BACKGROUND
the marine environment is characterized by a large degree of biodiversity, hosting more living organisms, especially microorganisms, than any other environment.
over the last decade, the introduction of novel and more efficient sequencing techniques has resulted in the acquisition of a wealth of genomic  and metagenomic data   <cit> .
their analysis is disclosing the hidden potential of this pandora’s box, strengthening our opinion on marine biodiversity, i.e.
that it is endowed of chemical diversity potentially exploitable for humankind benefit.
amongst bacterial genera that can be isolated from the marine environment, one of the most frequent is pseudoalteromonas  <cit> , a subgroup of gram-negative gammaproteobacteria that are highly diffuse obligatory marine bacteria.
in most cases, these rod-shaped bacteria display aerobic and chemoheterotrophic metabolism, and they are motile due to sheathed polar and/or unsheathed lateral flagella.
members of this genus have been isolated from almost all marine habitats, such as coastal, open and deep-sea waters, sediments, or in association with higher organisms, such as algae, invertebrates and fishes .
taking into account their frequent occurrence in polar marine samples, several species of pseudoalteromonas have also efficiently adapted to freezing lifestyle .
the genome analysis of the antarctic strain pseudoalteromonas haloplanktis tac <dig>  performed by medigue et al.
<cit>  was the first attempt to provide insights into the genetic basis of the remarkable versatility of this bacterium.
the combination of in silico and in vivo analyses allowed the identification of features related to its versatility and exceptional  fast growth.
among these, one notable trait is the resistance to reactive oxygen species , which represent a considerable stressor under cold conditions  <cit> .
other signatures of cold adaptation are the psychrophiles-specific codon usage bias, that is involved in resistance to protein aging features involving asparagine cyclization and deamidation  <cit> , and the high number of rrna and trna genes, which might explain its translational efficiency even in cold condition  <cit> .
this latter observation justifies an increasing use of phtac <dig> in biotechnological applications, such as for the high quality production of recombinant eukaryotic proteins .
this prompted a comprehensive characterization of this strains, through the analysis of its complete genome  <cit> , its proteome in different conditions  <cit> , detailed growth phenotypes in complex media  <cit> .
the integration of genomic and expression data together with detailed physiological data allowed for the formulation of a genome scale metabolic model  <cit> , that can be used to predict phenotypes and design experiments aimed at the optimization of phtac <dig> metabolic capabilities.
other full genome sequences of pseudoalteromonas representatives have since been reported, highlighting the specific strategies, which allow pseudoalteromonas sp.
sm <dig> strain to thrive in deep-sea sediments, or justify the p. tunicata d <dig> ability to efficiently colonize biotic surfaces  <cit> .
representatives of the genus pseudoalteromonas are also able to produce a broad array of bioactive molecules  and wide-spectrum enzymes with high specificity at low temperatures .
these molecules represent an interesting and important evolutionary strategy, since they are responsible for inhibitory interactions among bacteria inhabiting the same environment, to preserve limited resources   <cit> .
such interactions effectively shape the microbial community composition  <cit> ; in particular, it has been shown that antarctic sponges host specific bacterial communities whose members are able to inhibit the growth of bacteria inhabiting different sponges  <cit> .
it has been shown that some representatives of the main pseudoalteromonas clades  display differential degrees of bioactivity, in that pigmented strains are generally more active against a broad spectrum of organisms  <cit> .
although the non-pigmented strains generally have a minor bioactivity, those inhabiting extreme environments  have evolved adaptive mechanisms to cope with adverse conditions, resulting in competitive advantages.
in addition to modifications of the cellular physiology, the ability of inhibiting the growth of other microbes even at low temperatures could provide an advantage over other competitors  <cit> .
the extreme phenotypic variability observed in representatives of this genus is thus important for deriving evolutionary and functional insights and to guide future, rational, exploration and exploitation of these bacteria.
however, to date, no extensive analysis of the possible underlying genetic/genomic features was carried out.
herein, we have performed a deep comparative genomic analysis using almost every pseudoalteromonas genome currently available, mainly aimed at: i) the identification  of the major evolutionary events that led to the current structure of this genus and ii) the characterization of the genetic repertoire involved in biotechnologically relevant tasks for each strain.
the obtained results depict a complex evolutionary scenario in which horizontal gene transfer  has played  a central role in this genus, with a mechanism of action and spreading that might represent a general trend in marine microbiology.
RESULTS
pseudoalteromonas dataset
for the analysis of the pseudoalteromonas pangenome, a dataset comprising  <dig> genomes present in genbank,  <dig> of which belonging to strains isolated from different antarctic sea ecological niches  was assembled.
the general features of these genomes are reported in table  <dig>  more detailed statistics can be found in additional file  <dig>  briefly, the pseudoalteromonas genomes are, on average,  <dig> , <dig> base pairs long , harbor  <dig> genes , and have a gc composition of 41% .table  <dig>
pseudoalteromonas dataset
this table reports the main features of the pseudoalteromonas genomes
pseudoalteromonas phylogeny
a description of the phylogenetic relationships within the genus pseudoalteromonas, using different genome-wide methods, has been previously reported  <cit> .
here we report a phylogenetic tree of this genus , enriched with additional information , which was built using a concatamer of  <dig> genes, as described in the experimental methods section.fig.
<dig> ml phylogenetic tree of the pseudoalteromonas genus computed using a genome-scale set of genes.
the light blue shaded area marks the strains assigned to the p. haloplanktis-like group; the strain names and nodes are colored according to the literature records  about the pigmentation: strains with a red name are reported to be pigmented, where those with a blue name are non-pigmented.
support values for nodes, are reported when different from 100
one remarkable characteristic of the pseudoalteromonas genus is that it can be divided in two major clades, including the pigmented strains, with a greater bioactivity, and non-pigmented strains, which tend to have more unusual enzymatic activities  <cit> .
notably, in the reported phylogeny there is a clear distinction between pigmented and non-pigmented strains: i) the pigmented clade displays a phylogeny with a greater degree of variability, embedding most of the species included in the dataset; ii) the non-pigmented clade is characterized by a phylogenetic shallowness, consistently with the literature and, since it mostly embeds strains from the p. haloplanktis species , it will be referred to as p. haloplanktis group .
this phylogenetic shallowness suggests an apparently limited genomic diversity within the p. h.-group.
the genus pseudoalteromonas has an open pangenome
it has been argued that bacterial species and genera can be described in terms of their pangenome, that is, the collection of all the genes possessed by a group of bacteria belonging to the same taxon  <cit> .
the pangenome is defined on the basis of the analysis of cluster of orthologous genes  of each genome and it is divided into three categories: 1) core, 2) accessory, and 3) unique genome.
in this work we analyzed and compared the pangenome of: i) the pseudoalteromonas genus, ii) the group of pigmented strains and, iii) the p. h. group.
data obtained are shown in fig.
<dig>  whose analysis revealed thatfig.
<dig> pangenomes analysis.
the section a of this figure reports the number of genes in each pangenome category for: the pangenome of the pseudoalteromonas genus, the pigmented and the p.h.
–group.
the section b reports the rarefaction curves for the core genome and the pangenome, for each dataset.
for the pigmented and p.h.
–group, the dashed line represents the projection of the curve fit up to  <dig> genomes.
the panel c reports the new genes discovery rate for each dataset on a log-log scale.
the dashed line represents the correspondent curve for a closed pangenome
i) the pseudoalteromonas genus pangenome embeds a total of  <dig>  orthologous groups;
ii) the pangenome of pigmented strains contains  <dig>  gene families;
iii) the p. h. -group pangenome contains a total of  <dig>  gene families;
iv) each pangenome is split as follows:
core genome: pseudoalteromonas genus  <dig> genes ; pigmented strains  <dig> genes ; p. h.-group  <dig> genes .
dispensable genes: pseudoalteromonas genus,  <dig> accessory  and  <dig>  unique genes ; pigmented strains  <dig> accessory  and  <dig> unique genes ; p.h.
-group  <dig> accessory  and  <dig> unique genes .
interestingly, the proportion of dispensable genes is relatively high compared to available pangenomes from other taxa  <cit> .
if this might be expected on the basis of the variability of the genome size of pigmented strains and the whole set of pseudoalteromonas genomes , this might be in disagreement with the shallowness exhibited by the p. h.-group.
these findings strongly suggest that the three pseudoalteromonas pangenomes are open, in that the increase of the number of genome sequences results in a parallel increase of pangenome size.
to verify whether the pseudoalteromonas gene repertoire does not reach an asymptotic limit or, conversely, the number of novel genes discovered when adding a new genome to the pangenome is eventually negligible, an iterative sampling approach was used to compute a high number of pangenomes being made of random combinations of n genomes.
this analysis also allowed obtaining the values of i) the total genes, ii) the core genes, and iii) the novel  genes for the whole pseudoalteromonas genus.
data obtained are schematically shown in fig.
2a-c. plotting these values revealed clear trends of the point clouds when the number of considered genomes increases.
trying to capture the trends observed, two different functions were fitted onto this data, namely a double exponential decay function and the heap’s law function .
the fit for the core genome, pangenome size and new genes led to these parameters :i)
core genome size
genus: k
1= <dig>  k
2= <dig>  τ
1 =  <dig> , τ
2 =  <dig> , Ω = 1479
pigmented group: k
1= <dig>  k
2= <dig>  τ
1 =  <dig> , τ
2 =  <dig> , Ω = 2235
p.h.
-group: k
1= <dig> , k
2= <dig>  τ
1 =  <dig> , τ
2 =  <dig> , Ω = 1962
ii)
pangenome size
genus: k= <dig>  γ = <dig>
pigmented group: k= <dig>  γ= <dig>
p.h.
-group: k= <dig>  γ= <dig>
iii)
new genes rate
genus: k= <dig>  α =  <dig>
pigmented group: k= <dig>  α =  <dig>
p.h.
-group: k= <dig>  α =  <dig>
the finding that the γ value of the three pangenomes is greater than zero  indicates that they are open.
one of the main implications of an open pangenome is that the collection of strains with the sequenced genome available are not fully describing, in terms of gene content, the three groups.
regarding this concept, it has been argued  <cit>  how an open pangenome is typical of taxonomic groups, which are able to colonize multiple environments  and, therefore, have the opportunity to exchange genetic material with a variety of different sources, effectively enriching their gene pool with “alien” genes.
conversely, species living in isolation and with lower chances of acquire alien genes have a closed pangenome   <cit> .
the comparative analysis of data obtained for the three groups revealed that the pangenome of the whole genus embeds a more heterogeneous group of organisms than that of the pigmented group and/or p. h.-group; its rarefaction curves has a “more open” trend .
for the same reason, the number of core genes is considerably lower for the pseudoalteromonas pangenome .
however, the pangenome of the p. h.-group was larger than that of pigmented strains .
partially, this might be due to the higher number of genomes embedded in the p. h.-group.
however, these data  appeared to be in conflict with the phylogenetic shallowness of the p. h.-group mentioned above.
indeed, these phylogenetically related strains display a remarkable heterogeneity in terms of gene content, which is comparable to that of the group of pigmented representatives of multiple species .
this might have important evolutionary implications, in that it might suggest that a relatively high number of genes have been obtained through  events of hgt, which might have played a key role in shaping the genomic diversity between strains of this group.
for example, p. tunicata  acquired the gene encoding lipl <dig>  a protein involved in attachment to surface, from a leptospira bacterium  <cit> .
the functional characterization of the pseudoalteromonas pangenomes
as previously stated, the pangenome is a powerful concept that can be used to effectively represent a bacterial taxon, providing insights into the functional categories enriched in a pangenome.
therefore, a functional characterization has been performed by assigning for each pangenome the genes of each section  to a cog functional class .
data obtained regarding the distributions of the cog functional classes in the three pangenomes is shown in fig.
<dig> fig.
<dig> distributions of the cog categories in the different pangenomes.
for each of these, three colored lines indicate the distributions in each category
here, we present the results regarding: i) the differences observed between core and dispensable genome which are conserved across the pangenomes and ii) the functional differences among the pangenomes.
regarding the first point, as expected, the major portion of the core genes is mostly involved in housekeeping processes, including metabolic functions  and non-metabolic related central processes such as transcription, translation and replication.
conversely, in the dispensable genome, these categories are present, although less represented, whereas the majority of the dispensable genes were not assigned to any category.
such predominant genes could be either novel genes showing no homology in the cog database, or pseudogenes with severe primary amino acid sequence disruption.
indeed, it has been argued that pseudogenes are pervasively distributed in prokaryotes, and that a great portion of them derives from “failed” hgt events  <cit> , since genes acquired through hgt events may provide no advantage , or even be detrimental to the host .
in time, these sequences undergo mutations and are eventually lost, making it difficult to find them in the core genome.
comparing the distribution of cog categories between the three pangenomes, it can be argued the existence of few categories in which the genes are differentially distributed in the pangenome sections .
indeed, these partitions might be embedding genes responsible for functional differences between pseudoalteromonas groups.
for instance, considering the genes related to carbohydrate transport and metabolism , the pangenomes of pigmented strains and p. h. -group embed comparable number of genes associated to this category in the three pangenome sections, where the core genes are the most represented.
however, looking at the pangenome of the whole genus, there is a remarkable different distribution of the g genes, since the accessory genes are the most represented class.
this could be explained with the presence of a pool of group-specific genes that are shared by all the strains of a particular group, but not by all the strains of the other group.
to verify this hypothesis, the core genes of the p. h. group and the pigmented group were compared, revealing that all the pigmented strains share a pool of genes encoding enzymes involved in the production and degradation of glycogen .
this finding has some evolutionary implications, in that it suggests that this metabolic pathway has probably been lost during the evolutionary history of the p. h. group.
the most remarkable difference is observed for the cog category l , also including genes involved in hgt events, which is enriched for the unique genes of the p. h.-group , compared with the unique genes of the pangenome of the genus  and the pigmented group .
since the results of the pangenome analyses, i.e.
the open pangenomes and the high proportion of l genes, appear to suggest that the hgt had a role in the evolution of this genus, we investigated more in details these events, by searching the presence of genes involved in the exchange of genes and trying to estimate the events of gene losses and gains that have occurred during the evolution of pseudoalteromonas.
the contribution of horizontal gene transfer to pseudoalteromonas evolution
the pseudoalteromonas panmobilome
mobile genetic elements  are the main actors of the lateral exchange of genes and include plasmids, viruses  and transposons.
the mobilome of a strain is the repertoire of all the genes of that strain found to be associated with mges using in silico methods.
extending this concept to a whole taxon , we define the panmobilome as the collection of all the mges of that taxon.
the aclame database  <cit> , a collection of mges from various sources, was used to tentatively assign the pseudoalteromonas genes to three families of mobile elements  using a sequence similarity search approach, as described in the methods.
data obtained are shown in table  <dig>  which reports for each genome the total number of genes associated with each aclame family.
globally,  <dig>  genes associated with mges have been found with this approach, which, on average, account for an important fraction of the genomes of the pseudoalteromonas strains .
the comparative analysis of these genes allowed to identify a pool of shared genes , genes present in some but not all of the strains  and genes unique to some strains  consisting of  <dig>   <dig> and  <dig> genes, respectively .table  <dig> the pseudoalteromonas panmobilome
in this table are reported for each strain the number of genes assigned to each aclame family , along with the total
fig.
<dig> the pseudoalteromonas panmobilome.
in this figure are reported a the composition of the panmobilome and b the distribution of the aclame families in the panmobilome sections
the distribution of the aclame families in the panmobilome revealed that it mostly embeds genes associated with plasmids , with a minor fraction of genes associated to prophages  and virus .
interestingly, genes of different families are not all equally divided into the panmobilome sections .
in particular, genes belonging to the virus family are almost absent from the core mobilome , whereas they are more prevalent in the accessory and unique mobilome .
this result probably reflects the fact that different viruses infect preferentially some species/strains  <cit> .
another possible explanation could be that viral genes have been mostly lost during the evolution of the genus, since they are under negative selection.
to evaluate the impact of hgt-mediated events on the overall pseudoalteromonas phylogeny, a matrix of mge presence/absence was compiled using the data from aclame, where each column is an mge gene and each row is a pseudoalteromonas strain .
the dendrogram resulting from the clustering of the rows  was compared with the phylogenetic tree reported in fig.
<dig>  indeed, we identified a strong similarity between the two dendrograms, in that the p. h. -group  is clearly separated from the strains of the pigmented group.
the correlation between the pattern of mges presence and phylogeny is interesting and suggests that the mges harbor a strong phylogenetic signal, which, in principle, can discriminate the species.
this implies the presence of a set of mges which has been gained between the speciation of some pseudoalteromonas clades and that has followed the genus phylogeny.fig.
<dig> heatmap representation of the panmobilome.
each cell indicates whether a gene associated with a mge  is present  or not  in a strain .
each column is color labeled according to the three mge aclame families .
the dendrogram on the left has been produced from the hierarchical clustering of the rows
pseudoalteromonas genus has undergone genome reduction in evolution
the influence of the hgt events on the pseudoalteromonas evolution prompts a thorough investigation on the events of gene gain and loss occurred during the evolutionary history of the genus, raising the intriguing question on what was the gene repertoire of the pseudoalteromonas last common ancestor , and which evolutionary dynamics led to the structure of the present-day genomes.
using the phylogenetic tree reported in fig.
<dig>  it was possible to infer the events of gene gain/loss for each cluster of orthologous genes , by mapping the phyletic patterns of the genes on the tree.
in this way, it was possible to: i) assess the number of acquisitions and losses at each internal node of the tree, and ii) estimate the set of ancestral genes of the pseudoalteromonas lca, which are reported in the dendrogram in fig.
<dig> fig.
<dig> gene gains/losses reconstruction of the pseudoalteromonas genus.
for each node of the cladogram, the number of gene gains  and losses  are reported, along with the estimated ancestral genome size
according to the approach used to infer gene gain/loss events along the phylogenetic tree , we estimated that the pseudoalteromonas lca had  <dig> genes, a relatively small number considering the average number of genes in the present-day pseudoalteromonas genomes .
from this, we infer that the pseudoalteromonas strains have undergone genome expansion through a number of gene gain events.
interestingly, the greatest number of gene gains was found in the leaves of the tree, that is, the greatest contribution to the genomic variability comes from recent events.
looking in more detail at the gene gains, it can be observed how some strains  have recently gained a large number of genes.
the gene gains can be originated either from a combination of gene duplication and speciation events, or from the acquisition of a large number of genes through hgt.
since the horizontal evolution is more rapid than the vertical one, it is possible that most of the recent gene gains derive from hgt events.
hgt probably occurred via transduction
although we showed many evidences of hgt events in pseudoalteromonas, there are still no clues if there is a preferential molecular mechanism leading to the genetic recombination.
in principle, dna stretches can be horizontally transferred via conjugation, transduction and/or transformation .
it is quite possible that, for these marine bacteria, the transduction might represent the key mechanism responsible for hgt; indeed, conjugation requires the formation of the pilus, which might be unstable in an aqueous environment, unless it occurs when bacteria are associated with surfaces ; as for the transformation, it has been show how in the environment this is more likely to occur in the marine sediments rather than in the water column  <cit> .
based on these assumptions, it is unlikely that hgt occurred through these molecular mechanisms, considering the environment inhabited by these microbes.
to verify this hypothesis, the genome of  <dig> pseudoalteromonas was scanned using tra, mob and com genes  as seeds, but none of these genes was recovered.
indeed, since the molecular components necessary for the conjugation and transformation are missing, it is very likely that the hgt is mediated in pseudoalteromonas by transduction, or similar mechanisms.
in particular, it has been shown how the mobilization of genes through prophage-like elements known as gene transfer agents  is one of the major driving forces in the evolution of marine bacteria  <cit> .
these elements, which have been found to be common among the alphaproteobacteria, have the peculiarity of packaging random dna from the host genome, which is integrated into a recipient genome with a mechanism similar to the transduction  <cit> .
pseudoalteromonas metabolic processes
up to now, we have been focusing mainly on the evolutionary insights of the pseudoalteromonas.
however, bacteria belonging to this genus are known for possessing a remarkable biotechnological potential.
in particular, the ability to grow at low temperature and to produce antimicrobial molecules have important implications.
therefore, we focused on a thorough characterization of the genetic determinants of these processes.
the results of the analysis of genes related to xenobiotic resistance and molecule transport are reported in additional files  <dig> and  <dig>  respectively.
cold-adaptation proteins
since the marine environment is generally cold, it is reasonable that some pseudoalteromonas strains  might possess genes involved in the adaptation to cold temperatures , such as anti-freeze proteins , a class of proteins able to inhibit the ice nucleation or decrease the water freezing point temperature  <cit> .
for this reason, we searched caps in these genomes using a dataset of  <dig> caps.
results obtained  revealed how no clear distinction between antarctic and non-antarctic strains is observed, since most of the strains have 4– <dig> caps.
interestingly, we found one caps shared between all the strains, that is etfa, an electron transfer flavoprotein which has been found to be up-regulated at low temperature in p. haloplanktis tac <dig>  <cit> .
assuming that these proteins provide a strong advantage in a permanently cold environment, it could be expected that these are subject to purifying selection.
to test this hypothesis, we computed, for each group of caps, the ka/ks ratio.
the results of this analysis , showed remarkably low values of this ratio, thus confirming our hypothesis.
on average, the caps ka/ks ratio for the genus pseudoalteromonas is  <dig> .fig.
<dig> heatmap of caps presence/absence.
the strain names are colored according to the pigmentation
biosynthesis of secondary metabolites with antimicrobial activity
some pseudoalteromonas representatives are well known for their ability to antagonize the growth of bacteria, algae, fungi and parasites  <cit> , suggesting the presence of genes involved in the biosynthesis of antimicrobial compounds.
these genes have been searched in the pseudoalteromonas genomes.
there are few tools available to automatically identify operons encoding secondary metabolites, including clustscan  <cit> , the sbspks toolbox  <cit> , bagel <dig>  <cit> , clusean  <cit>  and antismash  <cit> .
since a systematic benchmarking is currently missing, we opted for antismash for two reasons.
first, it is not limited to pks and nrp operons, such as clustscan, sbspks and bagel <dig>  second, it’s extremely easy to use and produces clear results.
the results of antismash revealed a plethora of operons involved in the biosynthesis of molecules such as ferrins, bacteriocins, polyketides, lantipeptides and other non-ribosomial peptides.
data obtained  is reported in additional file  <dig>  moreover, a sequence similarity approach has been used to identify homologous clusters and each cluster was mapped onto the pseudoalteromonas phylogenetic tree .
our findings demonstrated that all the pseudoalteromonas genomes harbor at least one cluster involved in bioactive molecule synthesis.fig.
<dig> biosynthetic operons involved in the biosynthesis of secondary metabolites in  <dig> pseudoalteromonas genomes identified through antismash analysis.
the bar plot reports the number of the biosynthetic operon assigned to each pseudoalteromonas strain.
the phylogenetic tree is reported next to the bar plot.
the strain names are colored according to the pigmentation
therefore any pseudoalteromonas strain can, in theory, carry out an inhibitory activity, which is consistent with previous observation .
second, we observed a high variability of the biosynthetic operons, in terms of number and composition, across the different genomes .
in particular, while most of the strains have a similar number of biosynthetic operons , the genomes of five pigmented strains  are characterized by the presence of a high number of operons.
indeed, a large variability of the inhibition carried out by different strains was observed by holmstrom et al.
<cit> , with the pigmented strains  showing the largest inhibition.
thus, the results we obtained might explain the inhibition differences between strains, in that the pigmented strains are, in theory, able to produce a wider array of bioactive molecules, since they have a larger number of operons.
these results are also in agreement with more recent works describing the ability of pseudoalteromonas strains to completely inhibit the growth of human opportunistic pathogens belonging to the burkholderia cepacia complex .
in those works the authors found that one of the most active pseudoalteromonas strains was tb <dig>  which is the only one able to produce a polyketide synthase that very likely contributes  to its high inhibitory activity  <cit> .
all  <dig> strains share one operon; which suggests that this operon was present in the pseudoalteromonas lca.
concerning this issue, it is not clear whether the lca harbored just one operon  and the others have been acquired  by different strains or if the genome of the common ancestor embedded a higher number of different operons involved in the biosynthesis of different bioactive molecules.
according to this second scenario, one or more operons should have been lost during evolution by different pseudoalteromonas strains/species.
to test these hypotheses, we investigated where these genes have been acquired/lost, according to the ancestral gene states reconstruction shown in fig.
<dig>  although some of these genes were present in the pseudoalteromonas lca, the majority of these genes have been acquired through recent events, especially in the pigmented strains.
therefore, strain-specific gene acquisition led to the variability in the operon presence.
these results clearly show the biosynthetic potential of some of these strains, prompting for potential application, such as drug discovery and biocontrol of aquaculture.
as a proof of concept, some of the predicted operons correspond to clusters of genes, which have already been discovered and validated as antimicrobial.
for example, we found the gene cluster involved in the production of violacein  in both p. luteoviolacea and p. tunicata genomes, for which the production of this compound has been witnessed  <cit> .
CONCLUSIONS
in this work we have performed a comparative genomics analysis for a large portion of the pseudoalteromonas strains with the sequenced genome available in genbank, gaining new insights into the evolutionary history of this genus and trying to underline the main genomic features with an ecological relevance.
considering their pigmentation, the  <dig> strains under study can be classified in two major groups: one embedding  <dig> strains  and the other containing  <dig> strains , most of which isolated from different ecological niches of antarctica.
from a phylogenetic point of view, the pigmented group displays a greater degree of variability, whereas the non-pigmented one is characterized by a phylogenetic shallowness.
however, the analysis of the pangenome of the two groups revealed how they are characterized by an open pangenome with a  high percentage of unique genes.
this is remarkable for the non-pigmented strains, since this is in apparent contrast with the phylogenetic shallowness.
the analysis of the dynamics of gene gains and losses revealed how the evolution of the pseudoalteromonas strains is strongly affected by recent acquisitions of a large number of genes, most likely by means of hgt.
this idea is in agreement with the fact that most of these strains inhabit the antarctica and on the finding that the marine environment represents a key reservoir of horizontally transferred genes  <cit> .
this hypothesis was supported by the functional analyses of the pseudoalteromonas genomes, which revealed the existence of a large fraction of genes related to mges, most of which have a plasmid origin.
since the molecular actors of bacterial conjugation are absent in the pseudoalteromonas genomes, we speculate that the exchange of genetic material might occur via transduction, which could explain the considerable portion of phage genes found in the panmobilome.
the biological significance of such degree of hgt and the high number of unique genes might rely on the fact that most of these strains have been isolated from antarctica.
indeed, in such a harsh environment, could be favored those microorganisms that, by exploring many possible genetic/genomic combinations, find the more efficient phenotype for the colonization of a given ecological niche.
moreover, since all of the strains analyzed in this work are marine bacteria, which have been isolated from different world regions, their genomic variability could imply that marine-living bacteria have to cope with different niches and to be able to rapidly adapt to fast changes of the environmental conditions.
in this context the analyses of particular metabolic traits might shed some light on this issue.
indeed, in the marine environment it is very likely that microorganisms belonging to different species/genera have to compete for nutrients; accordingly, antagonistic interactions between bacteria isolated in antarctica from different niches  have been disclosed recently  <cit> .
these antagonistic interactions very likely rely on the production of antimicrobial compounds that can also inhibit the growth of human pathogens  <cit> .
according to this idea, the analyses performed in this work revealed the presence of gene clusters involved in the biosynthesis of secondary metabolites.
notably, as it might be expected, the higher the number of gene clusters, the higher the number of metabolites synthesized by pseudoalteromonas strains.
on the other side, bacteria living in a “competitive” environment, such as the marine one, have evolved different mechanisms to resist to antibiotics and/or other toxic compounds.
the remarkably high number of genes encoding efflux pumps in the pseudoalteromonas genomes fully supports this view.
the results reported in this work show how the pseudoalteromonas genus embeds a remarkable genomic diversity, with a large proportion of unique genes, underlining that our knowledge of this genus is still limited.
this, together with the fact that most of these strains seems to be very promising for applicative purposes, prompts for further explorative studies on these marine bacteria, with the collection of new strains and the sequencing of the genomes of other representatives of this genus.

 the marine environment is characterized by a large degree of biodiversity, hosting more living organisms, especially microorganisms, than any other environment. over the last decade, the introduction of novel and more efficient sequencing techniques has resulted in the acquisition of a wealth of genomic and metagenomic data. their analysis is disclosing the hidden potential of this pandora’s box, strengthening our opinion on marine biodiversity, i.e. that it is endowed of chemical diversity potentially exploitable for humankind benefit. amongst bacterial genera that can be isolated from the marine environment, one of the most frequent is pseudoalteromonas, a subgroup of gram-negative gammaproteobacteria that are highly diffuse obligatory marine bacteria. in most cases, these rod-shaped bacteria display aerobic and chemoheterotrophic metabolism, and they are motile due to sheathed polar and/or unsheathed lateral flagella. members of this genus have been isolated from almost all marine habitats, such as coastal, open and deep-sea waters, sediments, or in association with higher organisms, such as algae, invertebrates and fishes. taking into account their frequent occurrence in polar marine samples, several species of pseudoalteromonas have also efficiently adapted to freezing lifestyle. the genome analysis of the antarctic strain pseudoalteromonas haloplanktis tac performed by medigue et al. was the first attempt to provide insights into the genetic basis of the remarkable versatility of this bacterium. the combination of in silico and in vivo analyses allowed the identification of features related to its versatility and exceptional fast growth. among these, one notable trait is the resistance to reactive oxygen species, which represent a considerable stressor under cold conditions. other signatures of cold adaptation are the psychrophiles-specific codon usage bias, that is involved in resistance to protein aging features involving asparagine cyclization and deamidation, and the high number of rrna and trna genes, which might explain its translational efficiency even in cold condition. this latter observation justifies an increasing use of phtac in biotechnological applications, such as for the high quality production of recombinant eukaryotic proteins. this prompted a comprehensive characterization of this strains, through the analysis of its complete genome, its proteome in different conditions, detailed growth phenotypes in complex media. the integration of genomic and expression data together with detailed physiological data allowed for the formulation of a genome scale metabolic model, that can be used to predict phenotypes and design experiments aimed at the optimization of phtac metabolic capabilities. other full genome sequences of pseudoalteromonas representatives have since been reported, highlighting the specific strategies, which allow pseudoalteromonas sp. sm strain to thrive in deep-sea sediments, or justify the p. tunicata d ability to efficiently colonize biotic surfaces. representatives of the genus pseudoalteromonas are also able to produce a broad array of bioactive molecules and wide-spectrum enzymes with high specificity at low temperatures. these molecules represent an interesting and important evolutionary strategy, since they are responsible for inhibitory interactions among bacteria inhabiting the same environment, to preserve limited resources. such interactions effectively shape the microbial community composition; in particular, it has been shown that antarctic sponges host specific bacterial communities whose members are able to inhibit the growth of bacteria inhabiting different sponges. it has been shown that some representatives of the main pseudoalteromonas clades display differential degrees of bioactivity, in that pigmented strains are generally more active against a broad spectrum of organisms. although the non-pigmented strains generally have a minor bioactivity, those inhabiting extreme environments have evolved adaptive mechanisms to cope with adverse conditions, resulting in competitive advantages. in addition to modifications of the cellular physiology, the ability of inhibiting the growth of other microbes even at low temperatures could provide an advantage over other competitors. the extreme phenotypic variability observed in representatives of this genus is thus important for deriving evolutionary and functional insights and to guide future, rational, exploration and exploitation of these bacteria. however, to date, no extensive analysis of the possible underlying genetic/genomic features was carried out. herein, we have performed a deep comparative genomic analysis using almost every pseudoalteromonas genome currently available, mainly aimed at: i) the identification of the major evolutionary events that led to the current structure of this genus and ii) the characterization of the genetic repertoire involved in biotechnologically relevant tasks for each strain. the obtained results depict a complex evolutionary scenario in which horizontal gene transfer has played a central role in this genus, with a mechanism of action and spreading that might represent a general trend in marine microbiology.  pseudoalteromonas dataset for the analysis of the pseudoalteromonas pangenome, a dataset comprising genomes present in genbank, of which belonging to strains isolated from different antarctic sea ecological niches was assembled. the general features of these genomes are reported in table  more detailed statistics can be found in additional file briefly, the pseudoalteromonas genomes are, on average,, base pairs long, harbor genes, and have a gc composition of 41%.table pseudoalteromonas dataset this table reports the main features of the pseudoalteromonas genomes pseudoalteromonas phylogeny a description of the phylogenetic relationships within the genus pseudoalteromonas, using different genome-wide methods, has been previously reported. here we report a phylogenetic tree of this genus, enriched with additional information, which was built using a concatamer of genes, as described in the experimental methods section.fig. ml phylogenetic tree of the pseudoalteromonas genus computed using a genome-scale set of genes. the light blue shaded area marks the strains assigned to the p. haloplanktis-like group; the strain names and nodes are colored according to the literature records about the pigmentation: strains with a red name are reported to be pigmented, where those with a blue name are non-pigmented. support values for nodes, are reported when different from 100 one remarkable characteristic of the pseudoalteromonas genus is that it can be divided in two major clades, including the pigmented strains, with a greater bioactivity, and non-pigmented strains, which tend to have more unusual enzymatic activities. notably, in the reported phylogeny there is a clear distinction between pigmented and non-pigmented strains: i) the pigmented clade displays a phylogeny with a greater degree of variability, embedding most of the species included in the dataset; ii) the non-pigmented clade is characterized by a phylogenetic shallowness, consistently with the literature and, since it mostly embeds strains from the p. haloplanktis species, it will be referred to as p. haloplanktis group. this phylogenetic shallowness suggests an apparently limited genomic diversity within the p. h.-group. the genus pseudoalteromonas has an open pangenome it has been argued that bacterial species and genera can be described in terms of their pangenome, that is, the collection of all the genes possessed by a group of bacteria belonging to the same taxon. the pangenome is defined on the basis of the analysis of cluster of orthologous genes of each genome and it is divided into three categories: 1) core, 2) accessory, and 3) unique genome. in this work we analyzed and compared the pangenome of: i) the pseudoalteromonas genus, ii) the group of pigmented strains and, iii) the p. h. group. data obtained are shown in fig.  whose analysis revealed thatfig. pangenomes analysis. the section a of this figure reports the number of genes in each pangenome category for: the pangenome of the pseudoalteromonas genus, the pigmented and the p.h. –group. the section b reports the rarefaction curves for the core genome and the pangenome, for each dataset. for the pigmented and p.h. –group, the dashed line represents the projection of the curve fit up to genomes. the panel c reports the new genes discovery rate for each dataset on a log-log scale. the dashed line represents the correspondent curve for a closed pangenome i) the pseudoalteromonas genus pangenome embeds a total of orthologous groups; ii) the pangenome of pigmented strains contains gene families; iii) the p. h. -group pangenome contains a total of gene families; iv) each pangenome is split as follows: core genome: pseudoalteromonas genus genes; pigmented strains genes; p. h.-group genes. dispensable genes: pseudoalteromonas genus, accessory and unique genes; pigmented strains accessory and unique genes; p.h. -group accessory and unique genes. interestingly, the proportion of dispensable genes is relatively high compared to available pangenomes from other taxa. if this might be expected on the basis of the variability of the genome size of pigmented strains and the whole set of pseudoalteromonas genomes, this might be in disagreement with the shallowness exhibited by the p. h.-group. these findings strongly suggest that the three pseudoalteromonas pangenomes are open, in that the increase of the number of genome sequences results in a parallel increase of pangenome size. to verify whether the pseudoalteromonas gene repertoire does not reach an asymptotic limit or, conversely, the number of novel genes discovered when adding a new genome to the pangenome is eventually negligible, an iterative sampling approach was used to compute a high number of pangenomes being made of random combinations of n genomes. this analysis also allowed obtaining the values of i) the total genes, ii) the core genes, and iii) the novel genes for the whole pseudoalteromonas genus. data obtained are schematically shown in fig. 2a-c. plotting these values revealed clear trends of the point clouds when the number of considered genomes increases. trying to capture the trends observed, two different functions were fitted onto this data, namely a double exponential decay function and the heap’s law function. the fit for the core genome, pangenome size and new genes led to these parameters:i) core genome size genus: k 1= k 2= τ 1 = , τ 2 = , Ω = 1479 pigmented group: k 1= k 2= τ 1 = , τ 2 = , Ω = 2235 p.h. -group: k 1=, k 2= τ 1 = , τ 2 = , Ω = 1962 ii) pangenome size genus: k= γ = pigmented group: k= γ= p.h. -group: k= γ= iii) new genes rate genus: k= α =  pigmented group: k= α =  p.h. -group: k= α =  the finding that the γ value of the three pangenomes is greater than zero indicates that they are open. one of the main implications of an open pangenome is that the collection of strains with the sequenced genome available are not fully describing, in terms of gene content, the three groups. regarding this concept, it has been argued how an open pangenome is typical of taxonomic groups, which are able to colonize multiple environments and, therefore, have the opportunity to exchange genetic material with a variety of different sources, effectively enriching their gene pool with “alien” genes. conversely, species living in isolation and with lower chances of acquire alien genes have a closed pangenome. the comparative analysis of data obtained for the three groups revealed that the pangenome of the whole genus embeds a more heterogeneous group of organisms than that of the pigmented group and/or p. h.-group; its rarefaction curves has a “more open” trend. for the same reason, the number of core genes is considerably lower for the pseudoalteromonas pangenome. however, the pangenome of the p. h.-group was larger than that of pigmented strains. partially, this might be due to the higher number of genomes embedded in the p. h.-group. however, these data appeared to be in conflict with the phylogenetic shallowness of the p. h.-group mentioned above. indeed, these phylogenetically related strains display a remarkable heterogeneity in terms of gene content, which is comparable to that of the group of pigmented representatives of multiple species. this might have important evolutionary implications, in that it might suggest that a relatively high number of genes have been obtained through events of hgt, which might have played a key role in shaping the genomic diversity between strains of this group. for example, p. tunicata acquired the gene encoding lipl a protein involved in attachment to surface, from a leptospira bacterium. the functional characterization of the pseudoalteromonas pangenomes as previously stated, the pangenome is a powerful concept that can be used to effectively represent a bacterial taxon, providing insights into the functional categories enriched in a pangenome. therefore, a functional characterization has been performed by assigning for each pangenome the genes of each section to a cog functional class. data obtained regarding the distributions of the cog functional classes in the three pangenomes is shown in fig.  fig. distributions of the cog categories in the different pangenomes. for each of these, three colored lines indicate the distributions in each category here, we present the results regarding: i) the differences observed between core and dispensable genome which are conserved across the pangenomes and ii) the functional differences among the pangenomes. regarding the first point, as expected, the major portion of the core genes is mostly involved in housekeeping processes, including metabolic functions and non-metabolic related central processes such as transcription, translation and replication. conversely, in the dispensable genome, these categories are present, although less represented, whereas the majority of the dispensable genes were not assigned to any category. such predominant genes could be either novel genes showing no homology in the cog database, or pseudogenes with severe primary amino acid sequence disruption. indeed, it has been argued that pseudogenes are pervasively distributed in prokaryotes, and that a great portion of them derives from “failed” hgt events, since genes acquired through hgt events may provide no advantage, or even be detrimental to the host. in time, these sequences undergo mutations and are eventually lost, making it difficult to find them in the core genome. comparing the distribution of cog categories between the three pangenomes, it can be argued the existence of few categories in which the genes are differentially distributed in the pangenome sections. indeed, these partitions might be embedding genes responsible for functional differences between pseudoalteromonas groups. for instance, considering the genes related to carbohydrate transport and metabolism, the pangenomes of pigmented strains and p. h. -group embed comparable number of genes associated to this category in the three pangenome sections, where the core genes are the most represented. however, looking at the pangenome of the whole genus, there is a remarkable different distribution of the g genes, since the accessory genes are the most represented class. this could be explained with the presence of a pool of group-specific genes that are shared by all the strains of a particular group, but not by all the strains of the other group. to verify this hypothesis, the core genes of the p. h. group and the pigmented group were compared, revealing that all the pigmented strains share a pool of genes encoding enzymes involved in the production and degradation of glycogen. this finding has some evolutionary implications, in that it suggests that this metabolic pathway has probably been lost during the evolutionary history of the p. h. group. the most remarkable difference is observed for the cog category l, also including genes involved in hgt events, which is enriched for the unique genes of the p. h.-group, compared with the unique genes of the pangenome of the genus and the pigmented group. since the results of the pangenome analyses, i.e. the open pangenomes and the high proportion of l genes, appear to suggest that the hgt had a role in the evolution of this genus, we investigated more in details these events, by searching the presence of genes involved in the exchange of genes and trying to estimate the events of gene losses and gains that have occurred during the evolution of pseudoalteromonas. the contribution of horizontal gene transfer to pseudoalteromonas evolution the pseudoalteromonas panmobilome mobile genetic elements are the main actors of the lateral exchange of genes and include plasmids, viruses and transposons. the mobilome of a strain is the repertoire of all the genes of that strain found to be associated with mges using in silico methods. extending this concept to a whole taxon, we define the panmobilome as the collection of all the mges of that taxon. the aclame database, a collection of mges from various sources, was used to tentatively assign the pseudoalteromonas genes to three families of mobile elements using a sequence similarity search approach, as described in the methods. data obtained are shown in table  which reports for each genome the total number of genes associated with each aclame family. globally, genes associated with mges have been found with this approach, which, on average, account for an important fraction of the genomes of the pseudoalteromonas strains. the comparative analysis of these genes allowed to identify a pool of shared genes, genes present in some but not all of the strains and genes unique to some strains consisting of and genes, respectively.table the pseudoalteromonas panmobilome in this table are reported for each strain the number of genes assigned to each aclame family, along with the total fig. the pseudoalteromonas panmobilome. in this figure are reported a the composition of the panmobilome and b the distribution of the aclame families in the panmobilome sections the distribution of the aclame families in the panmobilome revealed that it mostly embeds genes associated with plasmids, with a minor fraction of genes associated to prophages and virus. interestingly, genes of different families are not all equally divided into the panmobilome sections. in particular, genes belonging to the virus family are almost absent from the core mobilome, whereas they are more prevalent in the accessory and unique mobilome. this result probably reflects the fact that different viruses infect preferentially some species/strains. another possible explanation could be that viral genes have been mostly lost during the evolution of the genus, since they are under negative selection. to evaluate the impact of hgt-mediated events on the overall pseudoalteromonas phylogeny, a matrix of mge presence/absence was compiled using the data from aclame, where each column is an mge gene and each row is a pseudoalteromonas strain. the dendrogram resulting from the clustering of the rows was compared with the phylogenetic tree reported in fig.  indeed, we identified a strong similarity between the two dendrograms, in that the p. h. -group is clearly separated from the strains of the pigmented group. the correlation between the pattern of mges presence and phylogeny is interesting and suggests that the mges harbor a strong phylogenetic signal, which, in principle, can discriminate the species. this implies the presence of a set of mges which has been gained between the speciation of some pseudoalteromonas clades and that has followed the genus phylogeny.fig. heatmap representation of the panmobilome. each cell indicates whether a gene associated with a mge is present or not in a strain. each column is color labeled according to the three mge aclame families. the dendrogram on the left has been produced from the hierarchical clustering of the rows pseudoalteromonas genus has undergone genome reduction in evolution the influence of the hgt events on the pseudoalteromonas evolution prompts a thorough investigation on the events of gene gain and loss occurred during the evolutionary history of the genus, raising the intriguing question on what was the gene repertoire of the pseudoalteromonas last common ancestor, and which evolutionary dynamics led to the structure of the present-day genomes. using the phylogenetic tree reported in fig.  it was possible to infer the events of gene gain/loss for each cluster of orthologous genes, by mapping the phyletic patterns of the genes on the tree. in this way, it was possible to: i) assess the number of acquisitions and losses at each internal node of the tree, and ii) estimate the set of ancestral genes of the pseudoalteromonas lca, which are reported in the dendrogram in fig.  fig. gene gains/losses reconstruction of the pseudoalteromonas genus. for each node of the cladogram, the number of gene gains and losses are reported, along with the estimated ancestral genome size according to the approach used to infer gene gain/loss events along the phylogenetic tree, we estimated that the pseudoalteromonas lca had genes, a relatively small number considering the average number of genes in the present-day pseudoalteromonas genomes. from this, we infer that the pseudoalteromonas strains have undergone genome expansion through a number of gene gain events. interestingly, the greatest number of gene gains was found in the leaves of the tree, that is, the greatest contribution to the genomic variability comes from recent events. looking in more detail at the gene gains, it can be observed how some strains have recently gained a large number of genes. the gene gains can be originated either from a combination of gene duplication and speciation events, or from the acquisition of a large number of genes through hgt. since the horizontal evolution is more rapid than the vertical one, it is possible that most of the recent gene gains derive from hgt events. hgt probably occurred via transduction although we showed many evidences of hgt events in pseudoalteromonas, there are still no clues if there is a preferential molecular mechanism leading to the genetic recombination. in principle, dna stretches can be horizontally transferred via conjugation, transduction and/or transformation. it is quite possible that, for these marine bacteria, the transduction might represent the key mechanism responsible for hgt; indeed, conjugation requires the formation of the pilus, which might be unstable in an aqueous environment, unless it occurs when bacteria are associated with surfaces; as for the transformation, it has been show how in the environment this is more likely to occur in the marine sediments rather than in the water column. based on these assumptions, it is unlikely that hgt occurred through these molecular mechanisms, considering the environment inhabited by these microbes. to verify this hypothesis, the genome of pseudoalteromonas was scanned using tra, mob and com genes as seeds, but none of these genes was recovered. indeed, since the molecular components necessary for the conjugation and transformation are missing, it is very likely that the hgt is mediated in pseudoalteromonas by transduction, or similar mechanisms. in particular, it has been shown how the mobilization of genes through prophage-like elements known as gene transfer agents is one of the major driving forces in the evolution of marine bacteria. these elements, which have been found to be common among the alphaproteobacteria, have the peculiarity of packaging random dna from the host genome, which is integrated into a recipient genome with a mechanism similar to the transduction. pseudoalteromonas metabolic processes up to now, we have been focusing mainly on the evolutionary insights of the pseudoalteromonas. however, bacteria belonging to this genus are known for possessing a remarkable biotechnological potential. in particular, the ability to grow at low temperature and to produce antimicrobial molecules have important implications. therefore, we focused on a thorough characterization of the genetic determinants of these processes. the results of the analysis of genes related to xenobiotic resistance and molecule transport are reported in additional files and respectively. cold-adaptation proteins since the marine environment is generally cold, it is reasonable that some pseudoalteromonas strains might possess genes involved in the adaptation to cold temperatures, such as anti-freeze proteins, a class of proteins able to inhibit the ice nucleation or decrease the water freezing point temperature. for this reason, we searched caps in these genomes using a dataset of caps. results obtained revealed how no clear distinction between antarctic and non-antarctic strains is observed, since most of the strains have 4– caps. interestingly, we found one caps shared between all the strains, that is etfa, an electron transfer flavoprotein which has been found to be up-regulated at low temperature in p. haloplanktis tac. assuming that these proteins provide a strong advantage in a permanently cold environment, it could be expected that these are subject to purifying selection. to test this hypothesis, we computed, for each group of caps, the ka/ks ratio. the results of this analysis, showed remarkably low values of this ratio, thus confirming our hypothesis. on average, the caps ka/ks ratio for the genus pseudoalteromonas is.fig. heatmap of caps presence/absence. the strain names are colored according to the pigmentation biosynthesis of secondary metabolites with antimicrobial activity some pseudoalteromonas representatives are well known for their ability to antagonize the growth of bacteria, algae, fungi and parasites, suggesting the presence of genes involved in the biosynthesis of antimicrobial compounds. these genes have been searched in the pseudoalteromonas genomes. there are few tools available to automatically identify operons encoding secondary metabolites, including clustscan, the sbspks toolbox, bagel, clusean and antismash. since a systematic benchmarking is currently missing, we opted for antismash for two reasons. first, it is not limited to pks and nrp operons, such as clustscan, sbspks and bagel second, it’s extremely easy to use and produces clear results. the results of antismash revealed a plethora of operons involved in the biosynthesis of molecules such as ferrins, bacteriocins, polyketides, lantipeptides and other non-ribosomial peptides. data obtained is reported in additional file moreover, a sequence similarity approach has been used to identify homologous clusters and each cluster was mapped onto the pseudoalteromonas phylogenetic tree. our findings demonstrated that all the pseudoalteromonas genomes harbor at least one cluster involved in bioactive molecule synthesis.fig. biosynthetic operons involved in the biosynthesis of secondary metabolites in pseudoalteromonas genomes identified through antismash analysis. the bar plot reports the number of the biosynthetic operon assigned to each pseudoalteromonas strain. the phylogenetic tree is reported next to the bar plot. the strain names are colored according to the pigmentation therefore any pseudoalteromonas strain can, in theory, carry out an inhibitory activity, which is consistent with previous observation. second, we observed a high variability of the biosynthetic operons, in terms of number and composition, across the different genomes. in particular, while most of the strains have a similar number of biosynthetic operons, the genomes of five pigmented strains are characterized by the presence of a high number of operons. indeed, a large variability of the inhibition carried out by different strains was observed by holmstrom et al., with the pigmented strains showing the largest inhibition. thus, the results we obtained might explain the inhibition differences between strains, in that the pigmented strains are, in theory, able to produce a wider array of bioactive molecules, since they have a larger number of operons. these results are also in agreement with more recent works describing the ability of pseudoalteromonas strains to completely inhibit the growth of human opportunistic pathogens belonging to the burkholderia cepacia complex. in those works the authors found that one of the most active pseudoalteromonas strains was tb which is the only one able to produce a polyketide synthase that very likely contributes to its high inhibitory activity. all strains share one operon; which suggests that this operon was present in the pseudoalteromonas lca. concerning this issue, it is not clear whether the lca harbored just one operon and the others have been acquired by different strains or if the genome of the common ancestor embedded a higher number of different operons involved in the biosynthesis of different bioactive molecules. according to this second scenario, one or more operons should have been lost during evolution by different pseudoalteromonas strains/species. to test these hypotheses, we investigated where these genes have been acquired/lost, according to the ancestral gene states reconstruction shown in fig.  although some of these genes were present in the pseudoalteromonas lca, the majority of these genes have been acquired through recent events, especially in the pigmented strains. therefore, strain-specific gene acquisition led to the variability in the operon presence. these results clearly show the biosynthetic potential of some of these strains, prompting for potential application, such as drug discovery and biocontrol of aquaculture. as a proof of concept, some of the predicted operons correspond to clusters of genes, which have already been discovered and validated as antimicrobial. for example, we found the gene cluster involved in the production of violacein in both p. luteoviolacea and p. tunicata genomes, for which the production of this compound has been witnessed.  in this work we have performed a comparative genomics analysis for a large portion of the pseudoalteromonas strains with the sequenced genome available in genbank, gaining new insights into the evolutionary history of this genus and trying to underline the main genomic features with an ecological relevance. considering their pigmentation, the strains under study can be classified in two major groups: one embedding strains and the other containing strains, most of which isolated from different ecological niches of antarctica. from a phylogenetic point of view, the pigmented group displays a greater degree of variability, whereas the non-pigmented one is characterized by a phylogenetic shallowness. however, the analysis of the pangenome of the two groups revealed how they are characterized by an open pangenome with a high percentage of unique genes. this is remarkable for the non-pigmented strains, since this is in apparent contrast with the phylogenetic shallowness. the analysis of the dynamics of gene gains and losses revealed how the evolution of the pseudoalteromonas strains is strongly affected by recent acquisitions of a large number of genes, most likely by means of hgt. this idea is in agreement with the fact that most of these strains inhabit the antarctica and on the finding that the marine environment represents a key reservoir of horizontally transferred genes. this hypothesis was supported by the functional analyses of the pseudoalteromonas genomes, which revealed the existence of a large fraction of genes related to mges, most of which have a plasmid origin. since the molecular actors of bacterial conjugation are absent in the pseudoalteromonas genomes, we speculate that the exchange of genetic material might occur via transduction, which could explain the considerable portion of phage genes found in the panmobilome. the biological significance of such degree of hgt and the high number of unique genes might rely on the fact that most of these strains have been isolated from antarctica. indeed, in such a harsh environment, could be favored those microorganisms that, by exploring many possible genetic/genomic combinations, find the more efficient phenotype for the colonization of a given ecological niche. moreover, since all of the strains analyzed in this work are marine bacteria, which have been isolated from different world regions, their genomic variability could imply that marine-living bacteria have to cope with different niches and to be able to rapidly adapt to fast changes of the environmental conditions. in this context the analyses of particular metabolic traits might shed some light on this issue. indeed, in the marine environment it is very likely that microorganisms belonging to different species/genera have to compete for nutrients; accordingly, antagonistic interactions between bacteria isolated in antarctica from different niches have been disclosed recently. these antagonistic interactions very likely rely on the production of antimicrobial compounds that can also inhibit the growth of human pathogens. according to this idea, the analyses performed in this work revealed the presence of gene clusters involved in the biosynthesis of secondary metabolites. notably, as it might be expected, the higher the number of gene clusters, the higher the number of metabolites synthesized by pseudoalteromonas strains. on the other side, bacteria living in a “competitive” environment, such as the marine one, have evolved different mechanisms to resist to antibiotics and/or other toxic compounds. the remarkably high number of genes encoding efflux pumps in the pseudoalteromonas genomes fully supports this view. the results reported in this work show how the pseudoalteromonas genus embeds a remarkable genomic diversity, with a large proportion of unique genes, underlining that our knowledge of this genus is still limited. this, together with the fact that most of these strains seems to be very promising for applicative purposes, prompts for further explorative studies on these marine bacteria, with the collection of new strains and the sequencing of the genomes of other representatives of this genus. 
these, most likely, included an extensive genome reduction and a strong contribution of horizontal gene transfer, which affected biotechnologically relevant gene sets and occurred in a strain-specific fashion. pseudoalteromonasmarine bacteriapangenomemicrobial evolutioncomparative genomicsantibioticsantarctic bacteriahorizontal gene transferpnrapnra 2013/b pnra az fondi marco fani renato pharmasea312184de pascale donatella issue-copyright-statement© the author 2017 the downstream analyses were conducted to describe the genomic diversity, both at genus and group levels. we drafted all the main evolutionary steps that led to the current structure and gene content of pseudoalteromonas representatives. furthermore, this study also identified the genomic determinants related to some of the most interesting features of the pseudoalteromonas representatives, such as the production of secondary metabolites, the adaptation to cold temperatures and the resistance to abiotic compounds. these sequences were used to reconstruct the largest pseudoalteromonas pangenome computed so far, including also the two main groups of pseudoalteromonas strains. indeed, biotechnological applications would greatly benefit from such analysis. a remarkable feature shared by these bacteria is their ability to produce secondary metabolites with a strong antimicrobial and antitumor activity. despite their biotechnological relevance, representatives of this genus are still lacking an extensive genomic characterization, including features involved in the evolution of secondary metabolites production.
 pseudoalteromonas is a genus of ubiquitous marine bacteria used as model organisms to study the biological mechanisms involved in the adaptation to cold conditions. a remarkable feature shared by these bacteria is their ability to produce secondary metabolites with a strong antimicrobial and antitumor activity. despite their biotechnological relevance, representatives of this genus are still lacking an extensive genomic characterization, including features involved in the evolution of secondary metabolites production. indeed, biotechnological applications would greatly benefit from such analysis.  here, we analyzed the genomes of strains belonging to different pseudoalteromonas species and isolated from diverse ecological niches, including extreme ones. these sequences were used to reconstruct the largest pseudoalteromonas pangenome computed so far, including also the two main groups of pseudoalteromonas strains. the downstream analyses were conducted to describe the genomic diversity, both at genus and group levels. this allowed highlighting a remarkable genomic heterogeneity, even for closely related strains. we drafted all the main evolutionary steps that led to the current structure and gene content of pseudoalteromonas representatives. these, most likely, included an extensive genome reduction and a strong contribution of horizontal gene transfer, which affected biotechnologically relevant gene sets and occurred in a strain-specific fashion. furthermore, this study also identified the genomic determinants related to some of the most interesting features of the pseudoalteromonas representatives, such as the production of secondary metabolites, the adaptation to cold temperatures and the resistance to abiotic compounds.  this study poses the bases for a comprehensive understanding of the evolutionary trajectories followed in time by this peculiar bacterial genus and for a focused exploitation of their biotechnological potential. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords pseudoalteromonasmarine bacteriapangenomemicrobial evolutioncomparative genomicsantibioticsantarctic bacteriahorizontal gene transferpnrapnra 2013/b pnra az fondi marco fani renato pharmasea312184de pascale donatella issue-copyright-statement© the author 2017 