BACKGROUND
vibrio parahaemolyticus naturally occurs in marine, estuarine and aquaculture environments worldwide and causes serious seafood-borne gastroenteritis and death in humans, particularly when raw, undercooked or mishandled seafood is consumed  <cit> .
v. parahaemolyticus was initially identified in  <dig> in osaka, japan, where an outbreak of acute gastroenteritis that was caused by the consumption of semidried juvenile sardines sickened  <dig> people and killed  <dig>  <cit> .
to date, over eighty v. parahaemolyticus serotypes have been described based on the somatic  and capsular  antigens  <cit> .
of these serotypes, complete genome sequences have been published for three v. parahaemolyticus strains—rimd <dig>   <cit> , bb22op   <cit>  and ucm-v <dig>  strains  <cit> .
additionally, two complete and multiple draft genome sequences for the v. parahaemolyticus strains are available in the genbank database  and online  .
v. parahaemolyticus is a gram-negative bacterium that is frequently isolated from raw seafood  <cit> .
seafood is often subjected to post-harvest processing and low-temperature storage, during which the bacterium is challenged to survive under detrimental cold conditions.
previous studies have indicated that the temperature decrease elicits complex cold shock responses in food-related bacteria , such as the regulation of uptake or synthesis of compatible solutes, dna supercoiling modifications, membrane fluidity maintenance, and cold shock protein  production .
csps comprise a group of low-molecular-weight proteins of approximately 7 kda.
csp families that contain between two and nine members have been identified in food-related bacteria and several food-borne pathogens, including escherichia coli, listeria monocytogenes, staphylococcus aureus, salmonella typhimurium and pseudomonas fragi  <cit> .
in e. coli, the csp family contains nine members , of which cspa is a well characterised rna chaperone that reduces low temperature-associated increases in rna secondary folding  <cit> .
although csps share a high degree of sequence similarity  with two conserved rna-binding motifs, it is surprising that not all csp members are cold-inducible, which implies that they may function in different cellular processes  <cit> .
cspd in e. coli reportedly plays a negative regulatory role in chromosomal replication in nutrient-depleted cells  <cit> .
recent studies have indicated that the mqsr/mqsa toxin/antitoxin pair directly regulates cspd, which may be involved in toxicity and biofilm formation in e. coli  <cit> .
despite its significance in human health and in the aquaculture industry, the molecular mechanisms that underlie the low-temperature survival of v. parahaemolyticus remain largely unknown.
previous studies have revealed three e. coli csp homologues in v. parahaemolyticus, including cspa, cspd and the cold shock dna-binding domain-containing protein  <cit> .
the cspa gene was up-regulated at the transcriptional level by over 30-fold after v. parahaemolyticus was treated for 60 min at 10 °c, a temperature below which bacterial growth was arrested  <cit> .
however, the genes that encoded the other two homologues were undetectable by dna microarray and real-time reverse transcription pcr   <cit> , which suggested that cspa could be a major csp in v. parahaemolyticus during low-temperature growth.
this study is the first to sequence, assemble and annotate the complete genome of v. parahaemolyticus chn <dig> , which has recently been isolated and characterised .
we constructed one dual-gene and two single-gene deletion mutants of the two main v. parahemolyticus chn <dig> csps  and determined the global-level gene expression profiles of the mutant and wild-type strains by illumina rna-sequencing.
these data will refine our grasp of the molecular mechanisms that underlie the low-temperature adaptation of the most common seafood-borne pathogen worldwide.
RESULTS
genomic features of v. parahaemolyticus chn25
the complete genome sequence of v. parahaemolyticus chn <dig> was determined by 454-pyrosequencing .
it consisted of two circular chromosomes that contained  <dig> ,467 bp and  <dig> ,316 bp .
the genome also contained three plasmids , all of which were absent from the other known v. parahaemolyticus genomes .
the complete v. parahaemolyticus chn <dig> genome contained  <dig> ,401bp with a  <dig> % g + c content;  <dig>  protein-encoding genes were predicted, of which approximately  <dig> % encoded hypothetical proteins with unknown functions in public databases.
additionally,  <dig> rrna operons and  <dig> ribosomal protein-encoding genes,  <dig> trna genes, and  <dig> pseudogenes were identified and annotated.
in marked contrast to the other known v. parahaemolyticus genomes, an integrative and conjugative element  was identified in the chn <dig> genome.
the  <dig> -kb element  contained sulfamethoxazole and streptomycin resistance genes.
mating assays demonstrated the active self-transmissibility of the antibiotic resistance from v. parahaemolyticus chn <dig> to e. coli mg <dig>  <cit> .
five prophage gene clusters that ranged from  <dig>  to  <dig>  kb were identified in the chn <dig> genome, and they displayed high degrees of sequence identity with vibrio phage martha 12b <dig> , vibrio phage vpusm  <dig> , vibrio phage henriette 12b <dig> , and vibrio phage n <dig>  <cit> .
additionally, five insertion sequences  were detected in the genome, including isshfr <dig> , isval <dig>  isvpa <dig>  and isvsa <dig> ; the latter existed as two copies in the genome, which suggested that it was probably active.
we concluded that the v. parahaemolyticus chn <dig> genome has undergone major rearrangements due to its mobile genetic elements.
consistent with the other v. parahaemolyticus genomes, most of the genes that encoded enzymes for the predicted central metabolic pathways were present in the chn <dig> strain, including those required for glycolysis, oxidative phosphorylation and tricarboxylic acid cycle .
additionally, the chn <dig> genome also contained genes for three restriction and modification  systems  and four dna repair systems , most of which were present in several other v. parahaemolyticus strains.
the high frequency of the horizontal gene transfer in the chn <dig> strain  may have led the bacteria to hijack the r-m and dna repair mechanisms to generate genetic diversity without losing genomic stability  <cit> .
construction of the Δvpacspa, Δvpacspd and Δvpacspad mutants of v. parahaemolyticus chn25
to investigate the low-temperature adaptation that was mediated by the predicted csps in v. parahaemolyticus chn <dig>  we constructed a deletion mutant of the vpacspa gene.
the upstream and downstream sequences  that flanked the vpacspa gene were obtained by pcr and cloned into a suicide vector, pds <dig>  to yield the recombinant vector, pds132 + Δvpacspa.
the inserted  <dig> -bp sequence was confirmed by dna sequencing .
the recombinant vector was transformed into e. coli β <dig>  and the chloramphenicol-resistant transformant was obtained and conjugated with v. parahaemolyticus chn <dig>  positive exconjugants were obtained using the two-step allelic exchange method  and validated by pcr.
dna sequencing of the pcr product further confirmed the in-frame deletion of the 213-bp sequence of the vpacspa gene from the v. parahaemolyticus chn <dig> genome .
similarly, the vpacspd gene that encoded a cold shock-like protein was deleted from the bacterial genome using the aforementioned method.
the Δvpacspd mutant with a 219-bp in-frame deletion was confirmed by dna sequencing .
furthermore, the vpacspd gene was also successfully deleted from the Δvpacspa mutant, yielding a dual-gene deletion mutant of Δvpacspad .
the genome-level transcriptome data provided direct evidence of the successful construction of the three mutants, in which expression of the corresponding vpacspa or vpacspd genes was undetectable .
survival of the Δvpacspa, Δvpacspd and Δvpacspad mutants at 10 °c
to gain insights into the possible effects of the csp-associated gene deletions on v. parahaemolyticus chn <dig> low-temperature survival, we determined growth curves for the Δvpacspa, Δvpacspd and Δvpacspad mutants, which were grown in lb broth  at 37 °c or 10 °c.
as shown in fig.
<dig> , no apparent differences in growth were observed between the mutant and wild-type strains at 37 °c, which was an optimal growth temperature.
however, the Δvpacspa mutant showed a longer lag phase  and grew more slowly compared with the wild-type strain at 10 °c , demonstrating that vpacspa was a crucial csp in v. parahaemolyticus chn <dig> low-temperature survival.
although vpacspd was identified as one of the three homologues of the e. coli csps  <cit> , the vpacspd gene deletion unexpectedly stimulated mutant growth at 10 °c in our study, which was notably faster than the wild-type strain , indicating that vpacspd likely functioned as a low-temperature bacterial growth inhibitor.
a blast analysis revealed that the vpacspd sequence shared a 70% amino acid identity with cspd in e. coli  , which has been proposed to function as a novel inhibitor of dna replication in nutrient-depleted cells  <cit> .
unlike eccspd, its null mutant grew well over a  <dig> to 42 °c temperature range with no detectable morphological changes.
our data indicated that vpacspd also functioned as a low-temperature induced-csp .
because only three csp-associated genes were identified in v. parahaemolyticus and because vpacspd only displayed a 48% amino acid sequence identity with vpacspa , vpacspd may have evolved to gain different biological functions.
interestingly, the Δvpacspad mutant also grew poorly at low temperature compared to the wild-type strain , indicating that the phenotype of the vpacspa gene deletion dominated that of the vpacspd gene .fig.
<dig> growth of v. parahaemolyticus chn <dig> and the Δvpacspa, Δvpacspd and Δvpacspad mutants in lb broth  at 37 °c  and 10 °c
fig.
<dig> a multi-sequence alignment of the csps from v. parahaemolyticus chn <dig> and e. coli.
the numbers above the alignments indicate the relative positions of the entirely aligned sequences.
identical and conserved  amino acid residues are highlighted in black and grey, respectively; the consensus sequence is shown below the alignment.
the rna-binding motifs  are boxed.
the eccspa and eccspd sequences were derived from e. coli jm <dig> , while the vpacspa  and vpacspd  sequences were obtained from v. parahaemolyticus chn <dig> in this study
construction of the reverse mutants Δvpacspa-com and Δvpacspd-com and complementary phenotypes at 10 °c
the cspa gene was amplified from the genomic dna of v. parahaemolyticus chn <dig> by pcr, and cloned into the expression vector pmmb <dig>  which yielded the recombinant vector pmmb207-vpacspa.
the inserted 213-bp sequence was confirmed by dna sequencing .
this recombinant vector was then electrotransformed into the Δvpacspa mutant competent cells, and generated the reverse mutant Δvpacspa-com .
similarly, the recombinant vector pmmb207-vpacspd carrying the 240-bp cspd gene was also constructed, and electrotransformed into the Δvpacspd mutant, yielding the reverse mutant Δvpacspd-com.
the growth curves for the reverse mutants Δvpacspa-com and Δvpacspd-com were also determined, which were incubated in lb broth  at 37 °c or 10 °c .
consistent with the results in fig.
1a, no obvious difference in growth at 37 °c was observed among the wild type, the mutants Δvpacspa and Δvpacspd, and the reverse mutants Δvpacspa-com and Δvpacspd-com .
however, at 10 °c, the reverse mutants displayed similar growth phenotype as the wild type , demonstrating that the distinct phenotypes of the mutants Δvpacspa and Δvpacspd were indeed resulted from the vpacspa and vpacspd gene deletions in v. parahaemolyticus chn <dig> fig.
<dig> growth of v. parahaemolyticus chn <dig>  the mutants , and the reverse mutants  at 37 °c  and 10 °c .
the wild type and the mutants were incubated in lb broth , and the reverse mutants in the lb supplemented with 5 μg/ml chloramphenicol
transcriptome profiles for the Δvpacspa, Δvpacspd and Δvpacspad mutants at 10 °c
to further investigate the vpacspa- and vpacspd-mediated low-temperature survival of v. parahaemolyticus chn <dig>  we determined global-level gene expression profiles for the Δvpacspa, Δvpacspd and Δvpacspad mutants that were grown at 10 °c, where distinct growth phenotypes were evident.
based on the complete genome sequence of v. parahaemolyticus chn <dig>  this analysis revealed numerous differentially expressed genes  in the mutants, indicating that vpacspa and vpacspd likely functioned as master or global regulators in low-temperature bacterial growth.
five hundred seventy-two genes were significantly altered in the Δvpacspa mutant compared with the wild-type strain; these genes represented approximately  <dig> % of the expressed genes in v. parahaemolyticus chn <dig>  of these,  <dig> genes showed higher transcriptional levels , while  <dig> genes were down-regulated .
the altered genes in the Δvpacspa mutant were grouped into eighty-three gene functional catalogues that were identified in the kyoto encyclopaedia of genes and genomes  database .
the vpacspd gene deletion elicited 10% of the differentially expressed genes in the bacterium, including  <dig> up-regulated and  <dig> down-regulated genes that were grouped into seventy-six gene functional catalogues .
additionally, the expression of  <dig> and  <dig> genes was up- and down-regulated, respectively, in the dual-gene deletion mutant , which accounted for  <dig> % of the expressed genes; they were grouped into seventy-four gene functional catalogues .
a complete list of the degs for the three mutants is available in the gene expression omnibus database  under accession number gse <dig>  to validate the transcriptome data, we examined ten representative genes for each of the three mutants by qrt-pcr.
the resulting data were correlated with data from the illumina rna-sequencing analysis, and there was no statistically significant difference between the two datasets  .
the major low-temperature survival-associated metabolic pathways that were altered in the Δvpacspa, Δvpacspd and Δvpacspad mutants
major altered metabolic pathways in the Δvpacspa mutant
based on the gene set enrichment analysis  of the transcriptome data against the kegg database, the following seven significantly altered metabolic pathways were identified in the Δvpacspa mutant at 10 °c: valine, leucine and isoleucine degradation; the propanoate, ascorbate and aldarate, sulphur, and glycerophospholipid metabolic pathways; atp-binding cassette  transporters; and bacterial secretion systems .
of these, the degs that were linked to valine, leucine and isoleucine degradation as well as propanoate metabolism were up-regulated , which may have resulted in increases in acetyl-coa and propanoyl-coa and subsequent entry into the tca and pyruvate metabolic cycles, respectively, by the Δvpacspa mutant.
*vpachn <dig>  chromosome 1; vpachn25a, chromosome 2
for the other five altered metabolic pathways, most of the degs were down-regulated in the Δvpacspa mutant, which was directly related to its remarkable low-temperature growth inhibition.
for example, the expression of twenty-four genes that were linked to abc transporters was reduced ; they included the glycine betaine /proline, oligopeptide, iron and zinc abc transporters.
this indicated the positive regulation of these abc transporters by vpacspa during low-temperature v. parahaemolyticus chn <dig> survival.
bacterial secretion systems play important roles in virulence, symbiosis, interbacterial interactions, and environmental stress  <cit> .
the genes that encoded components of the four secretion system types  were identified in the v. parahaemolyticus chn <dig> genome.
of these, eleven genes were differentially expressed in the Δvpacspa mutant at the low temperature.
activation of the tolc gene, which encodes an outer membrane protein of t1ss, has been reported in psychrobacter cryohalolentis k <dig> during growth at sub-zero temperatures  <cit> .
in this study, tolc gene expression  was down-regulated  in the Δvpacspa mutant, indicating that vpacspa positively regulated low-temperature tolc gene expression in v. parahaemolyticus chn <dig>  likewise, the yscqrstu genes , which encode the components of t3ss <dig>  were also highly down-regulated .
however, the expression of five genes that were required for t6ss <dig> was strongly enhanced  in the Δvpacspa mutant, which was inconsistent with previous speculation  <cit> .
future investigations into the biological significance of the secretion systems and their differential expression characteristics during low-temperature survival by v. parahaemolyticus will provide important insights on this topic.
major altered metabolic pathways in the Δvpacspd mutant
based on the gesa-kegg analysis, the following six significantly altered metabolic pathways were identified in the Δvpacspd mutant at 10 °c: the phosphotransferase system ; alanine, aspartate and glutamate metabolism; arginine and proline metabolism; the propanoate and nitrogen metabolic pathways; valine, leucine and isoleucine degradation.
consistent with its active low-temperature growth phenotype, several degs that were linked to pts, to nitrogen, arginine and proline metabolism and to alanine, aspartate and glutamate metabolism were significantly up-regulated in the Δvpacspd mutant.
a major barrier to protein function at low temperatures is the inability to maintain sufficient flexibility so that it can increase its interactions with substrates to reduce its required activation energy  <cit> .
in arginine and proline metabolism, all eight degs were up-regulated in the Δvpacspd mutant.
for example, expression of an arginine deiminase  and an arginine/ornithine succinyltransferase , which are required to convert l-arginine to l-citrulline and then to n2-succinyl-l-arginine, were up-regulated by  <dig> - and  <dig> -fold, respectively.
arginines are structurally stabilizing factors that contain side chains that form salt bridges and hydrogen bonds  <cit> .
our data indicated that a low-temperature decrease in l-arginine in the Δvpacspd mutant may have promoted increased protein flexibility.
moreover, the abundance of proline residues is related to increased protein stability due to the rigidity of the n-cα bond  <cit> .
in this study, a decrease in proline resulted from up-regulated proline metabolism-associated enzymes may have also enhanced protein flexibility in the Δvpacspd mutant.
to our knowledge, these genes have not been previously linked to low-temperature survival.
expression of a glutamine synthetase , which catalyses l-glutamate to l-glutamine, was up-regulated in the alanine, aspartate and glutamate metabolic pathways.
however, the genes that encoded a glutaminase  and a glucosamine-fructose-6-phosphate aminotransferase , which convert l-glutamine to l-glutamate and then to d-glucosamine, showed opposite expression profiles, which suggested a decrease in l-glutamate accumulation in the Δvpacspd mutant.
this was also suppressed in the psycrophilic proteins of vibrio salmonicida  <cit> .
unexpectedly, the comparative transcriptome analysis revealed very few genes that were up-regulated in Δvpacspd but down-regulated in the Δvpacspa mutant, indicating that these genes were specifically and negatively governed by vpacspd.
additionally, in the Δvpacspa mutant, vpacspd gene expression was increased  at the low temperature, which was validated by qrt-pcr analysis, but no significant change in vpacspa gene expression was observed in the Δvpacspd mutant.
the results indicated that vpacspd was inhibited by vpacspa at low temperatures, which was consistent with the growth phenotypes described above.
major altered metabolic pathways in the Δvpacspad mutant
similarly, the gesa-kegg analysis revealed the following nine significantly changed metabolic pathways in the Δvpacspad mutant at 10 °c: tca; pts; butanoate metabolism; fructose and mannose metabolism; the pyruvate and the cysteine and methionine metabolic pathways; arginine and proline metabolism; alanine, aspartate and glutamate metabolism; oxidative phosphorylation.
interestingly, these altered metabolic pathways were different from those that were induced in the Δvpacspa mutant, although both mutants demonstrated the slower-growth phenotype at the low temperature.
most of the degs that were linked to tca, oxidative phosphorylation, and pyruvate metabolism were inhibited in the Δvpacspad mutant, which may explain its slower growth at this low temperature.
the down-regulated central metabolic pathways were also observed in other bacteria that were grown at a low temperature  <cit> .
similar to the Δvpacspd mutant, the alanine, aspartate and glutamate metabolic pathways, pts, and the arginine and proline metabolic pathways were also significantly changed in the Δvpacspad mutant.
however, distinct expression patterns were detected in the two mutants.
for example, in contrast to the Δvpacspd mutant, all seven degs that were involved in arginine and proline metabolism were down-regulated  in the Δvpacspad mutant.
additionally, the phosphoenolpyruvate-dependent pts is a major sugar transport multicomponent system in bacteria, by which multiple sugars are transported into bacteria, concomitantly phosphorylated, and fed into glycolysis  <cit> .
in this study, expression of the genes that encoded the cellobiose- and trehalose -specific transporter subunits  also displayed opposite patterns between the Δvpacspad and Δvpacspd mutants.
these results highlighted the antagonistic regulatory effects by vpacspa and vpacspd on low-temperature survival of v. parahaemolyticus chn <dig>
in cysteine and methionine metabolism, a homocysteine synthase  and s-ribosylhomocysteinase , which are involved in converting o-acetyl-l-homoserine and s-ribosyl-l-homocysteine to l-homocysteine, were down-regulated  in the Δvpacspad mutant.
however, a 5-methyltetrahydropteroyltriglutamate-homocysteine s-methyltransferase  that catalyses l-homocysteine to l-methionine was up-regulated .
these results suggest the attenuation of l-homocysteine in the Δvpacspad mutant, which may reduce interference by l-homocysteine with amino acid metabolic and translation processes at low temperatures  <cit> .
differentially expressed regulators  that are involved in the low-temperature survival of the Δvpacspa, Δvpacspd and Δvpacspad mutants
the v. parahaemolyticus chn <dig> genome contains approximately two hundred and seventy-two genes that encode putative transcriptional or response regulators, which represent approximately  <dig> % of all protein-encoding genes in the bacterium.
changes in the expression of transcription factors, especially the master regulators, can modulate global regulatory networks that, in some cases, are essential for bacterial adaptation to changing environments  <cit> .
in this study, the genome-level transcriptome data revealed thirty, twenty-three and thirty-six ders in the Δvpacspa, Δvpacspd and Δvpacspad mutants at 10 °c, respectively .
they globally or specifically regulate various cellular processes, including cold-temperature survival in bacteria, by regulating transcriptional or response regulators that are involved in dna-binding, lysr-type transcriptional regulators, and gntr, arac/xyls, arsr, luxr, and deor regulator families.
of these regulators, several directly regulate gene expression in response to environmental signals in other bacteria.
for example, a recombination regulator, recx , which regulates dna recombination and protects the cell from ionising radiation and uv-irradiation in e. coli  <cit> , was notably down-regulated  in the Δvpacspa mutant; this indicates the positive regulation of recx by vpacspa in v. parahaemolyticus chn <dig> at low temperatures.
interestingly, a transcriptional regulator, beti , was also inhibited in the Δvpacspa mutant , which negatively regulated the bett and betiba genes that governed gb synthesis from choline in e. coli  <cit> .
moreover, two genes , which encode the osmotically inducible betaine-choline-carnitine transporters  that mediate the acquisition of preformed gb  <cit> , were also down-regulated in the Δvpacspa mutant.
these data indicate that vpacspa may stimulate an accumulation of cellular gb that adjusts the hydration level of the bacterial cell cytoplasm at low temperatures  <cit> .
additionally, expression of an important transcriptional regulator, pdhr , which belongs to the gntr family of transcriptional regulators, was repressed  in the Δvpacspa mutant.
pdhr regulates central metabolism by controlling transcription of the components that form the pyruvate dehydrogenase complex  <cit> .
among the ders that were elicited in the Δvpacspd mutant, two regulators  and a merr family transcriptional regulator ) were up-regulated in the Δvpacspd mutant at low temperatures .
the latter regulates gene transcription in response to different environmental signals, including signals from heavy metal ions, organic compounds, and oxidative stress  <cit> .
approximately  <dig> % of the ders in the Δvpacspd mutant were down-regulated, of which half were exclusively expressed in the Δvpacspd mutant  and sigma-e factor negative regulatory protein rsea )  <cit> .
transcriptome data comparisons revealed mosaic der expression profiles in the vpacspad mutant.
interestingly, three regulators of t3ss <dig> gene expression were inhibited in both Δvpacspd and Δvpacspad cells at the low temperature.
these included exsa  and exse , which belonged to the exsacde regulatory cascade, and a t3ss <dig> regulator , which was indicative of positive regulation of t3ss <dig> by vpacspd at low temperatures; this function was similar to that of vpacspa.
likewise, expression of the uhpc regulator , a membrane-bound sensor for external glucose-6-phosphate in e. coli  <cit> , was also decreased in the two mutants.
uhpc was reported to negatively modulate a ye <dig> gene in yersinia enterocolitica, which encoded a homologue of the fhac accessory protein; fhac was strongly expressed at 10 °c but not at 37 °c in bordetella pertussis  <cit> .
interestingly, three ders were detected in all three mutants, and the other five were synchronously induced in both Δvpacspa and Δvpacspd cells, indicating either similar regulatory functions that were shared between vpacspa and vpacspd or vpacspa/d-independent regulation in v. parahaemolyticus chn <dig> at low temperatures.
the molecular responses of bacteria to external environmental signals are complex, but two-component signal transduction systems reportedly play important roles in low-temperature adaptation by several bacteria .
in this study, the expression of a cytosolic response regulator, cpxr , which belongs to the two-component cpx-envelope stress system  <cit> , was repressed in the three mutants.
the cpx system responds to a broad range of environmental stimuli  that cause perturbation of the envelope  <cit> .
in this study, our data showed positive regulation of cpxr by both vpacspa and vpacspd, which may have protected envelope-bound proteins from low-temperature damage.
taken together, our transcriptome data revealed a complex molecular regulatory network that was specifically, coordinately or antagonistically modulated by vpacspa and vpacspd during low-temperature adaptation by v. parahaemolyticus.
numerous regulators, which act as activators or repressors in response to multiple environmental stressors in bacteria, were also elicited in the three mutants.
a future in-depth regulatory network analysis will improve our understanding of low-temperature adaptation mechanisms in v. parahaemolyticus.
possible low-temperature adaptation mechanisms that are mediated by vpacspa and vpacspd in v. parahaemolyticus chn25
the most common strategy that has been adopted by bacteria to survive a low-temperature environment is the accumulation of compatible solutes  by uptake or biosynthesis  <cit> .
in this study, a similar low-temperature strategy by v. parahaemolyticus chn <dig> was observed .
for example, seven genes that were associated with gb biosynthesis, bcct and gb-binding abc transporters were significantly inhibited in the Δvpacspa mutant, which indicated that vpacspa likely stimulated cellular gb accumulation to adjust the hydration level of the cytoplasm and to protect the bacterium from low-temperature damage.fig.
<dig> the possible vpacspa and vpacspd-mediated molecular mechanisms that underlie low-temperature adaptation by v. parahaemolyticus chn <dig>  t, trehalose; m, mannitol; gb, glycine betaine; c, camp regulator protein; tr, transducer; 16s and 23s, rrna subunits
interestingly, in this study, the glycerophospholipid metabolism-associated glpdfkq genes were more strongly inhibited in Δvpacspad than in the Δvpacspa or Δvpacspd mutants, which indicated a coordinated low-temperature activation of the genes by vpacspa and vpacspd.
for example, expression of the glpf gene , which encodes a glycerol uptake facilitator and functions in substrate equilibration between the extracellular and intracellular spaces  <cit> , was down-regulated in Δvpacspa , strongly suppressed in Δvpacspd , and suppressed in Δvpacspad .
similarly, the glpq and glpd genes encode a glycerophosphodiester phosphodiesterase  and a glycerol-3-phosphate dehydrogenase , and they catalyse sn-glycero-3-phosphocholine to choline and sn-glycerol-3-phosphate  and g3p to dihydroxyacetone phosphate , respectively.
expression of the glpq and glpd genes was also more strongly inhibited in Δvpacspad  than in Δvpacspa  or Δvpacspd , indicating a positively superposed regulation of the choline biosynthesis genes by vpacspa and vpacspd; this may have resulted in an increase in cellular compatible solutes to maintain cell membrane integrity at low temperatures.
however, the decreased dhap indirectly led to increased biofilm formation and contributed to several survival advantages under various environmental and energy insults in several other bacteria  <cit> .
moreover, the glpk gene, which encodes a glycerol kinase  that catalyses glycerol to g3p, showed similar expression profiles in all three mutants, which probably resulted in attenuated cellular g3p accumulation at low temperatures.
g3p has been reported to mediate catabolite repression through adenylate cyclase inhibition, which leads to decreases in 3’-5’-cyclic adenosine monophosphate  and inactivation of the camp receptor protein ; crp is a global regulator that participates in sugar metabolism and plays an important role in cold adaptation by e. coli  <cit> .
protective roles for trehalose in response to low-temperature, heat and osmotic stressors have been reported, including prevention of the denaturation and aggregation of specific proteins, in vivo activity as a free radical scavenger, and stabilisation of cell membrane fluidity  <cit> .
in this study, expression of the trehalose -specific transporter subunit ii bc components  was down-regulated in the Δvpacspa  and Δvpacspd  mutants, indicating the positive regulation of trehalose-specific transport by vpacspa and vpacspd to promote bacterial adaptation to a low-temperature environment.
nevertheless, the gene showed an opposite expression pattern in the Δvpacspad mutant , which implied unknown regulatory mechanisms in the Δvpacspad mutant by which trehalose was transported.
biofilm formation is related to bacterial survival in various environments.
it has been reported that type iv pili  played an important role in the biofilm formation of v. parahaemolyticus  <cit> .
in this study, the complete genome sequence analysis revealed a mannose-sensitive hemagglutinin gene cluster  that was required for tfp formation in v. parahaemolyticus chn <dig>  interestingly, the msh gene cluster was significantly down-regulated in the Δvpacspa mutant, which indicated a positive regulation of tfp by vpacspa.
the enhanced biofilm formation likely increased the persistence of v. parahaemolyticus in the aquatic environment by enhancing low-temperature colonisation of environmental surfaces  <cit> .
additionally, our transcriptome data also revealed several other molecular mechanisms that facilitated the low-temperature survival of v. parahaemolyticus chn <dig> .
for example, vpacspd negatively regulated arginine and proline metabolism, which likely resulted in increased cellular protein flexibility and stability so that efficient functionality could be maintained at the low temperature.
CONCLUSIONS
this study is the first to describe the complete  <dig> ,401-bp genome sequence  of v. parahaemolyticus chn <dig> , which consists of two circular chromosomes and three plasmids with  <dig>  predicted protein-encoding genes.
one dual-gene and two single-gene deletion mutants of the main csps, vpacspa and vpacspd, in v. parahaemolyticus chn <dig> were successfully constructed.
our data demonstrated that vpacspa was a primary csp in the bacterium, whereas vpacspd functioned as a growth inhibitor at 10 °c.
moreover, vpacspd gene expression was negatively regulated by vpacspa.
a global-level transcriptomic analysis revealed distinct gene expression profiles among the three mutants.
approximately  <dig> % of the expressed genes in v. parahaemolyticus chn <dig> were significantly altered in the Δvpacspa mutant at 10 °c, including those involved in amino acid degradation, abc transporters, secretion systems, sulphur metabolism and glycerophospholipid metabolism.
the low temperature elicited significant changes in expression of  <dig> % of the genes from the Δvpacspd mutant, including genes that were involved in the phosphotransferase system and in nitrogen and amino acid metabolism.
the following major altered metabolic pathways in the Δvpacspad mutant radically differed from those in the single-gene mutants at 10 °c: tca; pts; butanoate metabolism; fructose and mannose metabolism; pyruvate, cysteine and methionine metabolism; arginine and proline metabolism; alanine, aspartate and glutamate metabolism; and oxidative phosphorylation.
the transcriptome profile comparisons further revealed numerous degs that were shared among the three mutants and ders that were specifically, coordinately and or antagonistically mediated by vpacspa and vpacspd at a low temperature.
v. parahaemolyticus appears to have evolved several molecular strategies with a complex gene regulation network for coping with cold-induced damage.
the results from this study improve our understanding of the genetic basis for low-temperature survival of the most common seafood-borne pathogens worldwide.

 vibrio parahaemolyticus naturally occurs in marine, estuarine and aquaculture environments worldwide and causes serious seafood-borne gastroenteritis and death in humans, particularly when raw, undercooked or mishandled seafood is consumed. v. parahaemolyticus was initially identified in in osaka, japan, where an outbreak of acute gastroenteritis that was caused by the consumption of semidried juvenile sardines sickened people and killed. to date, over eighty v. parahaemolyticus serotypes have been described based on the somatic and capsular antigens. of these serotypes, complete genome sequences have been published for three v. parahaemolyticus strains—rimd, bb22op and ucm-v strains. additionally, two complete and multiple draft genome sequences for the v. parahaemolyticus strains are available in the genbank database and online. v. parahaemolyticus is a gram-negative bacterium that is frequently isolated from raw seafood. seafood is often subjected to post-harvest processing and low-temperature storage, during which the bacterium is challenged to survive under detrimental cold conditions. previous studies have indicated that the temperature decrease elicits complex cold shock responses in food-related bacteria, such as the regulation of uptake or synthesis of compatible solutes, dna supercoiling modifications, membrane fluidity maintenance, and cold shock protein production. csps comprise a group of low-molecular-weight proteins of approximately 7 kda. csp families that contain between two and nine members have been identified in food-related bacteria and several food-borne pathogens, including escherichia coli, listeria monocytogenes, staphylococcus aureus, salmonella typhimurium and pseudomonas fragi. in e. coli, the csp family contains nine members, of which cspa is a well characterised rna chaperone that reduces low temperature-associated increases in rna secondary folding. although csps share a high degree of sequence similarity with two conserved rna-binding motifs, it is surprising that not all csp members are cold-inducible, which implies that they may function in different cellular processes. cspd in e. coli reportedly plays a negative regulatory role in chromosomal replication in nutrient-depleted cells. recent studies have indicated that the mqsr/mqsa toxin/antitoxin pair directly regulates cspd, which may be involved in toxicity and biofilm formation in e. coli. despite its significance in human health and in the aquaculture industry, the molecular mechanisms that underlie the low-temperature survival of v. parahaemolyticus remain largely unknown. previous studies have revealed three e. coli csp homologues in v. parahaemolyticus, including cspa, cspd and the cold shock dna-binding domain-containing protein. the cspa gene was up-regulated at the transcriptional level by over 30-fold after v. parahaemolyticus was treated for 60 min at 10 °c, a temperature below which bacterial growth was arrested. however, the genes that encoded the other two homologues were undetectable by dna microarray and real-time reverse transcription pcr, which suggested that cspa could be a major csp in v. parahaemolyticus during low-temperature growth. this study is the first to sequence, assemble and annotate the complete genome of v. parahaemolyticus chn, which has recently been isolated and characterised. we constructed one dual-gene and two single-gene deletion mutants of the two main v. parahemolyticus chn csps and determined the global-level gene expression profiles of the mutant and wild-type strains by illumina rna-sequencing. these data will refine our grasp of the molecular mechanisms that underlie the low-temperature adaptation of the most common seafood-borne pathogen worldwide.  genomic features of v. parahaemolyticus chn25 the complete genome sequence of v. parahaemolyticus chn was determined by 454-pyrosequencing. it consisted of two circular chromosomes that contained,467 bp and,316 bp. the genome also contained three plasmids, all of which were absent from the other known v. parahaemolyticus genomes. the complete v. parahaemolyticus chn genome contained,401bp with a % g + c content; protein-encoding genes were predicted, of which approximately % encoded hypothetical proteins with unknown functions in public databases. additionally, rrna operons and ribosomal protein-encoding genes, trna genes, and pseudogenes were identified and annotated. in marked contrast to the other known v. parahaemolyticus genomes, an integrative and conjugative element was identified in the chn genome. the -kb element contained sulfamethoxazole and streptomycin resistance genes. mating assays demonstrated the active self-transmissibility of the antibiotic resistance from v. parahaemolyticus chn to e. coli mg. five prophage gene clusters that ranged from to  kb were identified in the chn genome, and they displayed high degrees of sequence identity with vibrio phage martha 12b, vibrio phage vpusm, vibrio phage henriette 12b, and vibrio phage n. additionally, five insertion sequences were detected in the genome, including isshfr, isval isvpa and isvsa; the latter existed as two copies in the genome, which suggested that it was probably active. we concluded that the v. parahaemolyticus chn genome has undergone major rearrangements due to its mobile genetic elements. consistent with the other v. parahaemolyticus genomes, most of the genes that encoded enzymes for the predicted central metabolic pathways were present in the chn strain, including those required for glycolysis, oxidative phosphorylation and tricarboxylic acid cycle. additionally, the chn genome also contained genes for three restriction and modification systems and four dna repair systems, most of which were present in several other v. parahaemolyticus strains. the high frequency of the horizontal gene transfer in the chn strain may have led the bacteria to hijack the r-m and dna repair mechanisms to generate genetic diversity without losing genomic stability. construction of the Δvpacspa, Δvpacspd and Δvpacspad mutants of v. parahaemolyticus chn25 to investigate the low-temperature adaptation that was mediated by the predicted csps in v. parahaemolyticus chn we constructed a deletion mutant of the vpacspa gene. the upstream and downstream sequences that flanked the vpacspa gene were obtained by pcr and cloned into a suicide vector, pds to yield the recombinant vector, pds132 + Δvpacspa. the inserted -bp sequence was confirmed by dna sequencing. the recombinant vector was transformed into e. coli β and the chloramphenicol-resistant transformant was obtained and conjugated with v. parahaemolyticus chn positive exconjugants were obtained using the two-step allelic exchange method and validated by pcr. dna sequencing of the pcr product further confirmed the in-frame deletion of the 213-bp sequence of the vpacspa gene from the v. parahaemolyticus chn genome. similarly, the vpacspd gene that encoded a cold shock-like protein was deleted from the bacterial genome using the aforementioned method. the Δvpacspd mutant with a 219-bp in-frame deletion was confirmed by dna sequencing. furthermore, the vpacspd gene was also successfully deleted from the Δvpacspa mutant, yielding a dual-gene deletion mutant of Δvpacspad. the genome-level transcriptome data provided direct evidence of the successful construction of the three mutants, in which expression of the corresponding vpacspa or vpacspd genes was undetectable. survival of the Δvpacspa, Δvpacspd and Δvpacspad mutants at 10 °c to gain insights into the possible effects of the csp-associated gene deletions on v. parahaemolyticus chn low-temperature survival, we determined growth curves for the Δvpacspa, Δvpacspd and Δvpacspad mutants, which were grown in lb broth at 37 °c or 10 °c. as shown in fig. , no apparent differences in growth were observed between the mutant and wild-type strains at 37 °c, which was an optimal growth temperature. however, the Δvpacspa mutant showed a longer lag phase and grew more slowly compared with the wild-type strain at 10 °c, demonstrating that vpacspa was a crucial csp in v. parahaemolyticus chn low-temperature survival. although vpacspd was identified as one of the three homologues of the e. coli csps, the vpacspd gene deletion unexpectedly stimulated mutant growth at 10 °c in our study, which was notably faster than the wild-type strain, indicating that vpacspd likely functioned as a low-temperature bacterial growth inhibitor. a blast analysis revealed that the vpacspd sequence shared a 70% amino acid identity with cspd in e. coli, which has been proposed to function as a novel inhibitor of dna replication in nutrient-depleted cells. unlike eccspd, its null mutant grew well over a to 42 °c temperature range with no detectable morphological changes. our data indicated that vpacspd also functioned as a low-temperature induced-csp. because only three csp-associated genes were identified in v. parahaemolyticus and because vpacspd only displayed a 48% amino acid sequence identity with vpacspa, vpacspd may have evolved to gain different biological functions. interestingly, the Δvpacspad mutant also grew poorly at low temperature compared to the wild-type strain, indicating that the phenotype of the vpacspa gene deletion dominated that of the vpacspd gene.fig. growth of v. parahaemolyticus chn and the Δvpacspa, Δvpacspd and Δvpacspad mutants in lb broth at 37 °c and 10 °c fig. a multi-sequence alignment of the csps from v. parahaemolyticus chn and e. coli. the numbers above the alignments indicate the relative positions of the entirely aligned sequences. identical and conserved amino acid residues are highlighted in black and grey, respectively; the consensus sequence is shown below the alignment. the rna-binding motifs are boxed. the eccspa and eccspd sequences were derived from e. coli jm, while the vpacspa and vpacspd sequences were obtained from v. parahaemolyticus chn in this study construction of the reverse mutants Δvpacspa-com and Δvpacspd-com and complementary phenotypes at 10 °c the cspa gene was amplified from the genomic dna of v. parahaemolyticus chn by pcr, and cloned into the expression vector pmmb which yielded the recombinant vector pmmb207-vpacspa. the inserted 213-bp sequence was confirmed by dna sequencing. this recombinant vector was then electrotransformed into the Δvpacspa mutant competent cells, and generated the reverse mutant Δvpacspa-com. similarly, the recombinant vector pmmb207-vpacspd carrying the 240-bp cspd gene was also constructed, and electrotransformed into the Δvpacspd mutant, yielding the reverse mutant Δvpacspd-com. the growth curves for the reverse mutants Δvpacspa-com and Δvpacspd-com were also determined, which were incubated in lb broth at 37 °c or 10 °c. consistent with the results in fig. 1a, no obvious difference in growth at 37 °c was observed among the wild type, the mutants Δvpacspa and Δvpacspd, and the reverse mutants Δvpacspa-com and Δvpacspd-com. however, at 10 °c, the reverse mutants displayed similar growth phenotype as the wild type, demonstrating that the distinct phenotypes of the mutants Δvpacspa and Δvpacspd were indeed resulted from the vpacspa and vpacspd gene deletions in v. parahaemolyticus chn fig. growth of v. parahaemolyticus chn the mutants, and the reverse mutants at 37 °c and 10 °c. the wild type and the mutants were incubated in lb broth, and the reverse mutants in the lb supplemented with 5 μg/ml chloramphenicol transcriptome profiles for the Δvpacspa, Δvpacspd and Δvpacspad mutants at 10 °c to further investigate the vpacspa- and vpacspd-mediated low-temperature survival of v. parahaemolyticus chn we determined global-level gene expression profiles for the Δvpacspa, Δvpacspd and Δvpacspad mutants that were grown at 10 °c, where distinct growth phenotypes were evident. based on the complete genome sequence of v. parahaemolyticus chn this analysis revealed numerous differentially expressed genes in the mutants, indicating that vpacspa and vpacspd likely functioned as master or global regulators in low-temperature bacterial growth. five hundred seventy-two genes were significantly altered in the Δvpacspa mutant compared with the wild-type strain; these genes represented approximately % of the expressed genes in v. parahaemolyticus chn of these, genes showed higher transcriptional levels, while genes were down-regulated. the altered genes in the Δvpacspa mutant were grouped into eighty-three gene functional catalogues that were identified in the kyoto encyclopaedia of genes and genomes database. the vpacspd gene deletion elicited 10% of the differentially expressed genes in the bacterium, including up-regulated and down-regulated genes that were grouped into seventy-six gene functional catalogues. additionally, the expression of and genes was up- and down-regulated, respectively, in the dual-gene deletion mutant, which accounted for % of the expressed genes; they were grouped into seventy-four gene functional catalogues. a complete list of the degs for the three mutants is available in the gene expression omnibus database under accession number gse to validate the transcriptome data, we examined ten representative genes for each of the three mutants by qrt-pcr. the resulting data were correlated with data from the illumina rna-sequencing analysis, and there was no statistically significant difference between the two datasets. the major low-temperature survival-associated metabolic pathways that were altered in the Δvpacspa, Δvpacspd and Δvpacspad mutants major altered metabolic pathways in the Δvpacspa mutant based on the gene set enrichment analysis of the transcriptome data against the kegg database, the following seven significantly altered metabolic pathways were identified in the Δvpacspa mutant at 10 °c: valine, leucine and isoleucine degradation; the propanoate, ascorbate and aldarate, sulphur, and glycerophospholipid metabolic pathways; atp-binding cassette transporters; and bacterial secretion systems. of these, the degs that were linked to valine, leucine and isoleucine degradation as well as propanoate metabolism were up-regulated, which may have resulted in increases in acetyl-coa and propanoyl-coa and subsequent entry into the tca and pyruvate metabolic cycles, respectively, by the Δvpacspa mutant. *vpachn chromosome 1; vpachn25a, chromosome 2 for the other five altered metabolic pathways, most of the degs were down-regulated in the Δvpacspa mutant, which was directly related to its remarkable low-temperature growth inhibition. for example, the expression of twenty-four genes that were linked to abc transporters was reduced; they included the glycine betaine /proline, oligopeptide, iron and zinc abc transporters. this indicated the positive regulation of these abc transporters by vpacspa during low-temperature v. parahaemolyticus chn survival. bacterial secretion systems play important roles in virulence, symbiosis, interbacterial interactions, and environmental stress. the genes that encoded components of the four secretion system types were identified in the v. parahaemolyticus chn genome. of these, eleven genes were differentially expressed in the Δvpacspa mutant at the low temperature. activation of the tolc gene, which encodes an outer membrane protein of t1ss, has been reported in psychrobacter cryohalolentis k during growth at sub-zero temperatures. in this study, tolc gene expression was down-regulated in the Δvpacspa mutant, indicating that vpacspa positively regulated low-temperature tolc gene expression in v. parahaemolyticus chn likewise, the yscqrstu genes, which encode the components of t3ss were also highly down-regulated. however, the expression of five genes that were required for t6ss was strongly enhanced in the Δvpacspa mutant, which was inconsistent with previous speculation. future investigations into the biological significance of the secretion systems and their differential expression characteristics during low-temperature survival by v. parahaemolyticus will provide important insights on this topic. major altered metabolic pathways in the Δvpacspd mutant based on the gesa-kegg analysis, the following six significantly altered metabolic pathways were identified in the Δvpacspd mutant at 10 °c: the phosphotransferase system; alanine, aspartate and glutamate metabolism; arginine and proline metabolism; the propanoate and nitrogen metabolic pathways; valine, leucine and isoleucine degradation. consistent with its active low-temperature growth phenotype, several degs that were linked to pts, to nitrogen, arginine and proline metabolism and to alanine, aspartate and glutamate metabolism were significantly up-regulated in the Δvpacspd mutant. a major barrier to protein function at low temperatures is the inability to maintain sufficient flexibility so that it can increase its interactions with substrates to reduce its required activation energy. in arginine and proline metabolism, all eight degs were up-regulated in the Δvpacspd mutant. for example, expression of an arginine deiminase and an arginine/ornithine succinyltransferase, which are required to convert l-arginine to l-citrulline and then to n2-succinyl-l-arginine, were up-regulated by - and -fold, respectively. arginines are structurally stabilizing factors that contain side chains that form salt bridges and hydrogen bonds. our data indicated that a low-temperature decrease in l-arginine in the Δvpacspd mutant may have promoted increased protein flexibility. moreover, the abundance of proline residues is related to increased protein stability due to the rigidity of the n-cα bond. in this study, a decrease in proline resulted from up-regulated proline metabolism-associated enzymes may have also enhanced protein flexibility in the Δvpacspd mutant. to our knowledge, these genes have not been previously linked to low-temperature survival. expression of a glutamine synthetase, which catalyses l-glutamate to l-glutamine, was up-regulated in the alanine, aspartate and glutamate metabolic pathways. however, the genes that encoded a glutaminase and a glucosamine-fructose-6-phosphate aminotransferase, which convert l-glutamine to l-glutamate and then to d-glucosamine, showed opposite expression profiles, which suggested a decrease in l-glutamate accumulation in the Δvpacspd mutant. this was also suppressed in the psycrophilic proteins of vibrio salmonicida. unexpectedly, the comparative transcriptome analysis revealed very few genes that were up-regulated in Δvpacspd but down-regulated in the Δvpacspa mutant, indicating that these genes were specifically and negatively governed by vpacspd. additionally, in the Δvpacspa mutant, vpacspd gene expression was increased at the low temperature, which was validated by qrt-pcr analysis, but no significant change in vpacspa gene expression was observed in the Δvpacspd mutant. the results indicated that vpacspd was inhibited by vpacspa at low temperatures, which was consistent with the growth phenotypes described above. major altered metabolic pathways in the Δvpacspad mutant similarly, the gesa-kegg analysis revealed the following nine significantly changed metabolic pathways in the Δvpacspad mutant at 10 °c: tca; pts; butanoate metabolism; fructose and mannose metabolism; the pyruvate and the cysteine and methionine metabolic pathways; arginine and proline metabolism; alanine, aspartate and glutamate metabolism; oxidative phosphorylation. interestingly, these altered metabolic pathways were different from those that were induced in the Δvpacspa mutant, although both mutants demonstrated the slower-growth phenotype at the low temperature. most of the degs that were linked to tca, oxidative phosphorylation, and pyruvate metabolism were inhibited in the Δvpacspad mutant, which may explain its slower growth at this low temperature. the down-regulated central metabolic pathways were also observed in other bacteria that were grown at a low temperature. similar to the Δvpacspd mutant, the alanine, aspartate and glutamate metabolic pathways, pts, and the arginine and proline metabolic pathways were also significantly changed in the Δvpacspad mutant. however, distinct expression patterns were detected in the two mutants. for example, in contrast to the Δvpacspd mutant, all seven degs that were involved in arginine and proline metabolism were down-regulated in the Δvpacspad mutant. additionally, the phosphoenolpyruvate-dependent pts is a major sugar transport multicomponent system in bacteria, by which multiple sugars are transported into bacteria, concomitantly phosphorylated, and fed into glycolysis. in this study, expression of the genes that encoded the cellobiose- and trehalose -specific transporter subunits also displayed opposite patterns between the Δvpacspad and Δvpacspd mutants. these results highlighted the antagonistic regulatory effects by vpacspa and vpacspd on low-temperature survival of v. parahaemolyticus chn in cysteine and methionine metabolism, a homocysteine synthase and s-ribosylhomocysteinase, which are involved in converting o-acetyl-l-homoserine and s-ribosyl-l-homocysteine to l-homocysteine, were down-regulated in the Δvpacspad mutant. however, a 5-methyltetrahydropteroyltriglutamate-homocysteine s-methyltransferase that catalyses l-homocysteine to l-methionine was up-regulated. these results suggest the attenuation of l-homocysteine in the Δvpacspad mutant, which may reduce interference by l-homocysteine with amino acid metabolic and translation processes at low temperatures. differentially expressed regulators that are involved in the low-temperature survival of the Δvpacspa, Δvpacspd and Δvpacspad mutants the v. parahaemolyticus chn genome contains approximately two hundred and seventy-two genes that encode putative transcriptional or response regulators, which represent approximately % of all protein-encoding genes in the bacterium. changes in the expression of transcription factors, especially the master regulators, can modulate global regulatory networks that, in some cases, are essential for bacterial adaptation to changing environments. in this study, the genome-level transcriptome data revealed thirty, twenty-three and thirty-six ders in the Δvpacspa, Δvpacspd and Δvpacspad mutants at 10 °c, respectively. they globally or specifically regulate various cellular processes, including cold-temperature survival in bacteria, by regulating transcriptional or response regulators that are involved in dna-binding, lysr-type transcriptional regulators, and gntr, arac/xyls, arsr, luxr, and deor regulator families. of these regulators, several directly regulate gene expression in response to environmental signals in other bacteria. for example, a recombination regulator, recx, which regulates dna recombination and protects the cell from ionising radiation and uv-irradiation in e. coli, was notably down-regulated in the Δvpacspa mutant; this indicates the positive regulation of recx by vpacspa in v. parahaemolyticus chn at low temperatures. interestingly, a transcriptional regulator, beti, was also inhibited in the Δvpacspa mutant, which negatively regulated the bett and betiba genes that governed gb synthesis from choline in e. coli. moreover, two genes, which encode the osmotically inducible betaine-choline-carnitine transporters that mediate the acquisition of preformed gb, were also down-regulated in the Δvpacspa mutant. these data indicate that vpacspa may stimulate an accumulation of cellular gb that adjusts the hydration level of the bacterial cell cytoplasm at low temperatures. additionally, expression of an important transcriptional regulator, pdhr, which belongs to the gntr family of transcriptional regulators, was repressed in the Δvpacspa mutant. pdhr regulates central metabolism by controlling transcription of the components that form the pyruvate dehydrogenase complex. among the ders that were elicited in the Δvpacspd mutant, two regulators and a merr family transcriptional regulator ) were up-regulated in the Δvpacspd mutant at low temperatures. the latter regulates gene transcription in response to different environmental signals, including signals from heavy metal ions, organic compounds, and oxidative stress. approximately % of the ders in the Δvpacspd mutant were down-regulated, of which half were exclusively expressed in the Δvpacspd mutant and sigma-e factor negative regulatory protein rsea ). transcriptome data comparisons revealed mosaic der expression profiles in the vpacspad mutant. interestingly, three regulators of t3ss gene expression were inhibited in both Δvpacspd and Δvpacspad cells at the low temperature. these included exsa and exse, which belonged to the exsacde regulatory cascade, and a t3ss regulator, which was indicative of positive regulation of t3ss by vpacspd at low temperatures; this function was similar to that of vpacspa. likewise, expression of the uhpc regulator, a membrane-bound sensor for external glucose-6-phosphate in e. coli, was also decreased in the two mutants. uhpc was reported to negatively modulate a ye gene in yersinia enterocolitica, which encoded a homologue of the fhac accessory protein; fhac was strongly expressed at 10 °c but not at 37 °c in bordetella pertussis. interestingly, three ders were detected in all three mutants, and the other five were synchronously induced in both Δvpacspa and Δvpacspd cells, indicating either similar regulatory functions that were shared between vpacspa and vpacspd or vpacspa/d-independent regulation in v. parahaemolyticus chn at low temperatures. the molecular responses of bacteria to external environmental signals are complex, but two-component signal transduction systems reportedly play important roles in low-temperature adaptation by several bacteria. in this study, the expression of a cytosolic response regulator, cpxr, which belongs to the two-component cpx-envelope stress system, was repressed in the three mutants. the cpx system responds to a broad range of environmental stimuli that cause perturbation of the envelope. in this study, our data showed positive regulation of cpxr by both vpacspa and vpacspd, which may have protected envelope-bound proteins from low-temperature damage. taken together, our transcriptome data revealed a complex molecular regulatory network that was specifically, coordinately or antagonistically modulated by vpacspa and vpacspd during low-temperature adaptation by v. parahaemolyticus. numerous regulators, which act as activators or repressors in response to multiple environmental stressors in bacteria, were also elicited in the three mutants. a future in-depth regulatory network analysis will improve our understanding of low-temperature adaptation mechanisms in v. parahaemolyticus. possible low-temperature adaptation mechanisms that are mediated by vpacspa and vpacspd in v. parahaemolyticus chn25 the most common strategy that has been adopted by bacteria to survive a low-temperature environment is the accumulation of compatible solutes by uptake or biosynthesis. in this study, a similar low-temperature strategy by v. parahaemolyticus chn was observed. for example, seven genes that were associated with gb biosynthesis, bcct and gb-binding abc transporters were significantly inhibited in the Δvpacspa mutant, which indicated that vpacspa likely stimulated cellular gb accumulation to adjust the hydration level of the cytoplasm and to protect the bacterium from low-temperature damage.fig. the possible vpacspa and vpacspd-mediated molecular mechanisms that underlie low-temperature adaptation by v. parahaemolyticus chn t, trehalose; m, mannitol; gb, glycine betaine; c, camp regulator protein; tr, transducer; 16s and 23s, rrna subunits interestingly, in this study, the glycerophospholipid metabolism-associated glpdfkq genes were more strongly inhibited in Δvpacspad than in the Δvpacspa or Δvpacspd mutants, which indicated a coordinated low-temperature activation of the genes by vpacspa and vpacspd. for example, expression of the glpf gene, which encodes a glycerol uptake facilitator and functions in substrate equilibration between the extracellular and intracellular spaces, was down-regulated in Δvpacspa, strongly suppressed in Δvpacspd, and suppressed in Δvpacspad. similarly, the glpq and glpd genes encode a glycerophosphodiester phosphodiesterase and a glycerol-3-phosphate dehydrogenase, and they catalyse sn-glycero-3-phosphocholine to choline and sn-glycerol-3-phosphate and g3p to dihydroxyacetone phosphate, respectively. expression of the glpq and glpd genes was also more strongly inhibited in Δvpacspad than in Δvpacspa or Δvpacspd, indicating a positively superposed regulation of the choline biosynthesis genes by vpacspa and vpacspd; this may have resulted in an increase in cellular compatible solutes to maintain cell membrane integrity at low temperatures. however, the decreased dhap indirectly led to increased biofilm formation and contributed to several survival advantages under various environmental and energy insults in several other bacteria. moreover, the glpk gene, which encodes a glycerol kinase that catalyses glycerol to g3p, showed similar expression profiles in all three mutants, which probably resulted in attenuated cellular g3p accumulation at low temperatures. g3p has been reported to mediate catabolite repression through adenylate cyclase inhibition, which leads to decreases in 3’-5’-cyclic adenosine monophosphate and inactivation of the camp receptor protein; crp is a global regulator that participates in sugar metabolism and plays an important role in cold adaptation by e. coli. protective roles for trehalose in response to low-temperature, heat and osmotic stressors have been reported, including prevention of the denaturation and aggregation of specific proteins, in vivo activity as a free radical scavenger, and stabilisation of cell membrane fluidity. in this study, expression of the trehalose -specific transporter subunit ii bc components was down-regulated in the Δvpacspa and Δvpacspd mutants, indicating the positive regulation of trehalose-specific transport by vpacspa and vpacspd to promote bacterial adaptation to a low-temperature environment. nevertheless, the gene showed an opposite expression pattern in the Δvpacspad mutant, which implied unknown regulatory mechanisms in the Δvpacspad mutant by which trehalose was transported. biofilm formation is related to bacterial survival in various environments. it has been reported that type iv pili played an important role in the biofilm formation of v. parahaemolyticus. in this study, the complete genome sequence analysis revealed a mannose-sensitive hemagglutinin gene cluster that was required for tfp formation in v. parahaemolyticus chn interestingly, the msh gene cluster was significantly down-regulated in the Δvpacspa mutant, which indicated a positive regulation of tfp by vpacspa. the enhanced biofilm formation likely increased the persistence of v. parahaemolyticus in the aquatic environment by enhancing low-temperature colonisation of environmental surfaces. additionally, our transcriptome data also revealed several other molecular mechanisms that facilitated the low-temperature survival of v. parahaemolyticus chn. for example, vpacspd negatively regulated arginine and proline metabolism, which likely resulted in increased cellular protein flexibility and stability so that efficient functionality could be maintained at the low temperature.  this study is the first to describe the complete,401-bp genome sequence of v. parahaemolyticus chn, which consists of two circular chromosomes and three plasmids with predicted protein-encoding genes. one dual-gene and two single-gene deletion mutants of the main csps, vpacspa and vpacspd, in v. parahaemolyticus chn were successfully constructed. our data demonstrated that vpacspa was a primary csp in the bacterium, whereas vpacspd functioned as a growth inhibitor at 10 °c. moreover, vpacspd gene expression was negatively regulated by vpacspa. a global-level transcriptomic analysis revealed distinct gene expression profiles among the three mutants. approximately % of the expressed genes in v. parahaemolyticus chn were significantly altered in the Δvpacspa mutant at 10 °c, including those involved in amino acid degradation, abc transporters, secretion systems, sulphur metabolism and glycerophospholipid metabolism. the low temperature elicited significant changes in expression of % of the genes from the Δvpacspd mutant, including genes that were involved in the phosphotransferase system and in nitrogen and amino acid metabolism. the following major altered metabolic pathways in the Δvpacspad mutant radically differed from those in the single-gene mutants at 10 °c: tca; pts; butanoate metabolism; fructose and mannose metabolism; pyruvate, cysteine and methionine metabolism; arginine and proline metabolism; alanine, aspartate and glutamate metabolism; and oxidative phosphorylation. the transcriptome profile comparisons further revealed numerous degs that were shared among the three mutants and ders that were specifically, coordinately and or antagonistically mediated by vpacspa and vpacspd at a low temperature. v. parahaemolyticus appears to have evolved several molecular strategies with a complex gene regulation network for coping with cold-induced damage. the results from this study improve our understanding of the genetic basis for low-temperature survival of the most common seafood-borne pathogens worldwide. 
these included genes that were involved in amino acid degradation, secretion systems, sulphur metabolism and glycerophospholipid metabolism along with atp-binding cassette transporters. in this study, we determined the complete genome sequence of v. parahaemolyticus chn. this study is the first to describe the complete genome sequence of v. parahaemolyticus. vibrio parahaemolyticuscomplete genome sequencecold shock proteingene deletiontranscriptomelow-temperature adaptationshanghai municipal science and technology commissionno.09320503600no.13yz098no.zzhy12028liu taigang http://dx.doi.org/ /501100003395shanghai municipal education commissionno.b-9500-10-0004chen lanming http://dx.doi.org/ /501100001809national natural science foundation of chinano.31271830no.31671946chen lanming issue-copyright-statement© the author 2017 the two main csp-encoding genes were deleted from the bacterial genome, and comparative transcriptomic analysis between the mutant and wild-type strains was performed to dissect the possible molecular mechanisms that underlie low-temperature adaptation by v. parahaemolyticus. the,401-bp v. parahaemolyticus chn genome consisted of two circular chromosomes and three plasmids with predicted protein-encoding genes. the transcriptome data revealed that % of the expressed genes in v. parahaemolyticus chn were significantly altered in the Δvpacspa mutant when it was grown at 10 °c. these results have improved our understanding of the genetic basis for low-temperature survival by the most common seafood-borne pathogen worldwide. vibrio parahaemolyticus causes serious seafood-borne gastroenteritis and death in humans. the complementary phenotypes were observed in the reverse mutants. to date, very little information is available regarding the biological functions of cold shock proteins in the low-temperature survival of the bacterium. the gene deletions, complementary insertions, and comparative transcriptomics demonstrate that vpacspa is a primary csp in the bacterium, while vpacspd functions as a growth inhibitor at 10 °c. the major metabolic pathways that were altered by the dual-gene deletion mutant radically differed from those that were altered by single-gene mutants. the growth of the Δvpacspa mutant was strongly inhibited at 10 °c, whereas the vpacspd gene deletion strongly stimulated bacterial growth at this low temperature compared with the wild-type strain. raw seafood is often subjected to post-harvest processing and low-temperature storage. one dual-gene and two single-gene deletion mutants were generated for vpacspa and vpacspd by homologous recombination. however, a low temperature elicited significant expression changes for % of the genes in the Δvpacspd mutant, including those involved in the phosphotransferase system and in the metabolism of nitrogen and amino acids. our data also revealed several possible molecular coping strategies for low-temperature adaptation by the bacterium.
 vibrio parahaemolyticus causes serious seafood-borne gastroenteritis and death in humans. raw seafood is often subjected to post-harvest processing and low-temperature storage. to date, very little information is available regarding the biological functions of cold shock proteins in the low-temperature survival of the bacterium. in this study, we determined the complete genome sequence of v. parahaemolyticus chn. the two main csp-encoding genes were deleted from the bacterial genome, and comparative transcriptomic analysis between the mutant and wild-type strains was performed to dissect the possible molecular mechanisms that underlie low-temperature adaptation by v. parahaemolyticus.  the,401-bp v. parahaemolyticus chn genome consisted of two circular chromosomes and three plasmids with predicted protein-encoding genes. one dual-gene and two single-gene deletion mutants were generated for vpacspa and vpacspd by homologous recombination. the growth of the Δvpacspa mutant was strongly inhibited at 10 °c, whereas the vpacspd gene deletion strongly stimulated bacterial growth at this low temperature compared with the wild-type strain. the complementary phenotypes were observed in the reverse mutants. the transcriptome data revealed that % of the expressed genes in v. parahaemolyticus chn were significantly altered in the Δvpacspa mutant when it was grown at 10 °c. these included genes that were involved in amino acid degradation, secretion systems, sulphur metabolism and glycerophospholipid metabolism along with atp-binding cassette transporters. however, a low temperature elicited significant expression changes for % of the genes in the Δvpacspd mutant, including those involved in the phosphotransferase system and in the metabolism of nitrogen and amino acids. the major metabolic pathways that were altered by the dual-gene deletion mutant radically differed from those that were altered by single-gene mutants. comparison of the transcriptome profiles further revealed numerous differentially expressed genes that were shared among the three mutants and regulators that were specifically, coordinately or antagonistically modulated by vpacspa and vpacspd. our data also revealed several possible molecular coping strategies for low-temperature adaptation by the bacterium.  this study is the first to describe the complete genome sequence of v. parahaemolyticus. the gene deletions, complementary insertions, and comparative transcriptomics demonstrate that vpacspa is a primary csp in the bacterium, while vpacspd functions as a growth inhibitor at 10 °c. these results have improved our understanding of the genetic basis for low-temperature survival by the most common seafood-borne pathogen worldwide. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords vibrio parahaemolyticuscomplete genome sequencecold shock proteingene deletiontranscriptomelow-temperature adaptationshanghai municipal science and technology commissionno.09320503600no.13yz098no.zzhy12028liu taigang http://dx.doi.org/ /501100003395shanghai municipal education commissionno.b-9500-10-0004chen lanming http://dx.doi.org/ /501100001809national natural science foundation of chinano.31271830no.31671946chen lanming issue-copyright-statement© the author 2017 