BACKGROUND
piezophilic microbes have been isolated from a variety of abyssal and hadal deep-sea environments and include several psychrophilic or psychrotolerant proteobacteria, and several high temperature euryarchaeota and crenarchaeota  <cit> .
while the study of these extremophiles is still in its infancy, both physiological and structural adaptations appear to be important for high-pressure life.
one moderately piezophilic, gamma-proteobacterial isolate, photobacterium profundum strain ss <dig>  has been the subject of a number of studies addressing the nature and regulation of genes important for pressure-sensing and high pressure adaptation, owing to the relative ease of its cultivation as well as its genetic tractability  <cit> .
here we make use of another important p. profundum feature, namely the availability of multiple closely related strains which differ in their pressure and temperature optima.
strain ss <dig> was isolated from an amphipod in the sulu trough at a depth of  <dig> m and displays optimum growth at  <dig> mpa and 15°c  <cit> .
p. profundum strain dsj <dig> was recovered from a sediment sample obtained from the ryukyu trench  at a depth of  <dig> m and displays its optimum growth at  <dig> mpa  and a temperature optimum of 10°c  <cit> .
p. profundum strain 3tck was isolated from a shallow sediment sample obtained from san diego bay  and exhibits optimal growth at atmospheric pressure and a broad temperature span for growth from below 0°c to above 20°c.
recently, the complete genome sequence of strain ss <dig> was obtained  <cit> .
this achievement has enabled the scaling up of the study of piezophily and, more generally, of adaptations to the deep sea , at the genomic level.
in this study a microarray covering nearly the complete ss <dig> genome was used to investigate both the flexible gene pool  and high pressure adaptation by means of two different post-genomic approaches:
1-using the ss <dig> genome as a reference, comparative genomic hybridization experiments were performed with dna extracted from the other two p. profundum strains  to identify the flexible gene pool in ss <dig>  to determine if these genes were obtained from lateral gene transfer events or, conversely, from genomic reduction events in the other strains, their gc content, codon usage and genomic signature was analyzed.
2-transcriptome analyses were performed as a function of pressure  and temperature .
although we have recently presented preliminary data on ss <dig> expression at  <dig>  and  <dig> mpa, in this study temperature effects on gene regulation were compared with pressure effects since increasing pressure exerts some common effects with decreasing temperature in terms of membrane microviscosity and with increasing temperature in terms of protein stability  <cit> .
moreover the transcriptional changes identified in the  <dig>  mpa vs.  <dig> mpa and  <dig> mpa vs.  <dig> mpa experiments were compared in order to reveal expression changes in a piezophilic bacterial species grown at supra-optimal pressure.
finally, the results obtained from comparative genomic analyses and expression profiling experiments were combined to identify genes shared among the p. profundum piezophiles, absent from the piezosensitive strain, and up-regulated at high pressure.
this allowed a few genes to be selected from a pool of approximately  <dig>  genes whose distribution and expression characteristics suggest possible function in high pressure adaptation and thus present themselves as candidates for future genetic investigation.
RESULTS
comparison of three p. profundum strains
amplification and analysis of the 16s rdna from strain 3tck revealed that it is  <dig> % identical at the 16s level with photobacterium profundum ss <dig> and  <dig> % identical to p. profundum dsj <dig>  suggesting that they are all members of the same species.
figure  <dig> shows a 16s rrna-based phylogenetic tree demonstrating the relationship among selected photobacterium species, including the three p. profundum strains selected for this study.
growth curves showed that 3tck is psychrotolerant and piezo-sensitive.
however it grows at higher temperatures than p. profundum ss <dig> and dsj <dig>  and has faster growth rates at  <dig>  mpa.
genomic comparison between different p. profundum strains
the first question that arises from the genomic comparison between ss <dig> and the other two strains is: "how many ss <dig> genes are missing or highly divergent in the 3tck and dsj <dig> genomes?".
<dig> orfs were determined to be absent in 3tck strain genome,  <dig>  belong to the ss <dig> chr <dig> and  <dig>  to chr <dig>   <dig> orfs are absent in the dsj <dig> genome,  <dig>  are located on ss <dig> chr <dig> and  <dig>  on chr <dig>
an interesting aspect of these data is that for both strains the percentage of missing/divergent regions is higher on chr <dig> than chr <dig>  this indicates that chr <dig>  contains a proportionally larger flexible gene pool and that it has been the target of more gene transfer events for its size  than chr <dig>  that contains the most "established" genes.
this is also true for other vibrionaceae genomes  <cit> .
in order to define if the regions absent in the 3tck-dsj <dig> strains could be considered to have been acquired by horizontal gene transfer we performed three different analyses: gc content variation , tetranucleotide composition    <cit>  and codon bias relative to the average gene versus s <dig> percentage    <cit> .
taken together these three different analyses were able to identify a large number of potentially laterally transferred regions.
for example, a region named chr <dig>   has an altered tetranucleotide composition but its gc content is similar to the surrounding regions and it has a "normal" codon bias relative to the average gene versus s <dig> percentage .
conversely, region chr <dig>  is only characterized by a slight gc content variation.
the results obtained from these analyses are discussed in-depth below.
a blastp similarity search of ss <dig> proteins identified various phage-related proteins, mostly encoded in three regions named chr <dig> , chr <dig>  and chr <dig> .
microarray data obtained by comparing the ss <dig>  3tck and dsj <dig> genomes  confirmed that these genomic portions are absent in both the 3tck and dsj <dig>  these regions present characteristics typical of a genomic island :  gc content anomalies,  altered codon bias,  insertion at the 3'-end of a trna gene  and  presence of a gene encoding an integrase at one end   <cit> .
the region chr <dig>  is located on chr <dig>  spans approximately  <dig>  kbp, has no gc content anomalies  but has a slightly altered tetranucleotide composition.
chr <dig>  contains twelve orfs that have similarity with phage proteins, one of these being a hypothetical integrase .
moreover it encodes a putative nadh oxidoreductase  and a putative trka family protein  .
furthermore pbprb <dig> gene has similarities with enterohemolysin  <dig>  a gene also present in the gifsy- <dig> prophage, and pbprb <dig> has similarities with exodeoxyribonuclease of the gifsy- <dig> prophage of salmonella typhimurium lt <dig>  <cit> .
chr <dig>  region appears to be completely absent in dsj <dig> whereas only the first part is absent in 3tck.
chr <dig>  contains a hypothetical integrase gene , twelve phage related proteins and various hypothetical proteins.
the higher gc content  of chr <dig>  suggests that it has been acquired more recently than chr <dig>  and chr <dig> .
a large part of the genes located in the chr <dig> , chr <dig>  and chr <dig>  regions clearly lacks orthologous genes in others bacteria  <cit>  such as v. cholerae, v. vulnificus , v. parahaemolyticus, v. fisheri, e. coli, and b. subtilis.
the high number of hypothetical proteins encoded in these regions suggests that these loci could have been acquired from bacteria still unknown.
consistent with its recent acquisition chr <dig>  presents an altered codon bias and a large percentage of its orfs are located on the right horn of a graph of the codon bias versus the gc content frequency in third position .
a large  <dig>  kbp element  is present only in strain ss <dig> and seems to be the result of plasmid integration into the chromosome.
this region has a high gc content and altered codon bias and genomic signature.
various bacterial conjugation factors  are present in this element.
these genes are typically found in widespread conjugationally transmitted plasmids  <cit> .
this element carries a large number of genes, but of particular interest is a multidrug efflux system .
ss <dig> also contains a  <dig> kbp plasmid  that shows similarity with v. vulnificus plasmid yj <dig> at least in the region spanning the genes related to conjugation.
this plasmid is absent in both dsj <dig> and 3tck and presents characteristics of horizontally transferred dna, in fact various orfs belonging to this element are localized to the right horn of figure  <dig> .
pcr examination for the presence of this plasmid in various laboratory derivatives of p. profundum ss <dig> has revealed that it can be lost: strain tw <dig>  <cit>  is a toxr- derivative of db <dig>  <cit>  which lacks the plasmid , yet tw <dig> exhibits no pressure or temperature growth defects.
this raises the question of the function of the genes located on the plasmid which must be playing some role in environmental adaptation for the plasmid to be retained  <cit> .
one of the most interesting results obtained from the annotation process was the finding that in the ss <dig> genome there are two flagellar clusters.
one of these  is located on chr <dig> between  <dig> and  <dig> bp and the second cluster  is localized on chr <dig> near the origin between  <dig> and  <dig> bp.
similarity searches revealed that the pf region contains most of the genes involved in polar flagellum assembly, with a gene organization typical of a vibrionaceae polar flagellar cluster  <cit> .
chr <dig> -lf contains all the genes involved in lateral flagellar synthesis and most of the genes localized in this region have similarity with the flagellar cluster of v. parahaemolyticus that codes for lateral flagella  <cit> .
this region is absent only in 3tck strain .
finally, the data on variable regions was compared with the transcriptome results to discern if any of the horizontally acquired genes might be indicated to perform a role in pressure or temperature adaptation.
the absolute fluorescence value from microarray analysis indicates that the expression levels of most of these genes was quite low.
indeed, only three regions in chr <dig>  and two in chr <dig>  had fluorescence values higher than the mean fluorescence level of the entire chromosome .
region chr <dig>  is absent in dsj <dig>  and has three genes, pbpra <dig> , pbpra <dig>  and pbpra <dig> , differentially expressed in pressure experiments.
region chr <dig>  is absent in both strains and contains a large gene cluster involved in the tricarboxylic acid fermentation and in the cleavage of citrate to oxaloacetate and acetate.
this region contains six genes up-regulated at 16°c and/or  <dig>  mpa  .
region chr <dig>  is absent in dsj <dig> and contains genes involved in pilus assembly, some of these are up-regulated at  <dig> mpa  and down-regulated at  <dig> mpa .
finally, region chr <dig>  is lacking in both of the comparison strains and contains genes having a high expression level at  <dig> mpa, some of which are differentially expressed at  <dig> mpa and 4°c .
this region contains flm genes that in other bacteria are involved in lps o-ag biosynthesis and flagellar filament assembly  <cit> .
interestingly changes in lps o-antigen structure have been observed in yersinia pestis km <dig> grown at low temperatures  <cit> .
this element has very low gc content , altered genomic signature and most part of its genes are localized to the left horn of the graph reported in supporting online material , thus supporting the idea that it could have been laterally acquired.
it is curious that some of the variable regions differentially expressed in pressure experiments are lacking or are highly divergent in both 3tck and dsj <dig>  this indicates that although genes located in these regions could be involved in the high pressure response of ss <dig>  they are not essential to it and other p. profundum strains will achieve piezophily with different strategies.
moreover, genes differentially expressed at  <dig> mpa or  <dig> mpa, but present in both dsj <dig> and 3tck, could be beneficial but not sufficient for high pressure adaptation.
considering only pressure regulated genes belonging to the group absent in 3tck and/or dsj <dig>   <dig> genes are absent in both strains but only  <dig> genes are absent in 3tck strain alone .
these data were obtained using a profile search with jexpress software  <cit> .
of these  <dig> genes,  <dig> are up-regulated at  <dig> mpa  and/or  <dig> mpa .
in the ss <dig> genome we found two genes for tors proteins .
only one of these  is differentially expressed at  <dig> mpa and this gene is also absent in 3tck strain.
tors is able to regulate various genes in response to trimethylamine n-oxide   <cit> , in particular it regulates tmao reductase  that is also up-regulated at  <dig> mpa.
it is conceivable that trimethylamine reduction increases the ph of the cytoplasm and, for this reason, other genes identified with microarray experiments, such as tryptophanase  increase their expression in order to counter this alcalinization.
alternatively, since no tmao was added to the ss <dig> cultures used for the microarray experiments, the second tors could also be responding to an as yet undiscovered signal.
in addition to these six genes, others could perform an important role in high pressure adaptation.
we expect that a high number of genes and proteins are regulated at the post-transcriptional level and have an important role in high pressure adaptation but these studies are beyond the scope of this paper.
moreover protein structural adaptations, that were not considered in this analysis, could also have great importance for ss <dig> piezophily.
transcriptome analysis of p. profundum strain ss <dig> under different pressure and temperature conditions
in a previous paper a preliminary analysis was presented for genes differentially expressed at  <dig> mpa  versus  <dig>  mpa, highlighting a stress response at low pressure, an heavy involvement of membrane transporters in pressure adaptation and an increased expression at  <dig> mpa of genes involved in the stickland reaction and tmao reduction.
nevertheless, additional transcriptional responses remain to be elucidated including the response to low temperatures and the effect of supraoptimal pressures  on ss <dig>  to better elucidate these points expression profiling was performed at different temperatures  and different pressures .
to clarify the role of specific biological processes on temperature and pressure adaptation, we also performed a gene ontology analysis  <cit>  on differentially expressed genes using specific software such as gominer  <cit>  and fatigo  <cit> .
analysis of co-regulated genes in pressure and temperature experiments
the rationale for temperature experiments was the comprehension of which genes are co-regulated during pressure and temperature changes.
it is known that low temperature and high pressure have similar effects on some biological structures   <cit> .
we found  <dig>  out of  <dig> genes that share a similar expression pattern between temperature  and pressure  experiments, a number higher that expected only by chance.
in fact, the number of up-regulated genes at  <dig> mpa and at 4°c is respectively  <dig> and  <dig> over  <dig> .
if the two conditions are independent, the expected number of genes that are up-regulated both at high pressure and low temperature could be estimated as ** <dig> =  <dig> .
similarly we could estimate the number of down-regulated genes at  <dig> mpa and 4°c as ** <dig> =  <dig> .
so the total number of genes expected to be up- or down-regulated in both the conditions considered  is approximately 1/10th of the differentially expressed genes observed.
therefore microarray experiments corroborate the hypothesis that high pressure and low temperature have a overlapping effects on gene expression.
the ss <dig> genome sequence reveals the presence of two iron transporters: one  that is up-regulated at  <dig> mpa  and at 4°c, while the other one  is up-regulated at  <dig>  mpa  and at 16°c.
iron accumulation in organisms that live in the ocean environment is difficult  <cit>  and the evolution of two alternative transporters could be important in order to survive under different physical conditions.
the presence of different isoforms of the same transporter that work at different pressure and temperature conditions is not limited to iron transporters.
orfs pbpra0098-pbpra <dig> code for a hypothetical oligopeptide transporter and are up-regulated at  <dig>  mpa, while orfs pbpra2251-pbpra <dig> code for a different oligopeptide transporter that is up-regulated at  <dig> mpa  and  <dig> mpa .
other oligopeptide transporters seem to have the same behaviour such as those codified by pbpra0521-pbpra <dig> and pbpra2934-pbpr <dig>  the first being up-regulated at  <dig> mpa  and 4°c, while the second is weakly up-regulated at  <dig>  mpa.
there is an entire region  containing a large number of genes that are up-regulated both at  <dig>  mpa and at 16°c.
this region was also identified in the above genome comparisons because it is absent in both 3tck and dsj <dig> strains.
analysis of co-regulated genes in  <dig> mpa and  <dig> mpa experiments
the expression profile of ss <dig> at  <dig> mpa  was also compared with that at  <dig> mpa.
interestingly the  <dig> mpa vs.  <dig> mpa expression profile comparison revealed only  <dig> differentially expressed genes , in contrast to the high number of differentially expressed genes between  <dig> mpa and  <dig>  mpa .
of these  <dig> differentially expressed genes, only  <dig> were specific for very high pressure adaptation, the remaining  <dig> also being expressed under other environmental conditions tested.
this result indicates that ss <dig> undergoes a heavy reorganization in gene expression between atmospheric pressure and  <dig> mpa, while this is not seen moving from  <dig> mpa to  <dig> mpa.
a gene ontology search with gominer software  <cit>  indicated that among the  <dig> genes specific for very high pressure adaptation there is an enrichment of genes involved in arginine metabolism , catabolism   and transport .
experiments at  <dig> mpa were also useful in identifying genes whose expression follows the direction of pressure variation, being up-regulated or down-regulated both at  <dig> mpa  and at  <dig> mpa .
twenty one genes matched this expression profile .
one of these genes encodes a putative delta- <dig> fatty acid desaturase .
high pressure increases the rigidity of membranes  <cit> , and for this reason genes such as the putative delta- <dig> fatty acid desaturase presumably were up-regulated in order to increase the membrane unsaturation and thus membrane fluidity.
despite the fact that much is known about membrane modification in response to pressure variation in ss <dig>  <cit> , these experiments reveal the possible involvement of a previously unrecognized gene in fatty acid unsaturation.
this is particularly noteworthy because fatty acid unsaturation is critical to high pressure growth of ss <dig>
a search performed using gominer software  <cit>  on differentially expressed genes obtained in the  <dig> mpa vs.  <dig>  mpa experiments and in the  <dig> mpa vs.  <dig> mpa experiments indicated that transport is one of the main biological processes involved .
similar result was obtained using fatigo software  <cit>  .
moreover six of the orfs that are up-regulated both at  <dig> mpa  and  <dig> mpa  are involved in transport processes  .
as described in the previously published  <dig> mpa versus  <dig>  mpa experiments  <cit> , transport is strongly influenced by pressure, probably due to the effect of pressure on membrane modification and because of the pressure influence on the activation volume Δv#  of the transport process.
discussion
in the microbial world genetic material can be transferred between species by several mechanisms involving conjugative plasmids, phages, phage-like elements or transposable elements  <cit> .
these elements allow exchange among bacteria of a flexible gene pool encoding additional functions that usually are not essential for bacterial growth, but which provide advantages under particular conditions.
despite the strong pressure on bacteria to maintain a small genome size by deleting the more expendable sequences from their genomes, advantages provided by some regions allow the maintenance of a flexible gene pool  <cit> .
recently  <cit>  it was demonstrated that in the case of xylella fastidiosa a large part of its flexible gene pool seems to be important in order to explain the broad host range of this phytopatogen.
this despite the lack of expression of these genes under the culture conditions examined.
using clones selected from the ss <dig> genome sequencing project and from a specific genomic library with short inserts, a microarray covering large part of the ss <dig> genomic sequence  was prepared and used to identify variable regions in three p. profundum strains.
the lower coverage of chr <dig> was due to the higher frequency of repeated regions present on this chromosome, identified using phred/phrap software during finishing step.
clones were selected which lacked these regions in order to avoid cross hybridization.
this type of analysis has previously been applied to other bacterial species  <cit>  but this is the first report regarding a microbial species containing members adapted to a high pressure environment.
in fact, using genomic dna derived from deep-sea  and coastal  isolates we were able to identify  <dig> regions that are absent or highly divergent in dsj <dig> and 3tck strains plus a large number of small regions scattered over the entire ss <dig> genome.
in silico analysis performed on the ss <dig> genome sequence reveals that some of these regions  show differences in gc content, codon usage and genomic signature .
there are also regions, such as chr <dig> 1634244– <dig> bp , that appear as alien dna from these analyses, but which are present in all three strains analyzed.
while pfa mutants are not pressure-sensitive in laboratory culture  <cit> , it is likely that the pfa operon is involved in high pressure adaptation under some deep-sea conditions because omega- <dig> polyunsaturated fatty acids  do increase in abundance in ss <dig> membranes with increasing pressure  <cit>  and because such fatty acids are known to modify membrane fluidity in response to hydrostatic pressure and temperature.
these results indicate that bioinformatics and genomic microarray analysis can be merged in order to obtain a more comprehensive picture of the flexible gene pool in bacteria.
moreover, some of the variable regions identified by microarray analysis but not by bioinformatic analysis could derive from genomic reduction and not from lateral gene transfer.
on the other hand, when the two separate analyses are in accordance with each other, lateral gene transfer is the most plausible explanation.
this is the case for the  <dig> kbp plasmid.
however, the adaptive value of this plasmid to ss <dig> remains a mystery.
it contains no obvious essential genes and can be lost during laboratory cultivation without a detectable phenotypic change.
a region that is particularly interesting is chr <dig> -lf, consisting mainly of a gene cluster present in ss <dig> and dsj <dig> strains and coding for a second flagellar motility system.
it is known that bacterial motility in the sea is a commonly expressed phenotype  <cit> .
this character is important in enhancing bacteria-organic-matter coupling.
moreover, high speed swimming in bacteria may reduce the ability of protozoa to graze on them  <cit> .
some bacteria, such as vibrio parahaemolyticus, posses dual flagellar systems that operate under different circumstances: a polar flagellum allows motility in liquid environments , while multiple lateral flagella allow translocation over surfaces or in viscous media  <cit> .
the role of the second motility system in ss <dig> and dsj <dig> motility is currently being investigated.
the three genomic islands chr <dig> , chr <dig>  and chr <dig>  are likely to be prophages in the p. profundum ss <dig> genome.
it has been suggested that prophages might carry genes beneficial for survival of the host in a selective environment  <cit> .
in this respect the presence, in these regions, of metabolic genes such as nadh oxidoreductase , a putative trka family protein   and tryptophanase  might be meaningful.
the latter gene has two other paralogues in the ss <dig> genome one of which  is pressure regulated suggesting some role in deep-sea adaptation.
recently  <cit>  we have shown that when moving from  <dig> mpa to  <dig>  mpa, ss <dig> undergoes various modifications in metabolic processes.
at  <dig> mpa up-regulation of genes involved in the stickland reaction  and of tmao reduction occurs.
another interesting metabolic modification involves genes of the citrate fermentation pathway located on region chr <dig> .
in leuconostoc paramesenteroides the citmcdefgrp operon, involved in citrate utilization, is located on a plasmid  <cit> .
in ss <dig> these genes are down-regulated both at  <dig> mpa and at 4°c, moreover this region is absent both in dsj <dig> and 3tck genomes.
the reason for the altered expression of this pathway in ss <dig> is not clear but pressure could favour some metabolic processes only on the basis of chemical-physical parameters.
some of these genes are absent in 3tck and dsj <dig> and this could reduce the growth rate of these strains at high pressure.
in fact reactions accompanied by large volume variation are greatly influenced by pressure, but even if the value of Δv  or Δv#  is known, it is still difficult to predict how elevated pressure will affect metabolic pathways in living organisms  <cit> .
as previously reported  <cit> , low pressure induces various stress responses reflected by the up-regulation of chaperones  and dna repair enzymes .
this stress response is not present at  <dig> mpa despite being supraoptimal for ss <dig> growth.
perhaps low pressure affects protein folding because these ss <dig> proteins are adapted to high pressure and this effect is not evident in high pressure experiments .
another curious result stemming from the  <dig> mpa transcriptome experiment is the apparent reduction in arginine biosynthesis and transport at high pressure relative to that at  <dig> mpa.
it could be that this large amino acid is selected against within much of the protein pool present at this high pressure, in contrast to the thermophilic and piezophilic archaea pyrococcus abyssi which has a higher arginine content in its proteins respect to the non-piezophilic bacterium pyrococcus furiosus  <cit> .
alternatively, other genes could govern arginine utilization at high pressure.
transport seems to be the cellular process most influenced by pressure, at least considering the number of regulated genes belonging to this category.
this influence could be due to volume changes associated with the transport process  <cit> .
also temperature variation influences the transport process, which could be due to alterations of transporters efficiency induced by membrane fluidity modifications.
it is interesting to note that some transporters are present in two or more copies in the ss <dig> genome and therefore could work at specific pressures and temperatures.
likewise, ss <dig> may choose among different metabolic processes  as a function of pressure in order to optimize energy gain.
other mechanisms previously described  <cit> , such as the influence of pressure on enzymes involved in complex polysaccharides utilization , support this hypothesis.
the ability of ss <dig> to choose between different transporters or metabolic strategies could also be related to the fact that ss <dig> is not an obligate and narrow spectrum piezophile, but is able to grow over a large range of pressures.
CONCLUSIONS
our findings on genome organization and transcriptional activity of different p. profundum strains depict a high level of genetic diversity, where variable regions influence extremely important processes such as motility and energy production.
but, due to the complexity of deep-sea environment, characterized by a peculiar combination of chemical-physical parameters and nutrient resources, it is difficult to assign a role to all the variable regions present only in the pressure adapted strains.
expression studies on p. profundum ss <dig> performed at different pressure and temperature conditions reveal a complex adaptation network involving a great number of membrane transporters, metabolic processes, and amino acid biosynthesis and membrane modification enzymes.
some of these genes are located on variable regions.
the pressure-regulated genes unique to piezophilic p. profundum strains are likely to be fruitful targets of future genetic investigations into genes which facilitate growth and survival under deep-sea conditions.

 piezophilic microbes have been isolated from a variety of abyssal and hadal deep-sea environments and include several psychrophilic or psychrotolerant proteobacteria, and several high temperature euryarchaeota and crenarchaeota. while the study of these extremophiles is still in its infancy, both physiological and structural adaptations appear to be important for high-pressure life. one moderately piezophilic, gamma-proteobacterial isolate, photobacterium profundum strain ss has been the subject of a number of studies addressing the nature and regulation of genes important for pressure-sensing and high pressure adaptation, owing to the relative ease of its cultivation as well as its genetic tractability. here we make use of another important p. profundum feature, namely the availability of multiple closely related strains which differ in their pressure and temperature optima. strain ss was isolated from an amphipod in the sulu trough at a depth of m and displays optimum growth at mpa and 15°c. p. profundum strain dsj was recovered from a sediment sample obtained from the ryukyu trench at a depth of m and displays its optimum growth at mpa and a temperature optimum of 10°c. p. profundum strain 3tck was isolated from a shallow sediment sample obtained from san diego bay and exhibits optimal growth at atmospheric pressure and a broad temperature span for growth from below 0°c to above 20°c. recently, the complete genome sequence of strain ss was obtained. this achievement has enabled the scaling up of the study of piezophily and, more generally, of adaptations to the deep sea, at the genomic level. in this study a microarray covering nearly the complete ss genome was used to investigate both the flexible gene pool and high pressure adaptation by means of two different post-genomic approaches: 1-using the ss genome as a reference, comparative genomic hybridization experiments were performed with dna extracted from the other two p. profundum strains to identify the flexible gene pool in ss to determine if these genes were obtained from lateral gene transfer events or, conversely, from genomic reduction events in the other strains, their gc content, codon usage and genomic signature was analyzed. 2-transcriptome analyses were performed as a function of pressure and temperature. although we have recently presented preliminary data on ss expression at and mpa, in this study temperature effects on gene regulation were compared with pressure effects since increasing pressure exerts some common effects with decreasing temperature in terms of membrane microviscosity and with increasing temperature in terms of protein stability. moreover the transcriptional changes identified in the mpa vs. mpa and mpa vs. mpa experiments were compared in order to reveal expression changes in a piezophilic bacterial species grown at supra-optimal pressure. finally, the results obtained from comparative genomic analyses and expression profiling experiments were combined to identify genes shared among the p. profundum piezophiles, absent from the piezosensitive strain, and up-regulated at high pressure. this allowed a few genes to be selected from a pool of approximately genes whose distribution and expression characteristics suggest possible function in high pressure adaptation and thus present themselves as candidates for future genetic investigation.  comparison of three p. profundum strains amplification and analysis of the 16s rdna from strain 3tck revealed that it is % identical at the 16s level with photobacterium profundum ss and % identical to p. profundum dsj suggesting that they are all members of the same species. figure shows a 16s rrna-based phylogenetic tree demonstrating the relationship among selected photobacterium species, including the three p. profundum strains selected for this study. growth curves showed that 3tck is psychrotolerant and piezo-sensitive. however it grows at higher temperatures than p. profundum ss and dsj and has faster growth rates at mpa. genomic comparison between different p. profundum strains the first question that arises from the genomic comparison between ss and the other two strains is: "how many ss genes are missing or highly divergent in the 3tck and dsj genomes?". orfs were determined to be absent in 3tck strain genome, belong to the ss chr and to chr orfs are absent in the dsj genome, are located on ss chr and on chr an interesting aspect of these data is that for both strains the percentage of missing/divergent regions is higher on chr than chr this indicates that chr contains a proportionally larger flexible gene pool and that it has been the target of more gene transfer events for its size than chr that contains the most "established" genes. this is also true for other vibrionaceae genomes. in order to define if the regions absent in the 3tck-dsj strains could be considered to have been acquired by horizontal gene transfer we performed three different analyses: gc content variation, tetranucleotide composition and codon bias relative to the average gene versus s percentage. taken together these three different analyses were able to identify a large number of potentially laterally transferred regions. for example, a region named chr has an altered tetranucleotide composition but its gc content is similar to the surrounding regions and it has a "normal" codon bias relative to the average gene versus s percentage. conversely, region chr is only characterized by a slight gc content variation. the results obtained from these analyses are discussed in-depth below. a blastp similarity search of ss proteins identified various phage-related proteins, mostly encoded in three regions named chr, chr and chr. microarray data obtained by comparing the ss 3tck and dsj genomes confirmed that these genomic portions are absent in both the 3tck and dsj these regions present characteristics typical of a genomic island: gc content anomalies, altered codon bias, insertion at the 3'-end of a trna gene and presence of a gene encoding an integrase at one end. the region chr is located on chr spans approximately kbp, has no gc content anomalies but has a slightly altered tetranucleotide composition. chr contains twelve orfs that have similarity with phage proteins, one of these being a hypothetical integrase. moreover it encodes a putative nadh oxidoreductase and a putative trka family protein. furthermore pbprb gene has similarities with enterohemolysin a gene also present in the gifsy- prophage, and pbprb has similarities with exodeoxyribonuclease of the gifsy- prophage of salmonella typhimurium lt. chr region appears to be completely absent in dsj whereas only the first part is absent in 3tck. chr contains a hypothetical integrase gene, twelve phage related proteins and various hypothetical proteins. the higher gc content of chr suggests that it has been acquired more recently than chr and chr. a large part of the genes located in the chr, chr and chr regions clearly lacks orthologous genes in others bacteria such as v. cholerae, v. vulnificus, v. parahaemolyticus, v. fisheri, e. coli, and b. subtilis. the high number of hypothetical proteins encoded in these regions suggests that these loci could have been acquired from bacteria still unknown. consistent with its recent acquisition chr presents an altered codon bias and a large percentage of its orfs are located on the right horn of a graph of the codon bias versus the gc content frequency in third position. a large kbp element is present only in strain ss and seems to be the result of plasmid integration into the chromosome. this region has a high gc content and altered codon bias and genomic signature. various bacterial conjugation factors are present in this element. these genes are typically found in widespread conjugationally transmitted plasmids. this element carries a large number of genes, but of particular interest is a multidrug efflux system. ss also contains a kbp plasmid that shows similarity with v. vulnificus plasmid yj at least in the region spanning the genes related to conjugation. this plasmid is absent in both dsj and 3tck and presents characteristics of horizontally transferred dna, in fact various orfs belonging to this element are localized to the right horn of figure. pcr examination for the presence of this plasmid in various laboratory derivatives of p. profundum ss has revealed that it can be lost: strain tw is a toxr- derivative of db which lacks the plasmid, yet tw exhibits no pressure or temperature growth defects. this raises the question of the function of the genes located on the plasmid which must be playing some role in environmental adaptation for the plasmid to be retained. one of the most interesting results obtained from the annotation process was the finding that in the ss genome there are two flagellar clusters. one of these is located on chr between and bp and the second cluster is localized on chr near the origin between and bp. similarity searches revealed that the pf region contains most of the genes involved in polar flagellum assembly, with a gene organization typical of a vibrionaceae polar flagellar cluster. chr -lf contains all the genes involved in lateral flagellar synthesis and most of the genes localized in this region have similarity with the flagellar cluster of v. parahaemolyticus that codes for lateral flagella. this region is absent only in 3tck strain. finally, the data on variable regions was compared with the transcriptome results to discern if any of the horizontally acquired genes might be indicated to perform a role in pressure or temperature adaptation. the absolute fluorescence value from microarray analysis indicates that the expression levels of most of these genes was quite low. indeed, only three regions in chr and two in chr had fluorescence values higher than the mean fluorescence level of the entire chromosome. region chr is absent in dsj and has three genes, pbpra, pbpra and pbpra, differentially expressed in pressure experiments. region chr is absent in both strains and contains a large gene cluster involved in the tricarboxylic acid fermentation and in the cleavage of citrate to oxaloacetate and acetate. this region contains six genes up-regulated at 16°c and/or mpa. region chr is absent in dsj and contains genes involved in pilus assembly, some of these are up-regulated at mpa and down-regulated at mpa. finally, region chr is lacking in both of the comparison strains and contains genes having a high expression level at mpa, some of which are differentially expressed at mpa and 4°c. this region contains flm genes that in other bacteria are involved in lps o-ag biosynthesis and flagellar filament assembly. interestingly changes in lps o-antigen structure have been observed in yersinia pestis km grown at low temperatures. this element has very low gc content, altered genomic signature and most part of its genes are localized to the left horn of the graph reported in supporting online material, thus supporting the idea that it could have been laterally acquired. it is curious that some of the variable regions differentially expressed in pressure experiments are lacking or are highly divergent in both 3tck and dsj this indicates that although genes located in these regions could be involved in the high pressure response of ss they are not essential to it and other p. profundum strains will achieve piezophily with different strategies. moreover, genes differentially expressed at mpa or mpa, but present in both dsj and 3tck, could be beneficial but not sufficient for high pressure adaptation. considering only pressure regulated genes belonging to the group absent in 3tck and/or dsj genes are absent in both strains but only genes are absent in 3tck strain alone. these data were obtained using a profile search with jexpress software. of these genes, are up-regulated at mpa and/or mpa. in the ss genome we found two genes for tors proteins. only one of these is differentially expressed at mpa and this gene is also absent in 3tck strain. tors is able to regulate various genes in response to trimethylamine n-oxide, in particular it regulates tmao reductase that is also up-regulated at mpa. it is conceivable that trimethylamine reduction increases the ph of the cytoplasm and, for this reason, other genes identified with microarray experiments, such as tryptophanase increase their expression in order to counter this alcalinization. alternatively, since no tmao was added to the ss cultures used for the microarray experiments, the second tors could also be responding to an as yet undiscovered signal. in addition to these six genes, others could perform an important role in high pressure adaptation. we expect that a high number of genes and proteins are regulated at the post-transcriptional level and have an important role in high pressure adaptation but these studies are beyond the scope of this paper. moreover protein structural adaptations, that were not considered in this analysis, could also have great importance for ss piezophily. transcriptome analysis of p. profundum strain ss under different pressure and temperature conditions in a previous paper a preliminary analysis was presented for genes differentially expressed at mpa versus mpa, highlighting a stress response at low pressure, an heavy involvement of membrane transporters in pressure adaptation and an increased expression at mpa of genes involved in the stickland reaction and tmao reduction. nevertheless, additional transcriptional responses remain to be elucidated including the response to low temperatures and the effect of supraoptimal pressures on ss to better elucidate these points expression profiling was performed at different temperatures and different pressures. to clarify the role of specific biological processes on temperature and pressure adaptation, we also performed a gene ontology analysis on differentially expressed genes using specific software such as gominer and fatigo. analysis of co-regulated genes in pressure and temperature experiments the rationale for temperature experiments was the comprehension of which genes are co-regulated during pressure and temperature changes. it is known that low temperature and high pressure have similar effects on some biological structures. we found out of genes that share a similar expression pattern between temperature and pressure experiments, a number higher that expected only by chance. in fact, the number of up-regulated genes at mpa and at 4°c is respectively and over. if the two conditions are independent, the expected number of genes that are up-regulated both at high pressure and low temperature could be estimated as ** =. similarly we could estimate the number of down-regulated genes at mpa and 4°c as ** =. so the total number of genes expected to be up- or down-regulated in both the conditions considered is approximately 1/10th of the differentially expressed genes observed. therefore microarray experiments corroborate the hypothesis that high pressure and low temperature have a overlapping effects on gene expression. the ss genome sequence reveals the presence of two iron transporters: one that is up-regulated at mpa and at 4°c, while the other one is up-regulated at mpa and at 16°c. iron accumulation in organisms that live in the ocean environment is difficult and the evolution of two alternative transporters could be important in order to survive under different physical conditions. the presence of different isoforms of the same transporter that work at different pressure and temperature conditions is not limited to iron transporters. orfs pbpra0098-pbpra code for a hypothetical oligopeptide transporter and are up-regulated at mpa, while orfs pbpra2251-pbpra code for a different oligopeptide transporter that is up-regulated at mpa and mpa. other oligopeptide transporters seem to have the same behaviour such as those codified by pbpra0521-pbpra and pbpra2934-pbpr the first being up-regulated at mpa and 4°c, while the second is weakly up-regulated at mpa. there is an entire region containing a large number of genes that are up-regulated both at mpa and at 16°c. this region was also identified in the above genome comparisons because it is absent in both 3tck and dsj strains. analysis of co-regulated genes in mpa and mpa experiments the expression profile of ss at mpa was also compared with that at mpa. interestingly the mpa vs. mpa expression profile comparison revealed only differentially expressed genes, in contrast to the high number of differentially expressed genes between mpa and mpa. of these differentially expressed genes, only were specific for very high pressure adaptation, the remaining also being expressed under other environmental conditions tested. this result indicates that ss undergoes a heavy reorganization in gene expression between atmospheric pressure and mpa, while this is not seen moving from mpa to mpa. a gene ontology search with gominer software indicated that among the genes specific for very high pressure adaptation there is an enrichment of genes involved in arginine metabolism, catabolism and transport. experiments at mpa were also useful in identifying genes whose expression follows the direction of pressure variation, being up-regulated or down-regulated both at mpa and at mpa. twenty one genes matched this expression profile. one of these genes encodes a putative delta- fatty acid desaturase. high pressure increases the rigidity of membranes, and for this reason genes such as the putative delta- fatty acid desaturase presumably were up-regulated in order to increase the membrane unsaturation and thus membrane fluidity. despite the fact that much is known about membrane modification in response to pressure variation in ss, these experiments reveal the possible involvement of a previously unrecognized gene in fatty acid unsaturation. this is particularly noteworthy because fatty acid unsaturation is critical to high pressure growth of ss a search performed using gominer software on differentially expressed genes obtained in the mpa vs. mpa experiments and in the mpa vs. mpa experiments indicated that transport is one of the main biological processes involved. similar result was obtained using fatigo software. moreover six of the orfs that are up-regulated both at mpa and mpa are involved in transport processes. as described in the previously published mpa versus mpa experiments, transport is strongly influenced by pressure, probably due to the effect of pressure on membrane modification and because of the pressure influence on the activation volume Δv# of the transport process. discussion in the microbial world genetic material can be transferred between species by several mechanisms involving conjugative plasmids, phages, phage-like elements or transposable elements. these elements allow exchange among bacteria of a flexible gene pool encoding additional functions that usually are not essential for bacterial growth, but which provide advantages under particular conditions. despite the strong pressure on bacteria to maintain a small genome size by deleting the more expendable sequences from their genomes, advantages provided by some regions allow the maintenance of a flexible gene pool. recently it was demonstrated that in the case of xylella fastidiosa a large part of its flexible gene pool seems to be important in order to explain the broad host range of this phytopatogen. this despite the lack of expression of these genes under the culture conditions examined. using clones selected from the ss genome sequencing project and from a specific genomic library with short inserts, a microarray covering large part of the ss genomic sequence was prepared and used to identify variable regions in three p. profundum strains. the lower coverage of chr was due to the higher frequency of repeated regions present on this chromosome, identified using phred/phrap software during finishing step. clones were selected which lacked these regions in order to avoid cross hybridization. this type of analysis has previously been applied to other bacterial species but this is the first report regarding a microbial species containing members adapted to a high pressure environment. in fact, using genomic dna derived from deep-sea and coastal isolates we were able to identify regions that are absent or highly divergent in dsj and 3tck strains plus a large number of small regions scattered over the entire ss genome. in silico analysis performed on the ss genome sequence reveals that some of these regions show differences in gc content, codon usage and genomic signature. there are also regions, such as chr 1634244– bp, that appear as alien dna from these analyses, but which are present in all three strains analyzed. while pfa mutants are not pressure-sensitive in laboratory culture, it is likely that the pfa operon is involved in high pressure adaptation under some deep-sea conditions because omega- polyunsaturated fatty acids do increase in abundance in ss membranes with increasing pressure and because such fatty acids are known to modify membrane fluidity in response to hydrostatic pressure and temperature. these results indicate that bioinformatics and genomic microarray analysis can be merged in order to obtain a more comprehensive picture of the flexible gene pool in bacteria. moreover, some of the variable regions identified by microarray analysis but not by bioinformatic analysis could derive from genomic reduction and not from lateral gene transfer. on the other hand, when the two separate analyses are in accordance with each other, lateral gene transfer is the most plausible explanation. this is the case for the kbp plasmid. however, the adaptive value of this plasmid to ss remains a mystery. it contains no obvious essential genes and can be lost during laboratory cultivation without a detectable phenotypic change. a region that is particularly interesting is chr -lf, consisting mainly of a gene cluster present in ss and dsj strains and coding for a second flagellar motility system. it is known that bacterial motility in the sea is a commonly expressed phenotype. this character is important in enhancing bacteria-organic-matter coupling. moreover, high speed swimming in bacteria may reduce the ability of protozoa to graze on them. some bacteria, such as vibrio parahaemolyticus, posses dual flagellar systems that operate under different circumstances: a polar flagellum allows motility in liquid environments, while multiple lateral flagella allow translocation over surfaces or in viscous media. the role of the second motility system in ss and dsj motility is currently being investigated. the three genomic islands chr, chr and chr are likely to be prophages in the p. profundum ss genome. it has been suggested that prophages might carry genes beneficial for survival of the host in a selective environment. in this respect the presence, in these regions, of metabolic genes such as nadh oxidoreductase, a putative trka family protein and tryptophanase might be meaningful. the latter gene has two other paralogues in the ss genome one of which is pressure regulated suggesting some role in deep-sea adaptation. recently we have shown that when moving from mpa to mpa, ss undergoes various modifications in metabolic processes. at mpa up-regulation of genes involved in the stickland reaction and of tmao reduction occurs. another interesting metabolic modification involves genes of the citrate fermentation pathway located on region chr. in leuconostoc paramesenteroides the citmcdefgrp operon, involved in citrate utilization, is located on a plasmid. in ss these genes are down-regulated both at mpa and at 4°c, moreover this region is absent both in dsj and 3tck genomes. the reason for the altered expression of this pathway in ss is not clear but pressure could favour some metabolic processes only on the basis of chemical-physical parameters. some of these genes are absent in 3tck and dsj and this could reduce the growth rate of these strains at high pressure. in fact reactions accompanied by large volume variation are greatly influenced by pressure, but even if the value of Δv or Δv# is known, it is still difficult to predict how elevated pressure will affect metabolic pathways in living organisms. as previously reported, low pressure induces various stress responses reflected by the up-regulation of chaperones and dna repair enzymes. this stress response is not present at mpa despite being supraoptimal for ss growth. perhaps low pressure affects protein folding because these ss proteins are adapted to high pressure and this effect is not evident in high pressure experiments. another curious result stemming from the mpa transcriptome experiment is the apparent reduction in arginine biosynthesis and transport at high pressure relative to that at mpa. it could be that this large amino acid is selected against within much of the protein pool present at this high pressure, in contrast to the thermophilic and piezophilic archaea pyrococcus abyssi which has a higher arginine content in its proteins respect to the non-piezophilic bacterium pyrococcus furiosus. alternatively, other genes could govern arginine utilization at high pressure. transport seems to be the cellular process most influenced by pressure, at least considering the number of regulated genes belonging to this category. this influence could be due to volume changes associated with the transport process. also temperature variation influences the transport process, which could be due to alterations of transporters efficiency induced by membrane fluidity modifications. it is interesting to note that some transporters are present in two or more copies in the ss genome and therefore could work at specific pressures and temperatures. likewise, ss may choose among different metabolic processes as a function of pressure in order to optimize energy gain. other mechanisms previously described, such as the influence of pressure on enzymes involved in complex polysaccharides utilization, support this hypothesis. the ability of ss to choose between different transporters or metabolic strategies could also be related to the fact that ss is not an obligate and narrow spectrum piezophile, but is able to grow over a large range of pressures.  our findings on genome organization and transcriptional activity of different p. profundum strains depict a high level of genetic diversity, where variable regions influence extremely important processes such as motility and energy production. but, due to the complexity of deep-sea environment, characterized by a peculiar combination of chemical-physical parameters and nutrient resources, it is difficult to assign a role to all the variable regions present only in the pressure adapted strains. expression studies on p. profundum ss performed at different pressure and temperature conditions reveal a complex adaptation network involving a great number of membrane transporters, metabolic processes, and amino acid biosynthesis and membrane modification enzymes. some of these genes are located on variable regions. the pressure-regulated genes unique to piezophilic p. profundum strains are likely to be fruitful targets of future genetic investigations into genes which facilitate growth and survival under deep-sea conditions. 
these data provide information on horizontal gene flow in the deep sea, provide additional details of p. profundum genome expression patterns and suggest genes which could perform critical functions for abyssal survival, including perhaps high pressure growth. this was verified by extensive analysis of gc content, codon usage and genomic signature of the ss genome. oceans cover approximately 70% of the earth's surface with an average depth of m and a pressure of mpa, thus a large part of the biosphere is occupied by high pressure environments. to better understand high pressure adaptation from a genomic point of view three different photobacterium profundum strains were compared. among these genes, six were found to also be up-regulated by high pressure. open reading frames were found to be specifically absent or highly divergent in the piezosensitive strain, but present in the two piezophilic strains. finally, the transcriptome of ss grown under different pressure and temperature conditions was analyzed taking into consideration the differentially expressed genes belonging to the flexible gene pool.
 oceans cover approximately 70% of the earth's surface with an average depth of m and a pressure of mpa, thus a large part of the biosphere is occupied by high pressure environments. piezophilic organisms are adapted to deep-sea life and grow optimally at pressures higher than mpa. to better understand high pressure adaptation from a genomic point of view three different photobacterium profundum strains were compared. using the sequenced piezophile p. profundum strain ss as a reference, microarray technology was used to identify the genomic regions missing in two other strains: a pressure adapted strain and a pressure-sensitive strain. finally, the transcriptome of ss grown under different pressure and temperature conditions was analyzed taking into consideration the differentially expressed genes belonging to the flexible gene pool.  these studies indicated the presence of a large flexible gene pool in ss characterized by various horizontally acquired elements. this was verified by extensive analysis of gc content, codon usage and genomic signature of the ss genome. open reading frames were found to be specifically absent or highly divergent in the piezosensitive strain, but present in the two piezophilic strains. among these genes, six were found to also be up-regulated by high pressure.  these data provide information on horizontal gene flow in the deep sea, provide additional details of p. profundum genome expression patterns and suggest genes which could perform critical functions for abyssal survival, including perhaps high pressure growth. 