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Guoping Zhao - One of the best experts on this subject based on the ideXlab platform.
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crispr cas12a assisted genome editing in Amycolatopsis Mediterranei
Frontiers in Bioengineering and Biotechnology, 2020Co-Authors: Yajuan Zhou, Guoping Zhao, Jiacheng Wu, Jin WangAbstract:Amycolatopsis Mediterranei U32 is an industrial producer of rifamycin SV, whose derivatives have long been the first-line antimycobacterial drugs. In order to perform genetic modification in this important industrial strain, a lot of efforts have been made in the past decades and a homologous recombination-based method was successfully developed in our laboratory, which, however, requires the employment of an antibiotic resistance gene for positive selection and did not support convenient markerless gene deletion. Here in this study, the clustered regularly interspaced short palindromic repeat (CRISPR) system was employed to establish a genome editing system in A. Mediterranei U32. Specifically, the Francisella tularensis subsp. novicida Cas12a (FnCas12a) gene was first integrated into the U32 genome to generate target-specific double-stranded DNA (dsDNA) breaks (DSBs) under the guidance of CRISPR RNAs (crRNAs). Then, the DSBs could be repaired by either the non-homologous DNA end-joining (NHEJ) system or the homology-directed repair (HDR) pathway, generating inaccurate or accurate mutations in target genes, respectively. Besides of A. Mediterranei, the present work may also shed light on the development of CRISPR-assisted genome editing systems in other species of the Amycolatopsis genus.
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glnr dominates rifamycin biosynthesis by activating the rif cluster genes transcription both directly and indirectly in Amycolatopsis Mediterranei
Frontiers in Microbiology, 2020Co-Authors: Guoping Zhao, Jin WangAbstract:: Because of the remarkable efficacy in treating mycobacterial infections, rifamycin and its derivatives are still first-line antimycobacterial drugs. It has been intensely studied to increase rifamycin yield from Amycolatopsis Mediterranei, and nitrate is found to provide a stable and remarkable stimulating effect on the rifamycin production, a phenomenon known as "nitrate-stimulating effect (NSE)". Although the NSE has been widely used for the industrial production of rifamycin, its detailed molecular mechanism remains ill-defined. And our previous study has established that the global nitrogen regulator GlnR may participate in the NSE, but the underlying mechanism is still enigmatic. Here, we demonstrate that GlnR directly controls rifamycin biosynthesis in A. Mediterranei and thus plays an essential role in the NSE. Firstly, GlnR specifically binds to the upstream region of rifZ, which leads us to uncover that rifZ has its own promoter. As RifZ is a pathway-specific activator for the whole rif cluster, GlnR indirectly upregulates the whole rif cluster transcription by directly activating the rifZ expression. Secondly, GlnR specifically binds to the upstream region of rifK, which is also characterized to have its own promoter. It is well-known that RifK is a 3-amino-5-hydroxybenzoic acid (AHBA, the starter unit of rifamycin) synthase, thus GlnR can promote the supply of the rifamycin precursor by directly activating the rifK transcription. Notably, GlnR and RifZ independently activate the rifK transcription through binding to different sites in rifK promoter region, which suggests that the cells have a sophisticated regulatory mechanism to control the AHBA biosynthesis. Collectively, this study reveals that GlnR activates the rif cluster transcription in both direct (for rifZ and rifK) and indirect (for the whole rif cluster) manners, which well interprets the phenomenon that the NSE doesn't occur in the glnR null mutant. Furthermore, this study deepens our understanding about the molecular mechanism of the NSE.
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glnr positive transcriptional regulation of the phosphate specific transport system pstscab in Amycolatopsis Mediterranei u32
Acta Biochimica et Biophysica Sinica, 2018Co-Authors: Yuhui Zhang, Guoping Zhao, Ying Wang, Zhi Hui Shao, Yixuan Zhang, Peng Li, Jing WangAbstract:: Amycolatopsis Mediterranei U32 is an important industrial strain for the production of rifamycin SV. Rifampicin, a derivative of rifamycin SV, is commonly used to treat mycobacterial infections. Although phosphate has long been known to affect rifamycin biosynthesis, phosphate transport, metabolism, and regulation are poorly understood in A. Mediterranei. In this study, the functional phosphate transport system pstSCAB was isolated by RNA sequencing and inactivated by insertion mutation in A. Mediterranei U32. The mycelium morphology changed from a filamentous shape in the wild-type and pstS1+ strains to irregular swollen shape at the end of filamentous in the ΔpstS1 strain. RT-PCR assay revealed that pstSCAB genes are co-transcribed as a polycistronic messenger. The pstSCAB transcription was significantly activated by nitrate supplementation and positively regulated by GlnR which is a global regulator of nitrogen metabolism in actinomycetes. At the same time, the yield of rifamycin SV decreased after mutation (ΔpstS1) compared with wild-type U32, which indicated a strong connection among phosphate metabolism, nitrogen metabolism, and rifamycin production in actinomycetes.
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a feedback regulatory model for rifq mediated repression of rifamycin export in Amycolatopsis Mediterranei
Microbial Cell Factories, 2018Co-Authors: Jingzhi Wang, Guoping Zhao, Jin WangAbstract:Due to the important role of rifamycin in curing tuberculosis infection, the study on rifamycin has never been stopped. Although RifZ, which locates within the rifamycin biosynthetic cluster, has recently been characterized as a pathway-specific regulator for rifamycin biosynthesis, little is known about the regulation of rifamycin export. In this work, we proved that the expression of the rifamycin efflux pump (RifP) was regulated by RifQ, a TetR-family transcriptional regulator. Deletion of rifQ had little impact on bacterial growth, but resulted in improved rifamycin production, which was consistent with the reverse transcription PCR results that RifQ negatively regulated rifP’s transcription. With electrophoretic mobility shift assay and DNase I Footprinting assay, RifQ was found to directly bind to the promoter region of rifP, and a typical inverted repeat was identified within the RifQ-protected sequences. The transcription initiation site of rifP was further characterized and found to be upstream of the RifQ binding sites, well explaining the RifQ-mediated repression of rifP’s transcription in vivo. Moreover, rifamycin B (the end product of rifamycin biosynthesis) remarkably decreased the DNA binding affinity of RifQ, which led to derepression of rifamycin export, reducing the intracellular concentration of rifamycin B as well as its toxicity against the host. Here, we proved that the export of rifamycin B was repressed by RifQ in Amycolatopsis Mediterranei, and the RifQ-mediated repression could be specifically relieved by rifamycin B, the end product of rifamycin biosynthesis, based on which a feedback model was proposed for regulation of rifamycin export. With the findings here, one could improve the antibiotic yield by simply inactivating the negative regulator of the antibiotic transporter.
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rifz amed_0655 is a pathway specific regulator for rifamycin biosynthesis in Amycolatopsis Mediterranei
Applied and Environmental Microbiology, 2017Co-Authors: Chen Li, Guoping Zhao, Ying Wang, Zhi Hui Shao, Jin Wang, Xiaoming DingAbstract:Rifamycin and its derivatives are particularly effective against the pathogenic mycobacteria Mycobacterium tuberculosis and Mycobacterium leprae . Although the biosynthetic pathway of rifamycin has been extensively studied in Amycolatopsis Mediterranei , little is known about the regulation in rifamycin biosynthesis. Here, an in vivo transposon system was employed to identify genes involved in the regulation of rifamycin production in A. Mediterranei U32. Totally, nine rifamycin-defecient mutants were isolated, among which three mutants had the transposon inserted in AMED_0655 (namely rifZ , encoding a LuxR_family regulator). The rifZ was further knocked out via homologous recombination, and the transcription of genes in the rifamycin biosynthetic gene cluster ( rif cluster) was remarkably reduced in rifZ null mutant. Based on the co-transcription assay results, genes within rif cluster were grouped into ten operons, sharing six promoter regions. With electrophoretic mobility shift assay and DNase I footprinting assay, RifZ was proved to specially bind to all six promoter regions, which was consistent with the fact that RifZ regulated the transcription of whole rif cluster. And the binding consensus sequence was further characterized through alignment using the RifZ-protected DNA sequences. With bioinformatics analysis, another five promoters containing the RifZ box (“CTACC-N8-GGATG”) were identified, among which the binding of RifZ to the promoter regions of both rifK and orf18 ( AMED_0645 ) was further verified. As RifZ directly regulates the transcription of all operons within the rif cluster, we propose that RifZ is a pathway-specific regulator for the rif cluster. Importance To this day, rifamycin and its derivatives are still the first-line anti-tuberculosis drugs. The biosynthesis of rifamycin has been extensively studied and most biosynthetic processes have been characterized. However, little is known about the regulation of the transcription of the rifamycin biosynthetic gene cluster ( rif cluster), and no direct regulator has been characterized before. Through the employment of transposon screening, we here characterized a LuxR-family regulator RifZ as a direct transcriptional activator for rif cluster. As RifZ directly regulates the transcription of all rif genes, it is considered as a pathway-specific regulator for the rif cluster. Therefore, as the first regulator characterized for direct regulation of rif transcription, RifZ may provide a new clue for further engineering of high-yield industrial strains.
Weihong Jiang - One of the best experts on this subject based on the ideXlab platform.
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nitrate stimulating effect in Amycolatopsis Mediterranei from discovery to mechanistic studies
Chinese Journal of Biotechnology, 2015Co-Authors: Zhi Hui Shao, Xiaoming Ding, Ying Wang, Weihong Jiang, Jin Wang, Guoping ZhaoAbstract:: Nitrate not only remarkably stimulates the rifamycinbiosynthesis in Amycolatopsis Mediterranei, but also influences the primary metabolisms, including the inhibition of fatty acids biosynthesis in the bacterial. This phenomenon has been designated as "Nitrate Stimulating Effect" by the late Prof. J.S. Chiaosince its discovery in the 1970's, and has been found in many other antibiotics-producing actinomycetes subsequently. Based on the research in his laboratory, we have revealed that the nitrate stimulation effect mainly manifests in two aspects over the last two decades. First, nitrate promotes the supply of rifamycin precursors, e.g., UDP-glucose, AHBA, malonyl-CoA and methylmalonyl-CoA. Specifically, the biosynthesis of fatty acids is inhibited by nitrate consequently the acetyl-CoA is shunted into malonyl-CoA. Second, nitrate facilitates the expression of genes in the rifclulsterthat encodes rifamycin biosynthetic enzymes. Following our current understanding, the future research will focus on the signals, the signal transduction pathway and the molecular mechanisms that dictate nitrate-mediated transcriptional and post-translational regulations.
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three of four glnr binding sites are essential for glnr mediated activation of transcription of the Amycolatopsis Mediterranei nas operon
Journal of Bacteriology, 2013Co-Authors: Ying Wang, Guoping Zhao, Weihong Jiang, Zhi Hui Shao, Yinhua Lu, Jingzhi Wang, Hua Yuan, Jing WangAbstract:In Amycolatopsis Mediterranei U32, genes responsible for nitrate assimilation formed one operon, nasACKBDEF, whose transcription is induced by the addition of nitrate. Here, we characterized GlnR as a direct transcriptional activator for the nas operon. The GlnR-protected DNA sequences in the promoter region of the nas operon were characterized by DNase I footprinting assay, the previously deduced Streptomyces coelicolor double 22-bp GlnR binding consensus sequences comprising a1, b1, a2, and b2 sites were identified, and the sites were then mutated individually to test their roles in both the binding of GlnR in vitro and the GlnR-mediated transcriptional activation in vivo. The results clearly showed that only three GlnR binding sites (a1, b1, and b2 sites) were required by GlnR for its specific binding to the nas promoter region and efficient activation of the transcription of the nas operon in U32, while the a2 site seemed unnecessary.
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a complex role of Amycolatopsis Mediterranei glnr in nitrogen metabolism and related antibiotics production
Archives of Microbiology, 2007Co-Authors: Hao Yu, Ruishen Jiao, Guoping Zhao, Weihong JiangAbstract:Amycolatopsis, genus of a rare actinomycete, produces many clinically important antibiotics, such as rifamycin and vancomycin. Although GlnR of Amycolatopsis Mediterranei is a direct activator of the glnA gene expression, the production of GlnR does not linearly correlate with the expression of glnA under different nitrogen conditions. Moreover, A. Mediterranei GlnR apparently inhibits rifamycin biosynthesis in the absence of nitrate but is indispensable for the nitrate-stimulating effect for its production, which leads to the hyper-production of rifamycin. When glnR of A. Mediterranei was introduced into its phylogenetically related organism, Streptomyces coelicolor, we found that GlnR widely participated in the host strain’s secondary metabolism, resemblance to the phenotypes of a unique S. coelicolorglnR mutant, FS2. In contrast, absence or increment in copy number of the native S. coelicolor glnR did not result in a detectable pleiotrophic effect. We thus suggest that GlnR is a global regulator with a dual functional impact upon nitrogen metabolism and related antibiotics production.
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identification and characterization of glna promoter and its corresponding trans regulatory protein glnr in the rifamycin sv producing actinomycete Amycolatopsis Mediterranei u32
Acta Biochimica et Biophysica Sinica, 2006Co-Authors: Hao Yu, Guoping Zhao, Weihong Jiang, Wentao Peng, Ting WuAbstract:The genetic requirements for the transcription of glnA, encoding the major glutamine synthetase in a rifamycin SV-producing Amycolatopsis Mediterranei strain, U32, were investigated. Primer extension experiments showed that the promoter of U32 glnA (pglnA) was likely to have two transcription initiation sites: P1 and P2, located 157 and 45 nucleotides (nt) upstream of the translational start codon, respectively. Gel mobility shift and DNase I footprinting analyses revealed a 30 bp cis-element located at 45 to 75 nt downstream of P1, or 38 to 68 nt upstream of P2. The sequence of the cis-element displayed high similarity to the corresponding regions of pglnA from Streptomyces coelicolor and S. roseosporus. With xylE as a reporter gene, the expression levels of U32 pglnA and its deletion derivatives under different nitrogen-source conditions were analyzed by detecting the catechol dioxygenase activities in S. lividans TK54, S. coelicolor J508 and S. coelicolor FS10 (glnR mutant). These in vivo studies showed that the activation of U32 pglnA in S. coelicolor required GlnR, and its binding to the U32 pglnA was further confirmed by the gel mobility shift assay. Cloning and heterologous expression of the U32 glnR allowed us to detect the in vitro interaction between the U32 GlnR and the corresponding pglnA cis-element. Further evidence shown by in vivo glnR inactivation and complementation indicated that GlnR is essential for the active transcription of glnA in U32.
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cloning and preliminary characterization of lh3 gene encoding a putative acetyltransferase from a rifamycin sv producing strain Amycolatopsis Mediterranei
Biotechnology Letters, 2005Co-Authors: Lei Chen, Hao Yu, Yinhua Lu, Weihong JiangAbstract:An ORF located immediately downstream of glnR gene was cloned from Amycolatopsis Mediterranei U32 and was named lh3. Sequence analysis revealed that lh3 encodes a putative acetyltransferase, which shows high amino acid sequence similarities to the mycothiol synthase (MshD) from other actinomycetes. For functional analysis, mutation in lh3 gene was generated by gene replacement with an apramycin resistance gene through homologous recombination. Compared with the wild type strain, the resulting mutant was more sensitive to H2O2, apramycin and erythromycin by two- to three-fold. These results suggest that the lh3 gene plays an important role in the course of detoxification in A. Mediterranei U32.
Juishen Chiao - One of the best experts on this subject based on the ideXlab platform.
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identification and functional analysis of a nitrate assimilation operon nasackbdef from Amycolatopsis Mediterranei u32
Archives of Microbiology, 2011Co-Authors: Zhi Hui Shao, Xiaoming Ding, Juishen Chiao, Jin Wang, Guoping ZhaoAbstract:Nitrate assimilation has been well studied for Gram-negative bacteria but not so much in the Gram-positive actinomycetes up to date. In a rifamycin SV-producing actinomycete, Amycolatopsis Mediterranei strain U32, nitrate not only can be used as a sole nitrogen source but also remarkably stimulates the antibiotic production along with regulating the related metabolic enzymes. A gene cluster of nasACKBDEF was cloned from a U32 genomic library by in situ hybridization screening with a heterogeneous nasB probe and confirmed later by whole genome sequence, corresponding to the protein coding genes of AMED_1121 to AMED_1127. These genes were co-transcribed as an operon, concomitantly repressed by ammonium while activated with supplement of either nitrate or nitrite. Genetic and biochemical analyses identified the essential nitrate/nitrite assimilation functions of the encoded proteins, orderly, the assimilatory nitrate reductase catalytic subunit (NasA), nitrate reductase electron transfer subunit (NasC), nitrate/nitrite transporter (NasK), assimilatory nitrite reductase large subunit (NasB) and small subunit (NasD), bifunctional uroporphyrinogen-III synthase (NasE), and an unknown function protein (NasF). Comparing rifamycin SV production and the level of transcription of nasB and rifE from U32 and its individual nas mutants in Bennet medium with or without nitrate indicated that nitrate assimilation function encoded by the nas operon played an essential role in the “nitrate stimulated” rifamycin production but had no effect upon the transcription regulation of the primary and secondary metabolic genes related to rifamycin biosynthesis.
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bacterial type i glutamine synthetase of the rifamycin sv producing actinomycete Amycolatopsis Mediterranei u32 is the only enzyme responsible for glutamine synthesis under physiological conditions
Acta Biochimica et Biophysica Sinica, 2006Co-Authors: Wentao Peng, Guoping Zhao, Juishen Chiao, Jin Wang, Ting Wu, Jianqiang HuangAbstract:The structural gene for glutamine synthetase, glnA, from Amycolatopsis Mediterranei U32 was cloned via screening a genomic library using the analog gene from Streptomyces coelicolor. The clone was functionally verified by complementing for glutamine requirement of an Escherichia coli glnA null mutant under the control of a lac promoter. Sequence analysis showed an open reading frame encoding a protein of 466 amino acid residues. The deduced amino acid sequence bears significant homologies to other bacterial type I glutamine synthetases, specifically, 71% and 72% identical to the enzymes of S. coelicolor and Myco- bacterium tuberculosis, respectively. Disruption of this glnA gene in A. Mediterranei U32 led to glutamine auxotrophy with no detectable glutamine synthetase activity in vivo. In contrast, the cloned glnA + gene can complement for both phenotypes in trans. It thus suggested that in A. Mediterranei U32, the glnA gene encoding glutamine synthetase is uniquely responsible for in vivo glutamine synthesis under our laboratory defined physiological conditions.
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a novel two component system amrb amkb involved in the regulation of central carbohydrate metabolism in rifamycin sv producing Amycolatopsis Mediterranei u32
Current Microbiology, 2004Co-Authors: Weiwu Wang, Guoping Zhao, Juishen Chiao, Weihong JiangAbstract:A novel two-component signal transduction system amrB-amkB was cloned from rifamycin SV-producing Amycolatopsis Mediterranei U32, and their biochemical functions as a response regulator and a histidine protein kinase, respectively, were proven. The amrB disruption mutant was generated by insertional inactivation with the aparmycin resistance gene. The metabolic response to the absence of amrB gene was determined by a biochemical profiling technique in which the concentration changes of metabolic intermediates were measured by gas chromatography with time-of-flight mass spectrometry (GC/TOF-MS). Although the phenotype analyses of the amrB gene disruption mutant showed no significant change with respect to rifamycin SV production and morphological differentiation, the global metabolomic analyses found the concentration levels of some key intermediates in the TCA cycle and glycolysis pathway were affected by an amrB gene disruption event. The primary results suggested that amrB-amkB genes might be involved in the regulation of central carbohydrate metabolism in A. Mediterranei U32.
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moea an enzyme in the molybdopterin synthesis pathway is required for rifamycin sv production in Amycolatopsis Mediterranei u32
Applied Microbiology and Biotechnology, 2002Co-Authors: Weiwu Wang, Guoping Zhao, Juishen Chiao, Wenquan Zhang, Jian Lu, Yunpeng Yang, Wei JiangAbstract:Rifamycin SV contains one amide nitrogen atom at its C7N moiety. Earlier labeling studies suggested that nitrogen might be incorporated from a pathway involved in a molybdenum-dependent nitrate reductase. However, no genetic evidence is available thus far. The structural gene moeA, which is involved in molybdopterin synthesis in various organisms, has been cloned from rifamycin SV-producing Amycolatopsis Mediterranei strain U32. The amino acid sequence deduced from the moeA gene showed significant similarity to members of the MoeA protein family and contains all the structural features that are highly conserved in the putative functional domains of MoeA proteins. Southern hybridization showed that there is only one moeA gene in the A. Mediterranei genome. To further investigate the possible physiological function of the moeA gene, a double crossover gene replacement was achieved by inserting an aparmycin resistance gene into moeA in the A. Mediterranei U32 chromosome. Phenotype analysis showed that the moeA gene is required for A. Mediterranei growth in a minimal medium with nitrate as sole nitrogen source, possibly because nitrate reductase activity is diminished due to disruption of the moeA gene. Compared to the wild type strain, moeA-disrupted mutants lost 95% of their rifamycin SV production capacity in complex fermentation media. The results demonstrate that the moeA gene is necessary for rifamycin SV production in A. Mediterranei, and that the nitrogen assimilation pathway involved in nitrate reductase is the major pathway for the genesis of the amide nitrogen atom in the rifamycin SV molecule.
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molecular and biochemical characterization of a novel two component signal transduction system amra amka involved in rifamycin sv production in Amycolatopsis Mediterranei u32
Archives of Microbiology, 2002Co-Authors: Weiwu Wang, Weiwen Zhang, Guoping Zhao, Juishen Chiao, Hui Chen, Weihong JiangAbstract:Two-component and phosphorelay signal transduction systems are the major means by which bacteria recognize and respond to a variety of environmental stimuli. Although several model systems, including sporulation in Bacillus subtilis and chemotaxis in Escherichia coli, have been extensively studied, the two-component signal transduction systems in industrially important actinomycetes are not well studied. We report the molecular and biochemical characterization of a novel two-component signal system, amrA-amkA, from the rifamycin-SV-producing Amycolatopsis Mediterranei U32. The deduced sequences of amkA and amrA contain all the structural features that are highly conserved in the typical bacterial histidine kinases and response regulators, respectively. BLAST analyses showed that AmrA and AmkA displayed high similarities to AfsQ1/AfsQ2 of Streptomyces coelicolor and MtrA/MtrB of Mycobacterium tuberculosis. The amrA and amkA genes were over-expressed and the gene products were purified from E. coli. Biochemical studies showed that AmkA is able to autophosphorylate, supporting its functional assignment as a histidine kinase. That AmrA functions as the cognate response regulator for histidine kinase AmkA was demonstrated by in vitro phosphotransfer from [γ-32P]ATP-labeled AmkA to AmrA. Rifamycin SV production was also decreased by 10–20% in amrA or amkA gene disruption mutants under the tested condition. Although the detailed regulatory mechanism is still unknown, this is the first report regarding the involvement of two-component signal systems in rifamycin biosynthesis in the genus Amycolatopsis.
Weiwen Zhang - One of the best experts on this subject based on the ideXlab platform.
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efficient isolation of total rna from antibiotic producing bacterium Amycolatopsis Mediterranei
Journal of Microbiological Methods, 2002Co-Authors: Weiwen Zhang, Ruishen Jiao, Guoping Zhao, Weihong JiangAbstract:Abstract RNA extraction from antibiotic-producing actinomycetes can be a difficult and time-consuming process due to their special peptidoglycans cell wall composition and the short life of RNA. Hence, the rapidity of cellular lysis and complete inhibition of RNase are of particular importance for isolating intact RNA of high quality. The genus of Amycolatopsis Mediterranei produces many clinically important antibiotics, such as rifamycin and vancomycin; however, the available methods for bacterial RNA isolation did not work very well with this genus. In this report, we described a new method for RNA isolation using the combination of LiCl, urea and guanidinium thiocyanate to disrupt the cell wall of Amycolatopsis . Compared with earlier published RNA isolation methods, the method gave higher yields of pure and intact RNA. About 1 μg total RNA free of DNA contamination can be obtained from 1 mg wet weight of A. Mediterranei . The integrity of the RNA was demonstrated by formaldehyde agarose gel electrophoresis and Northern blot analyses.
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molecular and biochemical characterization of a novel two component signal transduction system amra amka involved in rifamycin sv production in Amycolatopsis Mediterranei u32
Archives of Microbiology, 2002Co-Authors: Weiwu Wang, Weiwen Zhang, Guoping Zhao, Juishen Chiao, Hui Chen, Weihong JiangAbstract:Two-component and phosphorelay signal transduction systems are the major means by which bacteria recognize and respond to a variety of environmental stimuli. Although several model systems, including sporulation in Bacillus subtilis and chemotaxis in Escherichia coli, have been extensively studied, the two-component signal transduction systems in industrially important actinomycetes are not well studied. We report the molecular and biochemical characterization of a novel two-component signal system, amrA-amkA, from the rifamycin-SV-producing Amycolatopsis Mediterranei U32. The deduced sequences of amkA and amrA contain all the structural features that are highly conserved in the typical bacterial histidine kinases and response regulators, respectively. BLAST analyses showed that AmrA and AmkA displayed high similarities to AfsQ1/AfsQ2 of Streptomyces coelicolor and MtrA/MtrB of Mycobacterium tuberculosis. The amrA and amkA genes were over-expressed and the gene products were purified from E. coli. Biochemical studies showed that AmkA is able to autophosphorylate, supporting its functional assignment as a histidine kinase. That AmrA functions as the cognate response regulator for histidine kinase AmkA was demonstrated by in vitro phosphotransfer from [γ-32P]ATP-labeled AmkA to AmrA. Rifamycin SV production was also decreased by 10–20% in amrA or amkA gene disruption mutants under the tested condition. Although the detailed regulatory mechanism is still unknown, this is the first report regarding the involvement of two-component signal systems in rifamycin biosynthesis in the genus Amycolatopsis.
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Efficient isolation of total RNA from antibiotic-producing bacterium Amycolatopsis Mediterranei
Journal of Microbiological Methods, 2002Co-Authors: Yu-feng Yao, Ruishen Jiao, Weiwen Zhang, Guoping Zhao, Weihong JiangAbstract:RNA extraction from antibiotic-producing actinomycetes can be a difficult and time-consuming process due to their special peptidoglycans cell wall composition and the short life of RNA. Hence, the rapidity of cellular lysis and complete inhibition of RNase are of particular importance for isolating intact RNA of high quality. The genus of Amycolatopsis Mediterranei produces many clinically important antibiotics, such as rifamycin and vancomycin; however, the available methods for bacterial RNA isolation did not work very well with this genus. In this report, we described a new method for RNA isolation using the combination of LiCl, urea and guanidinium thiocyanate to disrupt the cell wall of Amycolatopsis. Compared with earlier published RNA isolation methods, the method gave higher yields of pure and intact RNA. About 1 μg total RNA free of DNA contamination can be obtained from 1 mg wet weight of A. Mediterranei. The integrity of the RNA was demonstrated by formaldehyde agarose gel electrophoresis and Northern blot analyses. © 2002 Elsevier Science B.V. All rights reserved.
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an eukaryotic type serine threonine protein kinase involved in the carbon source dependent pigment biosynthesis in Amycolatopsis Mediterranei u32
Biochemical and Biophysical Research Communications, 2001Co-Authors: Ling Yang, Weiwen Zhang, Guoping Zhao, Juishen Chiao, Weihong JiangAbstract:Abstract The structural gene, pkmA, was cloned and sequenced from a rifamycin SV-producing Amycolatopsis Mediterranei U32 strain. The N-terminal portion of the deduced amino acid sequence of pkmA showed significant similarity to the family of serine/threonine protein kinases. It contains all the structural features which are highly conserved in protein kinases, including the Gly-X-Gly-X-X-Gly motif of ATP binding and the essential amino acids known to be important for the recognition of the correct hydroxyamino acid in serine/threonine protein specific kinases. The protein possesses a region rich in Ala and Pro residues around the middle of pkmA open reading frame, which might be involved in the transmembrane function, as suggested by PhoA fusion protein analysis. The pkmA gene was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein, and the protein was found to have the activity of autophosphorylation. A double crossover gene replacement was achieved by inserting an aparmycin resistance gene into pkmA in A. Mediterranei chromosomal DNA. The phenotypic analysis of the mutant suggested that pkmA gene is involved in carbon source-dependent pigment formation in A. Mediterranei U32.
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An Eukaryotic-Type Serine/Threonine Protein Kinase Involved in the Carbon Source-Dependent Pigment Biosynthesis in Amycolatopsis Mediterranei U32
Biochemical and Biophysical Research Communications, 2001Co-Authors: Ling Yang, Weiwen Zhang, Guoping Zhao, Juishen Chiao, Weihong JiangAbstract:Abstract The structural gene, pkmA, was cloned and sequenced from a rifamycin SV-producing Amycolatopsis Mediterranei U32 strain. The N-terminal portion of the deduced amino acid sequence of pkmA showed significant similarity to the family of serine/threonine protein kinases. It contains all the structural features which are highly conserved in protein kinases, including the Gly-X-Gly-X-X-Gly motif of ATP binding and the essential amino acids known to be important for the recognition of the correct hydroxyamino acid in serine/threonine protein specific kinases. The protein possesses a region rich in Ala and Pro residues around the middle of pkmA open reading frame, which might be involved in the transmembrane function, as suggested by PhoA fusion protein analysis. The pkmA gene was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein, and the protein was found to have the activity of autophosphorylation. A double crossover gene replacement was achieved by inserting an aparmycin resistance gene into pkmA in A. Mediterranei chromosomal DNA. The phenotypic analysis of the mutant suggested that pkmA gene is involved in carbon source-dependent pigment formation in A. Mediterranei U32.
Yunliu Yang - One of the best experts on this subject based on the ideXlab platform.
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expression in escherichia coli purification and kinetic analysis of the aspartokinase and aspartate semialdehyde dehydrogenase from the rifamycin sv producing Amycolatopsis Mediterranei u32
Applied Microbiology and Biotechnology, 2000Co-Authors: Weiwen Zhang, Guoping Zhao, Weihong Jiang, Yunliu Yang, Juishen ChiaoAbstract:The operon encoding aspartokinase and aspartate semialdehyde dehydrogenase was cloned and sequenced from rifamycin-SV-producing Amycolatopsis Mediterranei U32 previously. In the present work, these two genes were introduced into the auxotrophic Escherichia coli strain CGSC5074 (ask−) and E. coli X6118 (asd −), respectively. The A. Mediterranei U32 aspartokinase and aspartate semialdehyde dehydrogenase genes can be functionally expressed in E. coli and the gene products are able to substitute for the E. coli enzymes. Histidine-tagged aspartokinase and aspartate semialdehyde dehydrogenase were partially purified from E. coli cellular extracts and their kinetic characteristics were studied. Both aspartokinase and aspartate semialdehyde dehydrogenase showed typical Michaelis-Menten type substrate saturation patterns. Aspartokinase has Km values of 3.4 mM for aspartate and 2.3 mM for ATP, while aspartate semialdehyde dehydrogenase has Km values of 1.25 mM for dl-aspartate semialdehyde and 0.73 mM for NADP, respectively. Aspartokinase was inhibited by l-threonine, l-lysine, and l-methionine, but not by l-isoleucine and diaminopimelate. Aspartate semialdehyde dehydrogenase was not inhibited by any of the end-product amino acids at a concentration of less than 5 mM. Hill plot analysis suggested that aspartokinase was subject to allosteric control by l-threonine. Repression of both aspartokinase and aspartate semialdehyde dehydrogenase gene transcription in A. Mediterranei U32 by l-lysine, l-methionine, l-threonine, and l-isoleucine were found. The network of regulation of aspartokinase and aspartate semialdehyde dehydrogenase in rifamycin SV-producing A. Mediterranei U32 is presented.
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A novel transmembrane serine/threonine protein kinase gene from a rifamycin SV‐producing Amycolatopsis Mediterranei U32
FEBS Journal, 2000Co-Authors: Weiwen Zhang, Guoping Zhao, Weihong Jiang, Yunliu Yang, Lei Li, Juishen ChiaoAbstract:Genomic DNA sequencing in the vicinity of methylmalonyl-CoA mutase gene (mutAB) from a rifamycin SV-producing Amycolatopsis Mediterranei U32 allowed us to clone, sequence, and identify a gene encoding a novel serine/threonine protein kinase (amk). The sequence contains a complete ORF of 1821 base pairs encoding a predicted protein of 606 amino acids in length. The N-terminal domain of the protein shows significant homology to the catalytic domain of other protein kinases from both prokaryotic and eukaryotic sources. It also contains all the structural features that are highly conserved in active protein kinases, including the Gly-X-Gly-X-X-Gly motif of ATP-binding and the essential amino acids known to be important for the recognition of the correct hydroxyamino acid in serine/threonine protein kinase. This protein kinase gene was expressed in Escherichia coli and was shown to have the ability of autophosphorylation. The autophosphorylated site was found to be the threonine at position 164 by labeled phosphoaminoacid analysis and site-directed mutagenesis. The C-terminal half of protein kinase was found to contain strong transmembrane structures by PhoA fusion protein analysis, suggesting that Amk protein kinase is a transmembrane protein. A Southern hybridization experiment showed that this type of protein kinase is distributed ubiquitously and might play significant physiological roles in the various species of streptomycetes. However, overexpression of amk gene in Streptomyces cinnamonensis showed no effect on methylmalonyl-CoA mutase activity, monensin production and the hyphae morphology. Although its biological role is still unknown, Amk protein kinase is the first transmembrane serine/threonine protein kinase described for genus Amycolatopsis.
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a novel transmembrane serine threonine protein kinase gene from a rifamycin sv producing Amycolatopsis Mediterranei u32
FEBS Journal, 2000Co-Authors: Weiwen Zhang, Guoping Zhao, Weihong Jiang, Yunliu Yang, Lei Li, Juishen ChiaoAbstract:Genomic DNA sequencing in the vicinity of methylmalonyl-CoA mutase gene (mutAB) from a rifamycin SV-producing Amycolatopsis Mediterranei U32 allowed us to clone, sequence, and identify a gene encoding a novel serine/threonine protein kinase (amk). The sequence contains a complete ORF of 1821 base pairs encoding a predicted protein of 606 amino acids in length. The N-terminal domain of the protein shows significant homology to the catalytic domain of other protein kinases from both prokaryotic and eukaryotic sources. It also contains all the structural features that are highly conserved in active protein kinases, including the Gly-X-Gly-X-X-Gly motif of ATP-binding and the essential amino acids known to be important for the recognition of the correct hydroxyamino acid in serine/threonine protein kinase. This protein kinase gene was expressed in Escherichia coli and was shown to have the ability of autophosphorylation. The autophosphorylated site was found to be the threonine at position 164 by labeled phosphoaminoacid analysis and site-directed mutagenesis. The C-terminal half of protein kinase was found to contain strong transmembrane structures by PhoA fusion protein analysis, suggesting that Amk protein kinase is a transmembrane protein. A Southern hybridization experiment showed that this type of protein kinase is distributed ubiquitously and might play significant physiological roles in the various species of streptomycetes. However, overexpression of amk gene in Streptomyces cinnamonensis showed no effect on methylmalonyl-CoA mutase activity, monensin production and the hyphae morphology. Although its biological role is still unknown, Amk protein kinase is the first transmembrane serine/threonine protein kinase described for genus Amycolatopsis.
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sequence analysis and expression of the aspartokinase and aspartate semialdehyde dehydrogenase operon from rifamycin sv producing Amycolatopsis Mediterranei
Gene, 1999Co-Authors: Weiwen Zhang, Guoping Zhao, Weihong Jiang, Yunliu Yang, Juishen ChiaoAbstract:Abstract A ∼4.8 kb KpnI fragment, from the upstream region of the methylmalonyl-CoA mutase gene (mutAB) of rifamycin SV-producing Amycolatopsis Mediterranei, was cloned and partially sequenced. Codon preference analysis showed three complete ORFs. ORF2 is internal to ORF1, and encodes a polypeptide corresponding to 172 amino acids, whereas ORF1 encodes a polypeptide of 421 amino acids. They were identified as the encoding genes of aspartokinase α- and β-subunits by comparing the amino acid sequences with those in the database. The downstream ORF3, whose start codon was overlapped with the stop codon of both ORF1 and ORF2 by 1 bp, was identified as the aspartate semialdehyde dehydrogenase gene (asd), encoding a polypeptide of 346 amino acids. Subclones containing either the ask gene or the asd gene were constructed, in which the genes could be expressed under Lac promoters. Two subclones could transform E. coli CGSC 5074 (ask-) and E. coli X6118 (asd-) to prototrophy, supporting the functional assignments. Southern hybridisation indicated that the ∼4.8 kb sequenced region represented a continuous segment in the A. Mediterranei chromosome. It is concluded that ask and asd genes are present in an operon in A. Mediterranei, and therefore that organisation of these two genes is the same as in most gram-positive bacteria, such as Mycobacteria, Corynebacterium glutamicum and Bacillus subtilis, but is different from Streptomyces akiyoshiensis.
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localization purification and characterization of nitrate reductase from Amycolatopsis Mediterranei u 32
Chinese Journal of Biochemistry and Molecular Biology, 1998Co-Authors: Guolong Li, Yunliu Yang, Ruishen JiaoAbstract:The localization of nitrate reductase was ascertained. Through osmotic lysis of protoplasts, nitrate reductase from AMediterranei U-32 was located in the cytoplasm. The enzyme was rather unstable, and inclusion of protective reagents in the buffer system greatly improved its stability. Electrophoretically purified enzyme was obtained through six steps of purification: Protamine sulfate precipitation, ammonium sulfate fractionation, Phenyl-Sepharose CL-4B, Bio-Gel A 1.5m, DEAE-Sephacel and Sephadex G-75 column chromatography. The electrophoretically pure enzyme was a monomer of 79 kD, each mol of enzyme contained 2.29 mol Mo, but no non-heme iron, acid-labil sulfur, FMN and FAD. Through isoelectric focusing, the isoelectric point of nitrate reductase was found to be 6.2. Its optimum pH was 7.2, and its optimum temperature 40℃. The K_(m) value for nitrate was 13.3 mumol/L, and the absorption spectrum of this enzyme was also analyzed.
