Sansanmycin生物合成基因簇的克隆和生物合成调节基因ssaA调控机制研究
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摘要
Sansanmycin (SS)是由我国贵州土壤中发现的链霉菌Streptomyces sp. SS所产生的一组尿苷肽类抗生素,此类抗生素家族还包括pacidamycin, napsamycin和mureidomycin等。Sansanmycin对铜绿假单胞菌Pseudomonas aeruginosa,结核分枝杆菌Mycobacterium tuberculosis及多重耐药结核分支杆菌具有很好的抑制作用。Pacidamycin和napsamycin的生物合成基因簇已经被克隆,pacidamycin基因簇中大部分生物合成酶的功能和生物合成步骤已经基本阐明,但是关于它们生物合成调控机制尚未报道。
本研究通过全基因组测序和构建大片段基因组文库相结合的方法首次克隆了sansanmycin的生物合成基因簇(ssa),其包含25个开放阅读框,与pacidamycin和mapsamycin的生物合成基因簇具有很高的序列相似性。利用HHpred对SsaA进行结构同源性比对分析,结果表明SsaA的N-端含有一个FHA(forkhead-accociated)结构域,C-端含有一个LuxR类型的螺旋-转角-螺旋(helix-turn-helix, HTH) DAN结合模体,预示着ssaA可能是sansanmycin生物合成转录调节基因。为了对ssaA的功能进行验证,本研究将ssaA的编码区克隆至含有强启动子ermE*p的质粒pL646中,通过接合转移导入野生型菌株中构建过表达菌株SS/pL-ssaA,生物学活性检测和高效液相色谱(high performance liquid chromatography, HPLC)分析结果表明sansanmycin的产量提高了约一倍,提示ssaA为sansanmycin生物合成正调节基因。为了对ssaA的调控功能进行进一步的确证,本研究采用PCR-targeting的方法构建了ssaA的阻断突变株SS/AKO,生物学活性检测和HPLC分析结果表明,SS/AKO不产sansanmycin,而导入ssaA基因可以使其恢复产生sansanmycin的能力,进一步确证ssaA为sansanmycin生物合成正调节基因。
利用实时荧光定量RT-PCR对阻断株SS/AKO中结构基因ssaH, ssaN, ssaP, ssaX, ssaC的转录水平进行分析,结果表明SS/AKO中这些基因的转录水平均明显下降,说明ssaA是通过控制结构基因的转录水平从而控制sansanmycin产量。
本研究利用E. coli BL21(DE3)表达并纯化了N-端融合了His标签的SsaA重组蛋白,进行凝胶阻滞实验(electrophoretic mobility shift assay, EMSA),结果表明SsaA可与基因簇内包括ssaHp, ssaN-Pp, ssaU-Ap, ssaC-Dp, ssaWp在内的多个结构基因启动子区结合,说明SsaA是通过直接结合于结构基因的启动子区从而控制sansanmycin的生物合成的。利用DNasel足迹法获得SsaA在这些启动子区上的结合位点,通过WebLogo对这些结合位点进行比对,获得了SsaA的一致结合序列GTMCTGAC AN2TGTC AGKAC,其特征为含两个9bp反向重复序列(inverted repeat,IR),中间间隔2bp。将一致结合序列进行突变和缺失,利用EMSA检测SsaA与它们的结合能力,结果表明SsaA均不能和它们结合形成复合物,验证了一致结合序列的正确性。
当EMSA实验反应体系中加入浓度逐渐升高的终产物sansanmycin A (SS-A)或sansanmycin H (SS-H)时,SsaA与目标基因启动子区ssaC-Dp和ssaU-A-1p的结合能力逐渐减弱,表面等离子体共振(Surface plasmon resonance, SPR)实验显示SS-A可以抑制SsaA与ssaC-Dp-3的结合,表明sansanmycin对SsaA的DNA结合活性具有调控作用。此外,SPR实验表明SS-A和SS-H可以与SsaA直接相互作用,说明sansanmycin可通过与SsaA相互作用从而改变SsaA的DNA结合活性。
综上所述,本研究首次克隆了sansanmycin生物合成基因簇,通过基因过表达和阻断实验证明ssaA为sansanmycin生物合成正调节基因,并且对ssaA的分子调控机制进行了深入研究,表明SsaA为一类新型转录因子,具有全新的调控机制,通过终产物负反馈调控机制控制sansanmycin的生物合成。
Sansanmycins, isolated from Streptomyces sp. SS, are members of a uridyl peptide antibiotic family including closely related pacidamycins, napsamycins and mureidomycins. Sansanmycins were reported to exert antibacterial activity against the highly refractory pathogen Pseudomonas aeruginosa, and more interesting, Mycobacterium tuberculosis H37RV and multidrug-resistant M. tuberculosis strains. The biosynthetic gene clusters for pacidamycin and napsamycin have been cloned, and most of the pacidamycin biosynthetic genes have been characterized. However, there were no reports about the transcriptional regulation of the uridyl peptide antibiotics prior to the present work.
The sansanmycin biosynthetic gene cluster (ssa) was identified in Streptomyces sp. SS by a combination of genome mining approach and conventional probing and cosmid sequencing, comprised of25ORFs showing considerable amino acid sequence identity to those of the pacidamycin and napsamycin gene cluster. The structure homology search of SsaA using the online program HHpred revealed that it combined an N-terminal forkhead-associated (FHA) domain with a C-terminal helix-turn-helix DNA-binding motif, suggesting it might be a pathway-specific regulator for sansanmycin biosynthesis. To investigate the contribution of ssaA to the regulation of sansanmycin biosynthesis, the coding region of ssaA was cloned into plasmid pL646under the control of a strong constitutive promoter ermE*p, and the recombinant plasmid were introduced into Streptomyces sp. SS by conjugation. Bioassay and HPLC analyses revealed that overexpression of ssaA increased sansanmycin production, suggesting ssaA is a positive regulator for sansanmycin biosynthesis. To confirm the role of ssaA in sansanmycin biosynthesis, the ssaA disrupted mutant SS/AKO was obtained via PCR-targeting method. Bioassay and HPLC analyses revealed that disruption of ssaA completely abolished sansanmycin production, which was restored by introducing intact ssaA into SS/AKO. These results comfirmed that ssaA is indeed a pivotal positive regulator for sansanmycin biosynthesis.
Quantitative RT-PCR was performed to investigate the relative mRNA level o sansanmycin biosynthetic genes in SS/AKO. The results showed that the relative mRNA level of five structural genes, ssaH, ssaN, ssaP, ssaX and ssaC was decreased, indicatinj that ssaA could positively regulate the sansanmycin biosynthesis by controlling the expression of the structural genes of ssa cluster.
SsaA was overexpressed in E. coli BL21(DE3) as His10-tagged protein. EMSA result: showed that His10-SsaA bound to several promoter regions of the ssa cluster, including ssaHp, ssaN-Pp, ssaU-Ap, ssaC-Dp and ssaWp, suggesting that SsaA controlleo sansanmycin biosynthesis by directly binding to the promoter regions of biosynthetio genes. The binding sites of SsaA in ssa promoter regions were identified by DNase footprinting analysis, and comparison of sequences of the binding sites allowed the identification of a consensus SsaA-binding sequence, GTMCTGACAN2TGTCAGKAC, which consisted of two9-bp inverted repeats (IRs) separated by a2-bp linker. The identified SsaA consensus binding site was validated by mutation or deletion of the most conserved CTGAC sequence in IRs.
EMSA results showed that SS-A or SS-H inhibited the band-shifting of ssaC-Dp anc ssaU-A-lp caused by SsaA in a concentration-dependent manner. SPR analysis results showed that a concentration-dependent inhibition of SsaA binding to ssaC-Dp-3fragment immobilized on an SA sensor chip was readily observed when the amount of SS-A was increased. All these results suggested that sansanmycins could modulate the binding activity of SsaA for its target DNA. These results suggested that sansanmycins could modulate the binding activity of SsaA for its target DNA. Further more, SPR analysis results showed that SsaA could direct interact with SS-A or SS-H. These results indicated that SsaA may strictly control the production of sansanmycins at transcriptional level in a feedback regulatory mechanism by sensing the accumulation of the end-products.
In conclusion, in this work, the biosynthetic gene cluster of uridyl peptide antibiotic sansanmycins was identified for the first time. It has comfirmed that SsaA is a novel class of pathway-specific transcriptional activator of sansanmycin biosynthesis, and that the end products can bind to and change the activity of SsaA to autoregulate the antibiotic production.
本研究通过全基因组测序和构建大片段基因组文库相结合的方法首次克隆了sansanmycin的生物合成基因簇(ssa),其包含25个开放阅读框,与pacidamycin和mapsamycin的生物合成基因簇具有很高的序列相似性。利用HHpred对SsaA进行结构同源性比对分析,结果表明SsaA的N-端含有一个FHA(forkhead-accociated)结构域,C-端含有一个LuxR类型的螺旋-转角-螺旋(helix-turn-helix, HTH) DAN结合模体,预示着ssaA可能是sansanmycin生物合成转录调节基因。为了对ssaA的功能进行验证,本研究将ssaA的编码区克隆至含有强启动子ermE*p的质粒pL646中,通过接合转移导入野生型菌株中构建过表达菌株SS/pL-ssaA,生物学活性检测和高效液相色谱(high performance liquid chromatography, HPLC)分析结果表明sansanmycin的产量提高了约一倍,提示ssaA为sansanmycin生物合成正调节基因。为了对ssaA的调控功能进行进一步的确证,本研究采用PCR-targeting的方法构建了ssaA的阻断突变株SS/AKO,生物学活性检测和HPLC分析结果表明,SS/AKO不产sansanmycin,而导入ssaA基因可以使其恢复产生sansanmycin的能力,进一步确证ssaA为sansanmycin生物合成正调节基因。
利用实时荧光定量RT-PCR对阻断株SS/AKO中结构基因ssaH, ssaN, ssaP, ssaX, ssaC的转录水平进行分析,结果表明SS/AKO中这些基因的转录水平均明显下降,说明ssaA是通过控制结构基因的转录水平从而控制sansanmycin产量。
本研究利用E. coli BL21(DE3)表达并纯化了N-端融合了His标签的SsaA重组蛋白,进行凝胶阻滞实验(electrophoretic mobility shift assay, EMSA),结果表明SsaA可与基因簇内包括ssaHp, ssaN-Pp, ssaU-Ap, ssaC-Dp, ssaWp在内的多个结构基因启动子区结合,说明SsaA是通过直接结合于结构基因的启动子区从而控制sansanmycin的生物合成的。利用DNasel足迹法获得SsaA在这些启动子区上的结合位点,通过WebLogo对这些结合位点进行比对,获得了SsaA的一致结合序列GTMCTGAC AN2TGTC AGKAC,其特征为含两个9bp反向重复序列(inverted repeat,IR),中间间隔2bp。将一致结合序列进行突变和缺失,利用EMSA检测SsaA与它们的结合能力,结果表明SsaA均不能和它们结合形成复合物,验证了一致结合序列的正确性。
当EMSA实验反应体系中加入浓度逐渐升高的终产物sansanmycin A (SS-A)或sansanmycin H (SS-H)时,SsaA与目标基因启动子区ssaC-Dp和ssaU-A-1p的结合能力逐渐减弱,表面等离子体共振(Surface plasmon resonance, SPR)实验显示SS-A可以抑制SsaA与ssaC-Dp-3的结合,表明sansanmycin对SsaA的DNA结合活性具有调控作用。此外,SPR实验表明SS-A和SS-H可以与SsaA直接相互作用,说明sansanmycin可通过与SsaA相互作用从而改变SsaA的DNA结合活性。
综上所述,本研究首次克隆了sansanmycin生物合成基因簇,通过基因过表达和阻断实验证明ssaA为sansanmycin生物合成正调节基因,并且对ssaA的分子调控机制进行了深入研究,表明SsaA为一类新型转录因子,具有全新的调控机制,通过终产物负反馈调控机制控制sansanmycin的生物合成。
Sansanmycins, isolated from Streptomyces sp. SS, are members of a uridyl peptide antibiotic family including closely related pacidamycins, napsamycins and mureidomycins. Sansanmycins were reported to exert antibacterial activity against the highly refractory pathogen Pseudomonas aeruginosa, and more interesting, Mycobacterium tuberculosis H37RV and multidrug-resistant M. tuberculosis strains. The biosynthetic gene clusters for pacidamycin and napsamycin have been cloned, and most of the pacidamycin biosynthetic genes have been characterized. However, there were no reports about the transcriptional regulation of the uridyl peptide antibiotics prior to the present work.
The sansanmycin biosynthetic gene cluster (ssa) was identified in Streptomyces sp. SS by a combination of genome mining approach and conventional probing and cosmid sequencing, comprised of25ORFs showing considerable amino acid sequence identity to those of the pacidamycin and napsamycin gene cluster. The structure homology search of SsaA using the online program HHpred revealed that it combined an N-terminal forkhead-associated (FHA) domain with a C-terminal helix-turn-helix DNA-binding motif, suggesting it might be a pathway-specific regulator for sansanmycin biosynthesis. To investigate the contribution of ssaA to the regulation of sansanmycin biosynthesis, the coding region of ssaA was cloned into plasmid pL646under the control of a strong constitutive promoter ermE*p, and the recombinant plasmid were introduced into Streptomyces sp. SS by conjugation. Bioassay and HPLC analyses revealed that overexpression of ssaA increased sansanmycin production, suggesting ssaA is a positive regulator for sansanmycin biosynthesis. To confirm the role of ssaA in sansanmycin biosynthesis, the ssaA disrupted mutant SS/AKO was obtained via PCR-targeting method. Bioassay and HPLC analyses revealed that disruption of ssaA completely abolished sansanmycin production, which was restored by introducing intact ssaA into SS/AKO. These results comfirmed that ssaA is indeed a pivotal positive regulator for sansanmycin biosynthesis.
Quantitative RT-PCR was performed to investigate the relative mRNA level o sansanmycin biosynthetic genes in SS/AKO. The results showed that the relative mRNA level of five structural genes, ssaH, ssaN, ssaP, ssaX and ssaC was decreased, indicatinj that ssaA could positively regulate the sansanmycin biosynthesis by controlling the expression of the structural genes of ssa cluster.
SsaA was overexpressed in E. coli BL21(DE3) as His10-tagged protein. EMSA result: showed that His10-SsaA bound to several promoter regions of the ssa cluster, including ssaHp, ssaN-Pp, ssaU-Ap, ssaC-Dp and ssaWp, suggesting that SsaA controlleo sansanmycin biosynthesis by directly binding to the promoter regions of biosynthetio genes. The binding sites of SsaA in ssa promoter regions were identified by DNase footprinting analysis, and comparison of sequences of the binding sites allowed the identification of a consensus SsaA-binding sequence, GTMCTGACAN2TGTCAGKAC, which consisted of two9-bp inverted repeats (IRs) separated by a2-bp linker. The identified SsaA consensus binding site was validated by mutation or deletion of the most conserved CTGAC sequence in IRs.
EMSA results showed that SS-A or SS-H inhibited the band-shifting of ssaC-Dp anc ssaU-A-lp caused by SsaA in a concentration-dependent manner. SPR analysis results showed that a concentration-dependent inhibition of SsaA binding to ssaC-Dp-3fragment immobilized on an SA sensor chip was readily observed when the amount of SS-A was increased. All these results suggested that sansanmycins could modulate the binding activity of SsaA for its target DNA. These results suggested that sansanmycins could modulate the binding activity of SsaA for its target DNA. Further more, SPR analysis results showed that SsaA could direct interact with SS-A or SS-H. These results indicated that SsaA may strictly control the production of sansanmycins at transcriptional level in a feedback regulatory mechanism by sensing the accumulation of the end-products.
In conclusion, in this work, the biosynthetic gene cluster of uridyl peptide antibiotic sansanmycins was identified for the first time. It has comfirmed that SsaA is a novel class of pathway-specific transcriptional activator of sansanmycin biosynthesis, and that the end products can bind to and change the activity of SsaA to autoregulate the antibiotic production.
引文
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12. Cosmina P, Rodriguez F, de FF, Grandi G, Perego M, Venema G, van SD.1993. Sequence and analysis of the genetic locus responsible for surfactin synthesis in Bacillus subtilis. Mol. Microbiol.8:821-831.
13. Pfennig F, Schauwecker F, Keller U.1999. Molecular characterization of the genes of actinomycin synthetase I and of a 4-methyl-3-hydroxyanthranilic acid carrier protein involved in the assembly of the acylpeptide chain of actinomycin in Streptomyces. J. Biol. Chem. 274:12508-12516.
14. Smith DJ, Earl AJ, Turner G.1990. The multifunctional peptide synthetase performing the first step of penicillin biosynthesis in Penicillium chrysogenum is a 421,073 dalton protein similar to Bacillus brevis peptide antibiotic synthetases. EMBO J.9:2743-2750.
15. Weber G, Schorgendorfer K, Schneider-Scherzer E, Leitner E.1994. The peptide synthetase catalyzing cyclosporine production in Tolypocladium niveum is encoded by a giant 45.8-kilobase open reading frame. Curr. Genet.26:120-125.
16. Haese A, Schubert M, Herrmann M, Zocher R.1993. Molecular characterization of the enniatin synthetase gene encoding a multifunctional enzyme catalysing N-methyldepsipeptide formation in Fusarium scirpi. Mol. Microbiol.7:905-914.
17. Gehring AM, Mori I, Walsh CT.1998. Reconstitution and characterization of the Escherichia coli enterobactin synthetase from EntB, EntE, and EntF. Biochemistry 37:2648-2659.
18. Kohli RM, Trauger JW, Schwarzer D, Marahiel MA, Walsh CT.200 L Generality of peptide cyclization catalyzed by isolated thioesterase domains of nonribosomal peptide synthetases. Biochemistry 40:7099-7108.
19. Keating TA, Ehmann DE, Kohli RM, Marshall CC, Trauger JW, Walsh CT.2001. Chain termination steps in nonribosomal peptide synthetase assembly lines:directed acyl-S-enzyme breakdown in antibiotic and siderophore biosynthesis. Chembiochem.2:99-107.
20. Chen RH, Buko AM, Whittern DN, McAlpine JB.1989. Pacidamycins, a novel series of antibiotics with anti-Pseudomonas aeruginosa activity. Ⅱ. Isolation and structural elucidation. J. Antibiot. (Tokyo) 42:512-520.
21. Isono F, Inukai M, Takahashi S, Haneishi T, Kinoshita T, Kuwano H.1989. Mureidomycins A-D, novel peptidylnucleoside antibiotics with spheroplast forming activity. II. Structural elucidation. J. Antibiot. (Tokyo) 42:667-673.
22. Chatterjee S, Nadkarni SR, Vijayakumar EK, Patel MV, Ganguli BN, Fehlhaber HW, Vertesy L.1994. Napsamycins, new Pseudomonas active antibiotics of the mureidomycin family from Streptomyces sp. HIL Y-82,11372. J. Antibiot. (Tokyo) 47:595-598.
23. Xie Y, Chen R, Si S, Sun C, Xu H.2007. A new nucleosidyl-peptide antibiotic, sansanmycin. J. Antibiot. (Tokyo) 60:158-161.
24. Lam WH, Rychli K, Bugg TD.2008. Identification of a novel beta-replacement reaction in the biosynthesis of 2,3-diaminobutyric acid in peptidylnucleoside mureidomycin A. Org. Biomol. Chem.6:1912-1917.
25. Gruschow S, Rackham EJ, Elkins B, Newill PL, Hill LM, Goss RJ.2009. New pacidamycin antibiotics through precursor-directed biosynthesis. Chembiochem.10:355-360.
26. Zhang W, Ostash B, Walsh CT.2010. Identification of the biosynthetic gene cluster for the pacidamycin group of peptidyl nucleoside antibiotics. Proc. Natl. Acad. Sci. U. S. A 107:16828-16833.
27. Rackham EJ, Gruschow S, Ragab AE, Dickens S, Goss RJ.2010. Pacidamycin biosynthesis: identification and heterologous expression of the first uridyl peptide antibiotic gene cluster. Chembiochem.11:1700-1709.
28. Kaysser L, Tang X, Wemakor E, Sedding K, Hennig S, Siebenberg S, Gust B.2011. Identification of a napsamycin biosynthesis gene cluster by genome mining. Chembiochem. 12:477-487.
29. Zhang W, Ntai I, Bolla ML, Malcolmson SJ, Kahne D, Kelleher NL, Walsh CT.2011. Nine enzymes are required for assembly of the pacidamycin group of peptidyl nucleoside antibiotics. J. Am. Chem. Soc.133:5240-5243.
30. Strieter ER, Vaillancourt FH, Walsh CT.2007. CmaE:a transferase shuttling aminoacyl groups between carrier protein domains in the coronamic acid biosynthetic pathway. Biochemistry 46:7549-7557.
31. Zhang W, Ntai I, Kelleher NL, Walsh CT.2011. tRNA-dependent peptide bond formation by the transferase PacB in biosynthesis of the pacidamycin group of pentapeptidyl nucleoside antibiotics. Proc. Natl. Acad. Sci. U. S. A 108:12249-12253.
32. Ginj C, Ruegger H, Amrhein N, Macheroux P.2005.3'-Enolpyruvyl-UMP, a novel and unexpected metabolite in nikkomycin biosynthesis. Chembiochem.6:1974-1976.
33. Rubio MA, Espinosa JC, Tercero JA, Jimenez A.1998. The Pur10 protein encoded in the gene cluster for puromycin biosynthesis of Streptomyces alboniger is an NAD-dependent ATP dehydrogenase. FEBS Lett.437:197-200.
34. Ragab AE, Gruschow S, Tromans DR, Goss RJ.2011. Biogenesis of the unique 4',5'-dehydronucleoside of the uridyl peptide antibiotic pacidamycin. J. Am. Chem. Soc. 133:15288-15291.