Biofilm forming bacteria are omnipresent in the marine environment.Pseudoalteromonas is one of the largest within the γ-proteobacteria class,and a member of marine bacteria.Species of Pseudoalteromonas are generally ...Biofilm forming bacteria are omnipresent in the marine environment.Pseudoalteromonas is one of the largest within the γ-proteobacteria class,and a member of marine bacteria.Species of Pseudoalteromonas are generally found in association with marine eukaryotes.In this work,we present the isolation and characterization of two strains forming biofilm on rock surface and associated with marine sponge.They were identified using 16SrDNA as Pseudoalteromonas prydzensis alex,and Pseudoalteromonas sp.alex.They showed the highest titer in biofilm formation quantified using the test tube method using crystal violet.展开更多
κ-carrageenan oligosaccharides exhibit various biological activities. Enzymatic degradation by κ-carrageenase is safe and controllable. Therefore, κ-carrageenases have captured more and more attentions. In this stu...κ-carrageenan oligosaccharides exhibit various biological activities. Enzymatic degradation by κ-carrageenase is safe and controllable. Therefore, κ-carrageenases have captured more and more attentions. In this study, a κ-carrageenase encoding gene, cgk X, was cloned from Pseudoalteromonas sp. QY203 with degenerate and inverse PCR. It comprised an ORF of 1194 bp in length, encoding a protein with 397 amino acid residues. Cgk X is a new member of glycoside hydrolase family 16. The deduced amino acid sequence shared a high similarity with Cgk X of Pseudoalteromonas κ-carrageenase; however, the recombinant Cgk X showed different biochemical characteristics. The recombinant enzyme was most active at p H 7.0 and 55℃ in the presence of 300 mmol L^(^(-1))Na Cl. It was stable in a broad range of acidity ranging from p H 3.0 to p H 10.0 when temperature was below 40℃. More than 80% of its activity was maintained after being incubated at p H 3.6–10.0 and 4℃ for 24 h. Cgk X retained more than 90% of activity after being incubated at 40℃ for 1 h. EDTA and SDS(1 mmol L^(-1)) did not inhibit its activity. Cgk X hydrolyzed κ-carrageenan into disaccharide and tetrasaccharide as an endo-cleaver. All these characteristics demonstrated that Cgk X is applicable to both κ-carrageenan oligosaccharide production and κ-carrageenase structure-function research.展开更多
Twenty-seven antarctic bacteria producing extracellular polysaccharide （EPS） were selected from 57 strains by staining technology. The effects of major environmental factors on the growth and EPS production of Pseud...Twenty-seven antarctic bacteria producing extracellular polysaccharide （EPS） were selected from 57 strains by staining technology. The effects of major environmental factors on the growth and EPS production of Pseudoalteromonas sp. S - 15 - 13 were investigated, and the EPS was separated and purified for characterization analysis. The results showed that the optimal conditions for the EPS production were culture period, 56 h; growth temperature, 8 ℃ ; carbon source, 1.0% glucose; NaCI concentration, 3.0% ; pH 6.0 - 7.0. The EPS was purified by cold ethanol precipitation, proteins removal, ion exchange chromatography and gel chromatography technology. The molecular mass of EPS - H was 62 kDa as determined by the high performance gel permeation chromatography. Its sugar composition was a homopolymer of marmose analyzed by gas chromatograph spectroscopy. After repeated freezing and thawing of the bacteria hiomass in the presence of EPS, the bacterial growth was much higher than that observed after freezing in the absence of EPS and the difference augmented with the increase of freeze-thaw cycles. It is hypothesized that the adaptation of Pseudoalteromonas sp. S- 15 - 13 to the antarctic marine conditions, characterized by low temperature, high NaCl concentration and repeated freeze-thaw cycles, might be related to the EPS production ability.展开更多
Pseudoalteromonas sp. SM9913 is a phychrotmphic bacterium isolated from the deep-sea sediment. The genes encoding chaperones DnaJ and DnaK of P. sp. SM9913 were cloned by normal PCR and TAIL - PCR （GenBank accession ...Pseudoalteromonas sp. SM9913 is a phychrotmphic bacterium isolated from the deep-sea sediment. The genes encoding chaperones DnaJ and DnaK of P. sp. SM9913 were cloned by normal PCR and TAIL - PCR （GenBank accession Nos DQ640312, DQ504163 ）. The chaperones DnaJ and DnaK from the strain SM9913 contain such conserved domains as those of many other bacteria, and show some cold-adapted characteristics in their structures when compared with those from psychro-, meso-and themophilic bacteria. It is indicated that chaperones DnaJ and DnaK of P. sp. SM9913 may be adapted to low temperature in deep-sea and function well in assisting folding, assembling and translocation of proteins at low temperature. This research lays a foundation for the further study on the cold-adapted mechanism of chaperones DnaJ and DnaK of cold-adapted microorganisms.展开更多
The psychrotropic bacterium, Pseudoalteromonas sp. QI-I, which produces extracellular cold-active protease, was isolated from Antarctic seawater. The genomic DNA of this bacterium was used to construct a plasmid genom...The psychrotropic bacterium, Pseudoalteromonas sp. QI-I, which produces extracellular cold-active protease, was isolated from Antarctic seawater. The genomic DNA of this bacterium was used to construct a plasmid genomic library with the goal of screening cold-active protease genes. Gene pro-2127 with an open reading frame of 2127 bp encoding protease PRO-2127 was cloned and sequenced. Alignment of amino acid sequences suggested that the precursor of PRO-2127 was a member of subfamily S8A, and that it might contain four domains： a signal peptide, an N-terminal prosequence, a catalytic domain and a C-terminal extension. Amino acids Asp185, His244 and Ser425 might form a catalytic triad. PRO-2127 showed some structural features common to psychrophilic enzymes, such as a decrease in Arg residues and the Arg/（Arg＋Lys） ratio. Heterologous expression of pro-2127 in Escherichia coli BL21 （DE3） by pColdlII was also successfully observed in this study.展开更多
Genus Pseudoalteromonas belongs to Family Pseudoalteromonadaceae in Gammaproteobacteria. A cold-adapted gram-negative bacterium, hydrocarbon-degrading Pseudoalteromonas sp. NJ289, was isolated from sea-ice of the Anta...Genus Pseudoalteromonas belongs to Family Pseudoalteromonadaceae in Gammaproteobacteria. A cold-adapted gram-negative bacterium, hydrocarbon-degrading Pseudoalteromonas sp. NJ289, was isolated from sea-ice of the Antarctica region, and sequenced the whole genome through the next generation sequencing platform. The assembly yielded three contigs representing two chromosomes and one plasmid with the sizes of 3.2 Mb, 636 kb and 1.8 kb, respectively. The G＋C contents of genome were 40.83% and included 3 589 ORFs. Functional annotation indicated some potential roles in enzymatic activity and environmental adaptability. This study may help for understanding the population diverse, evolutionary ecology and the microbial interaction.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
This study aimed to improve the thermostability of arylsulfatase from Pseudoalteromonas carrageenovora. A library of P. carrageenovora arylsulfatase mutants was constructed by introducing random mutagenesis using erro...This study aimed to improve the thermostability of arylsulfatase from Pseudoalteromonas carrageenovora. A library of P. carrageenovora arylsulfatase mutants was constructed by introducing random mutagenesis using error-prone PCR. After screening, two mutants of H260L and D84A/H260L showed enhanced thermal stability than the wild-type predecessor (WT). Site-directed mutagenesis demonstrated that only amino acid residue at Position 260 plays an important role in the thermostability of P. carrageenovora arylsulfatase. Thermal inactivation analysis showed that the half-life (t1/2) values at 55°C for H260L, H260I, H260Q, H260F and H260R were 40.6, 48.4, 30.9, 29.1 and 34.5 min, respectively, while that of WT was 9.1 min. Structure modeling demonstrated that the additional hydrogen bonds and/or optimization of surface charge-charge interactions could be responsible for the increased thermostability imparted by H260L, H260I, H260Q, H260F and H260R.展开更多
Oxidative stress is one of the major challenges faced by Arctic marine bacteria due to the high oxygen concentration of seawater, low temperatures and UV radiations. Transcriptome sequencing was performed to obtain th...Oxidative stress is one of the major challenges faced by Arctic marine bacteria due to the high oxygen concentration of seawater, low temperatures and UV radiations. Transcriptome sequencing was performed to obtain the key functional genes involved in the adaptation to oxidative stress induced by hydrogen peroxide in the Arctic bacterium Pseudoalteromonas sp. A2. Exposure to 1 mmol/L H2O2 resulted in large alterations of the transcriptome profile, including significant up-regulation of 109 genes and significant down-regulation of 174 genes. COG functional classification revealed that among the significantly regulated genes with known function categories, more genes belonging to posttranslational modification, protein turnover and chaperones were significantly up-regulated, and more genes affiliated with chaperones and amino acid transport and metabolism were significantly down-regulated. It was notable that the expressions of eighteen genes affiliated with flagella and four genes affiliated with heat shock proteins were significantly up-regulated. Meanwhile, the expression of nine genes belonging to cytochrome and cytochrome oxidase, and five genes belonging to Ton B-dependent receptor,were significantly down-regulated. Among the eighteen genes with antioxidant activity categorized by GO analysis, the expression of one gene was significantly up-regulated; however, the expressions of two genes were significantly down-regulated. Briefly, RNA-Seq indicated that, except for the classical anti-oxidative genes and stress proteins, genes affiliated with flagella and function unknown played important roles in coping with oxidative stress in Pseudoalteromonas sp. A2. This overall survey of transcriptome and oxidative stress-relevant genes can contribute to understand the adaptive mechanism of Arctic bacteria.展开更多
The genus Pseudoalteromonas is ubiquitous in the marine environment and can synthesize a wide range of extracellular compounds. Psychrotolerant Pseudoalteromonas sp. BSw20308 was isolated from the Chukchi Sea, a margi...The genus Pseudoalteromonas is ubiquitous in the marine environment and can synthesize a wide range of extracellular compounds. Psychrotolerant Pseudoalteromonas sp. BSw20308 was isolated from the Chukchi Sea, a marginal sea of the Arctic Ocean. It produces a number of extracellular enzymes that can degrade polysaccharides and proteins. The BSw20308 genome was sequenced to 38.1-fold coverage, and the sequences were assembled into 146 contigs （〉~500 bp）. In total, 4 172 open reading frames （ORFs） with an average gene length of 987 bp were detected. At least 86 ORFs were predicted by sequence analysis to encode a variety of catalytic modules involved in the degradation of polysaccharides, proteins, and lipids. In addition, 36 ORFs were predicted to encode catalytic modules involved in the degradation of organic pollutants and halogenated compounds, and in the production of bioactive compounds. The draft genome sequence of BSw20308 provides new information about the ecological function and adaptation of the genus Pseudoalteromonas in Arctic marine environments, and also indicates its potential applica- tions in the biotechnology industries （e.g., enzymology, and pollutant degradation）.展开更多
文摘Biofilm forming bacteria are omnipresent in the marine environment.Pseudoalteromonas is one of the largest within the γ-proteobacteria class,and a member of marine bacteria.Species of Pseudoalteromonas are generally found in association with marine eukaryotes.In this work,we present the isolation and characterization of two strains forming biofilm on rock surface and associated with marine sponge.They were identified using 16SrDNA as Pseudoalteromonas prydzensis alex,and Pseudoalteromonas sp.alex.They showed the highest titer in biofilm formation quantified using the test tube method using crystal violet.
基金supported by the National High-Tech R&D Program(No.2011AA090703)the National Natural Science Foundation of China(No.31070712)the Special Fund for Marine Scientific Research in the Public Interest(Nos.201105027 and 201005024)
文摘κ-carrageenan oligosaccharides exhibit various biological activities. Enzymatic degradation by κ-carrageenase is safe and controllable. Therefore, κ-carrageenases have captured more and more attentions. In this study, a κ-carrageenase encoding gene, cgk X, was cloned from Pseudoalteromonas sp. QY203 with degenerate and inverse PCR. It comprised an ORF of 1194 bp in length, encoding a protein with 397 amino acid residues. Cgk X is a new member of glycoside hydrolase family 16. The deduced amino acid sequence shared a high similarity with Cgk X of Pseudoalteromonas κ-carrageenase; however, the recombinant Cgk X showed different biochemical characteristics. The recombinant enzyme was most active at p H 7.0 and 55℃ in the presence of 300 mmol L^(^(-1))Na Cl. It was stable in a broad range of acidity ranging from p H 3.0 to p H 10.0 when temperature was below 40℃. More than 80% of its activity was maintained after being incubated at p H 3.6–10.0 and 4℃ for 24 h. Cgk X retained more than 90% of activity after being incubated at 40℃ for 1 h. EDTA and SDS(1 mmol L^(-1)) did not inhibit its activity. Cgk X hydrolyzed κ-carrageenan into disaccharide and tetrasaccharide as an endo-cleaver. All these characteristics demonstrated that Cgk X is applicable to both κ-carrageenan oligosaccharide production and κ-carrageenase structure-function research.
文摘Twenty-seven antarctic bacteria producing extracellular polysaccharide （EPS） were selected from 57 strains by staining technology. The effects of major environmental factors on the growth and EPS production of Pseudoalteromonas sp. S - 15 - 13 were investigated, and the EPS was separated and purified for characterization analysis. The results showed that the optimal conditions for the EPS production were culture period, 56 h; growth temperature, 8 ℃ ; carbon source, 1.0% glucose; NaCI concentration, 3.0% ; pH 6.0 - 7.0. The EPS was purified by cold ethanol precipitation, proteins removal, ion exchange chromatography and gel chromatography technology. The molecular mass of EPS - H was 62 kDa as determined by the high performance gel permeation chromatography. Its sugar composition was a homopolymer of marmose analyzed by gas chromatograph spectroscopy. After repeated freezing and thawing of the bacteria hiomass in the presence of EPS, the bacterial growth was much higher than that observed after freezing in the absence of EPS and the difference augmented with the increase of freeze-thaw cycles. It is hypothesized that the adaptation of Pseudoalteromonas sp. S- 15 - 13 to the antarctic marine conditions, characterized by low temperature, high NaCl concentration and repeated freeze-thaw cycles, might be related to the EPS production ability.
基金The work was supported by the Hi-Tech Research and Development Program of China under contract Nos 2006AA09Z414 and 2007AA091903；the China Ocean Mineral Resources R ＆ D Association under contract No. DYXM - 115 - 02 - 2 - 6；the National Natural Science Foundation of China under contract No. Z2004D02；the Natural Science Foundation of Shandong Province of China under contract No. Z2004D02；the Foundation for Young Excellent Scientists in Shandong Province of China under contract No. 2006BS02002；the Program for New Century Excellent Talents in University under contract No. NCET - 06 - 0578.
文摘Pseudoalteromonas sp. SM9913 is a phychrotmphic bacterium isolated from the deep-sea sediment. The genes encoding chaperones DnaJ and DnaK of P. sp. SM9913 were cloned by normal PCR and TAIL - PCR （GenBank accession Nos DQ640312, DQ504163 ）. The chaperones DnaJ and DnaK from the strain SM9913 contain such conserved domains as those of many other bacteria, and show some cold-adapted characteristics in their structures when compared with those from psychro-, meso-and themophilic bacteria. It is indicated that chaperones DnaJ and DnaK of P. sp. SM9913 may be adapted to low temperature in deep-sea and function well in assisting folding, assembling and translocation of proteins at low temperature. This research lays a foundation for the further study on the cold-adapted mechanism of chaperones DnaJ and DnaK of cold-adapted microorganisms.
基金supported by the National High Technology Research and Development Program of China (Grant no.2007AA091905)the Basic Scientific Research Operation Fund of China (Grant no. GY02-2007-T11)
文摘The psychrotropic bacterium, Pseudoalteromonas sp. QI-I, which produces extracellular cold-active protease, was isolated from Antarctic seawater. The genomic DNA of this bacterium was used to construct a plasmid genomic library with the goal of screening cold-active protease genes. Gene pro-2127 with an open reading frame of 2127 bp encoding protease PRO-2127 was cloned and sequenced. Alignment of amino acid sequences suggested that the precursor of PRO-2127 was a member of subfamily S8A, and that it might contain four domains： a signal peptide, an N-terminal prosequence, a catalytic domain and a C-terminal extension. Amino acids Asp185, His244 and Ser425 might form a catalytic triad. PRO-2127 showed some structural features common to psychrophilic enzymes, such as a decrease in Arg residues and the Arg/（Arg＋Lys） ratio. Heterologous expression of pro-2127 in Escherichia coli BL21 （DE3） by pColdlII was also successfully observed in this study.
基金The National Natural Science Foundation of China under contract Nos 31200097,41576187,U1406402-5 and31202024the Basic Scientific Fund for National Public Research Institutes of China under contract Nos 2013G33 and 2015G10
文摘Genus Pseudoalteromonas belongs to Family Pseudoalteromonadaceae in Gammaproteobacteria. A cold-adapted gram-negative bacterium, hydrocarbon-degrading Pseudoalteromonas sp. NJ289, was isolated from sea-ice of the Antarctica region, and sequenced the whole genome through the next generation sequencing platform. The assembly yielded three contigs representing two chromosomes and one plasmid with the sizes of 3.2 Mb, 636 kb and 1.8 kb, respectively. The G＋C contents of genome were 40.83% and included 3 589 ORFs. Functional annotation indicated some potential roles in enzymatic activity and environmental adaptability. This study may help for understanding the population diverse, evolutionary ecology and the microbial interaction.
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.
基金The National Natural Science Foundation of China under contract No.31401632the Program for New Century Excellent Talents in Fujian Province University,China under contract No.B15139
文摘This study aimed to improve the thermostability of arylsulfatase from Pseudoalteromonas carrageenovora. A library of P. carrageenovora arylsulfatase mutants was constructed by introducing random mutagenesis using error-prone PCR. After screening, two mutants of H260L and D84A/H260L showed enhanced thermal stability than the wild-type predecessor (WT). Site-directed mutagenesis demonstrated that only amino acid residue at Position 260 plays an important role in the thermostability of P. carrageenovora arylsulfatase. Thermal inactivation analysis showed that the half-life (t1/2) values at 55°C for H260L, H260I, H260Q, H260F and H260R were 40.6, 48.4, 30.9, 29.1 and 34.5 min, respectively, while that of WT was 9.1 min. Structure modeling demonstrated that the additional hydrogen bonds and/or optimization of surface charge-charge interactions could be responsible for the increased thermostability imparted by H260L, H260I, H260Q, H260F and H260R.
基金The National Natural Science Foundation of China under contract No.41176174the Public Science and Technology Funds for Ocean Projects under contract No.201205020-5
文摘Oxidative stress is one of the major challenges faced by Arctic marine bacteria due to the high oxygen concentration of seawater, low temperatures and UV radiations. Transcriptome sequencing was performed to obtain the key functional genes involved in the adaptation to oxidative stress induced by hydrogen peroxide in the Arctic bacterium Pseudoalteromonas sp. A2. Exposure to 1 mmol/L H2O2 resulted in large alterations of the transcriptome profile, including significant up-regulation of 109 genes and significant down-regulation of 174 genes. COG functional classification revealed that among the significantly regulated genes with known function categories, more genes belonging to posttranslational modification, protein turnover and chaperones were significantly up-regulated, and more genes affiliated with chaperones and amino acid transport and metabolism were significantly down-regulated. It was notable that the expressions of eighteen genes affiliated with flagella and four genes affiliated with heat shock proteins were significantly up-regulated. Meanwhile, the expression of nine genes belonging to cytochrome and cytochrome oxidase, and five genes belonging to Ton B-dependent receptor,were significantly down-regulated. Among the eighteen genes with antioxidant activity categorized by GO analysis, the expression of one gene was significantly up-regulated; however, the expressions of two genes were significantly down-regulated. Briefly, RNA-Seq indicated that, except for the classical anti-oxidative genes and stress proteins, genes affiliated with flagella and function unknown played important roles in coping with oxidative stress in Pseudoalteromonas sp. A2. This overall survey of transcriptome and oxidative stress-relevant genes can contribute to understand the adaptive mechanism of Arctic bacteria.
基金supported by the National Natural Science Foundation of China(Grant no.41076131)the Chinese Polar Environment Comprehensive Investigation and Assessment Program(Grant nos.CHI-NARE2013-02-01,CHINARE2013-04-01)the National High-Tech Re-search and Development Program of China(Grant no.2012AA021706)
文摘The genus Pseudoalteromonas is ubiquitous in the marine environment and can synthesize a wide range of extracellular compounds. Psychrotolerant Pseudoalteromonas sp. BSw20308 was isolated from the Chukchi Sea, a marginal sea of the Arctic Ocean. It produces a number of extracellular enzymes that can degrade polysaccharides and proteins. The BSw20308 genome was sequenced to 38.1-fold coverage, and the sequences were assembled into 146 contigs （〉~500 bp）. In total, 4 172 open reading frames （ORFs） with an average gene length of 987 bp were detected. At least 86 ORFs were predicted by sequence analysis to encode a variety of catalytic modules involved in the degradation of polysaccharides, proteins, and lipids. In addition, 36 ORFs were predicted to encode catalytic modules involved in the degradation of organic pollutants and halogenated compounds, and in the production of bioactive compounds. The draft genome sequence of BSw20308 provides new information about the ecological function and adaptation of the genus Pseudoalteromonas in Arctic marine environments, and also indicates its potential applica- tions in the biotechnology industries （e.g., enzymology, and pollutant degradation）.
