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
A convenient and widely applicable method has been developed to clone aniline metabolic gene cluster in this study. Three positive recombinant plasmids pDA1, pDB2 and pDB11 were cloned from genomic library of aniline ...A convenient and widely applicable method has been developed to clone aniline metabolic gene cluster in this study. Three positive recombinant plasmids pDA1, pDB2 and pDB11 were cloned from genomic library of aniline degradation strain AD9. The result of aniline dioxygenase (AD) activity and catechol 2,3-oxygenase (C23O) activity assay showed that pDA1 and pDB11 contain aniline dioxy-genase genes and catechol 2,3-dioxygenase genes, respectively. The sequence analysis of the total 24.7-kb region revealed that this region contains 25 ORFs, of which 17 genes involve metabolism of aniline. In the gene cluster, the first five genes (tadQTA1A2B) and the subsequent gene (tadR1) were pre-dicted to encode a multi-component aniline dioxygenase and a LysR-type regulator, respectively, while the others (tadD1C1D2C2EFGIJKL) were expected to encode meta- cleavage pathway enzymes for catechol degradation. The gene cluster was surrounded by two IS1071 sequences.展开更多
Pseudomonas stutzeri A1501, an associative nitrogen-fixing bacterium, was isolated from the rice paddy rhizosphere. This bacterium fixes nitrogen under microaerobic conditions. In this study, ge- nome-wide DNA microar...Pseudomonas stutzeri A1501, an associative nitrogen-fixing bacterium, was isolated from the rice paddy rhizosphere. This bacterium fixes nitrogen under microaerobic conditions. In this study, ge- nome-wide DNA microarrays were used to analyze the global transcription profile of A1501 under aerobic and microaerobic conditions. The expression of 135 genes was significantly altered by more than 2-fold in response to oxygen stress. Among these genes, 68 were down-regulated under aerobic conditions; these genes included those responsible for nitrogen fixation and denitrification. Sixty- seven genes were up-regulated under aerobic conditions; these genes included sodC, encoding a copper-zinc superoxide dismutase, PST2179, encoding an NAD(P)-dependent oxidoreductase, PST3584, encoding a 2OG-Fe(II) oxygenase, and PST3602, encoding an NAD(P)H-flavin oxidoreductase. Addi- tionally, seven genes involved in capsular polysaccharide and antigen oligosaccharide biosynthesis together with 17 genes encoding proteins of unknown function were up-regulated under aerobic con- ditions. The overall analysis suggests that the genes we identified are involved in the protection of the bacterium from oxygen, but the mechanisms of their action remain to be elucidated.展开更多
A mutagenesis library was constructed using GPS-LS system to insert a random 5 aa into the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoded by aroA gene. Active EPSPS pro- teins were identified by the abili...A mutagenesis library was constructed using GPS-LS system to insert a random 5 aa into the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoded by aroA gene. Active EPSPS pro- teins were identified by the ability to rescue growth of aroA-deleted mutant ER2799 on M9 minimal media. 12 unique sites, which can tolerate a 5-aa insertion, were identified. In all of the 12 sites, only F295/T296 site was found to split the G2-EPSPS properly by co-transformation of plasmids into E. coli ER2799. The G2-EPSPS gene was then divided into N-termi- nal and C-terminal from F295/T296 site which were fused to the N-terminal and C-terminal of Ssp.DnaE intein, respectively, creating two plasmids pMEPS- N295IN and pKEPSc296Ic. Co-transformation of plasmids, pMEPSN295IN and pKEPSc296Ic, rescu- ed growth of ER2799 in M9 minimal media, indicating that the intein splicing domains were bringing the EPSPS fragments together to generate activity. Re- consituted activity of splitted G2-EPSPS enzyme was 4.48 U/mg.展开更多
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.
基金This work was supported by 863 Project(Grant No.2005AA226030)of the Ministry of Science and Technologythe National Natural Science Foundation of China(Grant Nos.30470047&30200007).
文摘A convenient and widely applicable method has been developed to clone aniline metabolic gene cluster in this study. Three positive recombinant plasmids pDA1, pDB2 and pDB11 were cloned from genomic library of aniline degradation strain AD9. The result of aniline dioxygenase (AD) activity and catechol 2,3-oxygenase (C23O) activity assay showed that pDA1 and pDB11 contain aniline dioxy-genase genes and catechol 2,3-dioxygenase genes, respectively. The sequence analysis of the total 24.7-kb region revealed that this region contains 25 ORFs, of which 17 genes involve metabolism of aniline. In the gene cluster, the first five genes (tadQTA1A2B) and the subsequent gene (tadR1) were pre-dicted to encode a multi-component aniline dioxygenase and a LysR-type regulator, respectively, while the others (tadD1C1D2C2EFGIJKL) were expected to encode meta- cleavage pathway enzymes for catechol degradation. The gene cluster was surrounded by two IS1071 sequences.
基金the National Basic Research Program of China (Grant Nos. 2001CB108904 and 2007CB707805) High-Technology Research Development Program of China (Grant Nos. 2006AA020202 and 2006AA0Z229))
文摘Pseudomonas stutzeri A1501, an associative nitrogen-fixing bacterium, was isolated from the rice paddy rhizosphere. This bacterium fixes nitrogen under microaerobic conditions. In this study, ge- nome-wide DNA microarrays were used to analyze the global transcription profile of A1501 under aerobic and microaerobic conditions. The expression of 135 genes was significantly altered by more than 2-fold in response to oxygen stress. Among these genes, 68 were down-regulated under aerobic conditions; these genes included those responsible for nitrogen fixation and denitrification. Sixty- seven genes were up-regulated under aerobic conditions; these genes included sodC, encoding a copper-zinc superoxide dismutase, PST2179, encoding an NAD(P)-dependent oxidoreductase, PST3584, encoding a 2OG-Fe(II) oxygenase, and PST3602, encoding an NAD(P)H-flavin oxidoreductase. Addi- tionally, seven genes involved in capsular polysaccharide and antigen oligosaccharide biosynthesis together with 17 genes encoding proteins of unknown function were up-regulated under aerobic con- ditions. The overall analysis suggests that the genes we identified are involved in the protection of the bacterium from oxygen, but the mechanisms of their action remain to be elucidated.
文摘A mutagenesis library was constructed using GPS-LS system to insert a random 5 aa into the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoded by aroA gene. Active EPSPS pro- teins were identified by the ability to rescue growth of aroA-deleted mutant ER2799 on M9 minimal media. 12 unique sites, which can tolerate a 5-aa insertion, were identified. In all of the 12 sites, only F295/T296 site was found to split the G2-EPSPS properly by co-transformation of plasmids into E. coli ER2799. The G2-EPSPS gene was then divided into N-termi- nal and C-terminal from F295/T296 site which were fused to the N-terminal and C-terminal of Ssp.DnaE intein, respectively, creating two plasmids pMEPS- N295IN and pKEPSc296Ic. Co-transformation of plasmids, pMEPSN295IN and pKEPSc296Ic, rescu- ed growth of ER2799 in M9 minimal media, indicating that the intein splicing domains were bringing the EPSPS fragments together to generate activity. Re- consituted activity of splitted G2-EPSPS enzyme was 4.48 U/mg.
