half as “chromosome module” .
it has chromosomal features including thirteen trna genes that are functionally redundant, since the chromosome encoded all the trnas needed for protein synthesis.
from the albertsen list of orthologous markers of essential single-copy genes  <cit>  we found five within this module in both chromids -lysidine synthetase).
however, only one of them, coding for reca, had significant similarity  to the chromosomal homolog.
overall, the sequence of the chromids seems to have a hybrid nature originated by the insertion of a large segment  from a microbe distantly related to alteromonas .
the insertion of the large chromosomal like segment would have occurred next to a theta origin of replication    <cit> .
we compared the position and the number of conserved or similar proteins between the chromid and chromosome by blastp .
the result gave us a total of  <dig> shared proteins most of them located in the chromosomal half of the chromid .
although most of them encoded proteins with unknown function there was a cluster likely involved in the biosynthesis of the lipopolysaccharide, an important component of the cell outer membrane.
in order to determine the evolutionary origin of this cluster we performed several phylogenetic analyses using the amino acid sequences deduced from three essential genes  implicated in the production of this polysaccharide.
they all have homologs in marine microbes belonging to the gammaproteobacteria , indicating that at least this part of the chromid had been acquired from a related  bacterial cell.
in the plasmid module we found several genes encoding potential defense mechanisms such as a crispr/cas system , two toxin-antitoxin pairs and the components of the conjugative machinery .
however despite the differences described above, the analysis of both parts of the chromid showed no significant differences in genomic parameters such as gc content, codon adaptation index, coding density or tetranucleotide frequencies .fig.
<dig> circular representations of the pamcp48- <dig> chromid.
rings from outside to inside: circle 1: cds in the positive strand.
circle 2: cds in the negative strand.
circle 3: stretches in red have similarity to chromosomal proteins .
circle 4: in green, conjugation system.
circle 5: gc content.
circle 6: gc skew.
table indicates some general features of the two modules  that form the chromid
within the analyzed alteromonas plasmids we found only two crispr/cas systems, that according to the most recent classification both belong to the recently proposed type iv  <cit> , they were found in pamrg65- <dig> and the chromids .
this system that lacks cas <dig> and cas <dig> is rather rare and functionally uncharacterized.
seven and six unique spacers were identified using the crispr tool crisprfinder  <cit>  in the repeat-spacer array preceded by three cas genes  in the plasmid and chromid respectively.
we compared each crispr spacer sequence against the ncbi nr database as well as mobile genetic elements deposited in the aclame database  <cit>  and found that only spacer  <dig> of the chromid repeats matched at 93% identity a putative transposase gene in the a. macleodii hot1a <dig> plasmid , this isolate was obtained from a water sample collected from the pacific ocean  <cit> .
since only a small proportion of the total diversity of microorganism is available in public databases we have analysed the presence of spacers and also the direct repeats from both crispr/cas systems in the tara metagenomes.
we have found only two matches  in all the samples and both from the same sample and same spacer .
the sample corresponded to the station tara_ <dig>  the only sampling site where the chromid was detected .
in order to study the global distribution of the chromid we analyzed the recruitment along the tara oceans transect metagenomes  <cit>  using a cutoff at 98% nucleotide identity.
pamcp48-600/pamcp49- <dig> were only detected in the station tara_ <dig> from the western mediterranean sea , near the original place of isolation of the strain, indicating an endemic distribution of this conjugative element in this region of the mediterranean and/or a recent origin.
furthermore, the host of the chromids  also recruited highly in this metagenome .
stability and defense mechanisms
ces have developed systems to ensure their maintenance by postsegregational killing such as restriction-modification  and toxin-antitoxin   <cit> .
rebase database  <cit>  was used to identify rm genes within the plasmid sequences and their cognate chromosomes.
a total of  <dig> rm systems  were identified in the eight host chromosomes but only five in the plasmids.
rm systems were detected in all conjugative plasmids, except pamrg65- <dig>  associating their presence with mobility.
however, we have found some incomplete systems that have lost one of the two rm genes .
as has been described for bacterial genomes  <cit>  and phages  <cit>  mtases were more abundant than reases in plasmids, suggesting a selective degradation of the rease.
similarly, ta systems are also ubiquitous and comprise bicistronic operons encoding two small genes; one gene encoding a toxic protein and the other encoding a specific antitoxin.
we used the database rasta  <cit>  and ta finder  <cit>  to identified potential ta systems present in both, plasmids and chromosomes.
within the chromosomes we found  <dig> complete ta loci belonging to the five known ta gene families .
examinations of the plasmids revealed that there is at least one plasmid-encoded ta system in each of the eight plasmids.
two ta systems were particularly prevalent in the plasmids .
interestingly, strains belonging to a. mediterranea have twice the number of ta systems than the other species in the chromosome.
analysis of the location showed three hotspots of ta accumulation.
two of them corresponding to the integron and the third, that was only present in a. mediterranea, was located in the previously described mobilizable genomic island  <cit> .
these results indicate that ta systems are clearly associated to the flexible part of the chromosome, specifically of fgis, the most variable regions of the genome, suggesting that they are highly mobile and widespread.
integrative and conjugative elements
ices are also ces but, unlike plasmids, they are always integrated into the host chromosome.
they have been found in both gram-positive and gram-negative bacteria  <cit> .
we found six different ices in alteromonas , all belonging to the sxt/r <dig> ice family.
this family was first described in vibrio and all share the same chromosomal integration site into the 5′ end of the prfc gene, highly conserved within species of gammaproteobacteria  <cit> .
the sxt/r <dig> ice family has five variable hotspots .
iceameas <dig> had almost identical sequence in all the isolates of clonal frame  <dig> of a. mediterranea  <cit> .
nearly identical ices were also found in two other alteromonas strains belonging to two different genospecies : a. macleodii d <dig> and alteromonas sp.
rw2a <dig>  isolated from the andaman and the baltic sea, they were named iceamaans <dig> and iceaspbs <dig> respectively .
only the hotspot <dig>  region was highly variable among those ices .
this hs was variable even between two strains, a. mediterranea de <dig> and um4b, that belong to the same clonal frame   <cit> .
however, in addition to the previously described hs in fig.
<dig>  some ices contain additional variable regions  located at the left end  <cit> .
in these regions we found in iceamaans <dig> and iceaspbs <dig> several different components of the mer operon, that confers mercury resistance to bacteria, and a calcium:sodium antiporter.
alteromonas sp.
mex <dig> and a. mediterranea med <dig>  had the most diverging ices .
however, the gene cassettes of the hs <dig> region in iceameaes <dig> were identical to the same region in iceameas <dig> and the other related ices in the group, becoming the most conserved hs among these ices.fig.
<dig> different versions of the integrative conjugative element  found in the genus alteromonas.
the variable region  or hotspots  are highlighted by boxes with different colors.
the number of snps in a 1000 bp window is indicated by a red line.
ani and coverage between the strains containing the ices is indicated to the right of the double arrow
snp analyses showed that they were not evenly distributed throughout these ices and that they appeared concentrated in core genes being largely synonymous snps.
accumulation of large numbers of synonymous snps is a hallmark of high rates of homologous recombination  <cit> .
the high similarity among the common part of the different ices might lead to frequent recombination and replacement by double crossover of the hs genes .
on the other hand, the low amounts of snps in the hs genes indicates that they are very rapidly exchanged preventing synonymous snps to accumulate, as has been proposed for additive fgis in the chromosome  <cit> .
by contrast, the variability found in the vrs is likely mechanistically simpler, using is elements as drivers of the diversity.
genes in these modules are designated cargo genes.
most of cargo genes in alteromonas encode restriction modification or metal resistance systems and can confer diverse capabilities to the organisms that contain the ice.
contrastingly, in the pathogenic vibrio strains, cargo genes were mostly involved in transport and antibiotic resistance, probably critical for their clinical relevance.
to asses and compare the variability within the sxt/r <dig> family we used the six different ices found in alteromonas together with all the complete sxt/r <dig> ices available in the databases  <cit>  comprising both environmental and clinical isolates of several gammaproteobacteria species .
moreover, using iceameas <dig> as reference we were able to assemble another three new ices from the contigs of three marine members belonging to the recently reclassified glaciecola strains .
synteny of the sxt/r <dig> ices was well preserved having sizes ranging from  <dig> to  <dig> kb.
whole genome alignment among the  <dig> sxt/r <dig> ices revealed a total of  <dig> homologous regions representing a core of  <dig> kb, containing  <dig> genes, with  <dig> variables sites and a  <dig> % average identity at the nucleotide level.
in order to analyse the evolution of the sxt/r <dig> family we created a phylogenetic tree using the concatenated core .
the resulting tree showed that, leaving aside photobacterium damselae icepdaspa <dig>  there are two clusters that we designated a and b .
although most of the ices belonging to alteromonas species, together with other marine microbes, fell into group b, iceamaags <dig>  was grouped with most of the pathogenic vibrios.
in addition, three pathogenic enterobacteriaceae  showed close relationship with members of group b suggesting that saprophytic and free living microbes share the same pool of ices.
interestingly, in spite of the different locations and environmental origins , the ice sequences of a. mediterranea med <dig> iceameaes <dig> and v. cholera ahv <dig> were found to be almost identical .
the only difference at the level of hotspots was that both had different gene cassettes at hs <dig> .
in addition, ahv <dig> carried many antibiotic resistance genes in the four variable regions  that conferred resistance to tetracycline and erythromycin, critical to the clinical relevance of this strain.
we also performed pairwise blast comparison with all the ices showing that nearly identical cassettes were frequently detected regardless of the origin of the strain  and disconnected from the core degree of relatedness .fig.
<dig> phylogenetic tree constructed using a concatenate of the core  in all ices of the family sxt/r <dig> available.
the two clusters designated a and b are highlighted in different colour.
photobacterium damselae icepdaspa <dig> appears as an outgroup.
alteromonas ices are highlighted in red
discussion
we have studied genomes of alteromonas as a model to understand the evolutionary dynamics of a marine free-living bacterium  <cit> .
the fact that alteromonas is not known to associate to humans or marine animals simplify the interpretation and can shed light on other marine free-living microbes.
it can also help understanding the ones that are transient human pathogens such as v. cholera  <cit> .
here we have focused on the plasmids and ices that can provide the fastest way to mobilize genomic regions to create new combinations and expand the physio-ecological range of the microbe.
we have purposefully excluded lysogenic phages because, although they can transfer host dna, it is much more limited quantitatively and has other major role as a strategy of virus survival that is hard to discriminate from their role in host evolution.
gene transfer agents  <cit>  have been also obviated, although they could be carriers of large genomic fragments, but the comparative genomics approach used here would not be able to detect them.
we have found a relatively small number of plasmid and ices, only  <dig> among  <dig> strain genomes.
however, their role could be paramount in the networks of genomic communication among these microbes if they flow fast throughout the population.
although the plasmids described here seemed to be restricted to a single species, pamade1- <dig> was found in different strains of a. mediterranea pointing to a fast dispersion rate.
in addition, a clear connection was found between the plasmids of alteromonas and glaciecola, indicating networks of these elements that extend beyond a single genus.
actually, the identification of similar genes in isolates belonging to even different families  attests to a broad range of hosts at least of the ices described here.
although the core functions of the ices such as the mechanisms to promote their integration, excision, transfer and regulation have been well studied  <cit>  and mainly have been characterized as key vectors of antibiotic resistance, little is known about the evolutionary dynamics of these elements in aquatic environments.
our comparative analyses showed the importance of inter-element recombination producing hybrid ices.
the dynamics of variation of these elements based on the continuous change of the different modules recruited from an extensive pool, likely distant gammaproteobacteria, is fast enough to mask evolutionary history.
we have found examples that could illustrate how the two major kinds of fgis i.e.
additive and replacement  <cit>  can travel among relatively distant strains.
the case of the large hydrogenase-metal resistance gene cluster  <cit>  sharing nearly identical sequences in a conjugative plasmid and in the chromosome of different species of alteromonas is a paradigmatic example of how an additive fgi can be exchanged.
in the case of replacement fgis  <cit>  the evidence is less compelling but the polysaccharide cluster found in chromids pamcp48-600/pamcp49- <dig> could reflect an intermediate step in the process of exchange of a replacement fgi.
the chromid would provide an alternative gene cluster that eventually could replace the chromosomal one.
however, the lack of a similar cluster found in any alteromonas strain or other related microbe prevents concluding that this would be a viable path for this kind of fgi to be exchanged.
we have previously formulated a theory by which replacement fgis flow much more slowly throughout the clonal frames that constitute a species, allowing time for them to diverge at the level of microniche specialization  <cit> .
along these lines, it is not surprising that, with the relatively small numbers of strains analysed, no “smoking gun”, that is a replacement fgi caught in the process of being exchanged, would surface.
regarding the finding of the strains  containing the same chromid and crispr/cas system connected to two clearly divergent chromosomes, the most parsimonious explanation is that cp <dig> has received both the plasmid and the crispr cassette from a relative of cp <dig>  in both cases the cp <dig> fragments have small deletions that might have happened more easily than insertions in cp <dig> .
in addition, ar <dig> has a little insertion at the left hand side of the island.
exchange of the chromid and the crispr/cas system between strains cp <dig> and cp <dig> must have happened in a short timeframe given the nearly identical sequence of the chromids and the identical spacers shared by the crisprs.
although the plasmid was detected only in two strains isolated simultaneously, its recruitment from a distant location  illustrated that it can remain as a stable entity for a significant amount of time.
its presence in two widely divergent genomic backgrounds  illustrates how it can move fast in the population of divergent a. mediterranea clones, in spite of a significant decrease in growth rate  induced by the presence of the chromid.
CONCLUSIONS
findings presented here underscore the relevance of ces as main vectors in the exchange of gene cassettes in the chromosomal additive fgis.
we have found the first chromid described in alteromonas containing two clearly distinguishable parts designed as “chromosomal” and “plasmid” modules.
the chromid contained a lps biosynthesis cluster that provides a clue as to how replacement fgis might be exchanged among clonal lineages.
our analyses also revealed that the continuous change of the different modules by recombination is an important mechanism to produce ice variation and occur even at phylogenetic distances exceeding the family threshold, since we have found nearly identical ices in a mediterranea med <dig> and v. cholera ahv <dig>  a human pathogen.

 prokaryotic microbes have sex. this has been known for very long, individuals exchange genetic material to create hybrids that have different properties from the donor cells. however, contrastingly with eukaryotes genetic exchange is independent from reproduction and can act on smaller sections of the genome rather than the complete hybrid generated by meiosis in eukaryotes. as a consequence, there are fewer restrains regarding donors and receptors. one direct consequence of prokaryotic sex is that cells of the same species can have very different gene complements, what has led to a new paradigm, the pangenome, a theoretical catalog of the gene complement of the species at large. the pangenome includes a core of genes that is common to all of the strains in the taxon considered and a flexible pool that varies from strain to strain. since the discovery and latter proposal of this term, the numbers of genes in prokaryotic pangenomes have not stopped of amazing microbiologists. a recent census in escherichia coli reached close to different gene families with strain genomes analyzed and remarkably the discovery of new genes continue at a pace of ca. new per strain sequenced. however, in spite of the apparent enormous size of prokaryotic flexible genomes, there has to be rules that are applicable to how these genes circulate through the population. for some years we have studied the marine bacterium alteromonas with the focus on understanding its genomic make up and dynamics. this microbe is a typical r-selected specialist in which success is based in high growth rates and exploitation of transient niches. its species have large cells and genomes and can grow very fast when nutrients are available. the analyses of genome sequences of alteromonas species isolated from all around the world revealed remarkable conservation of synteny what has allowed us to study the physical arrangement of the core and flexible regions in the genomes of representatives of this genus. using comparative genomics we proposed a model of short term evolution in which clones diverge forming different clonal lineages, the smallest unit of differentiation of prokaryotes, by acquisition of specific glycosydic receptors that were called glycotypes. it has been already established that the flexible genome is largely collected in genomic islands that appear at equivalent positions in the genomes of the different isolates known as flexible genomic islands. there are two types of fgis with different mechanisms of variation: i) additive fgis vary by addition or subtraction of gene cassettes, largely by site directed recombination, which movement among lineages appears to be relatively fast, and ii) replacement fgis that code for major exposed structures of the cell and are exchanged by double crossover homologous recombination, using the neighboring highly conserved genes as substrate. they vary at a much slower pace and remain linked for relatively longer times preventing population-wide gene sweeps, making a specific clone, characterized by its surface glycotype and recognized by specific populations of phages, a single selection unit. to get a more complete picture of the mechanistic variation of alteromonas genomes an important element was missing. what are the mechanisms by which gene pools travel from one clone to another? alteromonas is not known to have natural transformation and transmission through phages, even specialized gene transfer agents, are unlikely to mediate the transfer of large genomic regions such as replacement fgis. for these large transfers conjugation mediated by plasmids or integrative and conjugative elements would be the most likely route in a gram negative bacterium such as alteromonas. both, conjugative plasmids and ices have been described before in alteromonas. plasmids are extra-chromosomal and auto-replicating dna molecules composed by a segment containing genetic information required for their replication and maintenance, as well as other accessory genes. plasmid genes are normally dispensable for the essential functions of the cell. they are transferred using the tra system coded by themselves or by other replicons and can mobilize large segments of the chromosome that can thus be moved to different chromosomal backgrounds. ices are also transferable by conjugation but, unlike plasmids, they are always integrated into the host chromosome and have been found in both gram-positive and gram-negative bacteria. integration in the chromosome occurs via site-specific recombination. although they are not as well-known as plasmids recently guglielmini et al. showed that they are the most abundant conjugative elements in practically all prokaryotic clades. these elements contain a group of core genes clustered into distinct modules required for integration/excision, conjugative transfer and regulation. in addition they can acquire new dna in specific sites proposed as hotspots by beaber et al.. most of cargo genes in these modules encode for restriction modification, antibiotic and metal resistance systems. the aim of this study was to make a detailed comparative analysis of ces obtained from different strains of the genus alteromonas to explore the evolutionary relationships not only among the different ces but also with the chromosomes and how they could play a role in the genomic variation detected at the genomic level among strains and species of the alteromonas genus. the patterns found might be extrapolated at least to other aquatic gram negative bacteria.  in order to characterize ces we examined all the strains available in databases of the genus alteromonas searching for plasmids or ices. in addition we are reporting here six new genomes of strains from different locations that have been fully sequenced and assembled and contain ces. in order to investigate the phylogenomic relationships of the new alteromonas strains within the alteromonas genus, whole-genome phylogeny was inferred from a concatenate of the core genome. presently, strains of the genus alteromonas are classified into six different species but using an average nucleotide identity similarity threshold of 95% the thirty-seven strains in the tree of fig.  can be classified into genospecies. our data also indicates that the new strains ar cp cp and rg are closely related and clearly belong to a. mediterranea. however, strains alteromonas sp. mex and alteromonas sp. rw2a had similarities ca. 75% to the closest genospecies and could be defined as distant and novel genospecies. the general features of the new strain genomes used in this study are shown in additional file 1: table s and fig.  fig. phylogenomic tree constructed using a concatenated of the core proteome in all available alteromonas genomes. pseudoalteromonas atlantica t6c was used as outgroup. strains with genomes reported here for the first time are highlighted in red. strains that belong to the same species are highlighted by the same colour. presence of either plasmids or ices are indicated with circles or triangles, same colour indicates same version of the genomic element. the number in the corner of the rectangles indicates the number of genospecies plasmids among the analysed alteromonas strains only had plasmids totalling nine different replicons that were found in only three different species. three of them are reported here for the first time. another plasmid has been described in the strain a. macleodii ad but we decided not to include it since it was identified as a putative defective phage. with the exception of pamde1- that was found with nearly identical sequence in different strains belonging to different a. mediterranea strains, and the pair pamcp48-600/pamcp49- that only differ in kb, the others were found only in one strain. plasmid size range was from kb to kb. the average gc content was always lower than the host chromosome with the highest difference observed for pamrg65-. based on their mobility, plasmids can be classified in three different classes. all alteromonas largest plasmids > kb can be classified as conjugative plasmids since they have a mob and mpf genes, except paste61- which has an incomplete t4ss and thus should be classified as mobilizable. the other three plasmids < kb, all of them belonging to a. macleodii strains, lack the conjugative modules and were classified as non-mobilizable. these small plasmids probably spread by natural transformation or transduction.table general features of the plasmids and ices found in alteromonas genomes ce conjugative element c conjugative mb mobilizable nmb non mobilizable sequence similarity among alteromonas plasmids within and between all the populations of plasmids described at the ncbi belonging to the order alteromonadales is displayed in fig. 2a, only showed any connection. we identified two major clusters of plasmids belonging to the genera shewanella and alteromonas. the shewanella plasmid network is made up by eight plasmids isolated from seven shewanella baltica strains and a megaplasmid from shewanella oneidensis mr-. the t4ss gene cluster was their main connection. shewanella ana- plasmid interconnects both networks through similarity with glaciecola sp. 4h-3-7 + ye- plasmid pglaag within the alteromonas plasmid network. apn contains all the alteromonas plasmids except the nearly identical couple pamcp48-600/pamcp49- and pamrg65- the last only share a few connections with the first two, all hypothetical proteins.fig. network representing protein sharing among all the alteromonadales plasmids. a a network representation of alteromonas plasmids within and between all the available plasmids described at the ncbi belonging to the order alteromonadales was produced using cytoscape at 90% identity. alteromonas plasmids are in red. b alignment of the plasmids associated with the alteromonas plasmid network in order to analyze more in detail the relationships among the members of the apn we performed an alignment of the sequences. the non-mobilizable pam1a had a region of high similarity with pglaag that corresponds nearly exactly with a large gene cassette coding for metal resistance and a hydrogenase that in a. mediterranea de is located in the chromosome at an additive fgi. several genomes of alteromonas contain different versions of this island at the same location. the presence in the plasmid of a series of gene cassettes nearly identical to some found in the a. mediterranea de chromosome, indicates that plasmids are important vectors for the dispersion of these cassettes among groups of strains even beyond the genus barriers, and also how a specific combination of different operons can be acquired in a single step. the other non-mobilizable plasmids pamad6- and pamad37- showed high number of small clusters in common. these plasmids were isolated from the same sample. they might share a common ancestor that lost the capacity to mobilize and since then each has diverged differentially. the two strains containing these two plasmids have an ani of ca. 97%. this is consistent with them belonging to different clonal frames within the same species. interestingly, pamde1- and paste61- showed a highly syntenic region with glaciecola plasmid pglaag. this region coincides with the presumed machinery for conjugal transfer and plasmid maintenance and could be designed as the plasmid “core”. all these plasmids contain also a flexible region that contains a high accumulation of is elements that are hotspots for different gene cluster integrations. as have been previously described, in this flexible region pamde1- contained a hybrid nrps-pks cluster and pglaag genes involved in metal resistance. however, paste61- seems that have lost not only most of the gene clusters but also some important tra genes suggesting that this plasmid alone cannot carry out conjugation. all this evidence illustrates how plasmids are modular, dynamic and flexible genetic scaffolds driving gene flux in their bacterial hosts over a range that might overlap at least a whole genus and beyond. to try to detect the relationships between plasmids and chromosomes, we have investigated the presence of homologs to alteromonas plasmid proteins in the genome of genomes belonging to the eight families of the alteromonadales order with an identity threshold set to 70%. the results revealed that out of plasmid proteins only appeared in chromosomes at this level of similarity. most of these homologs corresponded to the plasmid pam1a and were heavy metal efflux pumps and components of the copper resistance operon, present in the additive fgi mentioned above. the next plasmid with more homologs was pamcp48- in the latter we found that the homologs were housekeeping proteins, such as reca, the carbon storage regulator or several subunits of the ribonucleotide-diphosphate reductase. additional file 2: figure s shows the abundance of plasmid similar proteins of the alteromonas plasmids in all the genera within the alteromonadales order normalized by the number of genomes within each genus. in addition, we used a 16s rrna phylogenetic tree with sequences belonging to this order to infer phylogenetic relationships. most of the homologs are found among closely related strains and there seems to be a negative correlation with phylogenetic distance. these results support that these plasmids have been transferred preferentially among closely related genera. first alteromonas chromid the megaplasmids pamcp48- and pamcp49- were found in two strains of a. mediterranea cp and cp isolated from the same place in the western mediterranean sea. although the strain chromosomes are relatively divergent the plasmids were nearly identical, with the main difference being a segment of kb absent in pamcp48- containing three genes encoding hypothetical proteins. interestingly, we found another strain isolated from a slightly different location with a nearly identical chromosome to cp but that did not have the plasmid. this indicates that this replicon is not essential for cell survival. the major difference between these strains is that ar has lost a small genomic island containing two gene clusters, one of them the chromosomal crispr/cas system present in cp and cp. strain cp although clearly belonging to a different clonal frame from cp, possesses an identical crispr system inserted in the same position. the conservation of an identical set of spacers in both crispr clusters is indicative of very recent common ancestry. this island is located next to a single met-trna that probably acted facilitating the integration of the whole island, since we found the duplicated 3’-end of the trna gene at the other end, a hallmark of a single integration event. in this location strains cp also have a small deletion of kb containing three genes coding for a restriction modification system type i. the chromosomal crispr system belongs to subtype i-f based on the classification made by, sharing identical direct repeats with another strain belonging to a different species within the genus, a. australica de isolated from the south adriatic sea. however, no match was obtained in the blast searches of the cp spacers against ncbi nr databases. strain ar with a nearly identical genome to cp, had no trace of the island or the duplicated trna fragment.fig. whole-genome alignment of linearized a. mediterranea ar cp and cp genomes. the panel below indicates fragments recruited in err tara expedition metagenomes and their similarity to the homologous region of the cp chromosome and chromid. the two variables regions are indicated. crispr clusters are marked in red. arrows under the genome stretch represented indicate the 3’ end of the trna gene section that is duplicated and represents a hallmark of an integration event. annotations of proteins in the lower panel are only shown for the interesting regions these megaplasmids are by far the largest alteromonas plasmids published and one of the largest of all alteromonadales and contain all the hallmarks of a “chromid” including a plasmid-type replication system and paralog copies of housekeeping genes already present in the main chromosome. we found putative protein-coding sequences 75% of which were of unknown function. when analyzed in detail, these plasmids had actually two different segments. one that retains the typical features of a plasmid, containing the tra gene cluster, the plasmid origin of replication and multiple hypothetical proteins we have called this half “plasmid module”. we have defined the other half as “chromosome module”. it has chromosomal features including thirteen trna genes that are functionally redundant, since the chromosome encoded all the trnas needed for protein synthesis. from the albertsen list of orthologous markers of essential single-copy genes we found five within this module in both chromids -lysidine synthetase). however, only one of them, coding for reca, had significant similarity to the chromosomal homolog. overall, the sequence of the chromids seems to have a hybrid nature originated by the insertion of a large segment from a microbe distantly related to alteromonas. the insertion of the large chromosomal like segment would have occurred next to a theta origin of replication. we compared the position and the number of conserved or similar proteins between the chromid and chromosome by blastp. the result gave us a total of shared proteins most of them located in the chromosomal half of the chromid. although most of them encoded proteins with unknown function there was a cluster likely involved in the biosynthesis of the lipopolysaccharide, an important component of the cell outer membrane. in order to determine the evolutionary origin of this cluster we performed several phylogenetic analyses using the amino acid sequences deduced from three essential genes implicated in the production of this polysaccharide. they all have homologs in marine microbes belonging to the gammaproteobacteria, indicating that at least this part of the chromid had been acquired from a related bacterial cell. in the plasmid module we found several genes encoding potential defense mechanisms such as a crispr/cas system, two toxin-antitoxin pairs and the components of the conjugative machinery. however despite the differences described above, the analysis of both parts of the chromid showed no significant differences in genomic parameters such as gc content, codon adaptation index, coding density or tetranucleotide frequencies.fig. circular representations of the pamcp48- chromid. rings from outside to inside: circle 1: cds in the positive strand. circle 2: cds in the negative strand. circle 3: stretches in red have similarity to chromosomal proteins. circle 4: in green, conjugation system. circle 5: gc content. circle 6: gc skew. table indicates some general features of the two modules that form the chromid within the analyzed alteromonas plasmids we found only two crispr/cas systems, that according to the most recent classification both belong to the recently proposed type iv, they were found in pamrg65- and the chromids. this system that lacks cas and cas is rather rare and functionally uncharacterized. seven and six unique spacers were identified using the crispr tool crisprfinder in the repeat-spacer array preceded by three cas genes in the plasmid and chromid respectively. we compared each crispr spacer sequence against the ncbi nr database as well as mobile genetic elements deposited in the aclame database and found that only spacer of the chromid repeats matched at 93% identity a putative transposase gene in the a. macleodii hot1a plasmid, this isolate was obtained from a water sample collected from the pacific ocean. since only a small proportion of the total diversity of microorganism is available in public databases we have analysed the presence of spacers and also the direct repeats from both crispr/cas systems in the tara metagenomes. we have found only two matches in all the samples and both from the same sample and same spacer. the sample corresponded to the station tara_ the only sampling site where the chromid was detected. in order to study the global distribution of the chromid we analyzed the recruitment along the tara oceans transect metagenomes using a cutoff at 98% nucleotide identity. pamcp48-600/pamcp49- were only detected in the station tara_ from the western mediterranean sea, near the original place of isolation of the strain, indicating an endemic distribution of this conjugative element in this region of the mediterranean and/or a recent origin. furthermore, the host of the chromids also recruited highly in this metagenome. stability and defense mechanisms ces have developed systems to ensure their maintenance by postsegregational killing such as restriction-modification and toxin-antitoxin. rebase database was used to identify rm genes within the plasmid sequences and their cognate chromosomes. a total of rm systems were identified in the eight host chromosomes but only five in the plasmids. rm systems were detected in all conjugative plasmids, except pamrg65- associating their presence with mobility. however, we have found some incomplete systems that have lost one of the two rm genes. as has been described for bacterial genomes and phages mtases were more abundant than reases in plasmids, suggesting a selective degradation of the rease. similarly, ta systems are also ubiquitous and comprise bicistronic operons encoding two small genes; one gene encoding a toxic protein and the other encoding a specific antitoxin. we used the database rasta and ta finder to identified potential ta systems present in both, plasmids and chromosomes. within the chromosomes we found complete ta loci belonging to the five known ta gene families. examinations of the plasmids revealed that there is at least one plasmid-encoded ta system in each of the eight plasmids. two ta systems were particularly prevalent in the plasmids. interestingly, strains belonging to a. mediterranea have twice the number of ta systems than the other species in the chromosome. analysis of the location showed three hotspots of ta accumulation. two of them corresponding to the integron and the third, that was only present in a. mediterranea, was located in the previously described mobilizable genomic island. these results indicate that ta systems are clearly associated to the flexible part of the chromosome, specifically of fgis, the most variable regions of the genome, suggesting that they are highly mobile and widespread. integrative and conjugative elements ices are also ces but, unlike plasmids, they are always integrated into the host chromosome. they have been found in both gram-positive and gram-negative bacteria. we found six different ices in alteromonas, all belonging to the sxt/r ice family. this family was first described in vibrio and all share the same chromosomal integration site into the 5′ end of the prfc gene, highly conserved within species of gammaproteobacteria. the sxt/r ice family has five variable hotspots. iceameas had almost identical sequence in all the isolates of clonal frame of a. mediterranea. nearly identical ices were also found in two other alteromonas strains belonging to two different genospecies: a. macleodii d and alteromonas sp. rw2a isolated from the andaman and the baltic sea, they were named iceamaans and iceaspbs respectively. only the hotspot region was highly variable among those ices. this hs was variable even between two strains, a. mediterranea de and um4b, that belong to the same clonal frame. however, in addition to the previously described hs in fig.  some ices contain additional variable regions located at the left end. in these regions we found in iceamaans and iceaspbs several different components of the mer operon, that confers mercury resistance to bacteria, and a calcium:sodium antiporter. alteromonas sp. mex and a. mediterranea med had the most diverging ices. however, the gene cassettes of the hs region in iceameaes were identical to the same region in iceameas and the other related ices in the group, becoming the most conserved hs among these ices.fig. different versions of the integrative conjugative element found in the genus alteromonas. the variable region or hotspots are highlighted by boxes with different colors. the number of snps in a 1000 bp window is indicated by a red line. ani and coverage between the strains containing the ices is indicated to the right of the double arrow snp analyses showed that they were not evenly distributed throughout these ices and that they appeared concentrated in core genes being largely synonymous snps. accumulation of large numbers of synonymous snps is a hallmark of high rates of homologous recombination. the high similarity among the common part of the different ices might lead to frequent recombination and replacement by double crossover of the hs genes. on the other hand, the low amounts of snps in the hs genes indicates that they are very rapidly exchanged preventing synonymous snps to accumulate, as has been proposed for additive fgis in the chromosome. by contrast, the variability found in the vrs is likely mechanistically simpler, using is elements as drivers of the diversity. genes in these modules are designated cargo genes. most of cargo genes in alteromonas encode restriction modification or metal resistance systems and can confer diverse capabilities to the organisms that contain the ice. contrastingly, in the pathogenic vibrio strains, cargo genes were mostly involved in transport and antibiotic resistance, probably critical for their clinical relevance. to asses and compare the variability within the sxt/r family we used the six different ices found in alteromonas together with all the complete sxt/r ices available in the databases comprising both environmental and clinical isolates of several gammaproteobacteria species. moreover, using iceameas as reference we were able to assemble another three new ices from the contigs of three marine members belonging to the recently reclassified glaciecola strains. synteny of the sxt/r ices was well preserved having sizes ranging from to kb. whole genome alignment among the sxt/r ices revealed a total of homologous regions representing a core of kb, containing genes, with variables sites and a % average identity at the nucleotide level. in order to analyse the evolution of the sxt/r family we created a phylogenetic tree using the concatenated core. the resulting tree showed that, leaving aside photobacterium damselae icepdaspa there are two clusters that we designated a and b. although most of the ices belonging to alteromonas species, together with other marine microbes, fell into group b, iceamaags was grouped with most of the pathogenic vibrios. in addition, three pathogenic enterobacteriaceae showed close relationship with members of group b suggesting that saprophytic and free living microbes share the same pool of ices. interestingly, in spite of the different locations and environmental origins, the ice sequences of a. mediterranea med iceameaes and v. cholera ahv were found to be almost identical. the only difference at the level of hotspots was that both had different gene cassettes at hs. in addition, ahv carried many antibiotic resistance genes in the four variable regions that conferred resistance to tetracycline and erythromycin, critical to the clinical relevance of this strain. we also performed pairwise blast comparison with all the ices showing that nearly identical cassettes were frequently detected regardless of the origin of the strain and disconnected from the core degree of relatedness.fig. phylogenetic tree constructed using a concatenate of the core in all ices of the family sxt/r available. the two clusters designated a and b are highlighted in different colour. photobacterium damselae icepdaspa appears as an outgroup. alteromonas ices are highlighted in red discussion we have studied genomes of alteromonas as a model to understand the evolutionary dynamics of a marine free-living bacterium. the fact that alteromonas is not known to associate to humans or marine animals simplify the interpretation and can shed light on other marine free-living microbes. it can also help understanding the ones that are transient human pathogens such as v. cholera. here we have focused on the plasmids and ices that can provide the fastest way to mobilize genomic regions to create new combinations and expand the physio-ecological range of the microbe. we have purposefully excluded lysogenic phages because, although they can transfer host dna, it is much more limited quantitatively and has other major role as a strategy of virus survival that is hard to discriminate from their role in host evolution. gene transfer agents have been also obviated, although they could be carriers of large genomic fragments, but the comparative genomics approach used here would not be able to detect them. we have found a relatively small number of plasmid and ices, only among strain genomes. however, their role could be paramount in the networks of genomic communication among these microbes if they flow fast throughout the population. although the plasmids described here seemed to be restricted to a single species, pamade1- was found in different strains of a. mediterranea pointing to a fast dispersion rate. in addition, a clear connection was found between the plasmids of alteromonas and glaciecola, indicating networks of these elements that extend beyond a single genus. actually, the identification of similar genes in isolates belonging to even different families attests to a broad range of hosts at least of the ices described here. although the core functions of the ices such as the mechanisms to promote their integration, excision, transfer and regulation have been well studied and mainly have been characterized as key vectors of antibiotic resistance, little is known about the evolutionary dynamics of these elements in aquatic environments. our comparative analyses showed the importance of inter-element recombination producing hybrid ices. the dynamics of variation of these elements based on the continuous change of the different modules recruited from an extensive pool, likely distant gammaproteobacteria, is fast enough to mask evolutionary history. we have found examples that could illustrate how the two major kinds of fgis i.e. additive and replacement can travel among relatively distant strains. the case of the large hydrogenase-metal resistance gene cluster sharing nearly identical sequences in a conjugative plasmid and in the chromosome of different species of alteromonas is a paradigmatic example of how an additive fgi can be exchanged. in the case of replacement fgis the evidence is less compelling but the polysaccharide cluster found in chromids pamcp48-600/pamcp49- could reflect an intermediate step in the process of exchange of a replacement fgi. the chromid would provide an alternative gene cluster that eventually could replace the chromosomal one. however, the lack of a similar cluster found in any alteromonas strain or other related microbe prevents concluding that this would be a viable path for this kind of fgi to be exchanged. we have previously formulated a theory by which replacement fgis flow much more slowly throughout the clonal frames that constitute a species, allowing time for them to diverge at the level of microniche specialization. along these lines, it is not surprising that, with the relatively small numbers of strains analysed, no “smoking gun”, that is a replacement fgi caught in the process of being exchanged, would surface. regarding the finding of the strains containing the same chromid and crispr/cas system connected to two clearly divergent chromosomes, the most parsimonious explanation is that cp has received both the plasmid and the crispr cassette from a relative of cp in both cases the cp fragments have small deletions that might have happened more easily than insertions in cp. in addition, ar has a little insertion at the left hand side of the island. exchange of the chromid and the crispr/cas system between strains cp and cp must have happened in a short timeframe given the nearly identical sequence of the chromids and the identical spacers shared by the crisprs. although the plasmid was detected only in two strains isolated simultaneously, its recruitment from a distant location illustrated that it can remain as a stable entity for a significant amount of time. its presence in two widely divergent genomic backgrounds illustrates how it can move fast in the population of divergent a. mediterranea clones, in spite of a significant decrease in growth rate induced by the presence of the chromid.  findings presented here underscore the relevance of ces as main vectors in the exchange of gene cassettes in the chromosomal additive fgis. we have found the first chromid described in alteromonas containing two clearly distinguishable parts designed as “chromosomal” and “plasmid” modules. the chromid contained a lps biosynthesis cluster that provides a clue as to how replacement fgis might be exchanged among clonal lineages. our analyses also revealed that the continuous change of the different modules by recombination is an important mechanism to produce ice variation and occur even at phylogenetic distances exceeding the family threshold, since we have found nearly identical ices in a mediterranea med and v. cholera ahv a human pathogen. 
a nearly identical ice was found in a. mediterranea med and vibrio cholera ahv isolated from a human pathogen, indicating the potential exchange of these genes across phylogenetic distances exceeding the family threshold. the cargo genes found in these plasmids included cassettes found before in chromosome flexible genomic islands of alteromonas strains. integrative and conjugative elements were more common than plasmids and showed similar patterns of variation with cargo genes coding for components of additive flexible genomic islands. we have seen evidence of how ces can be vectors to transfer gene cassettes acquired in the chromosomal flexible genomic islands, both of the additive and replacement kind. they could represent the transport vectors for the previously described replacement flexible genomic islands. alteromonasplasmidspangenomeconjugative elementschromidintegrative and conjugative elementsicegenomic islandshttp://dx.doi.org/ /501100003359generalitat valencianaapostd/2016/051lópez-pérez mario issue-copyright-statement© the author 2017 however, the main genetic exchange drivers in these microbes, conjugative elements, have not been considered in detail thus far. to develop evolutionary models for the free living bacterium alteromonas the genome sequences of isolates of the genus have been extensively analyzed.
 to develop evolutionary models for the free living bacterium alteromonas the genome sequences of isolates of the genus have been extensively analyzed. however, the main genetic exchange drivers in these microbes, conjugative elements, have not been considered in detail thus far. in this work, ces have been searched in several complete alteromonas genomes and their sequence studied to understand their role in the evolution of this genus. six genomes are reported here for the first time.  we have found nine different plasmids of sizes ranging from to kb, most of them were found in a single strain. networks of gene similarity could be established among six of the plasmids that were also connected with another cluster of plasmids found in shewanella strains. the cargo genes found in these plasmids included cassettes found before in chromosome flexible genomic islands of alteromonas strains. we describe also the plasmids pamcp48- and pamcp49- the largest found in alteromonas thus far and containing all the hallmarks to be classified as chromids. we found in them some housekeeping genes and a cluster that code for an exocellular polysaccharide. they could represent the transport vectors for the previously described replacement flexible genomic islands. integrative and conjugative elements were more common than plasmids and showed similar patterns of variation with cargo genes coding for components of additive flexible genomic islands. a nearly identical ice was found in a. mediterranea med and vibrio cholera ahv isolated from a human pathogen, indicating the potential exchange of these genes across phylogenetic distances exceeding the family threshold.  we have seen evidence of how ces can be vectors to transfer gene cassettes acquired in the chromosomal flexible genomic islands, both of the additive and replacement kind. these ces showed evidence of how genetic material is exchanged among members of the same species but also across genus and family barriers. these gradients of exchange frequency are probably one of the main drivers of species origin and maintenance in prokaryotes and also provide these taxa with large genetic diversity. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords alteromonasplasmidspangenomeconjugative elementschromidintegrative and conjugative elementsicegenomic islandshttp://dx.doi.org/ /501100003359generalitat valencianaapostd/2016/051lópez-pérez mario issue-copyright-statement© the author 2017 