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Archives of Microbiology (v.191, #10)
Characterization of osmolyte betaine synthesizing sarcosine dimethylglycine N-methyltransferase from Methanohalophilus portucalensis by Shih-Ya Chen; Mei-Chin Lai; Shu-Jung Lai; Yu-Chien Lee (pp. 735-743).
To overcome the extracellular salt stress, Methanohalophilus portucalensis FDF1T synthesizes the compatible solute betaine through the methylation of glycine, sarcosine, and N,N-dimethylglycine. S-adenosylmethionine (AdoMet) is the methyl donor. The enzyme sarcosine dimethylglycine N-methyltransferase (SDMT) of M. portucalensis, that catalyzes the formation of N,N-dimethylglycine and glycine betaine, has been purified and characterized. SDMT, a monomer of 33 kDa with a pI at 5.03, has a narrow substrate specificity limited to using only sarcosine and dimethylglycine as substrates for the methyl transferase reaction. The K m values for sarcosine and AdoMet were 2.29 and 0.21 mM, respectively, with a V max of 0.83 μmol/mg-min (k cat value of 0.44 s−1). The K m values for dimethylglycine and AdoMet were 3.76 and 0.59 mM, respectively, with a V max of 4.88 μmol/mg-min (k cat of 2.68 s−1). A high concentration of the end product betaine (2.0 M) did not affect the SMT activity, but it slightly inhibited the DMT activity. Both activities were also not affected by potassium or sodium ions in concentrations of 200–1,000 mM. We compared this novel archaeal SDMT enzyme to other similar bacterial transferases as well as to the glycine sarcosine dimethylglycine methyltransferase found also in M. portucalensis.
Keywords: Compatible solutes; Betaine; Sarcosine dimethylglycine N-methyltransferase; Methanogen; Archaea
Characterization of osmolyte betaine synthesizing sarcosine dimethylglycine N-methyltransferase from Methanohalophilus portucalensis by Shih-Ya Chen; Mei-Chin Lai; Shu-Jung Lai; Yu-Chien Lee (pp. 735-743).
To overcome the extracellular salt stress, Methanohalophilus portucalensis FDF1T synthesizes the compatible solute betaine through the methylation of glycine, sarcosine, and N,N-dimethylglycine. S-adenosylmethionine (AdoMet) is the methyl donor. The enzyme sarcosine dimethylglycine N-methyltransferase (SDMT) of M. portucalensis, that catalyzes the formation of N,N-dimethylglycine and glycine betaine, has been purified and characterized. SDMT, a monomer of 33 kDa with a pI at 5.03, has a narrow substrate specificity limited to using only sarcosine and dimethylglycine as substrates for the methyl transferase reaction. The K m values for sarcosine and AdoMet were 2.29 and 0.21 mM, respectively, with a V max of 0.83 μmol/mg-min (k cat value of 0.44 s−1). The K m values for dimethylglycine and AdoMet were 3.76 and 0.59 mM, respectively, with a V max of 4.88 μmol/mg-min (k cat of 2.68 s−1). A high concentration of the end product betaine (2.0 M) did not affect the SMT activity, but it slightly inhibited the DMT activity. Both activities were also not affected by potassium or sodium ions in concentrations of 200–1,000 mM. We compared this novel archaeal SDMT enzyme to other similar bacterial transferases as well as to the glycine sarcosine dimethylglycine methyltransferase found also in M. portucalensis.
Keywords: Compatible solutes; Betaine; Sarcosine dimethylglycine N-methyltransferase; Methanogen; Archaea
Cyclic AMP regulates the biosynthesis of cellobiohydrolase in Cellulomonas flavigena growing in sugar cane bagasse by Jesús Antonio Herrera-Herrera; Odilia Pérez-Avalos; Luis M. Salgado; Teresa Ponce-Noyola (pp. 745-750).
Cellulomonas flavigena produces a battery of cellulase components that act concertedly to degrade cellulose. The addition of cAMP to repressed C. flavigena cultures released catabolic repression, while addition of cAMP to induced C. flavigena cultures led to a cellobiohydrolase hyperproduction. Exogenous cAMP showed positive regulation on cellobiohydrolase production in C. flavigena grown on sugar cane bagasse. A C. flavigena cellobiohydrolase gene was cloned (named celA), which coded for a 71- kDa enzyme. Upstream, a repressor celR1, identified as a 38 kDa protein, was monitored by use of polyclonal antibodies.
Keywords: Cellobiohydrolase; Cellulomonas flavigena ; cAMP regulation
Cyclic AMP regulates the biosynthesis of cellobiohydrolase in Cellulomonas flavigena growing in sugar cane bagasse by Jesús Antonio Herrera-Herrera; Odilia Pérez-Avalos; Luis M. Salgado; Teresa Ponce-Noyola (pp. 745-750).
Cellulomonas flavigena produces a battery of cellulase components that act concertedly to degrade cellulose. The addition of cAMP to repressed C. flavigena cultures released catabolic repression, while addition of cAMP to induced C. flavigena cultures led to a cellobiohydrolase hyperproduction. Exogenous cAMP showed positive regulation on cellobiohydrolase production in C. flavigena grown on sugar cane bagasse. A C. flavigena cellobiohydrolase gene was cloned (named celA), which coded for a 71- kDa enzyme. Upstream, a repressor celR1, identified as a 38 kDa protein, was monitored by use of polyclonal antibodies.
Keywords: Cellobiohydrolase; Cellulomonas flavigena ; cAMP regulation
Display of α-amylase on the surface of Corynebacterium glutamicum cells by using NCgl1221 as the anchoring protein, and production of glutamate from starch by Wenjuan Yao; Chunli Chu; Xiaozhao Deng; Yun Zhang; Miao Liu; Pu Zheng; Zhihao Sun (pp. 751-759).
We developed a new cell surface display system in Corynebacterium glutamicum based on the C-terminally truncated NCgl1221 anchor protein to increase l-glutamate production from starch directly. The C-terminally truncated NCgl1221 protein is a mutant NCgl1221 and leads to the constitutive export of l-glutamate. The N terminus of α-amylase (AmyA) was fused to truncated NCgl1221, and the resulting fusion protein was expressed on the cell surface by IPTG induction. Localization of the fusion protein was confirmed by immunofluorescence microscopy and flow cytometric analysis. The results of l-glutamate fermentation showed that the soluble starch was utilized to grow and produce l-glutamate by the recombinant strain displaying AmyA. The amount of soluble starch was reduced from 30.0 ± 2.8 to 4.5 ± 0.7 g/l under non-inducing condition and from 50.0 ± 2.4 to 12.5 ± 1.1 g/l under biotin limitation in 36 h. The glutamate concentration in the medium was transiently increased in 14 h under no induction, while under biotin-limiting condition, glutamate production was continuously elevated during fermentation. The amount of glutamate reached 19.3 ± 2.1 g/l after 26 h of fermentation with biotin limitation, which was greater than that produced by the strain using PgsA, one of the poly-γ-glutamate synthetase complexes, as the anchor protein under the same condition. Therefore, the truncated NCgl1221 anchor protein has more advantages than the PgsA anchor protein in glutamate fermentation because truncated NCgl1221 leads to the constitutive export of l-glutamate without any treatments.
Keywords: Corynebacterium glutamicum ; α-Amylase; Soluble starch; Glutamate production; Cell surface display
Display of α-amylase on the surface of Corynebacterium glutamicum cells by using NCgl1221 as the anchoring protein, and production of glutamate from starch by Wenjuan Yao; Chunli Chu; Xiaozhao Deng; Yun Zhang; Miao Liu; Pu Zheng; Zhihao Sun (pp. 751-759).
We developed a new cell surface display system in Corynebacterium glutamicum based on the C-terminally truncated NCgl1221 anchor protein to increase l-glutamate production from starch directly. The C-terminally truncated NCgl1221 protein is a mutant NCgl1221 and leads to the constitutive export of l-glutamate. The N terminus of α-amylase (AmyA) was fused to truncated NCgl1221, and the resulting fusion protein was expressed on the cell surface by IPTG induction. Localization of the fusion protein was confirmed by immunofluorescence microscopy and flow cytometric analysis. The results of l-glutamate fermentation showed that the soluble starch was utilized to grow and produce l-glutamate by the recombinant strain displaying AmyA. The amount of soluble starch was reduced from 30.0 ± 2.8 to 4.5 ± 0.7 g/l under non-inducing condition and from 50.0 ± 2.4 to 12.5 ± 1.1 g/l under biotin limitation in 36 h. The glutamate concentration in the medium was transiently increased in 14 h under no induction, while under biotin-limiting condition, glutamate production was continuously elevated during fermentation. The amount of glutamate reached 19.3 ± 2.1 g/l after 26 h of fermentation with biotin limitation, which was greater than that produced by the strain using PgsA, one of the poly-γ-glutamate synthetase complexes, as the anchor protein under the same condition. Therefore, the truncated NCgl1221 anchor protein has more advantages than the PgsA anchor protein in glutamate fermentation because truncated NCgl1221 leads to the constitutive export of l-glutamate without any treatments.
Keywords: Corynebacterium glutamicum ; α-Amylase; Soluble starch; Glutamate production; Cell surface display
Isolation of identical nitrilase genes from multiple bacterial strains and real-time PCR detection of the genes from soils provides evidence of horizontal gene transfer by Lee Coffey; Adrienne Clarke; Patrick Duggan; Karen Tambling; Serenia Horgan; David Dowling; Catherine O’Reilly (pp. 761-771).
Bacterial enzymes capable of nitrile hydrolysis have significant industrial potential. Microbacterium sp. AJ115, Rhodococcus erythropolis AJ270 and AJ300 were isolated from the same location in England and harbour identical nitrile hydratase/amidase gene clusters. Strain AJ270 has been well studied due to its nitrile hydratase and amidase activity. R. erythropolis ITCBP was isolated from Denmark and carries a very similar nitrile hydratase/amidase gene cluster. In this study, an identical nitrilase gene (nit1) was isolated from the four strains, and the nitrilase from strain AJ270 cloned and expressed in Escherichia coli. Analysis of the recombinant nitrilase has shown it to be functional with activity demonstrated towards phenylacetonitrile. A real-time PCR TaqMan® assay was developed that allowed nit1 detection directly from soil enrichment cultures without DNA extraction, with nit1 detected in all samples tested. Real-time PCR screening of isolates from these soils resulted in the isolation of nit1 and also very similar nitrilase gene nit2 from a number of Burkholderia sp. The genes nit1 and nit2 have also been detected in many bacteria of different genera but are unstable in these isolates. It is likely that the genes were acquired by horizontal gene transfer and may be widespread in the environment.
Keywords: Burkholderia ; Expression; Horizontal gene transfer; Nitrilase; Real-time PCR; Rhodococcus
Isolation of identical nitrilase genes from multiple bacterial strains and real-time PCR detection of the genes from soils provides evidence of horizontal gene transfer by Lee Coffey; Adrienne Clarke; Patrick Duggan; Karen Tambling; Serenia Horgan; David Dowling; Catherine O’Reilly (pp. 761-771).
Bacterial enzymes capable of nitrile hydrolysis have significant industrial potential. Microbacterium sp. AJ115, Rhodococcus erythropolis AJ270 and AJ300 were isolated from the same location in England and harbour identical nitrile hydratase/amidase gene clusters. Strain AJ270 has been well studied due to its nitrile hydratase and amidase activity. R. erythropolis ITCBP was isolated from Denmark and carries a very similar nitrile hydratase/amidase gene cluster. In this study, an identical nitrilase gene (nit1) was isolated from the four strains, and the nitrilase from strain AJ270 cloned and expressed in Escherichia coli. Analysis of the recombinant nitrilase has shown it to be functional with activity demonstrated towards phenylacetonitrile. A real-time PCR TaqMan® assay was developed that allowed nit1 detection directly from soil enrichment cultures without DNA extraction, with nit1 detected in all samples tested. Real-time PCR screening of isolates from these soils resulted in the isolation of nit1 and also very similar nitrilase gene nit2 from a number of Burkholderia sp. The genes nit1 and nit2 have also been detected in many bacteria of different genera but are unstable in these isolates. It is likely that the genes were acquired by horizontal gene transfer and may be widespread in the environment.
Keywords: Burkholderia ; Expression; Horizontal gene transfer; Nitrilase; Real-time PCR; Rhodococcus
Isolation of identical nitrilase genes from multiple bacterial strains and real-time PCR detection of the genes from soils provides evidence of horizontal gene transfer by Lee Coffey; Adrienne Clarke; Patrick Duggan; Karen Tambling; Serenia Horgan; David Dowling; Catherine O’Reilly (pp. 761-771).
Bacterial enzymes capable of nitrile hydrolysis have significant industrial potential. Microbacterium sp. AJ115, Rhodococcus erythropolis AJ270 and AJ300 were isolated from the same location in England and harbour identical nitrile hydratase/amidase gene clusters. Strain AJ270 has been well studied due to its nitrile hydratase and amidase activity. R. erythropolis ITCBP was isolated from Denmark and carries a very similar nitrile hydratase/amidase gene cluster. In this study, an identical nitrilase gene (nit1) was isolated from the four strains, and the nitrilase from strain AJ270 cloned and expressed in Escherichia coli. Analysis of the recombinant nitrilase has shown it to be functional with activity demonstrated towards phenylacetonitrile. A real-time PCR TaqMan® assay was developed that allowed nit1 detection directly from soil enrichment cultures without DNA extraction, with nit1 detected in all samples tested. Real-time PCR screening of isolates from these soils resulted in the isolation of nit1 and also very similar nitrilase gene nit2 from a number of Burkholderia sp. The genes nit1 and nit2 have also been detected in many bacteria of different genera but are unstable in these isolates. It is likely that the genes were acquired by horizontal gene transfer and may be widespread in the environment.
Keywords: Burkholderia ; Expression; Horizontal gene transfer; Nitrilase; Real-time PCR; Rhodococcus
Mutagenesis of the enolase–phosphatase gene in Xanthomonas oryzae pv. oryzae affects growth on methylthioadenosine and in vivo S-adenosylmethionine pools by Yan Zhang; Guiying Zhang; Jiahuan Zhang; Xiaoyu Wang; Jinsheng Wang (pp. 773-783).
Enolase–phosphatase (E1), as an enzyme, is involved in methionine salvage pathway in many prokaryotic and eukaryotic organisms. But the identity and function of E1 in Xanthomonas oryzae pv. oryzae (Xoo) remain undetermined. Here, we report the cloning and characterization of E1 gene, named xep, from Xoo. Sequence analysis shows that XEP is highly conserved among the six Xoo strains we investigated and all other Xanthomonas species. The strain with an insertion mutation in xep could not grow when methylthioadenosine (MTA) was used as the sole sulfur source, but its growth in rice leaves was comparable to that of wild-type strain. Furthermore, the mutant also showed less S-adenosylmethionine (SAM) and lower gene expression of sulfate reduction gene raxQ, compared to wild-type bacterial cells. Introduction of wild-type xep gene to the mutant resulted in the full restoration of growth on MTA, the SAM quantity and the expression level of raxQ. The results demonstrate that xep is involved in the predicted methionine salvage pathway and an inactive form of this gene results in a decreased SAM level in vivo. Our data also indicate that SAM may play a role in the regulation of sulfur reduction at the transcriptional level in Xoo.
Keywords: Xanthomonas oryzae pv. oryzae ; Enolase–phosphatase; Methionine salvage pathway; Bacterial growth; S-adenosylmethionine; raxQ
Mutagenesis of the enolase–phosphatase gene in Xanthomonas oryzae pv. oryzae affects growth on methylthioadenosine and in vivo S-adenosylmethionine pools by Yan Zhang; Guiying Zhang; Jiahuan Zhang; Xiaoyu Wang; Jinsheng Wang (pp. 773-783).
Enolase–phosphatase (E1), as an enzyme, is involved in methionine salvage pathway in many prokaryotic and eukaryotic organisms. But the identity and function of E1 in Xanthomonas oryzae pv. oryzae (Xoo) remain undetermined. Here, we report the cloning and characterization of E1 gene, named xep, from Xoo. Sequence analysis shows that XEP is highly conserved among the six Xoo strains we investigated and all other Xanthomonas species. The strain with an insertion mutation in xep could not grow when methylthioadenosine (MTA) was used as the sole sulfur source, but its growth in rice leaves was comparable to that of wild-type strain. Furthermore, the mutant also showed less S-adenosylmethionine (SAM) and lower gene expression of sulfate reduction gene raxQ, compared to wild-type bacterial cells. Introduction of wild-type xep gene to the mutant resulted in the full restoration of growth on MTA, the SAM quantity and the expression level of raxQ. The results demonstrate that xep is involved in the predicted methionine salvage pathway and an inactive form of this gene results in a decreased SAM level in vivo. Our data also indicate that SAM may play a role in the regulation of sulfur reduction at the transcriptional level in Xoo.
Keywords: Xanthomonas oryzae pv. oryzae ; Enolase–phosphatase; Methionine salvage pathway; Bacterial growth; S-adenosylmethionine; raxQ
Mutagenesis of the enolase–phosphatase gene in Xanthomonas oryzae pv. oryzae affects growth on methylthioadenosine and in vivo S-adenosylmethionine pools by Yan Zhang; Guiying Zhang; Jiahuan Zhang; Xiaoyu Wang; Jinsheng Wang (pp. 773-783).
Enolase–phosphatase (E1), as an enzyme, is involved in methionine salvage pathway in many prokaryotic and eukaryotic organisms. But the identity and function of E1 in Xanthomonas oryzae pv. oryzae (Xoo) remain undetermined. Here, we report the cloning and characterization of E1 gene, named xep, from Xoo. Sequence analysis shows that XEP is highly conserved among the six Xoo strains we investigated and all other Xanthomonas species. The strain with an insertion mutation in xep could not grow when methylthioadenosine (MTA) was used as the sole sulfur source, but its growth in rice leaves was comparable to that of wild-type strain. Furthermore, the mutant also showed less S-adenosylmethionine (SAM) and lower gene expression of sulfate reduction gene raxQ, compared to wild-type bacterial cells. Introduction of wild-type xep gene to the mutant resulted in the full restoration of growth on MTA, the SAM quantity and the expression level of raxQ. The results demonstrate that xep is involved in the predicted methionine salvage pathway and an inactive form of this gene results in a decreased SAM level in vivo. Our data also indicate that SAM may play a role in the regulation of sulfur reduction at the transcriptional level in Xoo.
Keywords: Xanthomonas oryzae pv. oryzae ; Enolase–phosphatase; Methionine salvage pathway; Bacterial growth; S-adenosylmethionine; raxQ
Cold-active halophilic bacteria from the ice-sealed Lake Vida, Antarctica by Lindsay J. Mondino; Marie Asao; Michael T. Madigan (pp. 785-790).
Lake Vida is a large, permanently ice-covered lake in the Victoria Valley of the McMurdo Dry Valleys, Antarctica and is unique among Dry Valley lakes because it is ice-sealed, with an ice-cover of nearly 19 m. Enrichment cultures of melt-water from Lake Vida 15.9 m ice yielded five pure cultures of aerobic, heterotrophic bacteria. Of these, one strain grew at −8°C and the four others at −4°C. All isolates were either halotolerant or halophilic, with two strains capable of growth at 15% NaCl. Phylogenetic analysis revealed the Lake Vida isolates to be Gammaproteobacteria, related to species of Psychrobacter and Marinobacter. This is the first report of pure cultures of bacteria from Lake Vida, and the isolates displayed a phenotype consistent with life in a cold hypersaline environment.
Keywords: Antarctica; Lake Vida; Psychrophiles; Psychrobacter ; Marinobacter
Cold-active halophilic bacteria from the ice-sealed Lake Vida, Antarctica by Lindsay J. Mondino; Marie Asao; Michael T. Madigan (pp. 785-790).
Lake Vida is a large, permanently ice-covered lake in the Victoria Valley of the McMurdo Dry Valleys, Antarctica and is unique among Dry Valley lakes because it is ice-sealed, with an ice-cover of nearly 19 m. Enrichment cultures of melt-water from Lake Vida 15.9 m ice yielded five pure cultures of aerobic, heterotrophic bacteria. Of these, one strain grew at −8°C and the four others at −4°C. All isolates were either halotolerant or halophilic, with two strains capable of growth at 15% NaCl. Phylogenetic analysis revealed the Lake Vida isolates to be Gammaproteobacteria, related to species of Psychrobacter and Marinobacter. This is the first report of pure cultures of bacteria from Lake Vida, and the isolates displayed a phenotype consistent with life in a cold hypersaline environment.
Keywords: Antarctica; Lake Vida; Psychrophiles; Psychrobacter ; Marinobacter
Production of diprenylated indole derivatives by tandem incubation of two recombinant dimethylallyltryptophan synthases by Han-Li Ruan; Edyta Stec; Shu-Ming Li (pp. 791-795).
Two dimethylallyltryptophan synthases, FgaPT2 and 7-DMATS, which catalysed the prenylation of l-tryptophan at positions C4 and C7, respectively, have been recently identified in Aspergillus fumigatus and proven biochemically. These enzymes were successfully used for the production of monoprenylated indole derivatives. In this study, we showed that C4,C7-diprenylated indole derivatives, e.g. 4,7-di-(dimethylallyl)-l-tryptophan, 4,7-di-(dimethylallyl)-l-abrine and 4,7-di-(dimethylallyl)-11-methyltryptophan, could be conveniently produced by tandem incubation of both enzymes. The structures of the isolated enzymatic products were elucidated by NMR and MS analyses. High conversion yields of up to 93% were achieved by an incubation sequence of FgaPT2 followed by 7-DMATS. The results reported in this study demonstrated the potential of secondary metabolite enzymes as promising tools for the production of designed compounds.
Keywords: Aspergillus fumigatus ; Chemoenzymatic synthesis; Dimethylallyltryptophan synthase; Prenylated indole derivatives; Prenyltransferase and prenylation
Production of diprenylated indole derivatives by tandem incubation of two recombinant dimethylallyltryptophan synthases by Han-Li Ruan; Edyta Stec; Shu-Ming Li (pp. 791-795).
Two dimethylallyltryptophan synthases, FgaPT2 and 7-DMATS, which catalysed the prenylation of l-tryptophan at positions C4 and C7, respectively, have been recently identified in Aspergillus fumigatus and proven biochemically. These enzymes were successfully used for the production of monoprenylated indole derivatives. In this study, we showed that C4,C7-diprenylated indole derivatives, e.g. 4,7-di-(dimethylallyl)-l-tryptophan, 4,7-di-(dimethylallyl)-l-abrine and 4,7-di-(dimethylallyl)-11-methyltryptophan, could be conveniently produced by tandem incubation of both enzymes. The structures of the isolated enzymatic products were elucidated by NMR and MS analyses. High conversion yields of up to 93% were achieved by an incubation sequence of FgaPT2 followed by 7-DMATS. The results reported in this study demonstrated the potential of secondary metabolite enzymes as promising tools for the production of designed compounds.
Keywords: Aspergillus fumigatus ; Chemoenzymatic synthesis; Dimethylallyltryptophan synthase; Prenylated indole derivatives; Prenyltransferase and prenylation