Comparative genomic and transcriptomic analyses reveal distinct response strategies to hypoxia by Vibrio parahaemolyticus isolates of clinical and aquatic animal origins

Purpose Vibrio parahaemolyticus is a leading seafood borne pathogen worldwide. The aim of this study was to decipher the response mechanism of V. parahaemolyticus isolates of clinical and aquatic animal origins to the hypoxic condition, which challenges the bacterial survival in the host and in the environment. Methods Growth profiles of V. parahaemolyticus isolates ( n = 5) of clinical and aquatic animal origins were examined at different stress conditions (osmolality, acid, temperature, and O 2 concentrations). Draft genomes of the V. para-haemolyticus isolates were determined using the Illumina sequencing technique. Comparative genomic analysis were performed to identify and validate the hypoxic tolerance-related genes. Results The V. parahaemolyticus isolates had an oxygen concentration-dependent growth mode, and the 10% O 2 condition strongly inhibited the bacterial growth, when incubated in TSB medium (pH 8.5, 3% NaCl) at 37 °C. Unex-pectedly, in marked contrast to the normal 21% O 2 condition, the 10% O 2 treatment for 24 h significantly increased biofilm formation of V. parahaemolyticus isolates ( p < 0.05). Draft genome sequences of four V. parahaemolyticus isolates of aquatic animal origins were determined (4.914–5.3530 Mb), which carried mobile genetic elements (n = 12–29). Genome-wide gene expression changes triggered by the hypoxic condition were further examined. Comparative transcriptomic analyses unveiled multiple molecular strategies employed by the bacterium to mitigate the cell damage caused by the hypoxia. Of note, the pathogenic V. parahaemolyticus ATCC17802 down-regulated and/or shut down ten metabolic pathways to reduce cell viability and maintain cell structure under the hypoxic stress. Conclusions The results of this study fill prior gaps in the response mechanism of V. parahaemolyticus to the hypoxic condition. Different tolerance to hypoxia contributes to the persistence of pathogenic V. parahaemolyticus in the niches.


Introduction
Vibrio parahaemolyticus is a Gram-negative bacterium that inhabits in marine and estuarine environments worldwide (Li et al. 2019).Consuming raw seafood, incomplete food processing, or cross-contamination during food processing can lead to V. parahaemolyticus infection in humans.The clinical manifestations are gastroenteritis, organ infection, septicaemia, and even death (Li et al. 2019).In the United States, more than 50% of foodborne gastrointestinal-Vibriosis cases are caused by V. parahaemolyticus (Karan et al. 2021), which leads to about 80,000 illnesses each year (https:// www.cdc.gov/ Vibrio/, accessed on November 1, 2022).In China, a national surveillance of 152,792 patients of all ages with acute diarrhea in 31 provinces was administered in 2009-2018.V. parahaemolyticus was found to be the third common bacterial pathogen and contributed to 10.83% of all positive detection (Wang et al. 2021).The pathogenicity of V. parahaemolyticus is strongly associated with the expression of thermostable direct hemolysin (TDH) and/ or TDH-related hemolysin (TRH) (Pazhani et al. 2021).
V. parahaemolyticus is frequently detected in seafood (Vu et al. 2022).The elevated seawater temperatures caused by rising global temperatures from climate change may affect transmission of waterborne pathogenic bacteria (Murray et al. 2020;Tong et al. 2022).For instance, the number of days per year suitable for Vibrio in the Baltic Sea and along the north east coast of the United States reached 107 in 2018, double the early 1980s baseline (Murray et al. 2020).As the sea temperature rises, oxygen (O 2 ) becomes less soluble via a process known as deoxygenation (Gao et al. 2012).Low oxygen condition refers to oxygen partial pressure of 0-10% (Chen et al. 2007).Compared to strictly aerobic or strictly anaerobic conditions, the hypoxic zone has unique biological properties in the environment (Pohl et al. 2022).
The respiration of oxygen is the main source of energy for biological cells (Bueno et al. 2020).The low oxygen condition impacts on cellular enzymatic activity, such as dehydrogenase and superoxide dismutase (Obbard et al. 1994;Reaney et al. 2005), and also change how quickly organisms metabolize and respire (Oschlies et al. 2018).Studies have indicated that waterborne pathogen Vibrio cholerae can exchange common oxygen respiratory terminal oxidases with high-oxygen-affinity terminal oxidases (H-O-ATOs), using the fermentation pathway, with some electron acceptors (EAs) such as fumarate, and nitrate (Bueno et al. 2018;Heidelberg et al. 2000).It has also been reported that biofilm formation of common pathogens Pseudomonas aeruginosa, Streptococcus aureus, and Escherichia coli induces the production of hypoxia-related enzymes such as H-O-ATOs, nitrate reductases, and anaerobic ribonucleotide reductases (Crespo et al. 2016;Jo et al. 2017;Létoffé et al. 2017).Biofilm, constructed primarily by autogenic extracellular polymeric substances, can protect bacteria from the environmental stress, hinder phagocytosis, and thus confer the capacity for long-term colonization and persistence in the host (Thi et al. 2020).The enteric pathogen V. parahaemolyticus can access, colonize, and proliferate within the human intestine with low oxygen level, causing severe diarrhea disease (Bueno et al. 2020;Sun et al. 2022).Nevertheless, to the best of our knowledge, how V. parahaemolyticus responses to the low oxygen condition has not yet been unveiled so far.
In our previous studies, a number of V. parahaemolyticus isolates of aquatic animal origins were isolated, identified and characterized (Su and Chen 2020;Sun et al. 2014;Xu et al. 2022a;Xu et al. 2022b;Yang et al. 2020;Yu et al. 2022;Zhu et al. 2020).Based on these studies, in the present study, we deciphered for the first time the fitness mechanism of V. parahaemolyticus of aquatic animal and human clinical origins under the hypoxic condition.The major objectives of this study were (1) to investigate growth features of V. parahaemolyticus isolates (n = 5) at different concentrations of oxygen (21-5% O 2 ); (2) to monitor dynamic process of biofilm formation, and observe morphological cell structure changes of the V. parahaemolyticus isolates at the hypoxic condition (e.g., 10% O 2 ); and (3) to decipher the molecular mechanism of V. parahaemolyticus response to the hypoxic condition by comparative genomics and transcriptomics analyses.The results of this study provide the first experimental evidence for multiple molecular strategies developed by V. parahaemolyticus to deal with the hypoxia.

V. parahaemolyticus strains and culture conditions
The V. parahaemolyticus B8-26, B1-21, N2-5, and L7-40 strains (Table 1) were individually inoculated (1%, v/v) into 5-mL Tryptone Soya Broth (TSB) (Beijing Land Bridge Technology, Beijing, China), and routinely incubated aerobically with shaking at 180 r/min at 37 °C incubator (Shanghai Zhichu Instrument, Shanghai, China).V. parahaemolyticus ATCC17802 was used as a positive control strain, which was first isolated in 1950, leading to the outbreak of human acute gastroenteritis in Japan (Fujino et al. 1953).The overnight cultures were re-inoculated in fresh TSB, incubated to middle logarithmic growth phase (mid-LGP), and used in the following assays.

Antibiotic resistance and heavy metal tolerance assays
The V. parahaemolyticus isolates was tested for antibiotic susceptibility using the standard disc diffusion method of Clinical and Laboratory Standards Institute (CLSI, M100-S28, 2018), USA.The Mueller-Hinton (MH) medium and ten antibiotic discs were purchased from OXOID, Basingstoke, UK.Escherichia coli ATCC25922 was used as a quality control strain (Su and Chen 2020).
Tolerance of the V. parahaemolyticus isolates to heavy metals was examined using the standard broth dilution testing (microdilution) (Su and Chen 2020;Xu et al. 2022a;Yu et al. 2022).Eight heavy metals (Analytical Reagents) were purchased from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China.E. coli K12 was used as a quality control strain (Su and Chen 2020).

Growth curve assay of the V. parahaemolyticus isolates at different concentrations of O 2
Growth curves of the V. parahaemolyticus isolates at different concentrations of O 2 were measured as described previously (Tian et al. 2020) with minor modifications.Briefly, the V. parahaemolyticus isolates were individually inoculated in the 5-mL TSB (pH8.5, 3% NaCl), which was adjusted and equilibrated to different concentrations of O 2 (21%, 18%, 15%, 10%, and 5% O 2 ) using the nitrogen-blowing instrument (Wen Dong Chemical, Shanghai, China).The O 2 concentrations were monitored using Ltd,Hangzhou,China).Under the different O 2 conditions (21-5% O 2 ), V. parahaemolyticus isolate was individually inoculated into thirty glass culture tubes (15 × 1.6 cm), which were sealed with sealing film (Beideng Medical Co., Ltd., Jiangsu, China), and incubated with shaking at 180 r/min at 37 °C.Instead of a bioreactor, the simple continuous culture condition allowed to measure OD 600 values every 4 h for 40 h from three independent tubes using the Multimode Microplate Reader (BioTek Instruments, USA).Meanwhile, O 2 concentrations in air within the tubes were measured correspondingly.Low oxygen condition refers to oxygen partial pressure of 0-10%, which corresponds to a range of dissolved oxygen in water body at room temperature and pressure of 0-4.5 mg/L (Chen et al. 2007).

Biofilm formation assay
Biofilm formation of the V. parahaemolyticus isolates was determined using the crystal violet staining method as described in our previous report (Yang et al. 2020).Briefly, 1 mL/well of each V. parahaemolyticus culture (adjusted OD 600 = 0.4) was inoculated into 12-well bacterial culture plates (Shanghai Sangon Biological Engineeing Technology and Service Co., Ltd., Shanghai, China).The plates were statically incubated at the 21% O 2 , and 10% O 2 conditions, respectively.Biofilms formed at 12 h, 24 h, 36 h, 48 h, and 60 h at 37 °C were individually stained, washed, fixed, and measured as described previously (Yang et al. 2020).The 0.1 M phosphate buffer saline (PBS) (pH 7.2-7.4),and 0.25% crystal violet were purchased from the Sangon (Shanghai, China).

Genome sequencing, assembly, and annotation
The V. parahaemolyticus isolates were routinely incubated in the TSB (3% NaCl, pH 8.5) at 37 °C to the mid- LGP.The genomic DNA was prepared using TIANamp Bacteria DNA Kit (Tiangen Biochemical Technology Co Ltd., Beijing, China).Three separately produced DNA samples were used for each of the V. parahaemolyticus isolates.Only high quality genomic DNA samples (A 260 / 280 = 1.8-2.0)were subjected to genome sequencing (Xu et al. 2022a).Whole-genome sequencing was conducted by Shanghai Majorbio Bio-Pharm Technology Co., Ltd.(Shanghai, China) using the Illumina Hiseq × 10 (Illumina, San Diego, CA, USA) platform (Xu et al. 2022a).The insert size of PE150 (pair-end) sequencing was 400 bp.Lowquality sequence filtering; high-quality sequence assembly; annotation of coding sequences (CDSs), rRNA genes, and tRNA genes; and prediction of Clusters of Orthologous Groups (COG) of proteins were performed using the same software with default parameters as described in our recent report (Xu et al. 2022a).
The virulence factor database (http:// www.mgc.ac.cn/ VFs) and antibiotic resistance gene database (http:// arpca rd.Mcmas ter.ca) were used to detect virulence-and antibiotic resistance-related genes in the V. parahaemolyticus genomes, respectively.Genomic isolands (GIs), prophages, integrons (INs), insertion sequences (ISs), and clustered regularly interspaced palindromic repeats (CRISPR)-Cas systems were predicted using the same software with default parameters as described in our recent report (Xu et al. 2022a) The MGEs were ordered according to the numbers of the Scaffolds on which they were present (see the corresponding Supplementary Tables).

Phylogenetic tree assay
A total of seventy-eight V. parahaemolyticus isolates were subjected for a phylogenetic tree, of which complete genome sequences of seventy-three V. parahaemolyticus isolates were downloaded from the GenBank database, together with draft genome sequence of V. parahaemolyticus ATCC17802 (Table S1).Amino acid data sets of single-copy orthologs of the V. parahaemolyticus genomes, coupled with the four genomes determined in this study, were analyzed using OrthoFinder (version 2.2.6) software (Emms and Kelly 2019).The phylogenetic tree was constructed using the RAxML (version 8) software (Stamatakis 2014) with 1,000 bootstrap replications and a cut-off threshold of ≥ 50% bootstrap values.

Illumina RNA sequencing and analysis
The V. parahaemolyticus isolates were individually incubated in the TSB (3% NaCl, pH 8.5) under the 10% condition at 37 °C for 24 h.Total RNA was extracted using RNeasy Protect Bacteria Mini Kit, QIAGEN RNeasy Mini Kit.The DNA was removed from the extracted RNA samples using RNase-Free DNase Set (QIAGEN Biotech Co. Ltd., Frankfurt, Germany).The Illumina RNA-sequencing was conducted by Shanghai Majorbio Bio-pharm Technology Co. Ltd. (Shanghai, China) using Illumina HiSeq 2500 platform (Illumina, USA).Three separately prepared RNA samples were used for each of the V. parahaemolyticus isolates.The V. parahaemolyticus isolates grown under the 21% O 2 condition were individually used as controls.

Reverse transcription real time-quantitative PCR (RT-qPCR) assay
Reverse transcription was performed using Prime-ScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time) (QIAGEN Biotech Co. Ltd., Hilden, Germany).The TB Green ® Premix Ex Taq ™ II (TliRNaseH Plus) (Takara Biomedical Technology Co., Ltd., Beijing, China) was used for relative quantitative PCR with 16 s RNA as the internal reference gene.The RT-qPCR reaction was performed using the Fast Real-Time analyzer (Applied Biosystems, Foster City, California, USA).with the following protocol: 50 °C for 2 min; 95 °C pre-denaturation for 10 min; and 95 °C denaturation for 15 s, 60 °C annealing for 1 min, for 40 cycles.The relative expression levels of target gene and internal reference gene were calculated by 2 −ΔΔCt (Yang et al. 2020).The primers targeting the representative DEGs were designed (Table S2), and synthesized by the Sangon (Shanghai, China).

Statistical analysis
The experimental data were analyzed using the SPSS software (version 17.0, SPSS Inc., Chicago, IL, USA).
Differences between the means and changes in the samples were compared by one-way analysis of variance using the least-significant difference (LSD) method, with the level of significance set at p < 0.05.All tests were conducted in triplicate.

Growth profiles of the V. parahaemolyticus isolates at different salinity concentrations, pH, temperatures, and oxygen concentrations
V. parahaemolyticus is a halophilic bacterium that inhabits in marine and estuarine environments worldwide (Li et al. 2019).We wondered growth features of the V. parahaemolyticus isolates of aquatic animal origins.Thus, we determined their growth curves in the TSB (pH 8.5) under the 0.5-5% NaCl conditions at 37 °C.As shown in Fig. S1 (A-E), all the V. parahaemolyticus isolates grew poorly at 0.5% NaCl.However, the increased NaCl concentration (1%) significantly promoted the bacterial growth (p < 0.05).Although all the isolates grew exuberantly at 2-5% NaCl, the highest biomass was observed when grew at 3% NaCl, showing the maximum OD 600 values (0.94-1.12) at stationary growth phase (SGP).Exceptionally, the growth of V. parahaemolyticus B1-21 was significantly decreased at 5% NaCl as compared to that at 3% NaCl (p < 0.05) (Fig. S1, B), suggesting that V. parahaemolyticus B1-21 was less tolerant to the higher NaCl concentration.These results indicated that the V. parahaemolyticus isolates grew optimally in the TSB at 3% NaCl.
The pH of human stomach normally ranges pH 1-3, but can rise above 6.0 after food consumption (Sun et al. 2014).The acidic stomach condition challenges the bacterial pass through to the gastrointestinal tract where it colonizes and causes the diarrhea disease.Thus, we determined growth curves of the V. parahaemolyticus isolates in the TSB (3% NaCl) under the pH 6.0-8.5 conditions at 37 °C.As shown in Fig. S2 (A-E), the growth of all the isolates was inhibited under the acidic conditions (pH 6.0-6.5),whereas the neutral condition (pH 7.0) promoted the bacterial growth, and all the isolates grew vigorously at pH 7.5-8.5,showing the maximum biomass at pH 8.5 (OD 600 = 0.94-1.20)at SGP.These results indicated that the V. parahaemolyticus isolates of aquatic animal origins grew optimally at pH 8.5, 3% NaCl in the TSB.
Growth curves of the V. parahaemolyticus isolates in the TSB (3% NaCl, pH 8.5) were also determined at 25 °C and 37 °C, respectively, wherein V. parahaemolyticus experiences during its life cycle in the environment and in the host.As shown in Fig. S3 (A-E), all the isolates could grow more exuberantly at 37 °C than at 25 °C.Therefore, the V. parahaemolyticus isolates were incubated in the TSB (pH 8.5, 3% NaCl) at 37 °C in the further analyses in this study, to avoid any condition influence other than the oxygen concentrations.
Growth of the V. parahaemolyticus isolates at different oxygen concentrations (21-5% O 2 ) was examined; and the results are presented in Fig. 1 A-E.Under the 5-10% O 2 conditions, the growth of all the isolates in the TSB (3% NaCl, pH 8.5) at 37 °C was repressed, and the maximum OD 600 values ranged 0.40-0.52 at SGP.Under the 12% O 2 condition, all the isolates grew better than at 10% O 2 .Upon the increased O 2 concentrations (15-21%), the V. parahaemolyticus isolates grew faster correspondingly, but reaching the maximum biomass (OD 600 = 0.90-1.28)under the normal 21% O 2 condition.Additionally, we observed that the growth of V. parahaemolyticus ATCC17802 of the clinical origin was the slowest under the lower O 2 conditions among the test strains, moreover, it still showed the lower biomass under the 18% O 2 condition.

Effects of the hypoxic condition on biofilm formation of the V. parahaemolyticus isolates.
The ability of Vibrio to form biofilm attributes to their survival in the host and withstanding in different aquatic environments (Khan et al. 2020).Therefore, we asked whether and how the hypoxic condition (10% O 2 ) would affect biofilm formation of the V. parahaemolyticus isolates.The dynamic process of biofilm formation was monitored under the 10% O 2 condition when the isolates were incubated in the TSB (pH 8.5, 3% NaCl) at 37 °C for 60 h.As shown in Fig. 2A-E, the V. parahaemolyticus isolates formed biofilms at three different stages, including the development, maturation, and diffusion, similar to those under the normal 21% O 2 condition.However, different biofilm formation patterns were observed among the isolates.
Unexpectedly, under the 10% O 2 condition, the biofilm biomass generated by all the V. parahaemolyticus isolates was significantly increased at the development and maturation stages (0-36 h) as compared to those under the 21% O 2 condition (p < 0.05).The highest increase (2.70fold) was observed in the V. parahaemolyticus B8-26 treatment group at 36 h (p < 0.001).Of note, the 10% O 2 condition significantly enhanced the biofilm formation of V. parahaemolyticus ATCC17802 for 48 h (p < 0.05) (Fig. 2, E).These results demonstrated that the hypoxic condition can enhance the biofilm formation of all the test V. parahaemolyticus isolates.

Cell morphological structure changes of the V. parahaemolyticus isolates under the hypoxic condition
Cell surface structure of the V. parahaemolyticus isolates under the hypoxic condition was further observed by the SEM assay.As shown in Fig. 3A-E, unexpectedly, in remarkable contrast to the control groups under the 21% O 2 condition, whose cell surface structure was broken with obvious crumpling, rupture and cellular contents leakage after 24-h incubation, the V. parahaemolyticus isolates showed rod cells with slightly shrinkage on the cell surface after being treated with 10% O 2 for 24 h.Particularly, V. parahaemolyticus ATCC17802 cells in the control group were severely shrunk and deformed, whereas those in the hypoxia treatment group were rod-shaped and less destroyed in cell structure (Fig. 3,  E).These results indicated that the 10% O 2 condition repressed the growth of the V. parahaemolyticus isolates.The cellular metabolisms-requiring O 2 were consequently down-regulated, thereby leading to the slower growth under the hypoxic condition.
Additionally, based on the draft genome of V. parahaemolyticus ATCC17802 in the GenBank database (accession numbers NZ_CP014046.2) (Yang et al. 2015), we also identified several MGEs, including GIs (n = 2), and INs (n = 7) in this isolate.

GIs
GIs can shape bacterial genomes and affect bacterial fitness to the environment (Ghazali et al. 2021).In this study, 36 GIs were identified in the four V. parahaemolyticus genomes of aquatic animal origins, which contained 3-14 GIs with 5,572-38,679 bp, and carried 7-45 predicted genes with diverse biological functions (Fig. 4, Table S3).

Comparative transcriptome analysis of the V. parahaemolyticus isolates in response to the hypoxic condition
Based on the V. parahaemolyticus B8-26, B1-21, N2-5, L7-40 genomes determined in this study, coupled with the V. parahaemolyticus ATCC17802 genome, we further examined genome-wide gene expression changes triggered by the hypoxic condition.The isolates were incubated in the TSB under the 10% O 2 condition at 37 °C for 24 h, at which the cell morphological structure changes of the V. parahaemolyticus isolates was the most obvious, therefore, their transcriptomes were determined.The complete lists of DEGs in the V. parahaemolyticus isolates are available in the NCBI SRA database (https://  Approximately 65.89% (2,722/4,131) of V. parahaemolyticus B8-26 genes were expressed differently under the hypoxic condition as compared to the control group.Among these, 413 differently expressed genes (DEGs) showed higher transcription levels (fold change, FC ≥ 2.0), whereas 2,309 DEGs were down-regulated (FC ≤ 0.5).
Remarkably, all DEGs in the aminoacyl-tRNA biosynthesis (n = 25), PG biosynthesis (n = 17), citrate cycle (n = 8), and ubiquinone and other terpenoid-quinone biosynthesis (n = 17) were significantly repressed at the transcription levels in V. parahaemolyticus B8-26 under the hypoxic condition (0.015-fold to 0.497-fold) (p < 0.05).Aminoacyl-tRNA synthetases are essential for protein synthesis (Rubio Gomez and Ibba 2020).The expression of many such enzymes and proteins was repressed in V. parahaemolyticus B8-26.For instance, the DEG encoding an alanyl-tRNA editing protein (Vp_B8-26_03025) was strongly inhibited (0.080-fold), suggesting that the stringent response shut down the translation to avoid toxic generation of mistranslated/misfolded proteins (Aggarwal et al. 2021).As the major component of bacterial cell wall, the biosynthesis of PG is closely related to cell wall growth and turnover (Wamp et al. 2020).In this study, seventeen DEGs involved in the PG biosynthesis were inhibited under the hypoxic condition.For example, the DEG encoding a D-alanyl-D-alanine carboxypeptidase (Vp_B8-26_24445) was significantly down-regulated (0.448-fold), which is involved in the reconstruction of newly synthesized PG in Francisella (Spidlova et al. 2018).The citrate cycle (Krebs cycle) is a major aerobic pathway for the final stages of carbohydrate and fatty acid Fig. 6 The Volcano plot of differential gene expression (A), and the major changed metabolic pathways (B) in V. parahaemolyticus strains under the hypoxic condition (10% O 2 ).A: The X and Y axes represented changes of the up/down-regulated genes, and the corresponding significant differences, respectively.B The X and Y axes represented the major changed metabolic pathways in V. parahaemolyticus strains under the hypoxic condition, and the ratios of the number of DEGs in the metabolic pathways to the total number of DEGs in all metabolic pathways.A1 and B1, A2 and B2, A3 and B3, A4 and B4: V. parahaemolyticus B8-26, B1-21, N2-5, and L7-40 strains, respectively oxidation, which provides NADH to be used for oxidative phosphorylation and other metabolic reactions.In this study, eight DEGs in the citrate cycle were down-regulated as well.For instance, the DEG encoding a malate dehydrogenases (MDH) (Vp_B8-26_01555) was strongly down-regulated (0.022-fold).The MDH catalyzes the NAD/NADH-dependent interconversion of malate and oxaloacetate.The malate/aspartate of this reaction is involved in tricarboxylic acid cycle (Minárik et al. 2002).Additionally, among the seventeen down-regulated DEGs in the ubiquinone and other terpenoid-quinone biosynthesis, the DEG encoding a NAD(P)H quinone oxidoreductase (NQO) (Vp_B8-26_12250) was significantly repressed (0.172-fold), which is a two-electron reductase responsible for detoxification of quinones and bioactivation of certain quinones (Zhang et al. 2018).These results signified that the hypoxic condition likely inhibited protein translation, bacterial cell wall synthesis, and energy supply in V. parahaemolyticus B8-26.
Sulfur is also a requirement for both the host and colonizing bacteria (Kies et al. 2022).In this study, fourteen DEGs were significantly down-regulated in the sulfur metabolism in V. parahaemolyticus B8-26 under the hypoxic condition (0.010-fold to 6.247-fold) (p < 0.05).For example, the NrfD-like subunits are found in many diverse membrane complexes, which may participate in the metabolism of oxygen, nitrogen, sulfur, arsenate or hydrogen (Calisto and Pereira 2021).Nevertheless, in this study, the expression of NrfD (Vp_B8-26_10960) was strongly down-regulated (0.007-fold), suggesting the inhibited sulfur metabolism, thereby energy conversion in V. parahaemolyticus B8-26 under the hypoxic condition.
Of note, approximately 85 DEGs in the ABC transport were also significantly inhibited in V. parahaemolyticus B8-26 under the hypoxic condition (0.003-fold to 0.498fold) (p < 0.05).The ATP-dependent ABC transporter proteins facilitate the import and/or export of various substrates, including lipids, sugars, amino acids and peptides, ions, and drugs (Thurm et al. 2021).For example, the DEG encoding the ATP-binding cassette domaincontaining protein (Vp_B8-26_06880) was greatly downregulated (0.003-fold), which contains two domains capable of ATP hydrolysis in order to fuel protein function (Alqahtani et al. 2019).The down-regulation of the ABC transport suggested the inactive cellular transport function under the hypoxia, which likely led to harmful substance accumulate and retarded bacterial growth.
Taken, the comparative transcriptome data revealed fourteen significantly changed metabolic pathways in V. parahaemolyticus B8-26 under the hypoxic condition.Notably, the down-regulated protein translation and transport, ABC transporters, bacterial cell wall synthesis and energy conversion likely contributed to the retarded cell growth of V. parahaemolyticus B8-26 under the hypoxic condition.
Bacterial chemotaxis is a critical ability to search for the optimal environment to ensure the survival of bacterial species (Jeong 2021).Three DEGs in the bacterial chemotaxis were significantly repressed in V. parahaemolyticus B1-21 under the hypoxic condition (0.188-fold to 0.465-fold) (p < 0.05), including a chemotactic protein Chew (Vp_B1-21_11985), a flagellar motor switch protein (Vp_B1-21_12100), and a chemotactic response regulator protein-glutamate methylase (Vp_B1-21_12000).For example, the DEG encoding the CheW (Vp_ B1-21_11985), which is involved in the transmission of sensory signals from the chemoreceptors to the flagellar motors (Rosario et al. 1994), was significantly repressed (0.465-fold).Meanwhile, the DEG encoding the flagellar motor switch protein (Vp_B1-21_12100) was also highly repressed (0.188-fold).These results indicated the down-regulated bacterial chemotaxis under the hypoxic condition, thereby inhibiting bacterial migration to the environment conducive to survival.
Similar to V. parahaemolyticus B8-26, the ABC transporters, sulfur metabolism, and pyruvate metabolism were also significantly changed in V. parahaemolyticus B1-21 under the hypoxic condition.
Taken together, the comparative transcriptome data revealed eleven significantly changed metabolic pathways in V. parahaemolyticus B1-21 under the hypoxic condition.Specially, V. parahaemolyticus B1-21 reduced protein translation, but enhanced transport and utilization of carbohydrates in response to the hypoxic condition.
Similar to V. parahaemolyticus B1-21, the ABC transport, and alanine, aspartate and glutamate metabolism were significantly changed in V. parahaemolyticus N2-5 as well.
Taken, the comparative transcriptome analysis revealed eleven significantly changed metabolic pathways in V. parahaemolyticus N2-5 under the hypoxic condition, some of which were also altered in V. parahaemolyticus B1-21 and V. parahaemolyticus B8-26.Specially, the protein translation, citrate cycle, propionate metabolism, oxidative phosphorylation, carbon fixation pathways in prokaryotes, and valine, leucine and isoleucine degradation were all repressed in V. parahaemolyticus N2-5 for growth under the hypoxic condition.
In the glyoxylate and dicarboxylate metabolism, the 20 of 24 DEGs were significantly down-regulated (0.019-fold to 0.491-fold) in V. parahaemolyticus L7-40 under the hypoxic condition (p < 0.05).Of these, the DEG encoding an acetate-CoA ligase (Vp_L7-40_16020) was highly down-regulated (0.019-fold).Acetyl-CoA is a vitally important and versatile metabolite used for many cellular processes, and can also deal with stress such as low nutritional availability and hypoxia (Miller et al. 2021).
In addition, similar to V. parahaemolyticus B8-26, and V. parahaemolyticus N2-5, the TCS, pyruvate metabolism, as well as alanine, aspartate and glutamate metabolism were also significantly changed in V. parahaemolyticus L7-40 under the hypoxic condition.
Taken, the comparative transcriptome analysis revealed fifteen significantly changed metabolic pathways in V. parahaemolyticus L7-40, some of which were also identified in V. parahaemolyticus B8-26, N2-5, and B1-21 isolates under the hypoxic condition.Specifically, V. parahaemolyticus L7-40 inhibited the glyoxylate and dicarboxylate metabolism, but up-regulated the glycolysis/gluconeogenesis, and changed the nitrogen metabolism, and the arginine biosynthesis in response to the hypoxic condition.
In the fatty acid biosynthesis, all DEGs (n = 17) were significantly down-regulated (0.047-fold to 0.427-fold) in V. parahaemolyticus ATCC17802 (p < 0.05).Fatty acid biosynthesis plays a central role in building cell membrane, reserving cell energy, and production of precursors to second messenger molecules (Günenc et al. 2022).For instance, the DEG encoding a trans-2-enoyl-CoA reductase family protein (Vp_17802_21460) was strongly down-regulated (0.047-fold) in V. parahaemolyticus ATCC17802, which is involved in chain lengthening of fatty acids (Uchida et al. 2021).
In the pyrimidine metabolism, most DEGs (26/30) were significantly down-regulated (0.026-fold to 0.450fold) in V. parahaemolyticus ATCC17802 (p < 0.05).Pyrimidines are structural elements of a wide range of essential compounds in the synthesis of DNA, RNA, lipids, and carbohydrates (Garavito et al. 2015).For instance, the DEG encoding a thymidine kinase (TK, Vp_17802_05700) was strongly down-regulated (0.051fold) in V. parahaemolyticus ATCC17802.The TK is involved in the pyrimidine nucleotide recovery pathway, a cell-proliferation marker (Fanelli et al. 2021).Interestingly, among the four up-regulated DEGs in Fig. 7 The Volcano plot of differential gene expression (A), and the major changed metabolic pathways (B) in V. parahaemolyticus ATCC17802 under the hypoxic condition the pyrimidine metabolism, the DEG encoding a 5' deoxynucleotidase (Vp_17802_04570, 4.317-fold) was highly up-regulated in V. parahaemolyticus ATCC17802 (p < 0.05).This enzyme is involved in the metabolism of cladribine (2CdA), by mediating phosphorolysis (deactivation) of 2CdA to prevent its accumulation in the cell, which leads to cell death (Carlini et al. 2022).
Taken, the comparative transcriptome analysis revealed ten significantly changed metabolic pathways in V. parahaemolyticus ATCC17802 under the hypoxic condition.Although some of the altered pathways were also observed in V. parahaemolyticus B8-26, B1-21, N2-5, and L7-40 isolates, however, wherein more repressed DEGs were elicited in V. parahaemolyticus ATCC1780 by the condition, consistent with the phenotypes of this isolate in the above results.In particular, compared to the other four V. parahaemolyticus isolates, V. parahaemolyticus ATCC17802 also down-regulated some metabolic pathways, including the pyrimidine metabolism, fatty acid biosynthesis, lipopolysaccharide biosynthesis, purine metabolism, as well as arginine and proline metabolism to survival under the hypoxic condition.
Additionally, to confirm the transcriptome data, thirty representative DEGs were analyzed by the RT-qPCR assay, and the resulting data were generally correlated with those by the transcriptome analysis (Table S12).

Possible molecular mechanisms of V. parahaemolyticus in response to the hypoxic condition
The transcriptome-wide analyses revealed a number of DEGs involved in multiple pathways of biosynthesis, degradation, salvage, interconversion, and transport of the compounds in V. parahaemolyticus B8-26, B1-21, N2-5, L7-40 and ATCC17802 isolates of aquatic animal and human clinical origins, suggesting a complex molecular regulation network in the bacterium in response to the hypoxic condition (Fig. S7, Table S13).
Given that the V. parahaemolyticus isolates, being from different origins, harbored different genome features, it was reasonable that the hypoxic condition triggered different transcriptomic profiles among the isolates.The transcriptome data, coupled with the phenotype results in this study, demonstrated that compared to the other isolates, V. parahaemolyticus ATCC17802 downregulated and/or shut down all the ten changed metabolic pathways to reduce cell viability and maintain cell structure integrated under the hypoxic condition.These results provided a novel mechanism for the persistence of the toxic strain in the environment and in the host.
Overall, V. parahaemolyticus developed multiple molecular strategies to efficiently mitigate the cell damage and/or cytotoxicity caused by the hypoxia: (1) downregulated the oxidative phosphorylation, carbon fixation pathways, citrate cycle, pyruvate metabolism, and propionate metabolism to balance the redox state of the cell and the energy conversion; (2) down-regulated the fatty acid biosynthesis, and lipopolysaccharide biosynthesis to inhibit the cell wall synthesis, thereby the cell proliferation; (3) decreased amino acid transport, aminoacyl-tRNA biosynthesis, and protein production to retard the cell growth, thus prolonging the growth cycle and maintaining the cell structure integrated; and (4) conversely, enhanced the expression of condition-related proteins (such as GapA), structurally stabilizing factors (such as arginine), and efflux RND transporters to reduce the cell damage for growth under the unfavorable hypoxic condition.

Phylogenetic relatedness of the V. parahaemolyticus isolates
To address the phylogenetic relatedness of the V. parahaemolyticus isolates, we constructed a phylogenetic tree based on a total of 1,921 homologous amino acid sequences identified from the seventy-eight V. parahaemolyticus genomes, seventy-four of which were derived from the GenBank database (Table S1).This analysis revealed ten different phylogenetic groups, designated as Groups 1-10 (Fig. 8).
Taken, these results demonstrated the diversity of V. parahaemolyticus genomes of the environmental and clinical origins.

Discussion
Infectious diseases caused by pathogenic bacteria continue to be a global concern for public health, which results in millions of deaths worldwide each year (Bueno et al. 2020).The sea foodborne illness caused by V. parahaemolyticus has shown a significant upward trend in Fig. 8 The phylogenetic tree showing the relationship of the seventy-eight V. parahaemolyticus genomes.Draft genome sequences of the V. parahaemolyticus isolates determined in this study were marked with red dots in the tree recent years (Zhai et al. 2021).Climate change is driving ocean oxygen declines (Jaccard and Galbraith 2012).On the other hand, the low oxygen level in the human gastrointestinal tract also challenges the survival and infection of V. parahaemolyticus.Thus, in the present study, we for the first time investigated how V. parahaemolyticus isolates of aquatic animal and human clinical origins response to the hypoxic condition.
V. parahaemolyticus B8-26, B1-21, N2-5, and L7-40 strains were isolated from aquatic animals S. strictus, C. aurea, O. oratoria, and C. idellus, respectively (Su and Chen 2020).Multiple antibiotic and heavy metal resistance profiles were derived from the four V. parahaemolyticus isolates, suggested possible antibiotic and heavy metal exposure or pollution sources in the aquaculture environments (Su and Chen 2020;Xu et al. 2022a).Moreover, we observed that these isolates grew optimally in the TSB medium with 3% NaCl and pH 8.5 at 37 °C, consistent with the previous report (Xu et al. 2022a).
V. cholerae is a member of the Vibrio genus and can cause pandemic cholerae in humans (Ramamurthy et al. 2020).V. cholerae was able to generate energy and maintain its physiological functions in the absence of oxygen using alternative electron acceptors (AEAs) (Bueno et al. 2020).In this study, we determined growth curves of the V. parahaemolyticus isolates in the TSB equalibrated with different oxygen concentrations (21-5% O 2 ) at 37 °C.Unlike V. cholerae, our data showed that the lower oxygen concentrations (5-10%) greatly inhibited the growth of the tested isolates.However, no significant difference in the growth was observed between 5% O 2 and 10% O 2 , suggesting that 10% O 2 was the cut-off point for the growth of V. parahaemolyticus at the low oxygen concentrations.Additionally, we observed that oxygen concentrations in air within the culture tubes fluctuated down by 0-1% within 24 h, and by 0-2% from 24 to 48 h, indicating the stable hypoxic condition maintained in our culture system.
Biofilm is critical for V. parahaemolyticus persistence in aquatic environments and pathogenicity in the host (Yildiz and Visick 2009).In this study, the results showed that the biofilm biomass generated by all the tested V. parahaemolyticus isolates under the 10% O 2 condition was significantly increased at the development and maturation stages (0-36 h) (p < 0.05), as compared to those under the normal 21% O 2 condition.It has been reported that biofilm formation of P. aeruginosa, S. aureus, and E. coli induced the production of hypoxia-related enzymes (Crespo et al.2016;Jo et al. 2017;Létoffé et al. 2017).Our results, coupled with these previous studies, suggested that biofilm formation was likely beneficial to the pathogenic bacteria to survival under lower oxygen niches.
Unexpectedly, we found that the pathogenic V. parahaemolyticus ATCC17802 was capable of maintaining more rod-shaped cells with no surface shrinkage than the other tested isolates, demonstrating that V. parahaemolyticus ATCC17802 was the most tolerant to the hypoxia.This may attribute to its survial in the host gastrointestinal tract with low oxygen level.
Draft genomes of the four V. parahaemolyticus isolates from aquatic animals were determined (4.91-5.35Mb), with no plasmid sequenced.Clean single peaks with a typical Poisson distribution in the frequency of observed unique 17-mers within the sequencing data indicated less repetitive DNA in the genomes, consistent with the fewer ISs identified in the V. parahaemolyticus genomes.Among the 4,458-4,924 predicted genes, 735 to 1,046 encoded unknown proteins.A large number of unknown genes have also been reported in seven V. parahaemolyticus genomes of aquatic animal origins in the previous report (Xu et al. 2022a).
In this study, a few antibiotic resistance-related genes, which are responsible for the fluoroquinolone (crp), TET (Tet ( 35)), and beta-lactam (bla CARB-18 ) resistance, were identified in the V. parahaemolyticus genomes, consistent with the observed phenotypes.Moreover, several heavy metal tolerance-related genes (e.g., cusARS, dsbABC, smtA, zntA) were found in these V. parahaemolyticus isolates as well.For example, the smtA and zntA genes, which are involved in bacterial tolerance to Zn, as well as Hg and Zn, respectively (Chen et al. 2021;Lee et al. 2001;Pal et al. 2014), were present in the V. parahaemolyticus B8-26, B1-21, L7-40, and ATCC17802 genomes.V. parahaemolyticus B8-26 also carried the cusARS and dsbABC gene clusters, which are involved in bacterial tolerance to Cu, as well as Cd, Zn, Hg and Cu, respectively.The antibiotic resistance-related (such as acrB, catB, hns, qnr, soxR), and heavy metal tolerance-related genes (e.g., copA, cusA, cusR, cusS, zntA) have also been reported in the V. parahaemolyticus genomes derived from six species of aquatic animals (Xu et al. 2022a).
The transcriptomic analyses revealed distinct response strategies to hypoxia by Vibrio parahaemolyticus isolates of clinical and aquatic animal origins.Different degrees of impact on the growth of V. parahaemolyticus isolates were observed, which were triggered by the hypoxic condition.For instance, all DEGs were significantly downregulated in the PG biosynthesis, as well as ubiquinone and other terpenoid-quinone biosynthesis in V. parahaemolyticus B8-26 (p < 0.05).In response to the hypoxic condition, V. parahaemolyticus L7-40 inhibited the glyoxylate and dicarboxylate metabolism, but up-regulated the glycolysis/gluconeogenesis, and changed the nitrogen metabolism, and the arginine biosynthesis (p < 0.05).The transcriptome data, coupled with the phenotype results in this study, demonstrated that V. parahaemolyticus ATCC17802 was the most tolerant to the hypoxia among the isolates.This isolate down-regulated and/or shut down all the ten changed metabolic pathways to reduce cell viability and maintain cell structure under the hypoxic condition.In particular, the pyrimidine metabolism, fatty acid biosynthesis, LPS biosynthesis, purine metabolism, as well as arginine and proline metabolism were all repressed in V. parahaemolyticus ATCC17802.
Fatty acids are essential for biosynthesis of phospholipids and also provide ATP, signaling molecules, and NADPH through β-oxidation (Chen et al. 2022).For example, the DEG encoding a β-ketoacyl-ACP synthase (Vp_17802_11840) in the fatty acid biosynthesis was significantly down-regulated (0.200-fold) in V. parahaemolyticus ATCC17802 (p < 0.05), which play a vital role in cell wall synthesis, biofilm formation and also pathogenesis (Hu et al. 2020).These results provided a novel mechanism for the persistence of the toxic strain in the environment and in the host.
Taken, V. parahaemolyticus developed multiple molecular strategies to balance the redox state of the cell and the energy conversion, to inhibit the cell wall synthesis, thereby the cell proliferation, to retard the cell growth, thus prolonging the growth cycle and maintaining the cell structure integrated, to enhance the expression of stressrelated proteins, structurally stabilizing factors, and efflux RND transporters, in order to efficiently mitigate the cell damage and/or cytotoxicity caused by the hypoxia.

Conclusions
In this study, we investigated, for the first time, the growth of V. parahaemolyticus B8-26, B1-21, N2-5, L7-40, and ATCC17802 isolates under the hypoxic condition.The former four were isolated from S. stritus, C. aurea, O. oratoria, and C. idellus, respectively, while V. parahaemolyticus ATCC17802 was of human clinical origin.These isolates harbored multiple antibiotic and heavy metal resistance phenotypes, and grew optimally in the TSB (3% NaCl, pH 8.5) at 37 °C.
V. parahaemolyticus B8-26, B1-21, N2-5, L7-40, and ATCC17802 isolates had an oxygen concentrationdependent growth mode, and the 10% O 2 condition strongly inhibited the growth of the isolates, when incubated in TSB medium at 37 °C.Unexpectedly, in marked contrast to the normal 21% O 2 condition, the V. parahaemolyticus isolates enhanced biofilm formation under the hypoxic condition for 24 h.
Genome-wide gene expression changes triggered by the hypoxic condition were further examined.Comparative transcriptomic analysis unveiled multiple significantly changed metabolic pathways in the isolates under the 10% O 2 condition for 24 h.V. parahaemolyticus developed multiple molecular strategies to efficiently mitigate the cell damage and/or cytotoxicity caused by the hypoxia.
In addition, V. parahaemolyticus ATCC17802 (serotype: O1:K1) of the clinical origin was the most tolerant to the hypoxia among the test isolates, and down-regulated and/or shut down ten metabolic pathways to reduce cell viability and prolong growth under the stress.
Overall, the results of this study fill prior gaps in V. parahaemolyticus response to the hypoxic condition, and provide a novel mechanism for the toxic V. parahaemolyticus strain to persist in the environment and in the host.This work was financially supported by Shanghai Municipal Science and Technology Commission, grant number 17050502200, and National Natural Science Foundation of China, grant number 31671946.

Availability of data and materials
Draft genome sequences of the four V. parahaemolyticus isolates of aquatic animal origins have been deposited in the GenBank database under the accession numbers: JAODUR000000000, JAOPTY000000000, JAODVT000000000, and JAODVU000000000.A complete list of the DEGs is available in the NCBI SRA database (https:// www.ncbi.nlm.nih.gov/) under the accession number PRJNA906699 and PRJNA767551.

Fig. 4
Fig. 4 Gene organizations of the GIs identified in the V. parahaemolyticus genomes.Different colors referred to COG functional classification and genes with unknown function were in grey

Fig. 5
Fig. 5 The structure diagram of the prophages identified in the V. parahaemolyticus genomes

Table 1
The phenotype features of the V. parahaemolyticus isolates used in this study

Table 2
Genome features of the V. parahaemolyticus isolates used in this study contained a truncated version (29,468 bp) of Enterobac-teria_phage_Mu (36,717 bp, NCBI accession number: NC_000929), carrying 47 genes encoding 32 unknown proteins, which suggested possible transmission of the Enterobacteria_phage_Mu between the Vibrio and Enterobacteria genera pathogens.

Table 3
The antibiotic and heavy metal resistance-related genes identified in the V. parahaemolyticus genomes