结核分枝杆菌生物膜形成相关基因的筛选与鉴定

中国生物工程杂志

2013, Vol. 33

Issue (4): 15-21

研究报告

结核分枝杆菌生物膜形成相关基因的筛选与鉴定

刘霞^1,2, 郭庆龙¹, 王若珺¹, 王洪海¹, 裴秀英², 张雪莲¹

1. 复旦大学生命科学学院遗传学研究所遗传工程国家重点实验室上海 200433;
2. 宁夏医科大学基础医学院生物化学与分子生物学系宁夏回族自治区生殖与遗传重点实验室医学科学技术研究中心银川 750004

Screening and Identification of Mycobacterium tuberculosis Biofilm Formation Related Genes

LIU Xia^1,2, GUO Qing-long¹, WANG Ruo-jun¹, WANG Hong-hai¹, PEI Xiu-ying², ZHANG Xue-lian¹

1. State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, China;
2. Biochemistry and Molecular Biology Department of School of Basic Medical Sciences, Ningxia Medical University Key Laboratory of Reproduction and Heredity of Ningxia Hui Autonomous Region, Medical Sci-Tech Research Center, Yinchuan 750004, China

全文: PDF(695 KB) HTML

摘要： 目的:通过菌落表型变化并结合生物膜生长缺陷筛选并鉴定可能与生物膜形成相关基因。方法:利用带有Himar1转座子的MycoMarT7转座子系统建立结核分枝杆菌H37Ra随机插入突变库;筛选细菌表面结构发生变化和生物膜形成有变化的突变菌株;运用T-A克隆法并结合抗性标记挽救法获得突变菌株的随机插入基因侧翼序列从而鉴定突变基因,并运用生物信息学方法分析预测突变基因的功能。结果:通过菌落形态变化及生物膜缺陷表型筛选出39株突变株,成功鉴定其中16株突变株,涉及16个基因发生突变,其中5个与脂质代谢相关,4个与细胞壁合成相关、2个与中间代谢和呼吸作用相关、1个调节蛋白相关基因,1个毒力相关基因,1个PE/PPE家族基因,还有2个功能未知基因。结合生物膜形成缺陷分析,其中8个基因可能与H37Ra体外生物膜的形成相关。结论:成功构建库容量约为1×10⁴结核分枝杆菌转座子随机插入突变文库,筛选获得生物膜生长受损突变株及可能与结核分枝杆菌生物膜形成相关的基因信息,为后续深入开展生物膜形成机制研究奠定基础。

关键词： 结核分枝杆菌; MycoMarT7噬菌体转座子; 菌落形态; 生物膜

Abstract: Objective:To screen and identify the related genes which may be associated with biofilm formation of Mycobacterium tuberculosis (M. tuberculosis) based on the changes of colony phenotype and biofilm defects. Methods:A random mutagenesis library was constructed by using the MycoMarT7 transposon system. Mutants with colony morphological changes on solid agar plate or with defects in pellicle biofilm formation in liquid culture medium were selected. In order to determine the locus of mutation gene, the flanking fragments of gene were obtained using T-A cloning and resistance marker recovery method. Then, the function of mutated genes was analyzed and predicted by bioinformatics methods. Results: 39 mutants with colony morphological changes or biofilm defects were selected and the loci interrupted of 16 mutants were successfully identified. Among the 16 genes, 5 genes were associated with the regulation of lipid metabolism, 4 were participated in cell wall and cell processes, 2 were related to intermediary metabolism and respiration, the product of 1 gene might be a regulatory protein, 1 was reported as virulence gene in previous studies, 1 was PE/PPE family gene and the function of other genes were unknown. Among these genes, 8 genes might be related to deficient in forming pellicle biofilm. Conclusions: A M. tuberculosis transposon insertion mutant library of approximately 10 000 mutants was successfully constructed, pellicle biofilm formation defects were selected and biofilm formation related genes were identified. All results will be the basis of the further research of biofilm formation mechanism in M. tuberculosis.

Key words: Mycobacterium tuberculosis MycoMarT7 transposon Colony morphology Bacterial biofilm

收稿日期: 2013-01-21 出版日期: 2013-04-25

ZTFLH:

Q819

基金资助: 国家自然科学基金资助项目(30901828,81261120558)

通讯作者: 裴秀英, 张雪莲 E-mail: xuelianzhang@fudan.edu.cn;peixy@nxmu.edu.cn

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	刘霞
	郭庆龙
	王若珺
	王洪海
	裴秀英
	张雪莲

引用本文:

刘霞, 郭庆龙, 王若珺, 王洪海, 裴秀英, 张雪莲. 结核分枝杆菌生物膜形成相关基因的筛选与鉴定[J]. 中国生物工程杂志, 2013, 33(4): 15-21.

LIU Xia, GUO Qing-long, WANG Ruo-jun, WANG Hong-hai, PEI Xiu-ying, ZHANG Xue-lian. Screening and Identification of Mycobacterium tuberculosis Biofilm Formation Related Genes. China Biotechnology, 2013, 33(4): 15-21.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I4/15

[1] World Health Organization. Tuberculosis Fact Sheet, 2012.No.104.
[2] Onyebujoh P, Rook G A. Tuberculosis. Nat Rev Microbiol,2004,2:930-932.
[3] Ojha A K, Anand M, Bhatt A, et al.GroEL1: a dedicated chaperone involved in mycolic acid biosynthesis during biofilm formation in mycobacteria. Cell,2005,123(5):861-873.
[4] Ojha A K, Baughn A D, Sambandan D, et al. Growth of Mycobacterium tuberculosis biofilms containing free mycolic acids and harbouring drug-tolerant bacteria. Mol Microbial,2008,69(1):164-174.
[5] Anil K Ojha, Xavier Trivelli, Yann Guerardel, et al. Enzymatic hydrolysis of trehalose dimycolate releases free mycolic acids during mycobacterial growth in biofilms. J Biol Chem,2010,285(23):17380-17389.
[6] 黄晓敏,郑秋桦,管文华,等. 细菌生物膜的自然属性与致病性. 韶关学院学报,自然科学,2010, 31 (9):77-79. Huang X M, Zheng Q H, Guang W H, et al. Natural attributes and pathogenicity of bacterial biofilm. Journal of Shaoguan University,Natural Science,2010, 31 (9):77-79.
[7] 李建华,宋丰贵. 细菌生物膜形成与细菌耐药机制研究进展. 中国新药与临床杂志,2008,27 (1):70-73. Li J H, Song F G. Research advance in bacterial biofilm formation and its antimicrobial resistance mechanisms.Chinese Journal of New Drugs and Clinical Remedies, 2008, 27 (1):70-73.
[8] Jennifer M Pang, Emilie Layre, Lindsay Sweet, et al. The polyketide Pks1 contributes to biofilm formation in Mycobacterium tuberculosis. J Bacteriol,2011,194(3):715-721.
[9] Stoyan Bardarov, Jordan Kriakov, Christian Carriere, et al. Conditionally replicating mycobacteriophages: a system for transposon delivery to Mycobacterium tuberculosis. Proc Natl Acad Sci USA,1997,94(20): 10961-10966.
[10] Eruc J Rubin, Brian J Akerley, Veronica N Novik, et al. In vivo transposition of mariner-based elements in enteric bacteria and mycobacterial. Proc Natl Acad Sci USA,1999,96(4): 1645-1650.
[11] Sassetti C M, Boyd D H. Comprehensive identification of conditionally essential genes in mycobacterial. Proc Natl Acad Sci USA,2001,98(22):12712-12717.
[12] 张玮祥,于佳,高谦. 从海分枝杆菌菌落形态变化筛选与毒力相关基因的方法.复旦大学报(医学版),2009,36(5):596-600. Zhang W X, Yu J, Gao Q. A novel method of screening for virulence genes from colony phenotype in Mycobacterium marinum.Fudan University Journal of Medical Sciences,2009,36(5):596-600.
[13] 方海红. 结核分枝杆菌二级信使环鸟苷酸的生理功能.安徽:中国科学技术大学,2011. Fang H H. Physiological function of second messenger cyclic diguanylate signaling in Mycobacterium tuberculosis. Anhui: University of Science and Technology of China,2011.
[14] Parsek M R, Singh P K. Bacterial biofilms: an emerging link to disease pathogenesis. Microbiol,2003,57:677-701.
[15] Yamazaki Y, Danelishvili L, Wu M, et al. The ability to form biofilm influences Mycobacterium avium invasion and translocation of bronchial epithelial cells. Cell Microbiol,2006,8(5): 806-814.
[16] Frieden T R, Sterling T R, Munsiff S S, et al. Tuberculosis. The Lancet,2003,362(9387):887-899.
[17] Griffin J E, Gawronski J D, Dejesus M A, et al. High-resolution phenotypic profiling defines genes essential for mycobacterial growth and cholesterol catabolism. PLoS Pathog,2011,7(9):1002251.
[18] 高阳赵春燕.分枝杆菌PE/PPE蛋白家族的结构和功能. 中华结核和呼吸杂志,2012,35(8):612-614. Gao Y, Zhao C Y. The structure and function of the mycobacterial PE/PPE protein family. Chinese Journal of Tuberculosis and Respiratory Diseases,2012,35(8):612-614.

[1]	邵映芝,车鉴,程驰,江志阳,薛闯. 分子生物学方法提高电活性微生物胞外电子传递效率的研究进展^*[J]. 中国生物工程杂志, 2021, 41(6): 50-59.
[2]	段海荣,魏赛金,黎循航. 铜绿假单胞菌中鼠李糖脂生物合成的研究进展^*[J]. 中国生物工程杂志, 2020, 40(9): 43-51.
[3]	谭杨,刘胜,罗凤玲,章晓联. 结核分枝杆菌H37Rv刺激巨噬细胞后差异表达lncRNA分析及鉴定 ^*[J]. 中国生物工程杂志, 2018, 38(5): 1-9.
[4]	陆健, 江佳稀, 刘建平, 王洪海. 结核分枝杆菌抗原重组酿酒酵母免疫诱导小鼠特异性免疫应答[J]. 中国生物工程杂志, 2014, 34(11): 47-53.
[5]	徐丹, 刘敏, 孔菊, 李校堃, 姜潮. CFP10-ESAT-6-MPB64在杆状病毒系统中的表达纯化及免疫原性鉴定[J]. 中国生物工程杂志, 2014, 34(06): 23-30.
[6]	李龙杰, 周刚, 施庆珊, 欧阳友生, 陈仪本, 胡文锋. 产生物膜菌株的分离鉴定及其产膜特性分析[J]. 中国生物工程杂志, 2013, 33(11): 38-43.
[7]	王晓娜, 米志强, 安小平, 李建彬, 范华昊, 张文慧, 张博, 黄勇, 周丽君, 童贻刚. 从大容量噬菌体抗体库中筛选抗Acr蛋白人源单链抗体[J]. 中国生物工程杂志, 2012, 32(09): 22-27.
[8]	丁笠, 王秀云, 齐海迪, 李海鑫, 周雅琼, 陈耀祖, 张娟, 王旻. 抗血管内皮生长因子受体2双价单链抗体的构建表达及其活性研究[J]. 中国生物工程杂志, 2011, 31(8): 1-6.
[9]	王建华, 权春善, 赵朋超, 范圣第. DKP对3株病原菌生物膜的抑制作用研究[J]. 中国生物工程杂志, 2011, 31(8): 61-65.
[10]	李浩, 殷瑛, 毛亚丽, 董大勇, 张军, 付玲, 郭继红, 徐俊杰, 陈薇. 结核分枝杆菌ESAT-6蛋白的重组表达及膜定位研究[J]. 中国生物工程杂志, 2011, 31(5): 55-59.
[11]	李浩殷瑛毛亚丽董大勇张军付玲郭继红徐俊杰陈薇. 结核分枝杆菌ESAT-6蛋白的重组表达及膜定位研究[J]. 中国生物工程杂志, 2011, 31(05): 0-0.
[12]	周爱萍陈艳炯李薇张旭燕徐纪茹. 结核杆菌DnaA蛋白在耻垢分枝杆菌中的同源表达[J]. 中国生物工程杂志, 2010, 30(08): 72-75.
[13]	田鑫,廖强,党楠,朱恂,王永忠. 营养及水力条件影响光合细菌生物膜生长特性实验[J]. 中国生物工程杂志, 2009, 29(04): 67-72.
[14]	柯为. 青枯假单胞菌致病性的分子生物学[J]. 中国生物工程杂志, 1987, 7(2): 57-57.

Viewed

Full text

Abstract

Cited

Shared

Discussed