ABSTRACT
In this study, a superoxide dismutase gene (PsSOD) from Pseudoalteromonas sp. ANT506 was cloned and over expressed in Escherichia coli. The PsSOD has an open reading frame of 582 bp with a putative product of 193 amino acid residue and an estimated molecular size of 21.4 kDa. His-tagged PsSOD was subsequently purified 12.6-fold by Ni-affinity chromatography and the yield of 22.9%. The characterization of the purified rPsSOD exhibited maximum activity at 30 °C and pH 8.0. The enzyme exhibited 13.9% activity at 0 °C and had high-thermo lability at higher than 50 °C. rPsSOD exhibited well capability to 2.5 M NaCl (62.4%). These results indicated that rPsSOD exhibited special catalytic properties.
Subject(s)
Bacterial Proteins/genetics , Escherichia coli/genetics , Pseudoalteromonas/enzymology , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Amino Acid Sequence , Antarctic Regions , Catalysis , Cloning, Molecular , Escherichia coli/metabolism , Hydrogen Peroxide/chemistry , Open Reading Frames/genetics , Pseudoalteromonas/genetics , Pseudoalteromonas/metabolism , Sequence AlignmentABSTRACT
An efficient conjugation method has been developed for the marine Actinomyces sp. isolate M048 to facilitate the genetic manipulation of the chandrananimycin biosynthesis gene cluster. A phiC31-derived integration vector pIJ8600 containing oriT and attP fragments was introduced into strain M048 by bi-parental conjugation from Escherichia coli ET12567 to strain M048. Transformation efficiency was (6.38+/-0.41)x10(-5) exconjugants per recipient spore. Analysis of eight exconjugants showed that the plasmid pIJ8600 was stably integrated at a single chromosomal site (attB) of the Actinomyces genome. The DNA sequence of the attB was cloned and shown to be conserved. The results of antimicrobial activity analysis indicated that the insertion of plasmid pIJ8600 seemed to affect the biosynthesis of antibiotics that could strongly inhibit the growth of E. coli and Mucor miehei (Tü284). HPLC-MS analysis of the extracts indicated that disruption of the attB site resulted in the complete abolition of chandrananimycin A-C production, proving the identity of the gene cluster. Instead of chandrananimycins, two bafilomycins were produced through disruption of the attB site from the chromosomal DNA of marine Actinomyces sp. M048.
Subject(s)
Actinomyces/genetics , Attachment Sites, Microbiological/genetics , DNA/genetics , Genetic Vectors/genetics , Transformation, Bacterial/genetics , Anti-Bacterial Agents , Conjugation, Genetic/genetics , Escherichia coli/genetics , Mutagenesis, Site-Directed , Oceans and Seas , Plasmids/genetics , Sequence Analysis, DNA , Substrate SpecificityABSTRACT
Marine Streptomyces are potential candidates for novel natural products and industrial catalysts. In order to set up biosynthesis approach for a holomycin-producing strain M095 isolated from Jiaozhou Bay, China, a genetic transformation system was established using intergeneric conjugation. The plasmid pIJ8600 consists of an origin of replication for Escherichia coli, a phage integrase directing efficient site-specific integration in bacterial chromosome, thiostrepton-induced promoter and an attP sequence. Using E. coli ET12567 (pUZ8002) carrying pIJ8600 as a conjugal donor, while it was mated with strain M095, pIJ8600 was mobilized to the recipient and the transferred DNA was also integrated into the recipient chromosome. The frequency of exconjugants was 1.9+/-0.13x10(-4) per recipient cell. Analysis of eight exconjugants showed pIJ8600 was stable integrated at a single chromosomal site (attB) of the Streptomyces genome. The DNA sequence of the attB was cloned and shown to be conserved. The results of growth and antimicrobial activity analysis indicated that the integration of pIJ8600 did not seem to affect the biosynthesis of antibiotics or other essential amino acids. To demonstrate the feasibility of above gene transfer system, the allophycocyanin gene (apc) from cyanobacterium Anacystis nidulans UTEX625 was expressed in strain M095, and the results indicated heterologous allophycocyanin could be expressed and folded effectively.
Subject(s)
Bacterial Proteins/metabolism , Conjugation, Genetic , Escherichia coli/genetics , Lactams/metabolism , Plasmids , Streptomyces/genetics , Attachment Sites, Microbiological , Base Sequence , China , Genome, Bacterial , Marine Biology , Molecular Sequence Data , Oceans and Seas , Sequence Alignment , Streptomyces/isolation & purification , Streptomyces/metabolism , Transformation, Bacterial , Water MicrobiologyABSTRACT
Marinomonas sp. NJ522, isolated from Antarctic sea ice, produces a cold-active iron superoxide dismutase (SOD; EC 1.15.1.1). The purified SOD was dimeric and had an approx. Mr of 48 kDa. Highest activity was detected from pH 8 to 10 and at 40 degrees C (assayed over 10 min). Activity at 0 degree C was nearly 35% of the maximum activity.
Subject(s)
Bacterial Proteins/isolation & purification , Gram-Negative Bacteria/enzymology , Superoxide Dismutase/isolation & purification , Bacterial Proteins/chemistry , Cold Temperature , Hot Temperature , Hydrogen-Ion Concentration , Superoxide Dismutase/chemistryABSTRACT
Colwellia sp. NJ341, isolated from Antarctic sea ice, secreted a cold-active serine protease. The purified protease had an apparent Mr of 60 kDa by SDS-PAGE and MALDI-TOF MS. It was active from pH 5-12 with maximum activity at 35 degrees C (assayed over 10 min). Activity at 0 degrees C was nearly 30% of the maximum activity. It was completely inhibited by phenylmethylsulfonyl fluoride.
Subject(s)
Cold Temperature , Gammaproteobacteria/classification , Gammaproteobacteria/metabolism , Serine Endopeptidases/chemistry , Serine Endopeptidases/isolation & purification , Catalysis , Enzyme Activation , Enzyme Stability , Hydrogen-Ion Concentration , Serine Endopeptidases/analysis , Species SpecificityABSTRACT
Glutathione (GSH) and GSH-related enzymes play a great role in protecting organisms from oxidative damage. The GSH level and GSH-related enzymes activities were investigated as well as the growth yield and malonyldialdehyde (MDA) content in the Antarctic ice microalga Chlamydomonas sp. ICE-L exposure to the different cadmium concentration in this paper. The results showed that the higher concentration Cd inhibited the growth of ICE-L significantly and Cd would induce formation of MDA. At the same time, it is clear that GSH level, glutathione peroxidases (GPx) activity and glutathione S-transferases (GST), activity were higher in ICE-L exposed to Cd than the control. But GR activity dropped notably when ICE-L were cultured in the medium containing Cd. Increase of GSH level, GPx and GST activities acclimate to oxidative stress induced by Cd and protect Antarctic ice microalga Chlamydomonas sp. ICE-L from toxicity caused by Cd exposure. These parameters may be used to assess the biological impact of Cd in the Antarctic pole region environment.