Objective: Based on a partialsubtilisin-like protease, Prl genomic sequence ofPythium guiyangense which has been cloned before, Panhandle PCR strategy was used to amplify the upstream flanking sequence adjacent to th...Objective: Based on a partialsubtilisin-like protease, Prl genomic sequence ofPythium guiyangense which has been cloned before, Panhandle PCR strategy was used to amplify the upstream flanking sequence adjacent to the known sequence of the Prl gene. Methods: The genomic DNA was firstly digested with BamH I and then treated with calf intestinal alkaline phosphatase(CIAP). Next, a 5' phosphorylated oligonucleotide was ligated to the 5' ends of BamH I -digested DNA. After denaturation, intmstrand annealing and polymemse extension, a pan with a handle was formed,and lastly the nested PCR was performed. Results: A 864 bp product was amplified,which was adjacent to the known sequence of Prl gene.The gene has been accessed by GenBank (Accession:JQ975036). Conclusion: Panhandle PCR is a quick and convenient approach for amplifying and identifying unknown partner genes,which facilitates cloning full-length Prl gene展开更多
A psychrophilic bacterium strain 547 producing cold-adaptive alkaline protease was isolated from the deep sea sediment of Prydz Bay, Antarctica. The organism was identified as a Planomicrobium species by 16S rRNA anal...A psychrophilic bacterium strain 547 producing cold-adaptive alkaline protease was isolated from the deep sea sediment of Prydz Bay, Antarctica. The organism was identified as a Planomicrobium species by 16S rRNA analysis. The optimal and highest growth temperatures for strain 547 were 15~C and 30~C, respectively. The extracellular protease was purified by ammonium sulfate precipitation and DEAE cellulose-52 chromatography. The optimal temperature and pH for the activity of the purified enzyme were 35~C and pH 9.0, respectively. The enzyme retained approximately 40% of its activity after 2 h of incubation at 50℃. The enzymatic activity was inhibited by 1 mmol/L phenylmethyl sulfonylfluoride (PMSF) and hydrochloride 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), indicating that it was a serine protease. The presence of Cae+ and Mnz+ increased the activity of the enzyme. The protease gene with a size of 1 269 bp was cloned from Planomicrobium sp. 547 using primers designed based on the conserved sequences of proteases in GenBank. The Planomicrobium sp. 547 protease contained a domain belonging to the peptidase S8 family, which has a length of 309 amino acid (AA) residues. The alignment and phylogenetic analysis of the AA sequence indicated that the protease belonged to the subtilisin family.展开更多
We report the isolation of a cold-adapted bacterium belonging to the genus Janthinobacterium (named AU 11), from a water sample collected in Lake Uruguay (King George Island, South Shetlands). AUI 1 (growth betwe...We report the isolation of a cold-adapted bacterium belonging to the genus Janthinobacterium (named AU 11), from a water sample collected in Lake Uruguay (King George Island, South Shetlands). AUI 1 (growth between 4℃ and 30℃) produces a single cold-active extracellular protease (ExPAU11), differentially expressed at low temperature. ExPAU11 was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) as an alkaline metallo-protease (70% coverage with an extracellular protease of Janthinobacterium sp. PI12), and by protease-inhibitor screening identified as a serine-protease. To the best of our knowledge this is the first experimental evidence of a cold-active extracellular protease produced by Janthinobacterium. Furthermore, we identified a serine-protease gene (named JSP8A) showing 60% identity (98% query coverage) to subtilisin peptidases belonging to the $8 family (S8A subfamily) of many cyanobacteria. A phylogenetic analysis of the JSP8A protease, along with related bacterial protein sequences, confirms that JSP8A clusters with S8A subtilisin sequences from different cyanobacteria, and is clearly separated from SSA bacterial sequences of other phyla (including its own). An analysis of the genomic organization around JSP8A suggests that this protease gene was acquired in an event that duplicated a racemase gene involved in transforming L- to D-amino acids. Our results suggest that AU11 probably acquired this subtilisin-like protease gene by horizontal gene transfer (HGT) from a cyanobacterittrn. We discuss the relevance of a bacterial protease-HGT in the Antarctic environment in light of this hypothesis.展开更多
基金Supported by the Guangxi Department of education scientific research funds,China(No.200103YB154)
文摘Objective: Based on a partialsubtilisin-like protease, Prl genomic sequence ofPythium guiyangense which has been cloned before, Panhandle PCR strategy was used to amplify the upstream flanking sequence adjacent to the known sequence of the Prl gene. Methods: The genomic DNA was firstly digested with BamH I and then treated with calf intestinal alkaline phosphatase(CIAP). Next, a 5' phosphorylated oligonucleotide was ligated to the 5' ends of BamH I -digested DNA. After denaturation, intmstrand annealing and polymemse extension, a pan with a handle was formed,and lastly the nested PCR was performed. Results: A 864 bp product was amplified,which was adjacent to the known sequence of Prl gene.The gene has been accessed by GenBank (Accession:JQ975036). Conclusion: Panhandle PCR is a quick and convenient approach for amplifying and identifying unknown partner genes,which facilitates cloning full-length Prl gene
基金supported by the National High Technology Research and Development Program of China (Grant no. 2007AA091407)the Project of China Ocean Mineral Resources Research and Development Association (COMRA, Grant no. DYXM-115-02-2-04)
文摘A psychrophilic bacterium strain 547 producing cold-adaptive alkaline protease was isolated from the deep sea sediment of Prydz Bay, Antarctica. The organism was identified as a Planomicrobium species by 16S rRNA analysis. The optimal and highest growth temperatures for strain 547 were 15~C and 30~C, respectively. The extracellular protease was purified by ammonium sulfate precipitation and DEAE cellulose-52 chromatography. The optimal temperature and pH for the activity of the purified enzyme were 35~C and pH 9.0, respectively. The enzyme retained approximately 40% of its activity after 2 h of incubation at 50℃. The enzymatic activity was inhibited by 1 mmol/L phenylmethyl sulfonylfluoride (PMSF) and hydrochloride 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), indicating that it was a serine protease. The presence of Cae+ and Mnz+ increased the activity of the enzyme. The protease gene with a size of 1 269 bp was cloned from Planomicrobium sp. 547 using primers designed based on the conserved sequences of proteases in GenBank. The Planomicrobium sp. 547 protease contained a domain belonging to the peptidase S8 family, which has a length of 309 amino acid (AA) residues. The alignment and phylogenetic analysis of the AA sequence indicated that the protease belonged to the subtilisin family.
基金supported by PEDECIBA (Programa De Desarrollo de las Ciencias Básicas), Uruguay, and IAU (Instituto Antártico Uruguayo)supported by ANII (Agencia Nacional de Investigación e Innovación)
文摘We report the isolation of a cold-adapted bacterium belonging to the genus Janthinobacterium (named AU 11), from a water sample collected in Lake Uruguay (King George Island, South Shetlands). AUI 1 (growth between 4℃ and 30℃) produces a single cold-active extracellular protease (ExPAU11), differentially expressed at low temperature. ExPAU11 was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) as an alkaline metallo-protease (70% coverage with an extracellular protease of Janthinobacterium sp. PI12), and by protease-inhibitor screening identified as a serine-protease. To the best of our knowledge this is the first experimental evidence of a cold-active extracellular protease produced by Janthinobacterium. Furthermore, we identified a serine-protease gene (named JSP8A) showing 60% identity (98% query coverage) to subtilisin peptidases belonging to the $8 family (S8A subfamily) of many cyanobacteria. A phylogenetic analysis of the JSP8A protease, along with related bacterial protein sequences, confirms that JSP8A clusters with S8A subtilisin sequences from different cyanobacteria, and is clearly separated from SSA bacterial sequences of other phyla (including its own). An analysis of the genomic organization around JSP8A suggests that this protease gene was acquired in an event that duplicated a racemase gene involved in transforming L- to D-amino acids. Our results suggest that AU11 probably acquired this subtilisin-like protease gene by horizontal gene transfer (HGT) from a cyanobacterittrn. We discuss the relevance of a bacterial protease-HGT in the Antarctic environment in light of this hypothesis.