Cytophaga hutchinsonii遗传操作体系的建立及应用
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摘要
工业革命大幅加速了人类文明前进的步伐,它的动力源泉是25亿年前开始形成的不可再生化石资源。在过去的两百多年里我们一直沉浸在它带来的各种喜悦里。直到几十年前,我们才逐渐意识到这些化石资源即将枯竭。同时我们还要不得不为自己以前的消费买单——治理环境污染,防止气候继续恶化。能源供应问题直接关系到人类的生存和发展,还涉及到世界和平、社会稳定。因此,寻找和开发环境友好的可再生资源已经成为各国迫在眉睫的主要任务。太阳能是地球上生命的最重要的能量来源,而生物质是固定化的太阳能。生物质能源储量丰富,有广阔的开发前景,每年全球植物光合作用生产的生物质能总量大于一千亿吨,仅陆地植物每年吸收固定的碳就有五百亿吨。生物质中最具开发价值的一部分是纤维素,它的生物转化技术条件温和、绿色环保、产品多样。然而其中存在的一个瓶颈问题是纤维素的酶解效率低,成本过高。而造成降解效率低的一个重要因素是纤维素中结晶区的难降解性。因此,结晶纤维素的高效降解是解决这一难题的关键。
噬纤维菌(Cytophaga)广泛存在于自然界中,它对结晶纤维素降解速度快,分解能力强,其模式菌株为Cytophaga hutchinsonii ATCC33406。哈氏噬纤维菌的结晶纤维素降解策略不同于已知的好氧真菌或厌氧细菌,菌体不分泌明显胞外纤维素酶,表面也不存在纤维小体结构,尚未得到阐明,参与降解的相关酶和蛋白尚缺乏研究。Wilson推测C. hutchinsonii存在第三种新型的结晶纤维素降解机制,对这种新型机制的阐明将会有助于对现有纤维素降解酶系的改进和提高。然而对C. hutchinsonii的研究进展一直非常缓慢,主要原因是缺乏成熟的遗传操作体系,难以进行遗传改造,不能深入地进行机理研究。本文的主要内容是以C. hutchinsonii为研究对象,建立一套可行的遗传操作体系,并进一步用该技术对某些相关基因的进行敲除研究。这套遗传操作体系的建立将为进一步揭示C. hutchinsonii未知的纤维素降解机制提供有力的工具。本文具体的研究内容和结果如下：
1、C. hutchinsonii的复制质粒的构建
质粒是遗传操作体系中不可缺少的工具,一些常用的广宿主质粒经过尝试均不能转化进入并在C. hutchinsonii中独立复制。本文利用C. hutchinsonii的基因组复制起点(oriC)和红霉素抗性基因(ermF)构建了一系列的复制质粒。它们可以通过接合转移或电击转化的方式,转化到C. hutchinsonii菌体内并进行复制。进一步对这些质粒的性质进行了研究,发现这些人工构建的质粒在C. hutchinsonii菌体中的拷贝数非常低,常规方法提取的质粒电泳不可见。Southern杂交结果显示,含有较长复制起点DNA片段的质粒更容易与基因组发生同源重组,含有标准长度复制起点DNA片段的质粒以游离形式存在的概率较大。
2、C. hutchinsonii电击转化技术的开发
针对拟杆菌门遗传操作体系开发的相关研究已经实现了转座子Tn4351通过接合转移的方式转入C. hutchinsonii。但是接合转化的条件要求比较苛刻,程序复杂,实验周期长,结果不稳定。化学转化和电击转化的方法虽然简单易行,但是并没有成功的报道。本文利用自己构建的oriC可复制质粒,首次成功实现了对C. hutchinsonii的电击转化。经过对培养条件和电击参数素的优化,初步确定了高效稳定的转化条件：用PYG培养40小时的菌体制备感受态细胞,电击参数采用12kV/cm、200Ω、25μF,电击后复苏4-8h,转化效率可达到103cfu/μg质粒,转化频率达到10-5。实验室其他人员在此基础上继续优化,已经把转化效率和频率各提高了十倍,为进一步对C. hutchinsonii的遗传改造创造了条件。
3、C. hutchinsonii蛋白表达质粒的构建
目前C. hutchinsonii中已报道的可用的报告基因只有红霉素抗性基因(ermF),而报告基因的多样性是一个完善的遗传操作体系所必需的。在oriC复制质粒的基础上构建了可分别更换表达元件和报告基因的质粒。拟杆菌的表达元件不同与常见的E. coli的,据信息学分析C. hutchinsonii的启动子而可能有保守的-33区和-7区序列,而不是-35和-10区,它的核糖体结合序列可能富含A,也不同于常见的RBS。基于本实验室蛋白组和转录组学的相关研究,选取一批本底高表达基因的表达元件用于构建表达质粒,并更换了一系列的报告基因。最终与实验室其他研究者共同发现了三个可用的报告基因：绿色荧光蛋白(gfp)、β－半乳糖苷酶(lacZ)和半乳糖激酶(galK),(其他研究者还发现ca,和tetA也可用)。加和lacZ的优点是直观,易检测定量,而galK作为反向筛选标记基因,可用于未来无痕敲除技术的开发。实验中筛选到的高效启动子(CHU_1284p)可以用于研究某些基因的过表达。另外,C. hutchinsonii的蛋白在E. coli表达系统进行异源表达困难一直很大,这或许与密码子使用偏好性,蛋白折叠差异等相关。若能将这种质粒开发为一种高效的表达载体,通过C.hutchinsonii表达自身的基因,便有可能获得易纯化、有活性的蛋白。
4、C. hutchinsonii基因敲除方法的开发
C. hutchinsonii同源重组效率低,利用常规的自杀质粒进行基因敲除非常困难。本文通过探索,建立了一种效率较高的单同源臂质粒插入敲除的方法,它基于复制质粒,通过两步筛选得到敲除突变株。第一步,利用红霉素抗性筛选出阳性转化子。因质粒可以独立复制,在菌体中存在的时间得到延长,重组到基因组的机会也相应地增加。第二步,利用氯霉素抗性筛选出敲除成功的突变子。质粒上的氯霉素抗性基因(cat)处于同源臂的下游,cat的读码框前端仅含有一段C. hutchinsonii的SD序列,不含启动子,所以游离的质粒不能赋予转化子氯霉素抗性。同源重组后,质粒整合到目的基因上,中断该基因,氯霉素抗性基因可以利用基因组上目的基因的转录元件进行转录,并通过自带的翻译元件进行独立表达。本文用这种敲除方法获得一批突变株,说明了它的高效性。
5、应用比较蛋白组学对C. hutchinson ii纤维素降解相关蛋白进行研究
利用蛋白质组学的手段,比较研究了以滤纸纤维素为唯一碳源和以葡萄糖为唯一碳源的条件下哈氏噬纤维菌全细胞蛋白的表达差异。差异表达蛋白的鉴定结果表明,以纤维素为唯一碳源时上调表达的蛋白质主要涉及氧化应激反应和氨基酸合成代谢。对这些蛋白的分析发现,它们可能与生物膜的形成相关。通过扫描电镜观察发现C. hutchinsonii在降解滤纸纤维素的时候均匀规则地排布在纤维素的表面,这很有可能就是一种生物膜的形式。另一个值得思考的问题是氧化压力的来源。褐腐菌被认为可以通过一种氧化机制降解木质纤维素,在它产生有氧化能力的物质的同时自身也必然会遇到氧化压力。C.hutchinsonii氧化压力来源是否也与氧化降解机制相关还有待探索研究。
6、对一系列可能参与纤维素降解的基因进行敲除,并对突变株的相关性状进行研究
C. hutchinsonii是滑动细菌,其运动形式比较特殊,它没有鞭毛、菌毛、纤毛等菌体表面附属运动器官,这种运动方式和Flavobacterium johnsoniae类似,可以在湿润固体表快速滑动(gliding),个体滑动是单菌落扩散(spreading)的必要不充分条件。信息学分析表明C. hutchinsonii具有全套滑动和扩散相关蛋白。最近的相关研究表明,这类蛋白在细菌表面的定位与一种新的Por分泌系统关系密切。研究人员猜测C. hutchinsonii在纤维素上降解时可能依靠这种滑动来“迁移觅食”。本文利用上面建立的敲除技术对可能在纤维素降解中起重要作用的基因进行了敲除,主要包括一些扩散(spr)和分泌系统(por)相关基因。另外一些敲除目标是实验室转录组中发现的相关基因,如与氨基酸合成和糖类运输相关的基因等。以这些突变株为材料初步研究了它们的相关性状,包括蛋白组分的变化、单菌落在琼脂表面的扩散、对纤维素降解效率、对纤维素的吸附等。初步实验结果显示,纤维素降解和单菌落的扩散能力并没有密切的相关性。
本文构建的遗传操作体系为C. hutchinsonii纤维素降解机制的研究提供了一种简便易行的工具。可复制质粒的构建为外源基因在C. hutchinsonii创造了条件,本实验室的研究人员已通过改变质粒上的抗性基因,成功构建了可以用于回补的质粒,实现了对转座突变子的功能回补。而基因敲除技术则对基因功能的研究意义重大,能够为揭示C. hutchinsonii独特的纤维素降解机制提良好的技术保障。
The industrial revolution has been accelerating the pace of progress of human civilization greatly, and the source of its power is the non-renewable fossil resources formed a hundred million years ago. In the past two hundred years we have been immersed in the joy it brings. Until a few decades ago. we gradually became aware of these fossil resources will soon exhausted. The same time, we still have to control environmental pollution and prevent climate continues to deteriorate. Energy supply problem is directly related to human survival and development, but also involves world peace and social stability. Therefore, to find and develop environmentally friendly renewable resources become an urgent task in countries. Solar energy is the most important source of energy for life on Earth, the biomass is immobilized solar energy. Biomass energy is abundant, there are broad prospects in bio fuel development. The most valuable part of biomass is cellulose. But there is a bottleneck problem, the cellulases' efficiency is too low. and the cost is too expensive. An important factor leading to the low efficiency is the presence of crystalline regions in the cellulose.
Cytophaga hutchinsonii is a Gram-negative bacterium belonging to the Cytophaga-Flavobacterium-Bacteroides (CFB) group which is widely distributed in earth (Walker and Warren1938; Xie et al.2007). C. hutchinsonii could degrade crystalline cellulose rapidly and completely (Stanier1942). However, its genome sequence does not code for any homologues of either processive cellulases, including exo-cellulases and processive endo-cellulases, or dockerin and cohesion (Wilson2009), which are important components of cellulosome (Bayer et al.2004). So, it utilizes an unknown strategy to degrade crystalline celluloses, which is different from both the free cellulases mechanism used by most aerobic microorganisms and the cellulosome mechanism used by most anaerobic bacteria (Wilson2009; Wilson2008). This unknown mechanism may be helpful to improve the efficiency from crystalline cellulose into biofuel. The complete sequencing of C. hutchinsonii has provided a large amount of information (Xie et al.2007), but functional genomics investigation have not been conducted so far because of the lack of genetic manipulation tools (Chen et al.2007b).
The only reported genetic manipulation technique for C. huichinsonii is the transposon-mediated mutagenesis by conjugation (McBride and Baker1996). C. hutchinsonii does not have any endogenous plasmid and no broad-host-range plasmid has been reported to replicate within it. Gene targeting by suicide vectors commonly used in other CFB group organisms does not work for C. hutchinsonii due to low rate of conjugate transformation and homologous recombination. Gene manipulation has been problematic for this bacterium. Here we developed some genetic techniques and tools to study the novel cellulose degradation mechanism of C. hutchinsonii.
1、This work developed replicative plasmids,carrying the replication origin of the C hutchinsonii chromosome. Plasmid is the most important tool, but we did not find any broad-host plasmid that can be used in C. hutchinsonii. Replicable plasmids have previously been constructed for bacteria lacking manipulation tools by cloning the replication origin of the chromosome (oriQ into artificial plasmids (Cordova et al.2002; Monteiro et al.2001; Ye et al.1994). We created a replicable plasmid according to this strategy for C. hutchinsonii, and as a result, they can be used as Escherichia coli-C. hutchinsonii shuttle vectors. Then there transformation efficiency and stability were compared to choose for different purposes. Such as plasmid containing the standard length of replication origin fragment has lower efficiency, but its probability of integration into the genome is also low, so it would be more suitable for the knockout.
2、Using the replicable plasmid, we developed a method for electro-transformation. Before this work, the only reported genetic manipulation technique for C. hutchinsonii is transposon-mediated mutagenesis by conjugation with Escherichia coli. Finally a transformation efficiency of about2×103transformants per microgram plasmid DNA or transformation frenquency of10-5as achieved. Higher transformation efficiency can be compensate for the low efficiency of the C. hutchinsonii homologous recombination defects.
3、Some heterologous genes, including green fluorescent protein, galactose kinase and β-galactosidase. were expressed successfully and proved functional in C. hutchinsonii under the control of the CHU_1284promoter in oriC plasmids. Studies on expression regulatory elements in Bacteroides is rare. Some reporter genes cannot be used in C. hutchinsonii directly. The expression elements such as the promoter must be modified in C. hutchinsonii. promoters in C. hutchinsonii are found very similar to Flavobacterium hibernum, they do not have the-35and-10conserved sequence, but-33and-7. The ribosome binding sequence in C. hutchinsonii is similar to Flavobacterium hibernum.
4、A comparative proteomic analysis of C. hutchinsonii was performed, and found some interesting proteins maybe taking part in cellulose degradation. We performed a comparative proteomic analysis of the total cell proteins of C. hutchinsonii cultured in Stanier's mineral base medium with either glucose or filter paper as the sole carbon source. The significantly up-regulated proteins induced by cellulose were identified by MS. The up-regulated proteins were mainly related to oxidative stress response and amino acid metabolism. The cells were under oxidative stress and might be in a kind of biofilm when they growing on cellulose fiber.
5、The replicative plasmid was successfully used for gene targeting. The homologous recombination efficiency in C. hutchinsonii is very low, so, gene targeting by ordinary suicide plasmid is very difficult. In this work, we developed an efficient knockout method, it is based on the oriC plasmid with two-step screening. The first step, screen of transformants having erythromycin resistance. Second step, the screen out chloramphenicol resistance colonies. The cat gene on the plasmid does not have its own promoter, so transformant only having free plasmid does not has chloramphenicol resistance. After homologous recombination, the plasmid integrates into the target gene, and the cat gene can be transcript by promoter upstream of target gene. These genetic tools will be useful in functional genomics investigations of the cellulolytic
6、Some genes were knocked out and some phenotypes of the mutants were investigated. Some genes, specially related to gliding and porSS, were selected for gene targeting. C. hutchinsonii does not have cell surface flagella or other similar motion organs, but could move fast on the wet surface, just like Flavobaclerium johnsoniae. Bio informatics analysis shows that C. hutchinsonii has a full set of gliding related proteins. The cellulose degradation of C. hutchinsonii was thought to be related to gliding motility before. But the phenotypes studies of these mutants reveals the relationship between cellulose degradation and spreading is not so close.
We believe these tools will be useful in the investigation of C. hutchinsonii.
噬纤维菌(Cytophaga)广泛存在于自然界中,它对结晶纤维素降解速度快,分解能力强,其模式菌株为Cytophaga hutchinsonii ATCC33406。哈氏噬纤维菌的结晶纤维素降解策略不同于已知的好氧真菌或厌氧细菌,菌体不分泌明显胞外纤维素酶,表面也不存在纤维小体结构,尚未得到阐明,参与降解的相关酶和蛋白尚缺乏研究。Wilson推测C. hutchinsonii存在第三种新型的结晶纤维素降解机制,对这种新型机制的阐明将会有助于对现有纤维素降解酶系的改进和提高。然而对C. hutchinsonii的研究进展一直非常缓慢,主要原因是缺乏成熟的遗传操作体系,难以进行遗传改造,不能深入地进行机理研究。本文的主要内容是以C. hutchinsonii为研究对象,建立一套可行的遗传操作体系,并进一步用该技术对某些相关基因的进行敲除研究。这套遗传操作体系的建立将为进一步揭示C. hutchinsonii未知的纤维素降解机制提供有力的工具。本文具体的研究内容和结果如下：
1、C. hutchinsonii的复制质粒的构建
质粒是遗传操作体系中不可缺少的工具,一些常用的广宿主质粒经过尝试均不能转化进入并在C. hutchinsonii中独立复制。本文利用C. hutchinsonii的基因组复制起点(oriC)和红霉素抗性基因(ermF)构建了一系列的复制质粒。它们可以通过接合转移或电击转化的方式,转化到C. hutchinsonii菌体内并进行复制。进一步对这些质粒的性质进行了研究,发现这些人工构建的质粒在C. hutchinsonii菌体中的拷贝数非常低,常规方法提取的质粒电泳不可见。Southern杂交结果显示,含有较长复制起点DNA片段的质粒更容易与基因组发生同源重组,含有标准长度复制起点DNA片段的质粒以游离形式存在的概率较大。
2、C. hutchinsonii电击转化技术的开发
针对拟杆菌门遗传操作体系开发的相关研究已经实现了转座子Tn4351通过接合转移的方式转入C. hutchinsonii。但是接合转化的条件要求比较苛刻,程序复杂,实验周期长,结果不稳定。化学转化和电击转化的方法虽然简单易行,但是并没有成功的报道。本文利用自己构建的oriC可复制质粒,首次成功实现了对C. hutchinsonii的电击转化。经过对培养条件和电击参数素的优化,初步确定了高效稳定的转化条件：用PYG培养40小时的菌体制备感受态细胞,电击参数采用12kV/cm、200Ω、25μF,电击后复苏4-8h,转化效率可达到103cfu/μg质粒,转化频率达到10-5。实验室其他人员在此基础上继续优化,已经把转化效率和频率各提高了十倍,为进一步对C. hutchinsonii的遗传改造创造了条件。
3、C. hutchinsonii蛋白表达质粒的构建
目前C. hutchinsonii中已报道的可用的报告基因只有红霉素抗性基因(ermF),而报告基因的多样性是一个完善的遗传操作体系所必需的。在oriC复制质粒的基础上构建了可分别更换表达元件和报告基因的质粒。拟杆菌的表达元件不同与常见的E. coli的,据信息学分析C. hutchinsonii的启动子而可能有保守的-33区和-7区序列,而不是-35和-10区,它的核糖体结合序列可能富含A,也不同于常见的RBS。基于本实验室蛋白组和转录组学的相关研究,选取一批本底高表达基因的表达元件用于构建表达质粒,并更换了一系列的报告基因。最终与实验室其他研究者共同发现了三个可用的报告基因：绿色荧光蛋白(gfp)、β－半乳糖苷酶(lacZ)和半乳糖激酶(galK),(其他研究者还发现ca,和tetA也可用)。加和lacZ的优点是直观,易检测定量,而galK作为反向筛选标记基因,可用于未来无痕敲除技术的开发。实验中筛选到的高效启动子(CHU_1284p)可以用于研究某些基因的过表达。另外,C. hutchinsonii的蛋白在E. coli表达系统进行异源表达困难一直很大,这或许与密码子使用偏好性,蛋白折叠差异等相关。若能将这种质粒开发为一种高效的表达载体,通过C.hutchinsonii表达自身的基因,便有可能获得易纯化、有活性的蛋白。
4、C. hutchinsonii基因敲除方法的开发
C. hutchinsonii同源重组效率低,利用常规的自杀质粒进行基因敲除非常困难。本文通过探索,建立了一种效率较高的单同源臂质粒插入敲除的方法,它基于复制质粒,通过两步筛选得到敲除突变株。第一步,利用红霉素抗性筛选出阳性转化子。因质粒可以独立复制,在菌体中存在的时间得到延长,重组到基因组的机会也相应地增加。第二步,利用氯霉素抗性筛选出敲除成功的突变子。质粒上的氯霉素抗性基因(cat)处于同源臂的下游,cat的读码框前端仅含有一段C. hutchinsonii的SD序列,不含启动子,所以游离的质粒不能赋予转化子氯霉素抗性。同源重组后,质粒整合到目的基因上,中断该基因,氯霉素抗性基因可以利用基因组上目的基因的转录元件进行转录,并通过自带的翻译元件进行独立表达。本文用这种敲除方法获得一批突变株,说明了它的高效性。
5、应用比较蛋白组学对C. hutchinson ii纤维素降解相关蛋白进行研究
利用蛋白质组学的手段,比较研究了以滤纸纤维素为唯一碳源和以葡萄糖为唯一碳源的条件下哈氏噬纤维菌全细胞蛋白的表达差异。差异表达蛋白的鉴定结果表明,以纤维素为唯一碳源时上调表达的蛋白质主要涉及氧化应激反应和氨基酸合成代谢。对这些蛋白的分析发现,它们可能与生物膜的形成相关。通过扫描电镜观察发现C. hutchinsonii在降解滤纸纤维素的时候均匀规则地排布在纤维素的表面,这很有可能就是一种生物膜的形式。另一个值得思考的问题是氧化压力的来源。褐腐菌被认为可以通过一种氧化机制降解木质纤维素,在它产生有氧化能力的物质的同时自身也必然会遇到氧化压力。C.hutchinsonii氧化压力来源是否也与氧化降解机制相关还有待探索研究。
6、对一系列可能参与纤维素降解的基因进行敲除,并对突变株的相关性状进行研究
C. hutchinsonii是滑动细菌,其运动形式比较特殊,它没有鞭毛、菌毛、纤毛等菌体表面附属运动器官,这种运动方式和Flavobacterium johnsoniae类似,可以在湿润固体表快速滑动(gliding),个体滑动是单菌落扩散(spreading)的必要不充分条件。信息学分析表明C. hutchinsonii具有全套滑动和扩散相关蛋白。最近的相关研究表明,这类蛋白在细菌表面的定位与一种新的Por分泌系统关系密切。研究人员猜测C. hutchinsonii在纤维素上降解时可能依靠这种滑动来“迁移觅食”。本文利用上面建立的敲除技术对可能在纤维素降解中起重要作用的基因进行了敲除,主要包括一些扩散(spr)和分泌系统(por)相关基因。另外一些敲除目标是实验室转录组中发现的相关基因,如与氨基酸合成和糖类运输相关的基因等。以这些突变株为材料初步研究了它们的相关性状,包括蛋白组分的变化、单菌落在琼脂表面的扩散、对纤维素降解效率、对纤维素的吸附等。初步实验结果显示,纤维素降解和单菌落的扩散能力并没有密切的相关性。
本文构建的遗传操作体系为C. hutchinsonii纤维素降解机制的研究提供了一种简便易行的工具。可复制质粒的构建为外源基因在C. hutchinsonii创造了条件,本实验室的研究人员已通过改变质粒上的抗性基因,成功构建了可以用于回补的质粒,实现了对转座突变子的功能回补。而基因敲除技术则对基因功能的研究意义重大,能够为揭示C. hutchinsonii独特的纤维素降解机制提良好的技术保障。
The industrial revolution has been accelerating the pace of progress of human civilization greatly, and the source of its power is the non-renewable fossil resources formed a hundred million years ago. In the past two hundred years we have been immersed in the joy it brings. Until a few decades ago. we gradually became aware of these fossil resources will soon exhausted. The same time, we still have to control environmental pollution and prevent climate continues to deteriorate. Energy supply problem is directly related to human survival and development, but also involves world peace and social stability. Therefore, to find and develop environmentally friendly renewable resources become an urgent task in countries. Solar energy is the most important source of energy for life on Earth, the biomass is immobilized solar energy. Biomass energy is abundant, there are broad prospects in bio fuel development. The most valuable part of biomass is cellulose. But there is a bottleneck problem, the cellulases' efficiency is too low. and the cost is too expensive. An important factor leading to the low efficiency is the presence of crystalline regions in the cellulose.
Cytophaga hutchinsonii is a Gram-negative bacterium belonging to the Cytophaga-Flavobacterium-Bacteroides (CFB) group which is widely distributed in earth (Walker and Warren1938; Xie et al.2007). C. hutchinsonii could degrade crystalline cellulose rapidly and completely (Stanier1942). However, its genome sequence does not code for any homologues of either processive cellulases, including exo-cellulases and processive endo-cellulases, or dockerin and cohesion (Wilson2009), which are important components of cellulosome (Bayer et al.2004). So, it utilizes an unknown strategy to degrade crystalline celluloses, which is different from both the free cellulases mechanism used by most aerobic microorganisms and the cellulosome mechanism used by most anaerobic bacteria (Wilson2009; Wilson2008). This unknown mechanism may be helpful to improve the efficiency from crystalline cellulose into biofuel. The complete sequencing of C. hutchinsonii has provided a large amount of information (Xie et al.2007), but functional genomics investigation have not been conducted so far because of the lack of genetic manipulation tools (Chen et al.2007b).
The only reported genetic manipulation technique for C. huichinsonii is the transposon-mediated mutagenesis by conjugation (McBride and Baker1996). C. hutchinsonii does not have any endogenous plasmid and no broad-host-range plasmid has been reported to replicate within it. Gene targeting by suicide vectors commonly used in other CFB group organisms does not work for C. hutchinsonii due to low rate of conjugate transformation and homologous recombination. Gene manipulation has been problematic for this bacterium. Here we developed some genetic techniques and tools to study the novel cellulose degradation mechanism of C. hutchinsonii.
1、This work developed replicative plasmids,carrying the replication origin of the C hutchinsonii chromosome. Plasmid is the most important tool, but we did not find any broad-host plasmid that can be used in C. hutchinsonii. Replicable plasmids have previously been constructed for bacteria lacking manipulation tools by cloning the replication origin of the chromosome (oriQ into artificial plasmids (Cordova et al.2002; Monteiro et al.2001; Ye et al.1994). We created a replicable plasmid according to this strategy for C. hutchinsonii, and as a result, they can be used as Escherichia coli-C. hutchinsonii shuttle vectors. Then there transformation efficiency and stability were compared to choose for different purposes. Such as plasmid containing the standard length of replication origin fragment has lower efficiency, but its probability of integration into the genome is also low, so it would be more suitable for the knockout.
2、Using the replicable plasmid, we developed a method for electro-transformation. Before this work, the only reported genetic manipulation technique for C. hutchinsonii is transposon-mediated mutagenesis by conjugation with Escherichia coli. Finally a transformation efficiency of about2×103transformants per microgram plasmid DNA or transformation frenquency of10-5as achieved. Higher transformation efficiency can be compensate for the low efficiency of the C. hutchinsonii homologous recombination defects.
3、Some heterologous genes, including green fluorescent protein, galactose kinase and β-galactosidase. were expressed successfully and proved functional in C. hutchinsonii under the control of the CHU_1284promoter in oriC plasmids. Studies on expression regulatory elements in Bacteroides is rare. Some reporter genes cannot be used in C. hutchinsonii directly. The expression elements such as the promoter must be modified in C. hutchinsonii. promoters in C. hutchinsonii are found very similar to Flavobacterium hibernum, they do not have the-35and-10conserved sequence, but-33and-7. The ribosome binding sequence in C. hutchinsonii is similar to Flavobacterium hibernum.
4、A comparative proteomic analysis of C. hutchinsonii was performed, and found some interesting proteins maybe taking part in cellulose degradation. We performed a comparative proteomic analysis of the total cell proteins of C. hutchinsonii cultured in Stanier's mineral base medium with either glucose or filter paper as the sole carbon source. The significantly up-regulated proteins induced by cellulose were identified by MS. The up-regulated proteins were mainly related to oxidative stress response and amino acid metabolism. The cells were under oxidative stress and might be in a kind of biofilm when they growing on cellulose fiber.
5、The replicative plasmid was successfully used for gene targeting. The homologous recombination efficiency in C. hutchinsonii is very low, so, gene targeting by ordinary suicide plasmid is very difficult. In this work, we developed an efficient knockout method, it is based on the oriC plasmid with two-step screening. The first step, screen of transformants having erythromycin resistance. Second step, the screen out chloramphenicol resistance colonies. The cat gene on the plasmid does not have its own promoter, so transformant only having free plasmid does not has chloramphenicol resistance. After homologous recombination, the plasmid integrates into the target gene, and the cat gene can be transcript by promoter upstream of target gene. These genetic tools will be useful in functional genomics investigations of the cellulolytic
6、Some genes were knocked out and some phenotypes of the mutants were investigated. Some genes, specially related to gliding and porSS, were selected for gene targeting. C. hutchinsonii does not have cell surface flagella or other similar motion organs, but could move fast on the wet surface, just like Flavobaclerium johnsoniae. Bio informatics analysis shows that C. hutchinsonii has a full set of gliding related proteins. The cellulose degradation of C. hutchinsonii was thought to be related to gliding motility before. But the phenotypes studies of these mutants reveals the relationship between cellulose degradation and spreading is not so close.
We believe these tools will be useful in the investigation of C. hutchinsonii.
引文
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