糖多孢红霉菌TetR家族转录调控子SACE_0012对形态分化的作用
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摘要
糖多孢红霉菌(Saccharopplysppra erythraea)是能形成气生菌丝的放线菌,产生的红霉素A在临床上有广泛的应用。目前,红霉素生物合成已经有较深入的研究,但是有关糖多孢红霉菌形态分化的报道还很少。TetR家族普遍存在于细菌中,主要参与多药抗性、抗生素的合成和形态分化等。实验室徐新强同学前期构建了SACE_0012基因敲除突变体ASACE_0012,通过实验发现TetR调控子SACE_0012可能负调控糖多孢红霉菌中菌丝体的形成。本研究在此基础上进一步验证并探究SACE_0012影响糖多孢红霉菌形态分化的分子机制。
为了验证SACE_0012的功能,接种ΔSACE_0012到R3M平板上,置于30℃培养箱中恒温培养,观察突变株的形态分化情况,发现ΔSACE_0012孢子生长比出发菌株A226提前24h,但继续培养到第六天,ΔSACE_0012与A226表型无明显差异,说明SACE_0012负责糖多孢红霉菌早期菌丝体的形成。利用HPLC检测红霉素A的产量,发现ΔSACE_0012相比糖多孢红霉菌A226没有明显差异,说明SACE_0012不影响糖多孢红霉菌红霉素的合成。为了证明ΔSACE_0012形态分化提前是由于SACE_0012的作用,将SACE_0012克隆到大肠杆菌-糖多孢红霉菌整合性质粒pZMW中,构建出回复菌株ΔSACE_0012/pZMW_0012。结果回复菌株与A226生长一致,说明糖多孢红霉菌ΔSACE_0012形态分化提前是由SACE_0012的缺失引起的。通过敲除和回补实验证明,SACE_0012特异性地调控糖多孢红霉菌早期菌丝体的形成,不参与红霉素的合成。
为了探究SACE_0012对糖多孢红霉菌形态分化的分子机制,对ΔSACE_0012的形态分化基因whi、bldD、amfC和红霉素合成基因eryA转录水平进行定量分析。首先接种ΔSACE_0012和糖多孢红霉菌A226到R3M平板上,置于30℃培养箱中恒温培养4天,用液氮研磨法破碎菌体,提取糖多孢红霉菌A226和ΔSACE_0012的总RNA。然后取适量的RNA进行DNA的消化,最后取适量消化完全的RNA反转录成cDNA作为荧光定量PCR的模板。实时荧光定量PCR结果显示相比出发菌株A226, ASACE_0012突变株中红霉素合成基因eryA和全局调控子bldD的转录量无明显变化；SACE_2141, SACE_6464, SACE_6040(链霉菌中参与孢子形成的调控基因whiA、whiB、whiG的同源基因)的转录量也无显著变化；而SACE—_7115(链霉菌菌丝体形成蛋白amfC的同源基因)的转录量是出发菌株的3倍,回复菌株ASACE_0012/pZMW_0012中SACE_7115的转录量降低到出发菌株的水平。表明SACE_0012与红霉素合成无关,SACE_0012可能是通过AmfC途径影响糖多孢红霉菌早期菌丝体形成。
实验室徐新强前期构建了AbldD/ASACE_0012和ASACE_7040/ΔSACE_0012双突变体,发现缺失SACE_0012不能恢复ΔbldD的孢子生长,也不能加快ASACE_7040的孢子生长。为了深入研究SACE_0012与糖多孢红霉菌BldD和SACE_7040调控系统之间的关系,利用实时荧光定量PCR技术分析ΔbldD、 ASACE_7040突变体中SACE_0012的转录量及ASACE_0012中bldD的转录量。结果显示,相比出发菌株A226,在ΔbldD和ASACE_7040突变体中SACE_0012的转录量有降低但无显著差异；在ASACE_0012中bldD的转录量也无显著差异。证实ASACE_0012突变株菌丝体形成提前是由于增加了amfC的转录,且SACE_0012是独立于BldD调控网络而参与糖多孢红霉菌早期菌丝体形成。
为了探讨SACE_0012和AmfC之间的关系,在糖多孢红霉菌A226中构建SACE_7115缺失突变株。构建方法如下：首先以糖多孢红霉菌A226基因组为模板,通过PCR方法扩增出SACE_7115两侧约1500bp的同源臂,并将SACE_0012的同源臂分别连接到质粒pUCTSR中硫链丝菌肽抗性基因tsr (thiostrepton resistance gene)的两侧,构建质粒pUCTSRΔ7115;然后以pUCTSRΔ7115为模板,PCR扩增片段,通过PEG介导的原生质体转化技术将大片段转入到糖多孢红霉菌A226原生质体中；在30μg/mL Thio的抗性条件下,筛选出有抗性的SACE_7115缺失突变体ASACE_7115。发现ASACE_7115形态分化相比A226延迟,这与ASACE_0012突变株的结果一致。更进一步证实SACE_0012是通过增加了SACE_7115的转录而作用于糖多孢红霉菌早期菌丝体形成。
综上所述,通过RT-PCR、基因敲除和回补分析,发现SACE_0012不影响糖多孢红霉菌的红霉素合成,独立于BldD调控系统,通过AmfC而调控糖多孢红霉菌早期菌丝体的形成。研究结论对全面理解糖多孢红霉菌形态分化调控机制具有重要意义。
Saccharopolyspora erythraea, a mycelium-forming actinomycete, produces erythromycin, a clinically important antibiotic. Extensive investigations have provided insights into erythromycin biosynthesis in Sac. erythraea, but knowledge of its morphogenesis remains limited so far. The TetR family regulates a wide range of cellular activities, such as multidrug resistance, antibiotic biosynthesis, and morphological differentiation, which is widely distributed among bacteria. It was reported that Sac. erythraea contains101TetR-like transcriptional regulators. TetR-family transcriptional regulator SACE_0012had been disrupted by Xinqiang Xu in our lab previously, and was identified to be a negative regulator of mycelium formation of Sac. erythraea. The aim of this study was to further explore the molecular mechanism of SACE_0012in mycelium formation of Sac. erythraea.
To verify the function of SACE_0012, ASACE_0012was inoculated into the flat of R3M at30℃. The results showed that ASACE0012mutant formed aerial mycelium24h earlier than the original strain A226. But after a longer cultivation to the sixth day, no significant phenotypic difference was observed between the wild-type strain A226and mutant ΔSACE_0012, revealing that SACE_0012was responsible for the early aerial hypha formation of Sac. erythraea. And by HPLC analysis, erythromycin A production was not apparently changed between ΔSACE_0012and Sac. erythraea A226. The results suggested that SACE_0012did not affect the synthesis of erythromycin. The ASACE_0012/pZMW-0012strain had restored the timing of aerial mycelium with a single copy of SACE_0012in pZMW to illustrate that it was the SACE_0012deletion that caused the aerial mycelium to form earlier in Sac. erythraea. By using gene inactivation and complementation strategies, the TetR-family transcriptional regulator SACE_0012was identified to be a negative regulator of early aerial hypha formation of Sac. erythraea, and not involved in erythromycin biosynthesis.
In order to explore the molecular mechanism of SACE_0012in mycelium formation, we compared A226and ΔSACE_0012for the transcriptional change to sporulation genes (whi, bldD, amfC) and the erythromycin structure gene eryA. ASACE_0012and Sac. erythraea A226were inoculated into the flat of R3M at30℃, the total RNA were extracted after the cells were finely ground in liguid nitrogen. Then, DNase1was used for degradation of genomic DNA from RNA preparations. RNA was reversely transcribed into cDNA, which was the template for quantitative real time PCR. The RT-PCR results showed that erythromycin structure gene eryA and global regulator gene bldD were not differentially expressed, the homologous genes of whi A, whiB, whiG involved in the regulation of sporulation in Streptomyces (SACE_2141, SACE_6464, SACE_6040, respectively) were also not differentially expressed, while the transcriptional level of SACE_7115(homologous to amfC) was2.0-fold higher in the mutant ΔSACE_0012than in the parent strain A226. The transcription of SACE_7115in the ΔSACE_0012/pZMW_0012complement mutant was back to the original level. The results showed that SACE_0012was not possiblly involved in the regulation of erythromycin biosynthesis, while affected the early aerial hypha formation of Sac.erythraea through AmfC pathway.
ΔbldD/ASACE_0012and ASACE_7040/ASACE_0012had constructed by Xinqiang Xu in our lab previously. The results showed that SACE_0012disruption failed to restore the defect in the mycelium formation of the bldD Mutant, and did not further accelerate the mycelium formation of the ASACE_7040mutant. To futher examine the relationship of SACE_0012and BldD or SACE_7040regulatory system, the transcriptional levels of SACE_0012in the AbldD and ΔSACE_7040strains and bldD in ASACE_0012strain were analysised by qRT-PCR. The results showed that SACE_0012transcriptions were slightly decreased but not obviously different in the AbldD and ASACE?_7040, and the transcriptional level of bldD in ΔSACE_0012were not differentially expressed compared to original strain A226, indicating that SACE_0012, although influencing morphological differentiation, was likely independent of the BldD regulatory system and the possible cause of the early aerial hyphae formation in ASACE_0012was the higher transcriptional level of SACE_7115.
The SACE_7115mutant was constructed to address the relationship between SACE_0012and AmfC. The method was as follows:two1.5kb DNA fragments that upstream and downstream fragments of SACE_7115of the adjacent regions were amplified from the genome of Sac. erythraea A226. Then the amplified products were digested and sequentially inserted into the corresponding sites of pUCTSR, yielding pUCTSRΔ7115. Secondly, the fragments, which carrying the thiostrepton resistance gene and the flanking fragments of SACE_7115, were introduced into protoplasts of Sac. erythraea A226through PEG-mediated transformation. SACE_7115disruption mutant were selected by growing on R3M agar medium flooded with thiostrepton (30μg/mL) selection. It was found that aerial hyphae mycelium of ASACE_7115was delayed compared to the original strain A226, and that was consistent with the results in SACE_0012deletion mutant, and the increased expression of SACE_7115in the ΔSACE_0012mutant. Those futher confirmed our conclusion that the early aerial hyphae formation in ΔSACE_0012is caused by the higher transcriptional level of amfC.
In summary, through gene inactivation and complementation with further transcriptional analysis, SACE_0012was shown not to be related to the BldD regulatory cascade and not to be related to erythromycin biosynthesis, and it regulated early aerial hypha formation of Sac.erythraea by AmfC. This conclusion was useful for comprehensive understanding regulatory mechanism of morphological differentiation in Sac. erythraea.
为了验证SACE_0012的功能,接种ΔSACE_0012到R3M平板上,置于30℃培养箱中恒温培养,观察突变株的形态分化情况,发现ΔSACE_0012孢子生长比出发菌株A226提前24h,但继续培养到第六天,ΔSACE_0012与A226表型无明显差异,说明SACE_0012负责糖多孢红霉菌早期菌丝体的形成。利用HPLC检测红霉素A的产量,发现ΔSACE_0012相比糖多孢红霉菌A226没有明显差异,说明SACE_0012不影响糖多孢红霉菌红霉素的合成。为了证明ΔSACE_0012形态分化提前是由于SACE_0012的作用,将SACE_0012克隆到大肠杆菌-糖多孢红霉菌整合性质粒pZMW中,构建出回复菌株ΔSACE_0012/pZMW_0012。结果回复菌株与A226生长一致,说明糖多孢红霉菌ΔSACE_0012形态分化提前是由SACE_0012的缺失引起的。通过敲除和回补实验证明,SACE_0012特异性地调控糖多孢红霉菌早期菌丝体的形成,不参与红霉素的合成。
为了探究SACE_0012对糖多孢红霉菌形态分化的分子机制,对ΔSACE_0012的形态分化基因whi、bldD、amfC和红霉素合成基因eryA转录水平进行定量分析。首先接种ΔSACE_0012和糖多孢红霉菌A226到R3M平板上,置于30℃培养箱中恒温培养4天,用液氮研磨法破碎菌体,提取糖多孢红霉菌A226和ΔSACE_0012的总RNA。然后取适量的RNA进行DNA的消化,最后取适量消化完全的RNA反转录成cDNA作为荧光定量PCR的模板。实时荧光定量PCR结果显示相比出发菌株A226, ASACE_0012突变株中红霉素合成基因eryA和全局调控子bldD的转录量无明显变化；SACE_2141, SACE_6464, SACE_6040(链霉菌中参与孢子形成的调控基因whiA、whiB、whiG的同源基因)的转录量也无显著变化；而SACE—_7115(链霉菌菌丝体形成蛋白amfC的同源基因)的转录量是出发菌株的3倍,回复菌株ASACE_0012/pZMW_0012中SACE_7115的转录量降低到出发菌株的水平。表明SACE_0012与红霉素合成无关,SACE_0012可能是通过AmfC途径影响糖多孢红霉菌早期菌丝体形成。
实验室徐新强前期构建了AbldD/ASACE_0012和ASACE_7040/ΔSACE_0012双突变体,发现缺失SACE_0012不能恢复ΔbldD的孢子生长,也不能加快ASACE_7040的孢子生长。为了深入研究SACE_0012与糖多孢红霉菌BldD和SACE_7040调控系统之间的关系,利用实时荧光定量PCR技术分析ΔbldD、 ASACE_7040突变体中SACE_0012的转录量及ASACE_0012中bldD的转录量。结果显示,相比出发菌株A226,在ΔbldD和ASACE_7040突变体中SACE_0012的转录量有降低但无显著差异；在ASACE_0012中bldD的转录量也无显著差异。证实ASACE_0012突变株菌丝体形成提前是由于增加了amfC的转录,且SACE_0012是独立于BldD调控网络而参与糖多孢红霉菌早期菌丝体形成。
为了探讨SACE_0012和AmfC之间的关系,在糖多孢红霉菌A226中构建SACE_7115缺失突变株。构建方法如下：首先以糖多孢红霉菌A226基因组为模板,通过PCR方法扩增出SACE_7115两侧约1500bp的同源臂,并将SACE_0012的同源臂分别连接到质粒pUCTSR中硫链丝菌肽抗性基因tsr (thiostrepton resistance gene)的两侧,构建质粒pUCTSRΔ7115;然后以pUCTSRΔ7115为模板,PCR扩增片段,通过PEG介导的原生质体转化技术将大片段转入到糖多孢红霉菌A226原生质体中；在30μg/mL Thio的抗性条件下,筛选出有抗性的SACE_7115缺失突变体ASACE_7115。发现ASACE_7115形态分化相比A226延迟,这与ASACE_0012突变株的结果一致。更进一步证实SACE_0012是通过增加了SACE_7115的转录而作用于糖多孢红霉菌早期菌丝体形成。
综上所述,通过RT-PCR、基因敲除和回补分析,发现SACE_0012不影响糖多孢红霉菌的红霉素合成,独立于BldD调控系统,通过AmfC而调控糖多孢红霉菌早期菌丝体的形成。研究结论对全面理解糖多孢红霉菌形态分化调控机制具有重要意义。
Saccharopolyspora erythraea, a mycelium-forming actinomycete, produces erythromycin, a clinically important antibiotic. Extensive investigations have provided insights into erythromycin biosynthesis in Sac. erythraea, but knowledge of its morphogenesis remains limited so far. The TetR family regulates a wide range of cellular activities, such as multidrug resistance, antibiotic biosynthesis, and morphological differentiation, which is widely distributed among bacteria. It was reported that Sac. erythraea contains101TetR-like transcriptional regulators. TetR-family transcriptional regulator SACE_0012had been disrupted by Xinqiang Xu in our lab previously, and was identified to be a negative regulator of mycelium formation of Sac. erythraea. The aim of this study was to further explore the molecular mechanism of SACE_0012in mycelium formation of Sac. erythraea.
To verify the function of SACE_0012, ASACE_0012was inoculated into the flat of R3M at30℃. The results showed that ASACE0012mutant formed aerial mycelium24h earlier than the original strain A226. But after a longer cultivation to the sixth day, no significant phenotypic difference was observed between the wild-type strain A226and mutant ΔSACE_0012, revealing that SACE_0012was responsible for the early aerial hypha formation of Sac. erythraea. And by HPLC analysis, erythromycin A production was not apparently changed between ΔSACE_0012and Sac. erythraea A226. The results suggested that SACE_0012did not affect the synthesis of erythromycin. The ASACE_0012/pZMW-0012strain had restored the timing of aerial mycelium with a single copy of SACE_0012in pZMW to illustrate that it was the SACE_0012deletion that caused the aerial mycelium to form earlier in Sac. erythraea. By using gene inactivation and complementation strategies, the TetR-family transcriptional regulator SACE_0012was identified to be a negative regulator of early aerial hypha formation of Sac. erythraea, and not involved in erythromycin biosynthesis.
In order to explore the molecular mechanism of SACE_0012in mycelium formation, we compared A226and ΔSACE_0012for the transcriptional change to sporulation genes (whi, bldD, amfC) and the erythromycin structure gene eryA. ASACE_0012and Sac. erythraea A226were inoculated into the flat of R3M at30℃, the total RNA were extracted after the cells were finely ground in liguid nitrogen. Then, DNase1was used for degradation of genomic DNA from RNA preparations. RNA was reversely transcribed into cDNA, which was the template for quantitative real time PCR. The RT-PCR results showed that erythromycin structure gene eryA and global regulator gene bldD were not differentially expressed, the homologous genes of whi A, whiB, whiG involved in the regulation of sporulation in Streptomyces (SACE_2141, SACE_6464, SACE_6040, respectively) were also not differentially expressed, while the transcriptional level of SACE_7115(homologous to amfC) was2.0-fold higher in the mutant ΔSACE_0012than in the parent strain A226. The transcription of SACE_7115in the ΔSACE_0012/pZMW_0012complement mutant was back to the original level. The results showed that SACE_0012was not possiblly involved in the regulation of erythromycin biosynthesis, while affected the early aerial hypha formation of Sac.erythraea through AmfC pathway.
ΔbldD/ASACE_0012and ASACE_7040/ASACE_0012had constructed by Xinqiang Xu in our lab previously. The results showed that SACE_0012disruption failed to restore the defect in the mycelium formation of the bldD Mutant, and did not further accelerate the mycelium formation of the ASACE_7040mutant. To futher examine the relationship of SACE_0012and BldD or SACE_7040regulatory system, the transcriptional levels of SACE_0012in the AbldD and ΔSACE_7040strains and bldD in ASACE_0012strain were analysised by qRT-PCR. The results showed that SACE_0012transcriptions were slightly decreased but not obviously different in the AbldD and ASACE?_7040, and the transcriptional level of bldD in ΔSACE_0012were not differentially expressed compared to original strain A226, indicating that SACE_0012, although influencing morphological differentiation, was likely independent of the BldD regulatory system and the possible cause of the early aerial hyphae formation in ASACE_0012was the higher transcriptional level of SACE_7115.
The SACE_7115mutant was constructed to address the relationship between SACE_0012and AmfC. The method was as follows:two1.5kb DNA fragments that upstream and downstream fragments of SACE_7115of the adjacent regions were amplified from the genome of Sac. erythraea A226. Then the amplified products were digested and sequentially inserted into the corresponding sites of pUCTSR, yielding pUCTSRΔ7115. Secondly, the fragments, which carrying the thiostrepton resistance gene and the flanking fragments of SACE_7115, were introduced into protoplasts of Sac. erythraea A226through PEG-mediated transformation. SACE_7115disruption mutant were selected by growing on R3M agar medium flooded with thiostrepton (30μg/mL) selection. It was found that aerial hyphae mycelium of ASACE_7115was delayed compared to the original strain A226, and that was consistent with the results in SACE_0012deletion mutant, and the increased expression of SACE_7115in the ΔSACE_0012mutant. Those futher confirmed our conclusion that the early aerial hyphae formation in ΔSACE_0012is caused by the higher transcriptional level of amfC.
In summary, through gene inactivation and complementation with further transcriptional analysis, SACE_0012was shown not to be related to the BldD regulatory cascade and not to be related to erythromycin biosynthesis, and it regulated early aerial hypha formation of Sac.erythraea by AmfC. This conclusion was useful for comprehensive understanding regulatory mechanism of morphological differentiation in Sac. erythraea.
引文
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