Cobetia marina is a model proteobacteria in researches on marine biofouling. Its taxonomic nomenclature has been revised many times over the past few decades. To better understand the role of the surface-associated li...Cobetia marina is a model proteobacteria in researches on marine biofouling. Its taxonomic nomenclature has been revised many times over the past few decades. To better understand the role of the surface-associated lifestyle of C. marina and the phylogeny of the family Halomonadaceae, we sequenced the entire genome of C. marina JCM 21022T using single molecule real-time sequencing technology （SMRT） and performed comparative genomics and phylogenomics analyses. The circular chromosome was 4 176 300 bp with an average GC content of 62.44% and contained 3 611 predicted coding sequences, 72 tRNA genes, and 21 rRNA genes. The C. marina JCM 2102U genome contained a set of crucial genes involved in surface colonization processes. The comparative genome analysis indicated the significant differences between C. marina JCM 21022T and Cobetia amphilecti KMM 296 （formerly named C. marina KMM 296） resulted from sequence insertions or deletions and chromosomal recombination. Despite these differences, pan and core genome analysis showed similar gene functions between the two strains. The phylogenomic study of the family Halomonadaceae is relationships were well resolved among every genera Cobetia, Kushneria, Zymobacter, and Halotalea. reported here for the first time. We found that the tested, including Chromohalobacter, Halomonas,展开更多
Pseudoalteromonas marina is one of the potential pathogens that cause green spot disease(GSD)in Pyropia yezoensis.To prevent GSD from development and spread,an effective method to detect the pathogen at early GSD infe...Pseudoalteromonas marina is one of the potential pathogens that cause green spot disease(GSD)in Pyropia yezoensis.To prevent GSD from development and spread,an effective method to detect the pathogen at early GSD infection stages need to be established.In this research,PCR methods were established targeting the dnaA gene(encoding chromosome replication initiator protein)and the dnaN gene(encodingβsliding clamp of DNA polymeraseⅢprotein)to detect P.marina with three primer pairs pws-dnaA2(Forward,5'-ACCGCATTAACGAACTACTCGTG-3';Reverse,5'-TGCCATTACCTACAGCATGG-3'),pcs-dnaN2(Forward,5'-CTTACAACGTTATCAGCGGC-3';Reverse,5'-GTTGAGTATTAAGTGATTGAGTAAGC-3')or pws-dnaN3(Forward,5'-ACTTACAA-CGTTATCAGCGGC-3';Reverse,5'-ACTGCTGTTTGAGTCTGCTAAC-3').Three PCR methods corresponding to the three primer pairs sufficiently distinguished P.marina from 22 bacterial species,thus resulting in detection limits of 4 to 4×10^2 CFU cells or 2.37×10^1 to 2.37×10^3 fg of P.marina DNA per PCR reaction.In an artificial infection experiment ofP.yezoensis infected with P.marina,all established PCRs successfully detected P.marina at early GSD infection stages.The results show that the established PCRs are specific and sensitive,and are potential for applications in early diagnosis of GSD in Pyropia.展开更多
Pyropia yezoensis(formerly Porphyra yezoensis)is an economically important red alga that is cultured extensively in China.The red rot disease occurs commonly during Pyropia cultivation,causing serious economic losses....Pyropia yezoensis(formerly Porphyra yezoensis)is an economically important red alga that is cultured extensively in China.The red rot disease occurs commonly during Pyropia cultivation,causing serious economic losses.An incidence of red rot disease was found in a P.yezoensis farm from mid-November to mid-December 2015 at Lianyungang,Jiangsu Province,China.Histopathological examination revealed that the naturally infected thalli were infected apparently by a pathogen,leading to red rot symptoms.The causative agent was isolated and identified as the oomycete Pythium chondricola by morphological analysis and sequence analysis of the internal transcribed spacer and cytochrome oxidase subunit 1(cox 1).In artifi cial infection experiments on the P.yezoensis blades,the P.chondricola isolate was able to cause the same characteristic histopathology seen in natural infections.P.chondricola grew well at a wide range of temperatures in the range 8-31℃,salinities at 0-45 and pH 5-9.In an orthogonal test used to determine the effects of environmental factors(temperature,salinity,and zoospore concentration)on infection,the data revealed that temperature was the most important factor to affect red rot disease development,with the optimal conditions for disease expansion being 20℃,35 salinity,and a zoospore concentration of 10^6 zoospores/mL.The results obtained from the present study prompted us to set up a comprehensive epidemiological study on Pyropia,which will provide support to maintain the healthy development of the Pyropia industry in China.展开更多
Dot enzyme-linked immunosorbent assay （dot-ELISA）, indirect ELISA and Westem blot were performed to detect the virulent protease secreted by Vibrio anguillarum which was isolated from the diseased left-eyed flounder...Dot enzyme-linked immunosorbent assay （dot-ELISA）, indirect ELISA and Westem blot were performed to detect the virulent protease secreted by Vibrio anguillarum which was isolated from the diseased left-eyed flounder, Paralichthys olivaceous. Sensitivity results showed that dot-ELISA is a more sensitive, rapid and simple technique for the protease detection. The minimal detectable amount of protease is about 7 pg in the dot-ELISA test, while 7.8 ng in the indirect ELISA and 6.25 ng in the Westem blot respectively. Protease could be detected 2 h after incubation of V. anguillarum in the 2216E liquid medium but enzyme activity was very low at that period. From 6 to 12 h, the amount and enzyme activity of protease increased markedly and reached maximum at stationary phase. Analysis of serum samples periodically collected from the infected flounders showed that after 2 h of infection by V. anguillarum, the pathogenic bacteria could be detected in the blood of the infected flounders but no protease was found. It was 5-6 h after infection that the protease was detected in blood and then the amount increased as infection advanced. Quantitative detection of protease either incubation in the medium or from the blood of infected flounders could be accomplished in virtue of positive controls of quantificational protease standards （＂marker＂） so that the alterations ofprotease secretion both in vitro and in vivo could be understood generally. In addition, the indirect ELISA and dot-ELISA were also performed to detect V. anguillarum cells. Results indicated that the sensitivity of indirect ELISA to bacteria cells is higher than that of the dot-ELISA, and that the minimal detectable amount is approximately 10^4 cell/mL in the indirect ELISA, while 10^5 cell/mL in the dot-ELISA.展开更多
EmpA is an extracellular metalloprotease secreted by Vibrio anguillarum.For better understanding its role in the patho-genicity of V.anguillarum strain M3,empA insertion mutant was constructed.In the mutant it decreas...EmpA is an extracellular metalloprotease secreted by Vibrio anguillarum.For better understanding its role in the patho-genicity of V.anguillarum strain M3,empA insertion mutant was constructed.In the mutant it decreased in extracellular proteolytic activity,swarming motility,hemolytic activity and virulence on turbot(Scophthalmus maximus).Significant decline(by 5-fold)of extracellular proteolytic activity and similar growth curve between mutant and wild type strains indicated that EmpA was the major extracellular protease of M3.LD50 of mutant increased by 38-fold compared with wild type.No pro-EmpA was detected in the su-pernatant of culture,indicating that EmpA autolyzed to mature protein after 24 h.Secretion of EmpA in M3 was similar to that in NB10 strain.Attenuated virulence of mutant was similar to that of M93Sm strain.It was demonstrated that specific operation of EmpA was different from that in previous studies and EmpA contributed to the swarming motility and hemolytic activity in V.an-guillarum strain M3.The results provides insight into understanding the function of EmpA and its potential application in vaccine development.展开更多
The bacteriophage P13 that infects Klebsiella serotype K13 contains a heat-stable depolymerase capable of effective degradation of exopolysaccharide(EPS) produced by this microorganism. In this study, the titer of pha...The bacteriophage P13 that infects Klebsiella serotype K13 contains a heat-stable depolymerase capable of effective degradation of exopolysaccharide(EPS) produced by this microorganism. In this study, the titer of phage P13, initially 2.0 × 107 pfu mL-1, was found increasing 20 min after infection and reached 5.0 × 109 pfu mL-1 in 60 min. Accordingly, the enzyme activity of depolymerase approached the maximum 60 min after infection. Treatment at 70℃ for 30 min inactivated all the phage, but retained over 90% of the depolymerase activity. Addition of acetone into the crude phage lysate led to precipitation of the protein, with a marked increase in bacterial EPS degradation activity and a rapid drop in the titer of phage. After partial purification by acetone precipitation and ultrafiltration centrifugation, the enzyme was separated from the phage particles, showing two components with enzyme activity on Q-Sepharose Fast Flow. The soluble enzyme had an optimum degradation activity at 60℃ and pH 6.5. Transmission electron microscopy demonstrated that the phage P13 particles were spherical with a diameter of 50 nm and a short stumpy tail. It was a double-strand DNA virus consisting of a nucleic acid molecule of 45976 bp. This work provides an efficient purification operation including thermal treatment and ultrafiltration centrifugation, to dissociate depolymerase from phage particles. The characterization of phage P13 and associated EPS depolymerase is beneficial for further application of this enzyme.展开更多
A method detecting pathogenic vibrio anguillarum and its virulent metalloprotease is reported. The metalloprotease is isolated from extracellular product of vibrio anguillarum by the cellophane plate technique and pur...A method detecting pathogenic vibrio anguillarum and its virulent metalloprotease is reported. The metalloprotease is isolated from extracellular product of vibrio anguillarum by the cellophane plate technique and purified by gel filtration and ion-exchange chromatography. Anti-sera are prepared by injecting vibrio anguillarum cells and metalloprotease into the rabbits. Slide agglutination assay is used to detect V. anguillarum in the infection experiment and enzyme linked immunosorbent assay (ELISA) is carried out to detect concentration of metalloprotease. The results show that bacterium strain M3 is able to diffuse into the viscera of infected fish through the blood circulating system 10 hours after intramuscular infection, and E-LASA is a sensitive method to detect the metalloprotease with detectable amount of 7.8 ng. The aim of this study is to establish a sensitive and specific method to observe the infection of vibrio anguillarum in the host.展开更多
基金Supported by the National Natural Science Foundation of China(Nos.41006082,31372517)
文摘Cobetia marina is a model proteobacteria in researches on marine biofouling. Its taxonomic nomenclature has been revised many times over the past few decades. To better understand the role of the surface-associated lifestyle of C. marina and the phylogeny of the family Halomonadaceae, we sequenced the entire genome of C. marina JCM 21022T using single molecule real-time sequencing technology （SMRT） and performed comparative genomics and phylogenomics analyses. The circular chromosome was 4 176 300 bp with an average GC content of 62.44% and contained 3 611 predicted coding sequences, 72 tRNA genes, and 21 rRNA genes. The C. marina JCM 2102U genome contained a set of crucial genes involved in surface colonization processes. The comparative genome analysis indicated the significant differences between C. marina JCM 21022T and Cobetia amphilecti KMM 296 （formerly named C. marina KMM 296） resulted from sequence insertions or deletions and chromosomal recombination. Despite these differences, pan and core genome analysis showed similar gene functions between the two strains. The phylogenomic study of the family Halomonadaceae is relationships were well resolved among every genera Cobetia, Kushneria, Zymobacter, and Halotalea. reported here for the first time. We found that the tested, including Chromohalobacter, Halomonas,
基金Supported by the China Agriculture Research System(No.CARS-50)the National High-Tech R&D Program of China(No.2012AA10A406)+1 种基金the National Science and Technology Infrastructure Platform Construction(No.2018DKA30470)the Aoshan Technology Innovation Program of Qingdao National Laboratory for Marine Science and Technology(No.2015ASKJ02)
文摘Pseudoalteromonas marina is one of the potential pathogens that cause green spot disease(GSD)in Pyropia yezoensis.To prevent GSD from development and spread,an effective method to detect the pathogen at early GSD infection stages need to be established.In this research,PCR methods were established targeting the dnaA gene(encoding chromosome replication initiator protein)and the dnaN gene(encodingβsliding clamp of DNA polymeraseⅢprotein)to detect P.marina with three primer pairs pws-dnaA2(Forward,5'-ACCGCATTAACGAACTACTCGTG-3';Reverse,5'-TGCCATTACCTACAGCATGG-3'),pcs-dnaN2(Forward,5'-CTTACAACGTTATCAGCGGC-3';Reverse,5'-GTTGAGTATTAAGTGATTGAGTAAGC-3')or pws-dnaN3(Forward,5'-ACTTACAA-CGTTATCAGCGGC-3';Reverse,5'-ACTGCTGTTTGAGTCTGCTAAC-3').Three PCR methods corresponding to the three primer pairs sufficiently distinguished P.marina from 22 bacterial species,thus resulting in detection limits of 4 to 4×10^2 CFU cells or 2.37×10^1 to 2.37×10^3 fg of P.marina DNA per PCR reaction.In an artificial infection experiment ofP.yezoensis infected with P.marina,all established PCRs successfully detected P.marina at early GSD infection stages.The results show that the established PCRs are specific and sensitive,and are potential for applications in early diagnosis of GSD in Pyropia.
文摘本研究分析了鳗弧菌(Vibrio anguillarum)O1/O2血清型二价灭活疫苗免疫大菱鲆后的抗体持续期和免疫保护期。以鳗弧菌O1血清型VAM003株和O2血清型VAM007株为抗原制备了福尔马林灭活二价疫苗,将疫苗按照三种剂量(10~7 cells/尾、10~8 cells/尾、10~9 cells/尾)以腹腔注射途径免疫大菱鲆,在免疫后3 d、7 d、14 d、30 d、60 d、90 d、120 d、150 d,用血清凝集实验检测了免疫鱼血清的VAM003和VAM007抗体效价,用攻毒实验检测了疫苗的免疫保护率(RPS)。结果显示,在免疫后7 d三个剂量组的大菱鲆均产生了特异抗体,并获得27%～60%的RPS。三个剂量组大菱鲆的O1血清型抗体持续期分别>90 d (10~7 cells/尾组)、>150 d (10~8 cells/尾组)、>150 d (10~9cells/尾组),而三个剂量组大菱鲆的O2血清型抗体持续期均>150 d。三个剂量组的大菱鲆获得的免疫保护持续期均>150 d;以RPS>75%为有效免疫保护,各剂量组大菱鲆抵抗O1血清型病原感染的有效免疫保护期为:14～120d(10~7 cells组)、14～120 d (10~8 cells/尾)、14～150 d (10~9 cells/尾),抵抗O2血清型病原感染的有效免疫保护期为:14～60 d (10~7 cells组)、14～120 d (10~8 cells/尾)、14～120 d (10~9 cells/尾)。研究结果表明鳗弧菌二价灭活疫苗可为大菱鲆提供有效而稳定的免疫保护,获得的抗体持续期和免疫保护期为该疫苗的临床中试研究提供了基础。
基金Supported by the China Agriculture Research System(No.CARS-50)the National Natural Sciences Foundation of China(No.31372517)+2 种基金the Fundamental Research Funds for the Central Universities(No.201562018)the National Infrastructure of Fishery Germplasm Resources(No.2017DKA30470)the Project of Aoshan Scientific and Technological Innovation Program of Qingdao National Laboratory for Marine Science and Technology(No.2015ASKJ02)
文摘Pyropia yezoensis(formerly Porphyra yezoensis)is an economically important red alga that is cultured extensively in China.The red rot disease occurs commonly during Pyropia cultivation,causing serious economic losses.An incidence of red rot disease was found in a P.yezoensis farm from mid-November to mid-December 2015 at Lianyungang,Jiangsu Province,China.Histopathological examination revealed that the naturally infected thalli were infected apparently by a pathogen,leading to red rot symptoms.The causative agent was isolated and identified as the oomycete Pythium chondricola by morphological analysis and sequence analysis of the internal transcribed spacer and cytochrome oxidase subunit 1(cox 1).In artifi cial infection experiments on the P.yezoensis blades,the P.chondricola isolate was able to cause the same characteristic histopathology seen in natural infections.P.chondricola grew well at a wide range of temperatures in the range 8-31℃,salinities at 0-45 and pH 5-9.In an orthogonal test used to determine the effects of environmental factors(temperature,salinity,and zoospore concentration)on infection,the data revealed that temperature was the most important factor to affect red rot disease development,with the optimal conditions for disease expansion being 20℃,35 salinity,and a zoospore concentration of 10^6 zoospores/mL.The results obtained from the present study prompted us to set up a comprehensive epidemiological study on Pyropia,which will provide support to maintain the healthy development of the Pyropia industry in China.
文摘Dot enzyme-linked immunosorbent assay （dot-ELISA）, indirect ELISA and Westem blot were performed to detect the virulent protease secreted by Vibrio anguillarum which was isolated from the diseased left-eyed flounder, Paralichthys olivaceous. Sensitivity results showed that dot-ELISA is a more sensitive, rapid and simple technique for the protease detection. The minimal detectable amount of protease is about 7 pg in the dot-ELISA test, while 7.8 ng in the indirect ELISA and 6.25 ng in the Westem blot respectively. Protease could be detected 2 h after incubation of V. anguillarum in the 2216E liquid medium but enzyme activity was very low at that period. From 6 to 12 h, the amount and enzyme activity of protease increased markedly and reached maximum at stationary phase. Analysis of serum samples periodically collected from the infected flounders showed that after 2 h of infection by V. anguillarum, the pathogenic bacteria could be detected in the blood of the infected flounders but no protease was found. It was 5-6 h after infection that the protease was detected in blood and then the amount increased as infection advanced. Quantitative detection of protease either incubation in the medium or from the blood of infected flounders could be accomplished in virtue of positive controls of quantificational protease standards （＂marker＂） so that the alterations ofprotease secretion both in vitro and in vivo could be understood generally. In addition, the indirect ELISA and dot-ELISA were also performed to detect V. anguillarum cells. Results indicated that the sensitivity of indirect ELISA to bacteria cells is higher than that of the dot-ELISA, and that the minimal detectable amount is approximately 10^4 cell/mL in the indirect ELISA, while 10^5 cell/mL in the dot-ELISA.
基金This project was funded by the 973 Program (2006CB101803)the National Natural Sciences Foundation of China (30871935)
文摘EmpA is an extracellular metalloprotease secreted by Vibrio anguillarum.For better understanding its role in the patho-genicity of V.anguillarum strain M3,empA insertion mutant was constructed.In the mutant it decreased in extracellular proteolytic activity,swarming motility,hemolytic activity and virulence on turbot(Scophthalmus maximus).Significant decline(by 5-fold)of extracellular proteolytic activity and similar growth curve between mutant and wild type strains indicated that EmpA was the major extracellular protease of M3.LD50 of mutant increased by 38-fold compared with wild type.No pro-EmpA was detected in the su-pernatant of culture,indicating that EmpA autolyzed to mature protein after 24 h.Secretion of EmpA in M3 was similar to that in NB10 strain.Attenuated virulence of mutant was similar to that of M93Sm strain.It was demonstrated that specific operation of EmpA was different from that in previous studies and EmpA contributed to the swarming motility and hemolytic activity in V.an-guillarum strain M3.The results provides insight into understanding the function of EmpA and its potential application in vaccine development.
基金supported by the National Natural Science Foundation of China(No.41076087)
文摘The bacteriophage P13 that infects Klebsiella serotype K13 contains a heat-stable depolymerase capable of effective degradation of exopolysaccharide(EPS) produced by this microorganism. In this study, the titer of phage P13, initially 2.0 × 107 pfu mL-1, was found increasing 20 min after infection and reached 5.0 × 109 pfu mL-1 in 60 min. Accordingly, the enzyme activity of depolymerase approached the maximum 60 min after infection. Treatment at 70℃ for 30 min inactivated all the phage, but retained over 90% of the depolymerase activity. Addition of acetone into the crude phage lysate led to precipitation of the protein, with a marked increase in bacterial EPS degradation activity and a rapid drop in the titer of phage. After partial purification by acetone precipitation and ultrafiltration centrifugation, the enzyme was separated from the phage particles, showing two components with enzyme activity on Q-Sepharose Fast Flow. The soluble enzyme had an optimum degradation activity at 60℃ and pH 6.5. Transmission electron microscopy demonstrated that the phage P13 particles were spherical with a diameter of 50 nm and a short stumpy tail. It was a double-strand DNA virus consisting of a nucleic acid molecule of 45976 bp. This work provides an efficient purification operation including thermal treatment and ultrafiltration centrifugation, to dissociate depolymerase from phage particles. The characterization of phage P13 and associated EPS depolymerase is beneficial for further application of this enzyme.
文摘A method detecting pathogenic vibrio anguillarum and its virulent metalloprotease is reported. The metalloprotease is isolated from extracellular product of vibrio anguillarum by the cellophane plate technique and purified by gel filtration and ion-exchange chromatography. Anti-sera are prepared by injecting vibrio anguillarum cells and metalloprotease into the rabbits. Slide agglutination assay is used to detect V. anguillarum in the infection experiment and enzyme linked immunosorbent assay (ELISA) is carried out to detect concentration of metalloprotease. The results show that bacterium strain M3 is able to diffuse into the viscera of infected fish through the blood circulating system 10 hours after intramuscular infection, and E-LASA is a sensitive method to detect the metalloprotease with detectable amount of 7.8 ng. The aim of this study is to establish a sensitive and specific method to observe the infection of vibrio anguillarum in the host.
