Objective:To investigate the contamination and distribution of Vibrio parahaemolyticus and Vibrio alginolyticus in seafood in Haikou City.Methods:Three types of seafood sold in Haikou from 2020 to 2022 were collected,...Objective:To investigate the contamination and distribution of Vibrio parahaemolyticus and Vibrio alginolyticus in seafood in Haikou City.Methods:Three types of seafood sold in Haikou from 2020 to 2022 were collected,Vibrio parahaemolyticus and Vibrio alginolyticus were detected according to the National Food Safety Standard Food Microbiological Examination of Vibrio parahaemolyticus(GB 4789.7-2013),and they were identified by real-time fluorescence PCR.The detection of Vibrio parahaemolyticus and Vibrio alginolyticus in different kinds of seafood,different years and different quarters was analyzed.Results:A total of 119 seafood samples were collected.Among them,24 samples were positive with Vibrio parahaemolyticus,with a positive rate of 20.1%;46 samples were positive with Vibrio alginolyticus,with a positive rate of 38.7%.Among various types of seafood,shrimp have the highest positivity rate for Vibrio parahaemolyticus at 50%,while shellfish have the highest positivity rate for Vibrio alginolyticus at 48%.Comparing between monitoring years,the positive rate of Vibrio alginolyticus was the highest in 2021(76.7%),while the positive rate of Vibrio parahaemolyticus was the highest in 2022(25%).Comparing between different quarters,the positivity rate for Vibrio alginolyticus was found to be highest in the second quarter at 80%,while the positivity rate for Vibrio parahaemolyticus was highest in the fourth quarter at 33.3%.There were statistically significant differences(P<0.05)in the positivity rate for Vibrio alginolyticus in different years and quarters,as well as in the positivity rate for Vibrio parahaemolyticus in different types and quarters.Conclusion:Vibrio parahaemolyticus and Vibrio alginolyticus were found in seafood products in Haikou City from 2020 to 2022.It is recommended that relevant departments strengthen supervision to ensure the safety of seafood products consumed by the public.展开更多
[Objectives]To study the effects of microplastics on antibiotic resistance genes and virulence genes of Vibrio alginolyticus,so as to provide a certain reference for controlling marine pollution,curbing the spread of ...[Objectives]To study the effects of microplastics on antibiotic resistance genes and virulence genes of Vibrio alginolyticus,so as to provide a certain reference for controlling marine pollution,curbing the spread of environmental antibiotic resistance genes and virulence genes,formulating environmental policies,and maintaining food safety.[Methods]After adding V.alginolyticus into the artificial seawater,they were divided into three groups,namely blank control group(BLK),polyvinyl chloride microplastic group(PVC group)and polyvinyl alcohol microplastic group(PVA group).Aerated culture experiments were carried out,and the effects of microplastics on the expression of resistance genes and virulence genes of V.alginolyticus were studied by PCR and qPCR methods.[Results]The presence of microplastics significantly changed the resistance gene structure of V.alginolyticus.Compared with the control group,the cfxA and cfr resistance genes were detected in the microplastic group.However,only PVC group detected blaZ resistance gene,and only PVA group did not detect aaC resistance gene.In addition,compared with the control group,the expressions of virulence genes in the microplastic group were all down-regulated(P<0.01).[Conclusions]This study provides some reference for curbing the spread of environmental antibiotic resistance genes and virulence genes,formulating environmental policies,and maintaining food safety,but the specific mechanisms of drug resistance and virulence need further research.展开更多
[Objectives]To amplify the h-ns gene of Vibrio alginolyticus and analyze it by bioinformatics.[Methods]According to the h-ns gene sequence of V.alginolyticus HY9901,a pair of specific primers were designed and amplifi...[Objectives]To amplify the h-ns gene of Vibrio alginolyticus and analyze it by bioinformatics.[Methods]According to the h-ns gene sequence of V.alginolyticus HY9901,a pair of specific primers were designed and amplified by PCR.[Results]The h-ns gene was 408 bp in length and 135 amino acids were encoded.The predicted theoretical protein molecular weight was about 14.98 kD,and the isoelectric point was 4.99.Protein subcellular localization,SignalP 5.0,TMHMM Server 2.0 and SoftBerry-Psite predictions showed that H-NS was located outside the cell membrane,and the protein was unstable and hydrophobic.There was no signal peptide cleavage site,no transmembrane region and no KEGG metabolic pathway.The amino acid sequence contained three phosphorylation sites,one N-terminal myristoylation site and three microsomal C-terminal target signal sites.Using MEGA 5.0,H-NS phylogenetic tree was constructed by ortho-connection method.The results showed that H-NS of V.alginolyticus was closer to H-NS of Vibrio diabolicus.Using SWISS-MODEL,the three-dimensional structure model of H-NS subunit was simulated,which was similar to the crystal structure of Salmonella typhimurium H-NS1-83.[Conclusions]This study lays a foundation for exploring the regulation mechanism of V.alginolyticus H-NS protein on bacterial virulence in the future.展开更多
[Objectives]The paper was to clone and analyze bioinformatics of ndk gene from Vibrio alginolyticus.[Methods]A pair of specific primers was designed based on the ndk gene sequence of V.alginolyticus HY9901.The full le...[Objectives]The paper was to clone and analyze bioinformatics of ndk gene from Vibrio alginolyticus.[Methods]A pair of specific primers was designed based on the ndk gene sequence of V.alginolyticus HY9901.The full length of ndk gene was amplified by PCR and bioinformatics analysis was performed.MEGA 5.0 software was used to construct NDK phylogenetic tree by neighbor-joining method.SWISS-MODEL program was used to obtain the three-dimensional structural model of single subunit from NDK protein.[Results]The ndk gene,molecular structural formula C702H1094N192O214S7,was 426 bp in total,encoding 141 amino acids,with the theoretical molecular weight of 15.87199 kD and the theoretical pI value of 5.13.The prediction results of protein subcellular localization,SignalP 5.0,TMHMM Server 2.0 and SoftBerry-Psite showed that NDK mainly existed in the cytoplasm,and the protein was unstable and hydrophobic.There was neither signal peptide cleavage site,nor transmembrane region and KEGG metabolic pathway.The amino acid sequence had two protein kinase C phosphorylation sites,a casein kinaseⅡphosphorylation site,a N-myristoylation site,three microbody C-terminal target signal sites,and a nucleoside diphosphate kinase active site.Homology analysis showed that the NDK of V.alginolyticus had high homology with that of V.diabolicus,with a similarity of 98.58%.Analysis of the structural functional domain revealed that the protein had one NDK structural functional domain.The prediction results of secondary structure showed that theα-helix,random coil,β-sheet and extended strand accounted for 53.19%,28.37%,7.09%and 11.35%,respectively.Analysis of NDK protein via STRING database demonstrated that the proteins interacting with NDK protein were NrdA,NrdB,GmK,CmK,TmK,PyrG,PyrH,RelA,FolE and SpoT.[Conclusions]The study plays a positive role in the prevention and control of vibriosis and the improvement of the current aquaculture environment.展开更多
[Objectives]To clone and analyze the vscB gene of Vibrio alginolyticus HY9901 by bioinformatics.[Methods]A pair of specific primers were designed according to the vscB gene sequence of Vibrio alginolyticus HY9901.The ...[Objectives]To clone and analyze the vscB gene of Vibrio alginolyticus HY9901 by bioinformatics.[Methods]A pair of specific primers were designed according to the vscB gene sequence of Vibrio alginolyticus HY9901.The full length of the primers was cloned by PCR and analyzed by bioinformatics.[Results]The vscB gene was 429 bp long,encoding 142 amino acids,with a theoretical molecular weight of 16.4 kDa and a pI value of 5.48.Amino acid sequence analysis of VscB showed that VscB was not a secretory protein,without signal peptide and transmembrane region,and there were protein kinase C phosphorylation site and casein kinase II phosphorylation site in the sequence.Homologous comparison of amino acid sequences showed that VscB of V.alginolyticus had the highest protein similarity with Vibrio Parahaemolyticus,reaching 91%.Phylogenetic tree analysis showed that the corresponding proteins of V.alginolyticus VscB,Vibrio Parahaemolyticus and Vibrio diabolicus were clustered in the same subfamily.Functional domain analysis showed that it had CesT family domain.Tertiary structure prediction showed that there were 3α-helices and 5β-turns in VscB protein.[Conclusions]This study provided a theoretical basis for further study on the function of chaperone of V.alginolyticus.展开更多
[Objectives]The paper was to clone and analyze bioinformatics of vscN gene from Vibrio alginolyticus.[Methods]A pair of specific primers was designed based on the vscN gene sequence of V.alginolyticus HY9901.The full ...[Objectives]The paper was to clone and analyze bioinformatics of vscN gene from Vibrio alginolyticus.[Methods]A pair of specific primers was designed based on the vscN gene sequence of V.alginolyticus HY9901.The full length of vscN gene was amplified by PCR and bioinformatics analysis was performed.[Results]The vscN gene was 1323 bp in total,encoding 440 amino acids,with the theoretical molecular weight of 47.86 kD and the theoretical pI value of 5.89.The online prediction showed that there was no signal peptide and no transmembrane region in VscN.The amino acid sequence had 10 N-myristoylation sites,8 phosphorylation sites(2 protein kinase C phosphorylation sites,6 casein kinase II phosphorylation sites),1 amidation site,11 microbody C-terminal target signal sites,1 ATP/GTP binding site motif A(P ring),and 1 ATPaseαandβsubunit specific site.Homology analysis showed that the VscN protein of V.alginolyticus had high homology with that of V.antiquarius,with a similarity of 95.14%.Phylogenetic tree analysis showed that the VscN of V.alginolyticus was clustered into the same subgroup as that of V.diabolicus and V.antiquarius.Functional domain analysis of VscN protein showed that it had Pfam and AAA domains,and involved in the regulation of bacterial virulence.The three-dimensional structure model of VscN simulated by SWISS-MODEL software was similar to the structure of flagellate-specific ATPase FliH-FliI complex.[Conclusions]The results lay a foundation for further study on the regulatory mechanism of VscN protein on bacterial virulence.展开更多
[Objectives]The purpose was to investigate the function and mechanism of exsA gene in type III secretion system of Vibrio alginolyticus.[Methods]The full length of exsA was cloned using molecular biology techniques to...[Objectives]The purpose was to investigate the function and mechanism of exsA gene in type III secretion system of Vibrio alginolyticus.[Methods]The full length of exsA was cloned using molecular biology techniques to analyze its biological information.Fluorescence quantitative PCR technology was used to analyze the expression of exsA after different media stress.[Results]The exsA gene contains an open reading frame(ORF)of 861 bp,encoding 286 amino acids.The physico-chemical analysis shows that the molecular structural formula is C1442H2267N393O441S12,the theoretical molecular weight is 32.549 kD,the theoretical pI value is 6.0,and the protein is non-hydrophilic and unstable.The gene does not contain a transmembrane region,and there is no obvious signal peptide.The prediction result of protein subcellular localization shows that the protein is inside the cell.The deduced amino acid sequence and constructed phylogenetic tree show that V.alginolyticus has a close relationship with Vibrio antiquarius.The qPCR results show that the expression level of exsA in different media is different,highest in TSB medium containing bile salts,followed by DMEM medium,and lowest in ordinary TSB medium.[Conclusions]The gene sequence,molecular structure and isoelectric point of exsA,as well as its expression in three different media were obtained.展开更多
The two-component regulatory system plays an important role in the growth and virulence of bacteria.In this study,the phosphate transcriptional regulatory protein (KdpE) gene of Vibrio alginolyticus strain HY9901 was ...The two-component regulatory system plays an important role in the growth and virulence of bacteria.In this study,the phosphate transcriptional regulatory protein (KdpE) gene of Vibrio alginolyticus strain HY9901 was cloned.Sequence analysis revealed that the length of kdp E gene is 732 bp and encodes a putative protein of 243 amino acids.The predicted molecular weight (MW) of Kdp E was 27.66 k D with an estimated p I of 5.01.Using SignalP 4.0 and TMHMM Server 2.0 software,it was predicted that the Kdp E protein was located in cytoplasm.It did not contain a signal peptide or a transmembranous region.A phylogenetic tree was constructed by MEGA 5.0 software,indicating that the Kdp E of V.alginolyticus had high genetic relationship with Vibrio campbellii and Vibrio parahaemolyticus.Using SWISS-MODEL work-space,the three-dimensional structures of conserved domain REC in the Kdp E was determined.These results can provide a basis for further studies on the two-component regulatory systems of V.alginolyticus.展开更多
In order to enrich the research about type Ⅲ secretion system(T3SS) injectisome of Vibrio alginolyticus,vscX gene was cloned from V.alginolyticus strain HY9901 for bioinformatics analysis and expression analysis.Spec...In order to enrich the research about type Ⅲ secretion system(T3SS) injectisome of Vibrio alginolyticus,vscX gene was cloned from V.alginolyticus strain HY9901 for bioinformatics analysis and expression analysis.Specific primers were designed according to the full-length genome sequence of V.alginolyticus in GenBank.vscX gene(GenBank accession number:FR780679) contained a 378 bp open reading frame(ORF),encoding a putative protein of 125 amino acids.The theoretical molecular weight was 14.209 2 kD and theoretical pI was 5.75.By using Signal 4.1 Server and TMHMM Server 2.0,it was predicted that VscX protein had no transmembrane domain or signal peptide.The results of prediction using SoftBerry-Psite software showed that VscX protein contained three casein kinase Ⅱ phosphorylation sites,one N-myristoylation site and three C-terminal targeting signal sites.Subcellular localization revealed that VscX might be located in cytoplasm with the possibility of 56.5%.According to SMART prediction,VscX had one Pfam(1-125 aa) domain.Phylogenetic analysis revealed that VscX from V.alginolyticus and VscX from V.parahemolyticus were clustered into the same group.Network interaction analysis showed that vscX was adjacent to vscY,vopB and sycN.By real-time fluorescent quantitative PGR technique and 2^(-△△Ct) method,the differences in expression levels of VscX mRNA in V.alginolyticus strain HY9901,T3 SS deletion strain AvscO and complementary strain C-vscO at different growth stages were analyzed.The results showed that the expression levels of VscX mRNA in V.alginolyticus strain HY9901 and C-vscO were significantly up-regulated at stable growth stage(P<0.01);the expression levels of VscX mRNA in deletion strain AvscO were significantly up-regulated at late growth stage(P <0.01).This study provided the basis for revealing the transport mechanism of T3 SS injectisome of V.alginolyticus.展开更多
[Objective]To clone araC gene of Vibrio alginolyticus HY9901 strain,and analyze bioinformatics.[Methods]the whole genome sequence of Vibrio alginolyticus on GenBank was used to design specific primers.According to the...[Objective]To clone araC gene of Vibrio alginolyticus HY9901 strain,and analyze bioinformatics.[Methods]the whole genome sequence of Vibrio alginolyticus on GenBank was used to design specific primers.According to the principle of PCR amplification sequence,the target gene araC was amplified,and then the sequence was further analyzed by bioinformatics method to establish the phylogenetic tree of araC gene and its corresponding subunit three-dimensional structure model.[Results]Sequence analysis revealed araC gene is 711 bp and encodes a putative protein of 236 amino acids.The predicted molecular mass of AraC was 26.92 ku.Using Signal P 4.0 and TMHMM Server 2.0 software for analysis,it was predicted that the AraC protein did not contain a signal peptide or a transmembranous region.The AraC protein had two cAMP and cGMP dependent protein kinase phosphorylation site,five protein kinase C phosphorylation sites,three casein kinase II phosphorylation sites,one prenyl group binding site(CAAX box)and five microbodies C-terminal targeting signal.The predicted results of protein subcellular localization showed that AraC was located in the mitochondria,nucleus and cytoplasm.Its protein is unstable and hydrophilic.The AraC protein is a transcriptional regulatory protein which belongs to HTH_18 superfamily.According to the prediction,secondary structure:a-helix(Alpha helix)accounted for 52.12%,random coil(31.78%),extended strand(11.02%),b-fold(Beta turn)accounted for 5.08%.V.alginolyticus,Vibrio parahaemolyticus and Vibrio palustris were clustered together,which implies that the genetic relationship between these three species was the closest.展开更多
[Objectives]To clone the pepck gene of Vibrio alginolyticus strain HY9901 and analyze its sequence by bioinformatics.[Methods]According to the complete gene sequence of V.alginolyticus on GenBank,specific primers were...[Objectives]To clone the pepck gene of Vibrio alginolyticus strain HY9901 and analyze its sequence by bioinformatics.[Methods]According to the complete gene sequence of V.alginolyticus on GenBank,specific primers were designed to amplify the target gene pepck by PCR.The sequence of the pepck gene was analyzed using bioinformatics.The phylogenic tree of pepck gene and the corresponding single-subunit three-dimensional structure were constructed.[Results]The pepck gene of V.alginolyticus strain HY9901 has a full length of 1629 bp,with theoretical molecular weight of 60.12 kD.The prediction results show that there is no signal peptide or transmembrane region at the N-terminus of the sequence,the amino acid sequence contains 11 phosphorylation sites of casein kinase II.The prediction results of protein subcellular localization indicate that PEPEK protein is localized in the cytoplasm.The protein is stable and hydrophobic.The tertiary structure of the PEPCK protein of V.alginolyticus is similar to that of Vibrio parahaemolyticus.It is predicted that PEPCK has a major functional domain PEPCK_ATP.In the secondary structure,alpha helix,random coil,and extended strand accounted for 21.96%,52.03%and 26.01%,respectively.The PEPCK homology between V.alginolyticus and Vibrio diabolicus is as high as 99%.[Conclusions]This study lays the foundation for further understanding the function of pepck gene in V.alginolyticus.展开更多
[Objectives]The crp gene in Vibrio alginolyticus was studied.[Methods]The crp gene of V.alginolyticus HY9901 was cloned and analyzed by bioinformatics.[Results]The ORF of the crp gene is 633 bp long and the predicted ...[Objectives]The crp gene in Vibrio alginolyticus was studied.[Methods]The crp gene of V.alginolyticus HY9901 was cloned and analyzed by bioinformatics.[Results]The ORF of the crp gene is 633 bp long and the predicted amino acid sequence encompasses 210 amino acid residues.The physicochemical property analysis indicated that the chemical formula of CRP is C_(1051)H_(1704)N_(290)O_(308)S_(10) with a molecular weight of 23.6514 kDa,and its theoretical pI is 7.74.Besides,the protein is stable and hydrophilic.The protein had three protein kinase C phosphorylation site,three casein kinase II phosphorylation site,one N-terminal myristoylation site.The BLAST analysis on the sequence revealed high homology with CRPs in other Vibrio species,and particularly the sequence shares about a homology of 99.52%with the CRP in V.parahaemolyticus.The SWISS-MODEL software simulated the subunit tertiary structural model of the CRP,and the similarity with template 3hif.1.A was 95.71%.[Conclusions]This study provides a reference for the search for efficient protective antigens against vibrosis.展开更多
[Objectives]To clone and analyze the TpiA gene of Vibrio alginolyticus HY9901.[Methods]According to the TpiA gene sequence of V.alginolyticus,a pair of specific primers was designed,and its full length was amplified b...[Objectives]To clone and analyze the TpiA gene of Vibrio alginolyticus HY9901.[Methods]According to the TpiA gene sequence of V.alginolyticus,a pair of specific primers was designed,and its full length was amplified by PCR.[Results]The full length of TpiA gene is 771 bp,encoding 256 amino acid residues in total,and the NCBI accession number is OM906798.According to the deduced amino acid sequence,its molecular weight was predicted to be about 26.97548 kDa,and its isoelectric point was 4.78.The amino acid sequence of the N-terminal signal peptide structure was predicted,and it was found that there was no obvious signal peptide cleavage site,no signal peptide,and no transmembrane region;the amino acid sequence contained 3 N-glycosylation sites,4 protein kinase C phosphorylation sites,2 casein kinase II phosphorylation sites,6 N-myristoylation sites,7 microbody C-terminal target signal site,and 1 triose phosphate isomerase active site.The prediction results of protein subcellular localization showed that TpiA may be located in mitochondria or cytoplasm,with probability of 39.1%and 34.8%,respectively.The amino acid sequence of the TpiA gene of V.alginolyticus shared 98.83%-99.61%homology with other Vibrio species,and it was clustered into the same subfamily with Vibrio parahaemolyticus and had a close relationship.In the secondary structure prediction,the proportions ofα-helix,random coil and extended chain were 44.53%,41.41%and 14.06%,respectively,and the similarity of its tertiary structure model to template 1aw1.1.A was 85.16%.[Conclusions]This study is intended to provide a basis for further research on the role of TpiA gene in the type III secretion system and related research on antibiotic resistance.展开更多
[Objectives]To amplify the DNA-binding response regulator PhoP in Vibrio alginolyticus and analyze its sequence characteristics and subunit structure.[Methods]According to the sequence of the DNA-binding response regu...[Objectives]To amplify the DNA-binding response regulator PhoP in Vibrio alginolyticus and analyze its sequence characteristics and subunit structure.[Methods]According to the sequence of the DNA-binding response regulator PhoP in V.alginolyticus,a pair of specific primers was designed for PCR amplification,and the bioinformatics of the sequence amplified was analyzed.Using MEGA 5.0 software,the phoP phylogenetic tree was constructed by the neighbor-joining method.Using SWISS-MODEL software,the three-dimensional structural model of the PhoP subunit was simulated.[Results]The full-length phoP gene was 732 bp,encoding a total of 243 amino acids.The predicted theoretical molecular weight of the protein is about 27.67 kD,and the isoelectric point is 5.09.The prediction results of protein subcellular localization,SignalP 4.0,TMHMM Server 2.0 and SoftBerry-Psite show that PhoP is located in the cytoplasm,and is stable and hydrophobic;there is a signal peptide cleavage site between amino acids 29 and 30,and there is no transmembrane region.The amino acid sequence contains one Asn-glycosylation site,one protein kinase C phosphorylation site,seven casein kinase II phosphorylation sites,one tyrosine kinase phosphorylation site,three myristoylation sites,and seven C-terminal microbody targeting signal sites.The PhoP of V.alginolyticus has high homology with that of Vibrio campbellii.The PhoP subunit of V.alginolyticus has similar configuration to the single-subunit RegX3 protein of Mycobacterium tuberculosis.[Conclusions]This study has a positive effect on the prevention and control of vibriosis and the improvement of the current aquatic economic animal breeding environment.展开更多
基金Major Science and Technology Project of Hainan Province(No.ZDKJ202003)。
文摘Objective:To investigate the contamination and distribution of Vibrio parahaemolyticus and Vibrio alginolyticus in seafood in Haikou City.Methods:Three types of seafood sold in Haikou from 2020 to 2022 were collected,Vibrio parahaemolyticus and Vibrio alginolyticus were detected according to the National Food Safety Standard Food Microbiological Examination of Vibrio parahaemolyticus(GB 4789.7-2013),and they were identified by real-time fluorescence PCR.The detection of Vibrio parahaemolyticus and Vibrio alginolyticus in different kinds of seafood,different years and different quarters was analyzed.Results:A total of 119 seafood samples were collected.Among them,24 samples were positive with Vibrio parahaemolyticus,with a positive rate of 20.1%;46 samples were positive with Vibrio alginolyticus,with a positive rate of 38.7%.Among various types of seafood,shrimp have the highest positivity rate for Vibrio parahaemolyticus at 50%,while shellfish have the highest positivity rate for Vibrio alginolyticus at 48%.Comparing between monitoring years,the positive rate of Vibrio alginolyticus was the highest in 2021(76.7%),while the positive rate of Vibrio parahaemolyticus was the highest in 2022(25%).Comparing between different quarters,the positivity rate for Vibrio alginolyticus was found to be highest in the second quarter at 80%,while the positivity rate for Vibrio parahaemolyticus was highest in the fourth quarter at 33.3%.There were statistically significant differences(P<0.05)in the positivity rate for Vibrio alginolyticus in different years and quarters,as well as in the positivity rate for Vibrio parahaemolyticus in different types and quarters.Conclusion:Vibrio parahaemolyticus and Vibrio alginolyticus were found in seafood products in Haikou City from 2020 to 2022.It is recommended that relevant departments strengthen supervision to ensure the safety of seafood products consumed by the public.
基金Supported by Outstanding Graduate Entering Laboratory Project of College of Fisheries,Guangdong Ocean UniversitySpecial Fund for Science and Technology Innovation Strategy of Guangdong Province(Undergraduate Science and Technology Innovation Cultivation)(pdjh2021b0239)+3 种基金National Natural Science Foundation of China(32073015)Natural Science Foundation of Guangdong Province(2021A1515011078)Undergraduate Innovation and Entrepreneurship Training Program of Guangdong Ocean University(CXXL2022005)Undergraduate Innovation Team of Guangdong Ocean University(CCTD201802).
文摘[Objectives]To study the effects of microplastics on antibiotic resistance genes and virulence genes of Vibrio alginolyticus,so as to provide a certain reference for controlling marine pollution,curbing the spread of environmental antibiotic resistance genes and virulence genes,formulating environmental policies,and maintaining food safety.[Methods]After adding V.alginolyticus into the artificial seawater,they were divided into three groups,namely blank control group(BLK),polyvinyl chloride microplastic group(PVC group)and polyvinyl alcohol microplastic group(PVA group).Aerated culture experiments were carried out,and the effects of microplastics on the expression of resistance genes and virulence genes of V.alginolyticus were studied by PCR and qPCR methods.[Results]The presence of microplastics significantly changed the resistance gene structure of V.alginolyticus.Compared with the control group,the cfxA and cfr resistance genes were detected in the microplastic group.However,only PVC group detected blaZ resistance gene,and only PVA group did not detect aaC resistance gene.In addition,compared with the control group,the expressions of virulence genes in the microplastic group were all down-regulated(P<0.01).[Conclusions]This study provides some reference for curbing the spread of environmental antibiotic resistance genes and virulence genes,formulating environmental policies,and maintaining food safety,but the specific mechanisms of drug resistance and virulence need further research.
基金Supported by Outstanding Graduate Entering Laboratory Project of College of Fisheries,Guangdong Ocean UniversityNational Natural Science Foundation of China(32073015)+2 种基金Natural Science Foundation of Guangdong Province(2021A1515011078)Undergraduate Innovation and Entrepreneurship Training Program of Guangdong Ocean University(CXXL2022005)Undergraduate Innovation Team of Guangdong Ocean University(CCTD201802).
文摘[Objectives]To amplify the h-ns gene of Vibrio alginolyticus and analyze it by bioinformatics.[Methods]According to the h-ns gene sequence of V.alginolyticus HY9901,a pair of specific primers were designed and amplified by PCR.[Results]The h-ns gene was 408 bp in length and 135 amino acids were encoded.The predicted theoretical protein molecular weight was about 14.98 kD,and the isoelectric point was 4.99.Protein subcellular localization,SignalP 5.0,TMHMM Server 2.0 and SoftBerry-Psite predictions showed that H-NS was located outside the cell membrane,and the protein was unstable and hydrophobic.There was no signal peptide cleavage site,no transmembrane region and no KEGG metabolic pathway.The amino acid sequence contained three phosphorylation sites,one N-terminal myristoylation site and three microsomal C-terminal target signal sites.Using MEGA 5.0,H-NS phylogenetic tree was constructed by ortho-connection method.The results showed that H-NS of V.alginolyticus was closer to H-NS of Vibrio diabolicus.Using SWISS-MODEL,the three-dimensional structure model of H-NS subunit was simulated,which was similar to the crystal structure of Salmonella typhimurium H-NS1-83.[Conclusions]This study lays a foundation for exploring the regulation mechanism of V.alginolyticus H-NS protein on bacterial virulence in the future.
基金Supported by Outstanding Graduate Entering Laboratory Project of College of Fisheries,Guangdong Ocean UniversityNational Natural Science Foundationof China(32073015)+2 种基金Natural Science Foundation of Guangdong Province(2021A1515011078)Undergraduate Innovation and Entrepreneurship Train-ing Program of Guangdong Ocean University(CXXL2022005)UndergraduateInnovation Team of Guangdong Ocean University(CCTD201802)。
文摘[Objectives]The paper was to clone and analyze bioinformatics of ndk gene from Vibrio alginolyticus.[Methods]A pair of specific primers was designed based on the ndk gene sequence of V.alginolyticus HY9901.The full length of ndk gene was amplified by PCR and bioinformatics analysis was performed.MEGA 5.0 software was used to construct NDK phylogenetic tree by neighbor-joining method.SWISS-MODEL program was used to obtain the three-dimensional structural model of single subunit from NDK protein.[Results]The ndk gene,molecular structural formula C702H1094N192O214S7,was 426 bp in total,encoding 141 amino acids,with the theoretical molecular weight of 15.87199 kD and the theoretical pI value of 5.13.The prediction results of protein subcellular localization,SignalP 5.0,TMHMM Server 2.0 and SoftBerry-Psite showed that NDK mainly existed in the cytoplasm,and the protein was unstable and hydrophobic.There was neither signal peptide cleavage site,nor transmembrane region and KEGG metabolic pathway.The amino acid sequence had two protein kinase C phosphorylation sites,a casein kinaseⅡphosphorylation site,a N-myristoylation site,three microbody C-terminal target signal sites,and a nucleoside diphosphate kinase active site.Homology analysis showed that the NDK of V.alginolyticus had high homology with that of V.diabolicus,with a similarity of 98.58%.Analysis of the structural functional domain revealed that the protein had one NDK structural functional domain.The prediction results of secondary structure showed that theα-helix,random coil,β-sheet and extended strand accounted for 53.19%,28.37%,7.09%and 11.35%,respectively.Analysis of NDK protein via STRING database demonstrated that the proteins interacting with NDK protein were NrdA,NrdB,GmK,CmK,TmK,PyrG,PyrH,RelA,FolE and SpoT.[Conclusions]The study plays a positive role in the prevention and control of vibriosis and the improvement of the current aquaculture environment.
基金Supported by Outstanding Graduate Entering Laboratory Project of College of Fisheries,Guangdong Ocean UniversityGrants from Shenzhen Science and Technology Project(JCYJ20190813104207152)+3 种基金National Natural Science Foundation of China(32073015)Natural Science Foundation of Guangdong Province(2021A1515011078)Undergraduate Innovation and Entrepreneurship Training Program of Guangdong Ocean University(CXXL2022005)Undergraduate Innovation Team of Guangdong Ocean University(CCTD201802).
文摘[Objectives]To clone and analyze the vscB gene of Vibrio alginolyticus HY9901 by bioinformatics.[Methods]A pair of specific primers were designed according to the vscB gene sequence of Vibrio alginolyticus HY9901.The full length of the primers was cloned by PCR and analyzed by bioinformatics.[Results]The vscB gene was 429 bp long,encoding 142 amino acids,with a theoretical molecular weight of 16.4 kDa and a pI value of 5.48.Amino acid sequence analysis of VscB showed that VscB was not a secretory protein,without signal peptide and transmembrane region,and there were protein kinase C phosphorylation site and casein kinase II phosphorylation site in the sequence.Homologous comparison of amino acid sequences showed that VscB of V.alginolyticus had the highest protein similarity with Vibrio Parahaemolyticus,reaching 91%.Phylogenetic tree analysis showed that the corresponding proteins of V.alginolyticus VscB,Vibrio Parahaemolyticus and Vibrio diabolicus were clustered in the same subfamily.Functional domain analysis showed that it had CesT family domain.Tertiary structure prediction showed that there were 3α-helices and 5β-turns in VscB protein.[Conclusions]This study provided a theoretical basis for further study on the function of chaperone of V.alginolyticus.
基金Supported by Outstanding Graduate Entering Laboratory Project of College of FisheriesGuangdong Ocean University+3 种基金National Natural Science Foundation of China (32073015)Natural Science Foundation of Guangdong Province(2021A1515011078)Undergraduate Innovation and Entrepreneurship Training Program of Guangdong Ocean University (CXXL2022005)Undergraduate Innovation Team of Guangdong Ocean University (CCTD201802)
文摘[Objectives]The paper was to clone and analyze bioinformatics of vscN gene from Vibrio alginolyticus.[Methods]A pair of specific primers was designed based on the vscN gene sequence of V.alginolyticus HY9901.The full length of vscN gene was amplified by PCR and bioinformatics analysis was performed.[Results]The vscN gene was 1323 bp in total,encoding 440 amino acids,with the theoretical molecular weight of 47.86 kD and the theoretical pI value of 5.89.The online prediction showed that there was no signal peptide and no transmembrane region in VscN.The amino acid sequence had 10 N-myristoylation sites,8 phosphorylation sites(2 protein kinase C phosphorylation sites,6 casein kinase II phosphorylation sites),1 amidation site,11 microbody C-terminal target signal sites,1 ATP/GTP binding site motif A(P ring),and 1 ATPaseαandβsubunit specific site.Homology analysis showed that the VscN protein of V.alginolyticus had high homology with that of V.antiquarius,with a similarity of 95.14%.Phylogenetic tree analysis showed that the VscN of V.alginolyticus was clustered into the same subgroup as that of V.diabolicus and V.antiquarius.Functional domain analysis of VscN protein showed that it had Pfam and AAA domains,and involved in the regulation of bacterial virulence.The three-dimensional structure model of VscN simulated by SWISS-MODEL software was similar to the structure of flagellate-specific ATPase FliH-FliI complex.[Conclusions]The results lay a foundation for further study on the regulatory mechanism of VscN protein on bacterial virulence.
基金National Natural Science Foundation of China(32073015).
文摘[Objectives]The purpose was to investigate the function and mechanism of exsA gene in type III secretion system of Vibrio alginolyticus.[Methods]The full length of exsA was cloned using molecular biology techniques to analyze its biological information.Fluorescence quantitative PCR technology was used to analyze the expression of exsA after different media stress.[Results]The exsA gene contains an open reading frame(ORF)of 861 bp,encoding 286 amino acids.The physico-chemical analysis shows that the molecular structural formula is C1442H2267N393O441S12,the theoretical molecular weight is 32.549 kD,the theoretical pI value is 6.0,and the protein is non-hydrophilic and unstable.The gene does not contain a transmembrane region,and there is no obvious signal peptide.The prediction result of protein subcellular localization shows that the protein is inside the cell.The deduced amino acid sequence and constructed phylogenetic tree show that V.alginolyticus has a close relationship with Vibrio antiquarius.The qPCR results show that the expression level of exsA in different media is different,highest in TSB medium containing bile salts,followed by DMEM medium,and lowest in ordinary TSB medium.[Conclusions]The gene sequence,molecular structure and isoelectric point of exsA,as well as its expression in three different media were obtained.
基金National Natural Science Foundation of China(31402344,31572656)College Student Innovation Training Program(201510566042)+1 种基金Major Program for the Fundamental Research of the Department of Education of Guangdong Province(2014GKXM046)International Cooperation Innovation Platform Project of Universities in Guangdong Province(2013gjhz0008)
文摘The two-component regulatory system plays an important role in the growth and virulence of bacteria.In this study,the phosphate transcriptional regulatory protein (KdpE) gene of Vibrio alginolyticus strain HY9901 was cloned.Sequence analysis revealed that the length of kdp E gene is 732 bp and encodes a putative protein of 243 amino acids.The predicted molecular weight (MW) of Kdp E was 27.66 k D with an estimated p I of 5.01.Using SignalP 4.0 and TMHMM Server 2.0 software,it was predicted that the Kdp E protein was located in cytoplasm.It did not contain a signal peptide or a transmembranous region.A phylogenetic tree was constructed by MEGA 5.0 software,indicating that the Kdp E of V.alginolyticus had high genetic relationship with Vibrio campbellii and Vibrio parahaemolyticus.Using SWISS-MODEL work-space,the three-dimensional structures of conserved domain REC in the Kdp E was determined.These results can provide a basis for further studies on the two-component regulatory systems of V.alginolyticus.
基金Supported by National Natural Science Foundation of China(31402344,31572656)Major Program of Natural Science Foundation of Guangdong Province(2015A030308020)
文摘In order to enrich the research about type Ⅲ secretion system(T3SS) injectisome of Vibrio alginolyticus,vscX gene was cloned from V.alginolyticus strain HY9901 for bioinformatics analysis and expression analysis.Specific primers were designed according to the full-length genome sequence of V.alginolyticus in GenBank.vscX gene(GenBank accession number:FR780679) contained a 378 bp open reading frame(ORF),encoding a putative protein of 125 amino acids.The theoretical molecular weight was 14.209 2 kD and theoretical pI was 5.75.By using Signal 4.1 Server and TMHMM Server 2.0,it was predicted that VscX protein had no transmembrane domain or signal peptide.The results of prediction using SoftBerry-Psite software showed that VscX protein contained three casein kinase Ⅱ phosphorylation sites,one N-myristoylation site and three C-terminal targeting signal sites.Subcellular localization revealed that VscX might be located in cytoplasm with the possibility of 56.5%.According to SMART prediction,VscX had one Pfam(1-125 aa) domain.Phylogenetic analysis revealed that VscX from V.alginolyticus and VscX from V.parahemolyticus were clustered into the same group.Network interaction analysis showed that vscX was adjacent to vscY,vopB and sycN.By real-time fluorescent quantitative PGR technique and 2^(-△△Ct) method,the differences in expression levels of VscX mRNA in V.alginolyticus strain HY9901,T3 SS deletion strain AvscO and complementary strain C-vscO at different growth stages were analyzed.The results showed that the expression levels of VscX mRNA in V.alginolyticus strain HY9901 and C-vscO were significantly up-regulated at stable growth stage(P<0.01);the expression levels of VscX mRNA in deletion strain AvscO were significantly up-regulated at late growth stage(P <0.01).This study provided the basis for revealing the transport mechanism of T3 SS injectisome of V.alginolyticus.
基金National Natural Science Foundation of China(32073015).
文摘[Objective]To clone araC gene of Vibrio alginolyticus HY9901 strain,and analyze bioinformatics.[Methods]the whole genome sequence of Vibrio alginolyticus on GenBank was used to design specific primers.According to the principle of PCR amplification sequence,the target gene araC was amplified,and then the sequence was further analyzed by bioinformatics method to establish the phylogenetic tree of araC gene and its corresponding subunit three-dimensional structure model.[Results]Sequence analysis revealed araC gene is 711 bp and encodes a putative protein of 236 amino acids.The predicted molecular mass of AraC was 26.92 ku.Using Signal P 4.0 and TMHMM Server 2.0 software for analysis,it was predicted that the AraC protein did not contain a signal peptide or a transmembranous region.The AraC protein had two cAMP and cGMP dependent protein kinase phosphorylation site,five protein kinase C phosphorylation sites,three casein kinase II phosphorylation sites,one prenyl group binding site(CAAX box)and five microbodies C-terminal targeting signal.The predicted results of protein subcellular localization showed that AraC was located in the mitochondria,nucleus and cytoplasm.Its protein is unstable and hydrophilic.The AraC protein is a transcriptional regulatory protein which belongs to HTH_18 superfamily.According to the prediction,secondary structure:a-helix(Alpha helix)accounted for 52.12%,random coil(31.78%),extended strand(11.02%),b-fold(Beta turn)accounted for 5.08%.V.alginolyticus,Vibrio parahaemolyticus and Vibrio palustris were clustered together,which implies that the genetic relationship between these three species was the closest.
基金Shenzhen Science and Technology Project(JCYJ2019081310-4207152,JCYJ20170818111629778)Undergraduate Innovative and Entrepreneurial Team Project。
文摘[Objectives]To clone the pepck gene of Vibrio alginolyticus strain HY9901 and analyze its sequence by bioinformatics.[Methods]According to the complete gene sequence of V.alginolyticus on GenBank,specific primers were designed to amplify the target gene pepck by PCR.The sequence of the pepck gene was analyzed using bioinformatics.The phylogenic tree of pepck gene and the corresponding single-subunit three-dimensional structure were constructed.[Results]The pepck gene of V.alginolyticus strain HY9901 has a full length of 1629 bp,with theoretical molecular weight of 60.12 kD.The prediction results show that there is no signal peptide or transmembrane region at the N-terminus of the sequence,the amino acid sequence contains 11 phosphorylation sites of casein kinase II.The prediction results of protein subcellular localization indicate that PEPEK protein is localized in the cytoplasm.The protein is stable and hydrophobic.The tertiary structure of the PEPCK protein of V.alginolyticus is similar to that of Vibrio parahaemolyticus.It is predicted that PEPCK has a major functional domain PEPCK_ATP.In the secondary structure,alpha helix,random coil,and extended strand accounted for 21.96%,52.03%and 26.01%,respectively.The PEPCK homology between V.alginolyticus and Vibrio diabolicus is as high as 99%.[Conclusions]This study lays the foundation for further understanding the function of pepck gene in V.alginolyticus.
基金Supported by Project of Outstanding Undergraduates Entering Laboratory of Fisheries College,Guangdong Ocean UniversityNational Natural Science Foundation of China(32073015)+3 种基金Natural Science Foundation of Guangdong Province(2021A1515011078)Special Fund for Science and Technology Innovation Strategy in Guangdong Province(Undergraduates Science and Technology Innovation Cultivation)(pdjh2021b0239)Undergraduate Innovation and Enterpreneurship Training Program of Guangdong Ocean University(CXXL2021122)Undergraduate Innovation Team of Guangdong Ocean University(CCTD201802).
文摘[Objectives]The crp gene in Vibrio alginolyticus was studied.[Methods]The crp gene of V.alginolyticus HY9901 was cloned and analyzed by bioinformatics.[Results]The ORF of the crp gene is 633 bp long and the predicted amino acid sequence encompasses 210 amino acid residues.The physicochemical property analysis indicated that the chemical formula of CRP is C_(1051)H_(1704)N_(290)O_(308)S_(10) with a molecular weight of 23.6514 kDa,and its theoretical pI is 7.74.Besides,the protein is stable and hydrophilic.The protein had three protein kinase C phosphorylation site,three casein kinase II phosphorylation site,one N-terminal myristoylation site.The BLAST analysis on the sequence revealed high homology with CRPs in other Vibrio species,and particularly the sequence shares about a homology of 99.52%with the CRP in V.parahaemolyticus.The SWISS-MODEL software simulated the subunit tertiary structural model of the CRP,and the similarity with template 3hif.1.A was 95.71%.[Conclusions]This study provides a reference for the search for efficient protective antigens against vibrosis.
基金Project for Outstanding Undergraduates Entering the Laboratory in Fisheries College of Guangdong Ocean UniversityNational Natural Science Foundation of China (32073015)+3 种基金Natural Science Foundation of Guangdong Province (2021A1515011078)Special Fund for Science and Technology Innovation Strategy of Guangdong Province(Scientific and Technological Innovation Cultivation of College Students)(pdjh2021b0239)Students’Platform for Innovation and Entrepreneurship Training Program of Guangdong Ocean University (CXXL2021122)Undergraduate Innovation Team Project of Guangdong Ocean University(CCTD201802)。
文摘[Objectives]To clone and analyze the TpiA gene of Vibrio alginolyticus HY9901.[Methods]According to the TpiA gene sequence of V.alginolyticus,a pair of specific primers was designed,and its full length was amplified by PCR.[Results]The full length of TpiA gene is 771 bp,encoding 256 amino acid residues in total,and the NCBI accession number is OM906798.According to the deduced amino acid sequence,its molecular weight was predicted to be about 26.97548 kDa,and its isoelectric point was 4.78.The amino acid sequence of the N-terminal signal peptide structure was predicted,and it was found that there was no obvious signal peptide cleavage site,no signal peptide,and no transmembrane region;the amino acid sequence contained 3 N-glycosylation sites,4 protein kinase C phosphorylation sites,2 casein kinase II phosphorylation sites,6 N-myristoylation sites,7 microbody C-terminal target signal site,and 1 triose phosphate isomerase active site.The prediction results of protein subcellular localization showed that TpiA may be located in mitochondria or cytoplasm,with probability of 39.1%and 34.8%,respectively.The amino acid sequence of the TpiA gene of V.alginolyticus shared 98.83%-99.61%homology with other Vibrio species,and it was clustered into the same subfamily with Vibrio parahaemolyticus and had a close relationship.In the secondary structure prediction,the proportions ofα-helix,random coil and extended chain were 44.53%,41.41%and 14.06%,respectively,and the similarity of its tertiary structure model to template 1aw1.1.A was 85.16%.[Conclusions]This study is intended to provide a basis for further research on the role of TpiA gene in the type III secretion system and related research on antibiotic resistance.
基金Outstanding Graduate Entering Laboratory Project of College of Fisheries,Guangdong Ocean UniversityNational Natural Science Foundation of China(32073015)+3 种基金Natural Science Foundation of Guangdong Province(2021A1515011078)Special Fund for Science and Technology Innovation Strategy of Guangdong Province(Undergraduate Science and Technology Innovation Cultivation)(pdjh2021b0239)Undergraduate Innovation and Entrepreneurship Training Program of Guangdong Ocean University(CXXL2021122)Undergraduate Innovation Team of Guangdong Ocean University(CCTD-201802).
文摘[Objectives]To amplify the DNA-binding response regulator PhoP in Vibrio alginolyticus and analyze its sequence characteristics and subunit structure.[Methods]According to the sequence of the DNA-binding response regulator PhoP in V.alginolyticus,a pair of specific primers was designed for PCR amplification,and the bioinformatics of the sequence amplified was analyzed.Using MEGA 5.0 software,the phoP phylogenetic tree was constructed by the neighbor-joining method.Using SWISS-MODEL software,the three-dimensional structural model of the PhoP subunit was simulated.[Results]The full-length phoP gene was 732 bp,encoding a total of 243 amino acids.The predicted theoretical molecular weight of the protein is about 27.67 kD,and the isoelectric point is 5.09.The prediction results of protein subcellular localization,SignalP 4.0,TMHMM Server 2.0 and SoftBerry-Psite show that PhoP is located in the cytoplasm,and is stable and hydrophobic;there is a signal peptide cleavage site between amino acids 29 and 30,and there is no transmembrane region.The amino acid sequence contains one Asn-glycosylation site,one protein kinase C phosphorylation site,seven casein kinase II phosphorylation sites,one tyrosine kinase phosphorylation site,three myristoylation sites,and seven C-terminal microbody targeting signal sites.The PhoP of V.alginolyticus has high homology with that of Vibrio campbellii.The PhoP subunit of V.alginolyticus has similar configuration to the single-subunit RegX3 protein of Mycobacterium tuberculosis.[Conclusions]This study has a positive effect on the prevention and control of vibriosis and the improvement of the current aquatic economic animal breeding environment.
