【作者】 韩蓓；

【导师】 袁志明；

【作者基本信息】 中国科学院研究生院（武汉病毒研究所） ， 微生物学， 2007， 博士

【摘要】 球形芽胞杆菌(Bacillus sphaericus)是一种自然界中广泛分布的好氧芽胞杆菌。由于其对蚊幼虫具有的特异性毒杀作用，在世界范围内被成功地应用于疾病媒介蚊虫的生物防治。所有的球形芽胞杆菌不能代谢除N-乙酰基葡萄糖胺外的其它糖类物质，只能利用蛋白质类物质生长和发育。目前对球形芽胞杆菌这种独特的产能代谢途径还缺少了解。早期的研究表明球形芽胞杆菌缺少葡萄糖激酶、己糖激酶活性，还缺少糖酵解途径和磷酸戊糖途径的早期酶：磷酸葡萄糖异构酶、磷酸果糖激酶和葡萄糖-6-磷酸脱氢酶，这些关键酶的缺乏是其不能代谢糖类物质的根本原因。本研究是在完成球形芽胞杆菌C3-41全基因组测序的基础上，通过生物信息学分析绘制了代谢网络图，重点研究细胞内的糖酵解途径及其相关酶—葡萄糖激酶、磷酸果糖激酶和磷酸葡萄糖异构酶的功能。在球形芽胞杆菌菌株中首次发现有活性的营养期高表达的葡萄糖激酶，其编码基因glcK广泛分布于球形芽胞杆菌不同血清型的菌株中。从C3-41中克隆出glcX基因，并完成其在大肠杆菌中表达及表达产物特性分析。glcK由876 bp核苷酸组成，编码33 kDa蛋白，纯化的酶动力学分析表明它对ATP和葡萄糖的K_m值分别为0.52 mM和0.31 mM。该酶具有广泛的底物选择性，可以磷酸化葡萄糖、果糖、甘露糖等，是一种己糖激酶。GlcK氨基酸序列的N末端为典型的ATP结合单元，C末端为α-螺旋-转角-α-螺旋的DNA结合单元，这些保守序列和其他细菌的GlcK序列有很高的同源性，并显示其属于ROK家族蛋白。磷酸果糖激酶PFK催化的反应是EMP途径中的一个限速步骤。实验证实磷酸果糖激酶基因(pfk)以单拷贝形式广泛分布于球形芽胞杆菌不同血清型的菌株中。pfk基因由960 bp核苷酸组成，编码42 kDa的PFK蛋白，其重组表达质粒可以使大肠杆菌(Escherichia coli)PFK缺陷型菌株DF1020回复糖代谢能力，序列分析表明其氨基酸序列上存在保守的底物结合氨基酸位点和ATP结合域。球形芽胞杆菌C3-41全基因组序列分析和特异性PCR检测分析表明，所有球形芽胞杆菌缺少磷酸葡萄糖异构酶编码基因pgi，也无磷酸葡萄糖异构酶活性。在完成蜡状芽胞杆菌(B.cereus)ATCC 33018 pgi基因克隆、表达和纯化的基础上，并将构建的外源pgi穿梭表达质粒，分别转化球形芽胞杆菌高毒力菌株2297和2362。结果表明重组菌株能在二元毒素启动子和cry3A启动子的调控下分别表达PGI蛋白。体外的糖代谢分析试验证实，尽管重组菌株在以糖为碳源的无机盐培养基中生长很慢，但其细胞抽提液能将葡萄糖转化为有机酸物质。通过对球形芽胞杆菌C3-41全基因组测序初步注释结果的分析，发现在碳源的磷酸烯醇式磷酸转移酶转移(PEP-PTS)系统中，缺失细胞膜上特异的碳源结合蛋白EⅡ；而且ATP-Binding Cassette(ABC)转运系统绝大部分注释结果是寡肽的结合蛋白和通透酶，只有少数是碳源的结合蛋白和通透酶。结合实验结果分析，证明球形芽胞杆菌中pgi基因的缺失是其糖酵解途径中断的分子基础，同时球形芽胞杆菌膜转运系统的缺失、突变或者代谢调控系统的作用导致代谢工程菌株不能很好的利用糖类物质生长发育。本研究为进一步研究球形芽胞杆菌特殊产能代谢和保守的进化提供了重要信息，为定向选育，以便获得杀虫毒力高、发酵特性好的工程菌株奠定了基础。更多还原

【Abstract】 Bacillus sphaericus is a Gram-positive, spore-forming, aerobic mosquito pathogenicbacterium. Due to its specific activity against mosquito larvae, B. sphaericus has beensuccessfully used for mosquito control worldwide. Except N-acetylglucosamine, all B.sphaericus could not metabolize any sugars as carbon resource, thus hindering the furtherapplication of B. sphaericus as larvicidal agent because of high production cost offormulations. Previous research indicated that the inability of B. sphaericus to metabolizecarbohydrates could be attributed to the absence of key enzyme activities in the EMP(glucokinase, glucose phosphate isomerase, phosphofructokinase), HMP(phosphogluconate dehydratase) and ED (6-phospho-2-keto-3-deoxyglyconate aldolase)pathways. Unfortunately, little was known on this special sugar metabolism in B.sphaericus untill now. In this research, the glycolysis related enzymes-glucokinase,phosphofructokinase and phosphate glucose isomerase in B. sphaericus had been studied.Our results confirmed the presence of a glucokinase encoding gene glcK on thebacterial chromosome and the expression of glucokinase during the vegetative growth ofB. sphaericus strains. Furthermore, the glcK was cloned from strain C3-41 and expressedin Escherichia coli. Biochemical analysis revealed that this gene encoded a protein withmolecular weight of 33 kDa and the purified recombinant glucokinase had a K_m value of0.52 mM and 0.31 mM for ATP and glucose, respectively. It has been proved this ATPdependent glucokinase could also phosphorylate fructose and mannose. Sequencealignment of glcK indicated that it had a ATP-binding motif in the N-terminal and aα-helix-turn-α-helix DNA-binding motif in the C-terminal, and these conservatived motifs had a high similiary with Glck of other bacterial, and it belongs to the ROKprotein family.A phosphofructokinase encoding gene pfk was found to be widely distributed in B.sphaericus, it had a sigle copy on chromosome and was composed of 960 bp nucleitidesencoding a protein about 42 kDa. Futhermore, a pfk ORF was cloned from B. sphaericusstrain C3-41 and expressed in E. coli. The expression of pfk in recombinant E. coli straincould complement the PFK activity of a pfk mutated E. coli strain DF1020. And the PFKsequece analysis showed it had the conservative amino acids-binding sites and anATP-bingding domain.Genomic sequence of B. sphaericus C3-41 and the specific PCR analysis revealedthat all B. sphaericus strains lacked phosphoglucose isomerase gene pgi, and could notproduce phosphoglucose isomerase activity. It is postulated that the absence of pgi in B.sphaericus might be one of the reasons for bacterial inability to metabolize carbohydrates.For restoring the EMP pathway of B. sphaericus, a pgi ORF was cloned from B. cereusATCC 33018, and then have it expressed in B. sphaericus 2297 and 2362 under thecontrol of binary toxin promoter and cry3A promoter seperately. All recombinant B.sphaericus strains could expressed PGI during bacterial vegetative and spore stage. Evenif the recombinant could not grow and develop well on a inorganic medium with sugar asa carbon resource as expected, the cell extracts could convert glucose into acid in vitro.Primary anonatation result of the C3-41 genome sequence revealed that B.sphaericus lacked EⅡin the PEP-PTS system as well as the dominance ofoligopiptide-specific binding protein and permease protein in ATP-Binding Cassette(ABC) transporter. Supported by our experimental results, it is obvious that the interruptof EMP in B. sphaericus was ascribed to the absence of pgi gene and PGI production.And the partly absence of transporter system, or some effect of the metabolism regulatedsystem likely result in the inability of sugar metabolism in B. sphaericus recombinants.Our findings provide additional data to further elucidate the specific metabolicpathway and for genetic-improvement of B. sphaericus for further mosquito control.更多还原