Pseudomonas stutzeri caused an outbreak of freshwater fish in Luwuk Banggai （tilapia and catfish）, Bali （tilapia）, Jambi （tilapia and catfish） and Tanjung Pinang （catfish）. The study was purposed to comprehens...Pseudomonas stutzeri caused an outbreak of freshwater fish in Luwuk Banggai （tilapia and catfish）, Bali （tilapia）, Jambi （tilapia and catfish） and Tanjung Pinang （catfish）. The study was purposed to comprehensively identify special phenotypic and genotypic characteristics of P. stutzeri isolated from several areas in Indonesia, including its morphometric and biochemical characteristics and molecular variation. Bacteria were isolated from internal organs （kidney, ulcer and eye） of fish. They were then identified using morphology and biochemical test. DNA isolates were entirely extracted, amplified and reversed on 16S rRNA region, and further then were sequenced. Phylogenetic trees of bacteria were constructed using neighbor-joining and maximum-parsimony methods. The colony were similar, such as rod shape （Jambi, Tanjung Pinang, Bali）, bacil shape （Luwuk Banggai）, transparant in tryptic soy agar （TSA） （Luwuk Banggai）, creamy beige in glutamate starch phenol red （GSP） （Bali）, gram negative, motile, no reaction in the oxidative-fermentative test, positive result in catalase and oxidase test, negative in lysine decarboxylase and ornithine decarboxylase test and positive result in indole test; gelatin was degraded （only Bali）, urea was not degraded, no color change in Methyl-red and Voges-proskaeur （MR-VP） test; acid not produce from glucose, inositol or sucrose. Citrate was utilized by some isolates： positive （Jambi, Tanjung Pinang） and negative （Bali, Luwuk Banggai）. Results showed us that isolates of Jambi, Bali and Tanjung Pinang were monophyletic species with P. stutzeri $8 and ZH-1 comparing to gen bank. However, merely phenotypic analysis among Pseudomonas sp. was confused compared to each other.展开更多
C4-dicarboxylate transport proteins of di-azotroph Pseudomonas stutzeri were encoded by dctPQM genes. Nucleotide sequence analysis indicated that dctP, dctQ, and dctM grouped together. Its nucleotide and amino acid se...C4-dicarboxylate transport proteins of di-azotroph Pseudomonas stutzeri were encoded by dctPQM genes. Nucleotide sequence analysis indicated that dctP, dctQ, and dctM grouped together. Its nucleotide and amino acid sequence shared high homology with that of dctP gene en-coding periplasmic C4-dicarboxylate-binding protein and dctQM genes encoding C4-dicarboxylate transport proteins from the free-living nitrogen-fixing Aotobacter vinelandii. Structural analysis showed that DctP of P. stutzeri did not include membrane-spanning regions, and DctQ and DctM contained 5 and 12 transmembrane segments, respectively. The fragment containing the complete dctPQM genes was cloned into the Tn5 transposon region of suicide mobiliza-tion plasmid pSZ21. The resultant plasmid was named pSZY6. By triparental mating, Tn5 transposon carrying the dctPQM genes inserted into the genome of the wild type strain A1501, randomly. The recombinant strain A-142 which harboured an extra copy of dctPQM genes was con-structed and identified by PCR amplification of npt II gene. When A-142 was grown in minimal medium with different concentrations (20, 10 and 5 mmol/L) of C4-dicarboxylates succinate, malate, or fumarate as the sole carbon source, the rate of nitrogen fixation assayed by acetylene reduction was significantly higher than that of the wild-type strain A1501. This result was established that an extra copy of dctPQM genes could increase the activity of nitrogen fixation of P. stutzeri strain A1501.展开更多
文摘Pseudomonas stutzeri caused an outbreak of freshwater fish in Luwuk Banggai （tilapia and catfish）, Bali （tilapia）, Jambi （tilapia and catfish） and Tanjung Pinang （catfish）. The study was purposed to comprehensively identify special phenotypic and genotypic characteristics of P. stutzeri isolated from several areas in Indonesia, including its morphometric and biochemical characteristics and molecular variation. Bacteria were isolated from internal organs （kidney, ulcer and eye） of fish. They were then identified using morphology and biochemical test. DNA isolates were entirely extracted, amplified and reversed on 16S rRNA region, and further then were sequenced. Phylogenetic trees of bacteria were constructed using neighbor-joining and maximum-parsimony methods. The colony were similar, such as rod shape （Jambi, Tanjung Pinang, Bali）, bacil shape （Luwuk Banggai）, transparant in tryptic soy agar （TSA） （Luwuk Banggai）, creamy beige in glutamate starch phenol red （GSP） （Bali）, gram negative, motile, no reaction in the oxidative-fermentative test, positive result in catalase and oxidase test, negative in lysine decarboxylase and ornithine decarboxylase test and positive result in indole test; gelatin was degraded （only Bali）, urea was not degraded, no color change in Methyl-red and Voges-proskaeur （MR-VP） test; acid not produce from glucose, inositol or sucrose. Citrate was utilized by some isolates： positive （Jambi, Tanjung Pinang） and negative （Bali, Luwuk Banggai）. Results showed us that isolates of Jambi, Bali and Tanjung Pinang were monophyletic species with P. stutzeri $8 and ZH-1 comparing to gen bank. However, merely phenotypic analysis among Pseudomonas sp. was confused compared to each other.
基金supported by the National Basic Research Priorities Programme(Grant No.001CB108904)the National Natural Science Foundation of China(Grant No.39580001).
文摘C4-dicarboxylate transport proteins of di-azotroph Pseudomonas stutzeri were encoded by dctPQM genes. Nucleotide sequence analysis indicated that dctP, dctQ, and dctM grouped together. Its nucleotide and amino acid sequence shared high homology with that of dctP gene en-coding periplasmic C4-dicarboxylate-binding protein and dctQM genes encoding C4-dicarboxylate transport proteins from the free-living nitrogen-fixing Aotobacter vinelandii. Structural analysis showed that DctP of P. stutzeri did not include membrane-spanning regions, and DctQ and DctM contained 5 and 12 transmembrane segments, respectively. The fragment containing the complete dctPQM genes was cloned into the Tn5 transposon region of suicide mobiliza-tion plasmid pSZ21. The resultant plasmid was named pSZY6. By triparental mating, Tn5 transposon carrying the dctPQM genes inserted into the genome of the wild type strain A1501, randomly. The recombinant strain A-142 which harboured an extra copy of dctPQM genes was con-structed and identified by PCR amplification of npt II gene. When A-142 was grown in minimal medium with different concentrations (20, 10 and 5 mmol/L) of C4-dicarboxylates succinate, malate, or fumarate as the sole carbon source, the rate of nitrogen fixation assayed by acetylene reduction was significantly higher than that of the wild-type strain A1501. This result was established that an extra copy of dctPQM genes could increase the activity of nitrogen fixation of P. stutzeri strain A1501.
