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1、基于代谢组学的芽胞杆菌属间胞外代谢物异质性研究基金项目：国家“973”前期项目（2011CB111607），国家“948”项目（2011-G25），福建省青年科技创新基金（2011J05053）。作者简介：陈峥（1982-），男，博士，主要从事化学生态学和生物活性物质分析研究通讯作者：刘波（1957-），男，博士、研究员，主要从事微生物生物技术及农业生物药物研究（E-mail：Fzliubo163com）陈峥，刘波*，史怀，朱育菁，刘国红，潘志针，陈梅春(福建省农业科学院农业生物资源研究所，福建 福州 350003)摘要：采用基于气相色谱质谱联用（GC/MS）的代谢组学方法研究了芽胞杆菌三个属
2、胞外代谢物，得到代谢物共229种。并对结果做了PCA和分层聚类分析，结果表明供试不同属的芽胞杆代谢物种类和含量不同有所不同，而同属的菌株代谢物相似，同属的菌株能聚成一类。其中类芽胞杆菌属和芽胞杆菌属又聚为一个亚类，而短芽胞杆菌属聚为另一个亚类。通过对三个属芽胞杆菌所分析的数据中，初步鉴定得到3个差异性代谢物。关键词：芽胞杆菌，代谢组学，属，胞外代谢物，异质性Heterogeneity research of extracellular metabolites of three Bacillus genera based on metabonomicsCHEN Zheng, LIU Bo*, S
3、HI Huai, ZHU Yu-jing，LIU Guo-Hong，PAN Zhi-zhen，CHEN Mei-chun(Institute of Agrobiological Resources, Fujian Academy of Agricultural Sicences, Fuzhou, Fujian 350003, China)Abstract：The extracellular metabolites of three Bacillus genera were analyzed by gas chromatography/ mass spectrometry(GC/MS) base
4、d on metabonomics. 229 metabolites were indentified from these genera. The result of PCA and hierarchical cluster analysis show that the metabonomics in different generic were different. The metabonomics in the same generic were similar. The strains of the same genera could be clustered into one gro
5、up. The strains of Paenibacillus and Bacillus clustered into one subgroup, and stains of Brevibacillus clustered into one subgroup. Three metabolic markers were identified from the metabolites of three Bacillus genera.Key words: Bacillus，metabonomics，genera，extracellular metabolites，heterogeneity芽胞杆
6、菌属Bacillus是一类产芽孢的革兰氏阳性细菌，产生具有抗逆性球形或椭圆形芽孢，芽孢对高温、紫外、干燥、电离辐射和很多有毒化学物质都有很强的抗性1。芽胞杆菌是一类重要的资源微生物，与人类生活关系密切，如在工业领域用于环境污染治理等、在农业领域用于生物农药等、医学领域用于新型药物载体等。代谢组学（metabonomics）是对限定条件下特定生物样品中所有代谢组分的定性和定量2，是进行生物表型研究的主要手段之一，是系统生物学研究不可或缺的部分3。代谢组学主要的研究平台包括气相色谱质谱联用（GC/MS）、液相色谱质谱联用(LC/MS)、毛细管电泳质谱联用(CE/MS)和核磁共振(NMR)等。代谢组
7、学在生物，农业，医学，环境等领域得到了或将会得到广泛的应用，有着非常重要的意义4。芽胞杆菌的代谢物研究有过许多报道，如对芽胞杆菌挥发性物质的研究，包括枯草芽胞杆菌E20 5、BL026、G86、短短芽胞杆菌FJAT-0809-GLX7挥发性物质的研究等；对芽胞杆菌发酵液成分研究，包括多粘类芽胞杆菌HY96-28；多粘类芽胞杆菌BS049；枯草芽胞杆菌B3610；枯草芽胞杆菌BY-211；短短芽胞杆菌FJAT-0809-GLX12,13等；芽胞杆菌脂肪酸成分的研究，包括需氧芽胞杆菌芽胞脂肪酸分析14。然而，对该短短芽胞杆菌的代谢物差异的研究未见报道，本文通过对三个芽胞杆菌属胞外代谢物异质性研究，
8、揭示芽胞杆菌属间分类的代谢组学分析方法，分析芽胞杆菌属间差异的代谢组学基础，为研究芽胞杆菌次生代谢物提供基础。1 材料与方法1.1 材料供试菌株7株，均为DSMZ标准菌种，分别为：莫海威芽胞杆菌FJAT-10005（Bacillus mojavensis）；植物内芽胞杆菌FJAT-10010（Bacillus endophyticus）；蜂房类芽胞杆菌FJAT-10002（Paenibacillus alvei）；解凝乳类芽胞杆菌FJAT-10009（Paenibacillus curdlanolyticus）；波茨坦短芽胞杆菌FJAT-10006（Brevibacillus borstele
9、nsis）；桥石短芽胞杆菌FJAT-10008（Brevibacillus choshinensis）；美丽短芽胞杆菌FJAT-10011（Brevibacillus formosus）。保鲜菌种子培养基：淀粉 1.0 %，牛肉膏 0.5 %，蛋白胨 0.3 %，蔗糖 1.0 %，酵母粉 0.5 %，CaCl2 0.5 %，pH 7.0。发酵培养基：TSB培养基（美国BD公司）。实验试剂：分析纯丙酮。实验仪器：主要实验仪器采用美国Agilent公司制造的7890A/5975C气质联用仪（GC/MS）；电子天平（Navigator NO4120）；恒温摇床（HZQ-F160）；pH计。 1.2
10、实验方法1.2方法1.2.1样品制备方法 各取200 mL 芽胞杆菌菌株发酵液于1 L三角瓶中，加入200 mL丙酮溶液，充分搅拌后，隔8 h搅拌一次，24 h后，离心取上清液A。对离心后的残余部分，加入等体积的丙酮，萃取24h后离心得上清液B，与原来的萃取液A合并。最后取部分溶液加无水硫酸钠处理，离心取上清液加入GC小瓶，置于4 冰箱保存，等待进样。1.2.2 色谱质谱条件色谱条件：采用HP5-MS色谱柱，进样口温度250 ，压力5.5977 psi，总流量104 mL/min，隔垫吹扫流量3 mL/min，不分流。升温程序为：50 保持2 min，以6 /min上升到250 保持5 min
11、，然后以20 /min上升到280 保持5 min。质谱条件：采集模式：全扫描；EMV模式：相对值；全扫描参数：开始时候的质量数50.00 amu，结束时的质量数550.00 mu；MS温度：离子源230 ，MS四级杆150 。1.2.3 数据采集及统计 对气相质谱所得到的数据，先用Agilent GC/MS Translator将数据转换为“.D”格式后，用Agilent MassHunter Workstation对所得数据进行色谱图解卷积提取（Find by chromatogram Deconvolution）后导出“.CEF”格式文件，用Agilent Mass Profiler P
12、rofessional进行数据处理统计。 气相质谱数据采集分析采用Agilent MSD Chemstation E.02.00.493；数据转换采用软件Agilent GC/MS Translator；数据分子特征提取采用软件Agilent MassHunter Workstation Software Qualitative Analysis;数据统计采用软件Agilent Mass Profiler Professional B.02.00。对所获得的数据进行分子特征提取、背景扣除、数据筛选，采用ANOVA和PCA进行统计分析，分类预测模型构建，分子式推断和数据库检索等。2 结果与分析2
13、.1 芽胞杆菌代谢物提取与筛选利用Agilent MassHunter Workstation对所得数据进行色谱图解卷积提取（Find by chromatogram Deconvolution），从三个属七个菌株中得到代谢物共229个（见表1）。分别从样品FJAT-10005（Bacillus mojavensis）、FJAT-10010（Bacillus endophyticus）、FJAT-10006（Brevibacillus borstelensis）、FJAT-10008（Brevibacillus choshinensis）、FJAT-10011（Brevibacillus fo
14、rmosus）、FJAT-10002（Paenibacillus alvei）、FJAT-10009（Paenibacillus curdlanolyticus）中分别得到代谢物58、49、81、68、40、77、72种。而存在于七个菌株的总体代谢物，而未在FJAT-10005（Bacillus mojavensis）、FJAT-10010（Bacillus endophyticus）、FJAT-10006（Brevibacillus borstelensis）、FJAT-10008（Brevibacillus choshinensis）、FJAT-10011（Brevibacillus fo
15、rmosus）、FJAT-10002（Paenibacillus alvei）、FJAT-10009（Paenibacillus curdlanolyticus）中存在的代谢物分别为171、180、148、161、189、152、157种。表1 经色谱图解卷积提取后得到的三个属芽胞杆菌代谢物数量Sample NameSampleinformationCompoundsPresentCompoundsAbsenttotalFJAT-10005Bacillus mojavensis58171229FJAT-10010Bacillus endophyticus49180FJAT-10006Brevi
16、bacillus borstelensis81148FJAT-10008Brevibacillus choshinensis68161FJAT-10011Brevibacillus formosus40189FJAT-10002Paenibacillus alvei77152FJAT-10009Paenibacillus curdlanolyticus72157图1 不同代谢物在保留时间上的分布情况2.2 芽胞杆菌属间代谢物PCA分析以芽胞杆菌三个属7个菌株为例，进行PCA分析，结果见图2。试验结果表明类芽胞杆菌属和芽胞杆菌属内的样品点比较集中，而短芽胞杆菌属内的样品点较为松散。但种间差异大于
17、组内差异，各组在PCA图上的区分较为明显。样品点莫海威芽胞杆菌FJAT-10005（Bacillus mojavensis）、植物内芽胞杆菌FJAT-10010（Bacillus endophyticus）聚为一组为芽胞杆菌属；样品点蜂房类芽胞杆菌FJAT-10002（Paenibacillus alvei）、解凝乳类芽胞杆菌FJAT-10009（Paenibacillus curdlanolyticus）聚为一组为类芽胞杆菌属。样品点波茨坦短芽胞杆菌FJAT-10006（Brevibacillus borstelensis）；桥石短芽胞杆菌FJAT-10008（Brevibacillus c
18、hoshinensis）；美丽短芽胞杆菌FJAT-10011（Brevibacillus formosus）聚为一组为短芽胞杆菌属。X轴：组分1（80.09%）Y轴：组分2（19.71%）Z轴：组分3（0.19%）X轴：组分1（80.09%）Y轴：组分2（19.71%）图2 基于代谢组学三个属芽胞杆菌代谢物PCA分析（上图为3D PCA score，下图为2D PCA score）2.3 芽胞杆菌属间代谢物聚类分析以芽胞杆菌三个属7个菌株为例，对芽胞杆菌代谢物进行聚类分析，结果见图3。对不同菌株发酵液中测定出次生代谢物进行聚类分析，将七个芽胞杆菌的产生的次生代谢物可以分为2大类群。类群1包括莫
19、海威芽胞杆菌FJAT-10005（Bacillus mojavensis）；植物内芽胞杆菌FJAT-10010（Bacillus endophyticus）；蜂房类芽胞杆菌FJAT-10002（Paenibacillus alvei）；解凝乳类芽胞杆菌FJAT-10009（Paenibacillus curdlanolyticus）。该类群的特点为：菌株分别为芽胞杆菌属及类芽胞杆菌属。类群2包括波茨坦短芽胞杆菌FJAT-10006（Brevibacillus borstelensis）；桥石短芽胞杆菌FJAT-10008（Brevibacillus choshinensis）；美丽短芽胞杆菌F
20、JAT-10011（Brevibacillus formosus）。该类群特点为：菌株都为短芽胞杆菌属。供试不同属的芽胞杆代谢物种类和含量不同有所不同，而同属的菌株代谢物相似，同属的菌株能聚成一类。其中类芽胞杆菌属和芽胞杆菌属又聚为一个亚类。图3 基于代谢组学芽胞杆菌菌种分类聚类分析2.4 芽胞杆菌属间代谢标记物鉴定以芽胞杆菌三个属7个菌株为例，对发生显著性变化(P0.05,倍数变化2)的代谢物经过在NIST数据中搜索（IDBrowser）比对后，得到差异性代谢物3个，初步鉴定结果见表2。差异性代谢物分别为N-methy-Propanamide、2,5,6-Trimethyl-1,3-oxat
21、hiane、1H-Indole-7-carboxylic acid。表2 三个属芽胞杆菌代谢标记物CompoundFJAT-10005FJAT-10010FJAT-10006FJAT-10008FJAT-10011FJAT-10002FJAT-10009AnnotationsPropanamide, N-methyl-1111139593504158002311 C4H9NO, overall=58.48, CAS ID=1187-58-2 2,5,6-Trimethyl-1,3-oxathiane112176394343558929111 C7H14OS, overall=52.08, CA
22、S ID=114376-06-6 1H-Indole-7-carboxylic acid7-吲哚甲酸116970674377232976630649704 C9H7NO2, overall=59.51, CAS ID=1670-83-3 3 讨论近年来，关于芽胞杆菌脂肪酸类型14、挥发性物质5-7、发酵液成分8-13的报道较多，而芽胞杆菌的代谢组学研究较少，Bundy JG等15（2005）应用基于NMR的代谢轮廓分析，区分了蜡状芽胞杆菌脑膜炎致病性菌株和无致病性菌株。基于GC-MS的芽胞杆菌代谢组学分析未见报道。本实验以芽胞杆菌标准菌株为样品，采用气相色谱质谱联用（GC/MS）的方法对三个属
23、七个种芽胞杆菌标准菌种的代谢图谱进行分析比较，研究其胞外代谢产物的差异。并对结果做了PCA和分层聚类分析，结果表明供试不同属的芽胞杆代谢物种类和含量有所不同，同属的菌株能聚成一类。其中类芽胞杆菌属和芽胞杆菌属又聚为一个亚类，短芽胞杆菌属单独聚为另一个亚类。代谢组学分析可以区别短芽胞杆菌属，类芽胞杆菌属，芽胞杆菌属，属间的物质差异较大。通过对三个属芽胞杆菌所分析的数据中，初步鉴定得到3个差异性代谢物，但是否其就是生物标记物（Biomarker）还需要做二级质谱（MS/MS）的分析，差异性化合物的初步鉴定为进一步分析芽胞杆菌属间差异和芽胞杆菌代谢组学提供基础。本文通过对三个芽胞杆菌属胞外代谢物异质
24、性研究，揭示芽胞杆菌属间分类的代谢组学分析方法，分析芽胞杆菌属间差异的代谢组学基础，为研究芽胞杆菌次生代谢物提供基础。 本实验基于GC-MS的方法，GC-MS由于兼备气相色谱的高分辨率和质谱的高灵敏度16，且有固定的谱库可提供检索，多被用于对代谢物定性定量和复杂样品的分离鉴定。但GC-MS只能检测到易气化，热稳定性强，分子量相对较低的代谢物；而不易被气化，热稳定性差，分子量相对较大，不易衍生化的代谢物需进一步用LC-MS的数据进行补充，对阐明芽胞杆菌属间异质性提供更全面的参考依据。参考文献参考文献1 刘国红,林营志,林乃铨,刘波.芽胞杆菌分类研究进展.福建农业学报.2012,26(5):911
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27、3-456. 12 陈峥,刘波,车建美,等.龙眼微生物保鲜菌FJAT-0809-GLX发酵液丙酮萃取物的成分分析,2012,27(3):294-298.13 陈峥,刘波,朱育菁,等. pH条件对短短芽胞杆菌FJAT-0809-GLX次生代谢物产生的影响. 2012,27(1）:71-76.14 宋亚军,杨瑞馥,郭兆彪,等. 若干需氧芽胞杆菌芽胞脂肪酸成分分析微生物通报,2001,28(1）:23-28.15 Bundy JG,Willey TL,Castel RS,et alDiscrimination of pathogenic clinical isolates and laborator
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29、Moen et al. Fractionation and characterization of bio-oil from microwave-assisted pyrolysis of corn stover. Int J Agric & Biol Eng.2010,3(3):1-8.20 J. Liu, Y. Lei, F. Wang, Y. Yi, Y. Liu, G. Wang. Immunostimulatory activities of specific bacterial secondary metabolite of Anoxybacillus flavithermus s
30、train SX-4 on carp, Cyprinus carpio. Journal of Applied Microbiology,2011,110(4）:1056-1064.Editors note: Judson Jones is a meteorologist, journalist and photographer. He has freelanced with CNN for four years, covering severe weather from tornadoes to typhoons. Follow him on Twitter: jnjonesjr (CNN)
31、 - I will always wonder what it was like to huddle around a shortwave radio and through the crackling static from space hear the faint beeps of the worlds first satellite - Sputnik. I also missed watching Neil Armstrong step foot on the moon and the first space shuttle take off for the stars. Those
32、events were way before my time.As a kid, I was fascinated with what goes on in the sky, and when NASA pulled the plug on the shuttle program I was heartbroken. Yet the privatized space race has renewed my childhood dreams to reach for the stars.As a meteorologist, Ive still seen many important weath
33、er and space events, but right now, if you were sitting next to me, youd hear my foot tapping rapidly under my desk. Im anxious for the next one: a space capsule hanging from a crane in the New Mexico desert.Its like the set for a George Lucas movie floating to the edge of space.You and I will have
34、the chance to watch a man take a leap into an unimaginable free fall from the edge of space - live.The (lack of) air up there Watch man jump from 96,000 feet Tuesday, I sat at work glued to the live stream of the Red Bull Stratos Mission. I watched the balloons positioned at different altitudes in t
35、he sky to test the winds, knowing that if they would just line up in a vertical straight line we would be go for launch.I feel this mission was created for me because I am also a journalist and a photographer, but above all I live for taking a leap of faith - the feeling of pushing the envelope into
36、 uncharted territory.The guy who is going to do this, Felix Baumgartner, must have that same feeling, at a level I will never reach. However, it did not stop me from feeling his pain when a gust of swirling wind kicked up and twisted the partially filled balloon that would take him to the upper end
37、of our atmosphere. As soon as the 40-acre balloon, with skin no thicker than a dry cleaning bag, scraped the ground I knew it was over.How claustrophobia almost grounded supersonic skydiverWith each twist, you could see the wrinkles of disappointment on the face of the current record holder and capc
38、om (capsule communications), Col. Joe Kittinger. He hung his head low in mission control as he told Baumgartner the disappointing news: Mission aborted.The supersonic descent could happen as early as Sunday.The weather plays an important role in this mission. Starting at the ground, conditions have
39、to be very calm - winds less than 2 mph, with no precipitation or humidity and limited cloud cover. The balloon, with capsule attached, will move through the lower level of the atmosphere (the troposphere) where our day-to-day weather lives. It will climb higher than the tip of Mount Everest (5.5 mi
40、les/8.85 kilometers), drifting even higher than the cruising altitude of commercial airliners (5.6 miles/9.17 kilometers) and into the stratosphere. As he crosses the boundary layer (called the tropopause), he can expect a lot of turbulence.The balloon will slowly drift to the edge of space at 120,0
41、00 feet (22.7 miles/36.53 kilometers). Here, Fearless Felix will unclip. He will roll back the door.Then, I would assume, he will slowly step out onto something resembling an Olympic diving platform.Below, the Earth becomes the concrete bottom of a swimming pool that he wants to land on, but not too
42、 hard. Still, hell be traveling fast, so despite the distance, it will not be like diving into the deep end of a pool. It will be like he is diving into the shallow end.Skydiver preps for the big jumpWhen he jumps, he is expected to reach the speed of sound - 690 mph (1,110 kph) - in less than 40 se
43、conds. Like hitting the top of the water, he will begin to slow as he approaches the more dense air closer to Earth. But this will not be enough to stop him completely.If he goes too fast or spins out of control, he has a stabilization parachute that can be deployed to slow him down. His team hopes
44、its not needed. Instead, he plans to deploy his 270-square-foot (25-square-meter) main chute at an altitude of around 5,000 feet (1,524 meters).In order to deploy this chute successfully, he will have to slow to 172 mph (277 kph). He will have a reserve parachute that will open automatically if he l
45、oses consciousness at mach speeds.Even if everything goes as planned, it wont. Baumgartner still will free fall at a speed that would cause you and me to pass out, and no parachute is guaranteed to work higher than 25,000 feet (7,620 meters).It might not be the moon, but Kittinger free fell from 102,800 feet in 1960 - at the dawn of an infamous space race that captured the hearts of many. Baumgartner will attempt to break that record, a feat that boggles the mind. This is one of those monumental moments I will always remember, because there is no way Id miss this.
