the shewanella piezotolerans wp3 ( hereafter referred to as wp3 ) strains were cultured in modified 2216e marine medium ( 2216e ) ( 5  g / l tryptone , 1  g / l yeast extract , 0.1  g / l fepo4 , 34  g / l nacl ) .
the single clone of wp3 strains were inoculated into a 5  ml test tube with shaking ( 220  rpm ) at 0.1  mpa ( 1  atm ) and 20  c .
the culture was diluted 1000-fold in the same medium and was grown to late - exponential phase ( od600    2.0 ) , and then 100  l cultures were transferred into 5 syringes ( 50  ml ) , which were sealed with no air space and placed inside pressure vessels .
pin closure pressure vessels   were used in this study ( feiyu petrochemical instrument equipment inc . ,
pressure was applied using a hand - operated pump with a quick - fit connector to the pressure vessel .
the cells were then incubated at a hydrostatic pressure of 20  mpa ( 200  atm ) at 4  c .
the growth of the wp3 strains was determined using turbidity measurements at 600  nm with a spectrophotometer ( uv-2550 , shimadzu , kyoto , japan ) .
the culture of wp3 wild - type stain ( wp3-wt ) and lexa gene mutant ( wp3lexa ) was collected immediately when the cells reached late - exponential phase ( od600    0.15 ) .
the samples were centrifuged for 30  s at the maxim speed ( 16,000    g ) .
total rna was isolated from the wp3 culture growing at different temperatures with tri reagent - rna / dna / protein isolation kit ( molecular research center , cincinnati , usa ) according to the manufacturer 's instructions as described previously , .
the quality of rna samples were determined by visualizing the nearly 2:1 ratio of 23s:16s ribosomal rna by running a 1.0% tae ( tris - acitate - edta ) agarose gel ( fig . 
the total rna was treated with dnase i at 37  c for 1  h to remove dna contamination and the purity was checked by pcr amplification with rna as template .
the quantity and integrity of rna was evaluated with a uv spectrophotometer ( nanodrop 2000c , thermo scientific ) . in general , the ratio of 260  nm/280  nm  >  2 and 260  nm/280  nm    2.2 indicate the rna is pure and could be used for the follow - up microarray analysis .
pcr primers for 4744 of the 4945 predicted orfs in the wp3 genome ( excluding 200 cdss shorter than 150  bp ) were designed using primer version 5.0 and then synthesized ( bioasia biotech , shanghai , china ) .
the following criteria were used to identify the optimal forward and reverse primers to generate pcr products specific for each selected orf : ( 1 ) the entire orf was used as a probe if it was <  75% similar to all other genes in the genome ; ( 2 ) for homologous genes , the maximal portion of the genes showing <  75% similarity was selected as specific probes ; ( 3 ) for homologous genes where no specific fragments could be identified , one of the genes was selected as a probe to represent the entire gene group ; ( 4 ) each oligonucleotide primer contained 20 to 25 bases . to simplify the pcr amplification ,
most of the primer sets were designed to have annealing temperatures of ~  60  c .
orf - specific fragments were amplified by taq dna polymerase with the following cycling conditions : denaturation for 30  s at 95  c , annealing for 1  min at 60  c and extension for 1.5  min at 72  c , along with an initial 5  min denaturation at 95  c and a final extension of 10  min at 72  c .
the quality of the amplified products was checked by 1.5% agarose gel electrophoresis and ethidium bromide staining . amplified dna fragments were considered correct if the pcr results contained a single product of the expected size .
the pcr for 94 genes consistently failed to yield satisfactory products ( e.g. , no product , product of the wrong size , multiple or faint bands ) .
we used specific 70-mer oligonucleotides to represent the 39 orfs which were not successfully amplified . in total , the pcr amplicons and oligonucleotide probes represented 95% of the total predicted gene content of wp3 .
the pcr products and microarray reagents were arrayed from 384-well microtiter probes printed in triplicate onto telechem superamine slides ( telechem , sunnyvale , usa ) .
the printed slides were dried and subjected to uv cross - linking ( scientz , ningbo , china ) .
the total rnas were reverse transcribed with superscript ii ( invitrogen , carlsbad , usa ) and the cdnas were labeled with cy3 and cy5 by using a klenow enzyme ( takara bio inc . , shiga , japan ) according to the manufacturer 's instructions .
labeled cdna was purified with a pcr purification kit ( macherey - nagel , dren , germany ) and resuspended in elution buffer .
the labeling efficiency was evaluated with a uv spectrophotometer ( nanodrop 2000c , thermo scientific ) , and the florescence value should be >  150  pmol
. labeled controls and test samples were quantitatively adjusted based on the efficiency of the cy - dye incorporation and mixed with 30  l of hybridization solution ( 50% formamide , 1   hybridization buffer ; amersham biosciences ) .
the dna in hybridization solution was denatured at 95  c for 3  min prior to loading onto a microarray .
the arrays were hybridized overnight at 42  c and washed with 2 consecutive solutions ( 0.2% sds , 2   ssc for 5  min at 42  c , and 0.2   ssc for 5  min at rt ) .
the microarray slides were hybridized with cdna prepared from 3 biological replicate samples . as a measure of technical replication , the dye - swap experiment was performed on each sample so that a total of 6 data points were available for every orf on the microarrays .
a luxscan 10  k scanner and microarray scanner 2.3 software ( capitalbio , beijing , china ) were used for the array image acquisition .
we quantified the signal intensities of individual spots from the 24-bit tiff images using spotdata pro 2.2 ( capitalbio , beijing , china ) .
the linear normalization method was used for data analysis , based on the expression levels of wp3 housekeeping genes in combination with the yeast external controls .
the normalized data were log - transformed and loaded into maanova under r environment for multiple testings , by fitting a mixed - effects anova model .
microarray spots with p values  <  0.001 in the f - test were regarded as differentially expressed genes ( degs ) ( fig . 
in addition , all of the degs were confirmed with significance analysis of microarrays ( sam ) software . to validate the microarray data , 7 genes , including those that were up - regulated , down - regulated or unchanged ,
were selected for quantitative real - time pcr ( qpcr ) ( abi , foster city , ca , usa ) .
it is a need to be pointed out that the same samples were used for the microarray and qpcr .
the correlation coefficient ( r ) between the data obtained by microarray and qpcr was 0.9599 ( fig .
the shewanella piezotolerans wp3 ( hereafter referred to as wp3 ) strains were cultured in modified 2216e marine medium ( 2216e ) ( 5  g / l tryptone , 1  g / l yeast extract , 0.1  g / l fepo4 , 34  g / l nacl ) .
the single clone of wp3 strains were inoculated into a 5  ml test tube with shaking ( 220  rpm ) at 0.1  mpa ( 1  atm ) and 20  c .
the culture was diluted 1000-fold in the same medium and was grown to late - exponential phase ( od600    2.0 ) , and then 100  l cultures were transferred into 5 syringes ( 50  ml ) , which were sealed with no air space and placed inside pressure vessels .
pin closure pressure vessels   were used in this study ( feiyu petrochemical instrument equipment inc . ,
pressure was applied using a hand - operated pump with a quick - fit connector to the pressure vessel .
the cells were then incubated at a hydrostatic pressure of 20  mpa ( 200  atm ) at 4  c .
the growth of the wp3 strains was determined using turbidity measurements at 600  nm with a spectrophotometer ( uv-2550 , shimadzu , kyoto , japan ) .
the culture of wp3 wild - type stain ( wp3-wt ) and lexa gene mutant ( wp3lexa ) was collected immediately when the cells reached late - exponential phase ( od600    0.15 ) .
the samples were centrifuged for 30  s at the maxim speed ( 16,000    g ) .
total rna was isolated from the wp3 culture growing at different temperatures with tri reagent - rna / dna / protein isolation kit ( molecular research center , cincinnati , usa ) according to the manufacturer 's instructions as described previously , .
the quality of rna samples were determined by visualizing the nearly 2:1 ratio of 23s:16s ribosomal rna by running a 1.0% tae ( tris - acitate - edta ) agarose gel ( fig . 
the total rna was treated with dnase i at 37  c for 1  h to remove dna contamination and the purity was checked by pcr amplification with rna as template .
the quantity and integrity of rna was evaluated with a uv spectrophotometer ( nanodrop 2000c , thermo scientific ) . in general , the ratio of 260  nm/280  nm  >  2 and 260  nm/280  nm    2.2 indicate the rna is pure and could be used for the follow - up microarray analysis .
pcr primers for 4744 of the 4945 predicted orfs in the wp3 genome ( excluding 200 cdss shorter than 150  bp ) were designed using primer version 5.0 and then synthesized ( bioasia biotech , shanghai , china ) .
the following criteria were used to identify the optimal forward and reverse primers to generate pcr products specific for each selected orf : ( 1 ) the entire orf was used as a probe if it was <  75% similar to all other genes in the genome ; ( 2 ) for homologous genes , the maximal portion of the genes showing <  75% similarity was selected as specific probes ; ( 3 ) for homologous genes where no specific fragments could be identified , one of the genes was selected as a probe to represent the entire gene group ; ( 4 ) each oligonucleotide primer contained 20 to 25 bases . to simplify the pcr amplification ,
most of the primer sets were designed to have annealing temperatures of ~  60  c .
orf - specific fragments were amplified by taq dna polymerase with the following cycling conditions : denaturation for 30  s at 95  c , annealing for 1  min at 60  c and extension for 1.5  min at 72  c , along with an initial 5  min denaturation at 95  c and a final extension of 10  min at 72  c .
the quality of the amplified products was checked by 1.5% agarose gel electrophoresis and ethidium bromide staining . amplified dna fragments were considered correct if the pcr results contained a single product of the expected size .
the pcr for 94 genes consistently failed to yield satisfactory products ( e.g. , no product , product of the wrong size , multiple or faint bands ) .
we used specific 70-mer oligonucleotides to represent the 39 orfs which were not successfully amplified . in total , the pcr amplicons and oligonucleotide probes represented 95% of the total predicted gene content of wp3 .
the pcr products and microarray reagents were arrayed from 384-well microtiter probes printed in triplicate onto telechem superamine slides ( telechem , sunnyvale , usa ) .
the printed slides were dried and subjected to uv cross - linking ( scientz , ningbo , china ) .
the total rnas were reverse transcribed with superscript ii ( invitrogen , carlsbad , usa ) and the cdnas were labeled with cy3 and cy5 by using a klenow enzyme ( takara bio inc . , shiga , japan ) according to the manufacturer 's instructions .
labeled cdna was purified with a pcr purification kit ( macherey - nagel , dren , germany ) and resuspended in elution buffer .
the labeling efficiency was evaluated with a uv spectrophotometer ( nanodrop 2000c , thermo scientific ) , and the florescence value should be >  150  pmol .
labeled controls and test samples were quantitatively adjusted based on the efficiency of the cy - dye incorporation and mixed with 30  l of hybridization solution ( 50% formamide , 1   hybridization buffer ; amersham biosciences ) .
the dna in hybridization solution was denatured at 95  c for 3  min prior to loading onto a microarray .
the arrays were hybridized overnight at 42  c and washed with 2 consecutive solutions ( 0.2% sds , 2   ssc for 5  min at 42  c , and 0.2   ssc for 5  min at rt ) .
the microarray slides were hybridized with cdna prepared from 3 biological replicate samples . as a measure of technical replication
, the dye - swap experiment was performed on each sample so that a total of 6 data points were available for every orf on the microarrays .
a luxscan 10  k scanner and microarray scanner 2.3 software ( capitalbio , beijing , china ) were used for the array image acquisition .
we quantified the signal intensities of individual spots from the 24-bit tiff images using spotdata pro 2.2 ( capitalbio , beijing , china ) .
the linear normalization method was used for data analysis , based on the expression levels of wp3 housekeeping genes in combination with the yeast external controls .
the normalized data were log - transformed and loaded into maanova under r environment for multiple testings , by fitting a mixed - effects anova model .
microarray spots with p values  <  0.001 in the f - test were regarded as differentially expressed genes ( degs ) ( fig . 
in addition , all of the degs were confirmed with significance analysis of microarrays ( sam ) software . to validate the microarray data , 7 genes , including those that were up - regulated , down - regulated or unchanged , were selected for quantitative real - time pcr ( qpcr ) ( abi , foster city , ca , usa ) .
it is a need to be pointed out that the same samples were used for the microarray and qpcr .
the correlation coefficient ( r ) between the data obtained by microarray and qpcr was 0.9599 ( fig .
here we describe the data of differentially expressed genes in lexa deletion mutant by compared to wild - type strain using our custom designed genome - wide s. piezotolerans array .
the microarray data is of high quality as evidenced by the sam analysis and qpcr validation .
the shewanella species are well - known for their versatile respiration ability and widely distributed in aquatic environment including deep - sea , . meanwhile , the lexa protein is the key transcriptional regulator of sos pathway .
the present data thus would significantly assist investigation of the function of sos response and adaptation mechanism of bacterium in the extreme deep - sea environment .
 addressing dna lesion , the sos response is conserved in bacterial domain and governed by dna binding protein lexa , which have been well characterized in model microorganism such as escherichia coli . 
 however , our understanding of the roles of sos pathway in deep - sea bacteria is limited . to indentify the composition of sos regulon and function of lexa , we performed whole genome transcriptional profiling using a custom designed microarray which contains 95% open reading frames of shewanella piezotolerans wp3 . here 
 we describe the experimental procedures and methods in detail to reproduce the results ( available at gene expression omnibus database under gse66790 ) and provide resource to be employed for comparative analyses of sos response in microorganisms which inhabited in different environments , and thus broaden our understanding of life strategy of bacteria against various environment stresses . 
Direct link to deposited data
Experimental design, materials and methods
Bacterial culture conditions and RNA isolation
Preparation of fluorescent dye-labeled DNA and hybridizations
Image acquisition, data processing and validation
Discussion
Conflict of interest